Frontiers in Clinical Drug Research - CNS and Neurological Disorders: Volume 4 [1 ed.] 9781681082950, 9781681082967

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Frontiers in Clinical Drug Research - CNS and Neurological Disorders Volume 4 Edited By Atta-ur-Rahman, FRS Kings College University of Cambridge Cambridge UK

 

Frontiers in Clinical Drug Research - CNS and Neurological Disorders Volume # 4 ISSN (Online): 2214-7527 ISSN (Print): 2451-8883

Frontiers in Clinical Drug Research - CNS and Neurological Disorders Editor: Atta-ur-Rahman, FRS ISBN (eBook): 978-1-68108-295-0 ISBN (Print): 978-1-68108-296-7 ©[2016], Bentham eBooks imprint. Published by Bentham Science Publishers – Sharjah, UAE. All Rights Reserved. 

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CONTENTS PREFACE .................................................................................................................................................................... i LIST OF CONTRIBUTORS ..................................................................................................................................... iii CHAPTER 1 MULTIPLE SCLEROSIS DRUG THERAPY: FROM PHARMACEUTICAL DOWN TO CELLULAR AND MOLECULAR APPROACH

THE CLASSICAL .............................. 3

5REHUWD5LJROLR(OLVD%DOODULQL0DULD*ULPROGL0DUJKHULWD*DUGLQHWWLand*DEULHOH'L6DQWH MULTIPLE SCLEROSIS ................................................................................................................................... 4 Epidemiology, Environmental Agents and Genetics .................................................................................... 5 Pathogenesis ................................................................................................................................................... 8 Animal Models ............................................................................................................................................. 11 Different Immune System Players on the MS Stage .................................................................................... 14 T-cells ................................................................................................................................................... 14 B-cells ................................................................................................................................................... 17 Other Immune System Cells and the Innate Immune Response ........................................................... 18 Histopathology ............................................................................................................................................ 20 Active Lesions ....................................................................................................................................... 22 Chronic Plaques ................................................................................................................................... 22 Remyelinated Plaques .......................................................................................................................... 23 Clinical Features and Diagnostic Criteria .................................................................................................... 24 DISEASE-MODIFYING DRUGS .................................................................................................................... 29 Steroids ......................................................................................................................................................... 30 Injectable Drugs ........................................................................................................................................... 31 Beta-Interferons ................................................................................................................................... 31 Glatiramer Acetate ............................................................................................................................... 33 Oral Drugs .................................................................................................................................................... 34 Teriflunomide ....................................................................................................................................... 34 Dimethyl Fumarate (DMF, BG-12) ..................................................................................................... 35 Fingolimod (FTY720) ........................................................................................................................... 36 Conventional Immunosuppressants .............................................................................................................. 38 Cyclophosphamide ............................................................................................................................... 38 Azathioprine ........................................................................................................................................ 39 Mitoxantrone ........................................................................................................................................ 40 Laquinimod .......................................................................................................................................... 40 Methotrexate ........................................................................................................................................ 41 Biologics (Monoclonal Antibodies) ............................................................................................................. 41 Natalizumab ......................................................................................................................................... 43 Alemtuzumab (Campath-1H) ................................................................................................................ 45 RECENT EMERGING BIOLOGICAL AND EXPERIMENTAL THERAPEUTICAL APPROACHES ....................................................................................................................................................................... 46 B-Cells ......................................................................................................................................................... 47 Rituximab ............................................................................................................................................ 47 Ocrelizumab and Ofatumumab ............................................................................................................ 49 MEDI-551 ............................................................................................................................................ 52 Mast Cells ..................................................................................................................................................... 53 Masitinib mesylate ................................................................................................................................ 53 Cytokines and Chemokines .......................................................................................................................... 54 Daclizumab .......................................................................................................................................... 54 Tabalumab (LY2127399) ...................................................................................................................... 57

MOR103 .............................................................................................................................................. Secukinumab ......................................................................................................................................... Tolerogenic Vaccines for MS ...................................................................................................................... Stem Cells .................................................................................................................................................... Haematopoietic Stem Cells (HSCs) ...................................................................................................... Mesenchymal Stem Cells (MSCs) ......................................................................................................... Helminths .................................................................................................................................................... Vitamin D ..................................................................................................................................................... Remyelination Strategies in MS ................................................................................................................... BIIB033 ................................................................................................................................................ rHIgM22 ............................................................................................................................................... CONFLICT OF INTEREST ............................................................................................................................. ACKNOWLEDGEMENTS ............................................................................................................................... ABBREVIATIONS ............................................................................................................................................ REFERENCES ...................................................................................................................................................

58 59 61 65 65 68 70 73 74 75 77 79 80 80 82

CHAPTER 2 PROSPECTIVE THERAPIES FOR ALZHEIMER DISEASE: BIOMARKERS, CLINICAL TRIALS AND PRECLINICAL RESEARCH ...................................................................................................... 114 -RIUH*HOO%RVFK*LVHOD(VTXHUGD&DQDOV/DLD0RQWROLX*D\Dand6DQGUD9LOOHJDV I. INTRODUCTION ........................................................................................................................................ 115 I.1. Background .......................................................................................................................................... 115 I.2. Alzheimer's Disease and the Need for New Drugs .............................................................................. 116 I.3. The Amyloid Cascade Hypothesis (ACH) ........................................................................................... 117 I.4. Correlation among Aβ and other AD Hallmarks ................................................................................. 120 I.5. Genetics of AD .................................................................................................................................... 123 I.5.1. APP ........................................................................................................................................... 123 I.5.2. PSEN1/PSEN2 .......................................................................................................................... 125 I.5.3. APOE ....................................................................................................................................... 125 I.5.4 . APOJ/CLU ............................................................................................................................... 130 I.5.5. The Role of Genetic Testing in Clinical Care ........................................................................... 130 II. THE ROLE OF BIOMARKERS IN EARLY DIAGNOSIS AND CLINICAL TRIALS ASSESSMENT ..................................................................................................................................................................... 131 II.1. Biochemical Markers .......................................................................................................................... 132 II.1.1. Cerebrospinal Fluid Biomarkers ............................................................................................. 132 II.1.2. Blood Biomarkers .................................................................................................................... 135 II.2. Imaging-based Biomarkers ................................................................................................................. 137 II.2.1. Structural Magnetic Resonance Imaging ................................................................................ 137 II.2.2. Diffusion Tensor Imaging ........................................................................................................ 137 II.2.3. Functional Magnetic Resonance Imaging ............................................................................... 138 II.2.4. Proton Magnetic Resonance Spectroscopy ............................................................................. 138 II.2.5. Fluorodeoxyglucose-Positron Emission Tomography ............................................................ 139 II.2.6. Amyloid-Positron Emission Tomography ................................................................................ 139 II.3. The Continuum of AD: Diagnosis and Prognosis .............................................................................. 140 II.4. Regulatory Framework ....................................................................................................................... 142 III. PROSPECTIVE THERAPIES ................................................................................................................. 144 III.1. Non-immunologic Therapies ............................................................................................................. 144 III.1.1. Clinical Trials ........................................................................................................................ 145 III.1.2. Preclinical Studies ................................................................................................................. 151 III.2. Immunotherapy ................................................................................................................................ 153 III.2.1. Active Immunotherapy ........................................................................................................... 155 III.2.2. Passive Immunotherapy ......................................................................................................... 159 CONCLUSION AND FUTURE PERSPECTIVES ...................................................................................... 168

CONFLICT OF INTEREST ........................................................................................................................... FUNDING ......................................................................................................................................................... ACKNOWLEDGMENTS ............................................................................................................................... REFERENCES .................................................................................................................................................

171 171 171 171

CHAPTER 3 AT THE CROSSROAD BETWEEN NEURONAL HYPEREXCITABILITY AND NEUROINFLAMMATION: NEW THERAPEUTIC OPPORTUNITIES FOR ALZHEIMER’S DISEASE? 192 &KHOVHD&DYDQDJKand6ODYLFD.UDQWLF INTRODUCTION ............................................................................................................................................ 1. BACKGROUND ON AD ............................................................................................................................. 1.1. APP Processing ................................................................................................................................... 1.2. Genetics of AD .................................................................................................................................... 1.3. Clinical Trials & Treatment Strategies ................................................................................................ 2. PRECLINICAL AVENUES ........................................................................................................................ 2.1. Neuroinflammation & Tumor Necrosis Factor-α ............................................................................... 2.2. Network Hyperexcitability .................................................................................................................. 3. CROSS-TALK BETWEEN SYNAPTIC HYPEREXCITABILITY & TNF ................................ CONCLUSION ................................................................................................................................................. CONFLICT OF INTEREST ........................................................................................................................... ACKNOWLEDGEMENTS ............................................................................................................................. FUNDING ......................................................................................................................................................... REFERENCES .................................................................................................................................................

193 194 194 195 198 200 201 205 209 212 212 212 213 213

CHAPTER 4 TREATMENT OF DIABETIC NEUROPATHY – CURRENT POSSIBILITIES AND PERSPECTIVES .................................................................................................................................................. 227 -DUPLOD9RMWNRYi0LULDPýLOMDNRYiand3HWHU%iQRYþLQ INTRODUCTION ............................................................................................................................................ DIABETES MELLITUS .................................................................................................................................. CHRONIC COMPLICATIONS OF DIABETES MELLITUS ................................................................... DIABETIC NEUROPATHY ........................................................................................................................... ETIOPATHOGENESIS OF DIABETIC NEUROPATHY .......................................................................... Advanced Glycation End Products ............................................................................................................ Oxidative Stress .......................................................................................................................................... Gene Polymorphisms Of Antioxidant Enzymes And Chronic Diabetic Complications ..................... Polyol Pathway ........................................................................................................................................... Protein Kinase C ......................................................................................................................................... Hexosamine Pathway ................................................................................................................................. Low C-peptide Concentration .................................................................................................................... Proinflammatory Cytokines ....................................................................................................................... Neurotrophic Factors .................................................................................................................................. Angiotensin-converting Enzyme ................................................................................................................ Kinin B1 Receptor ...................................................................................................................................... TREATMENT OF DIABETIC NEUROPATHY ......................................................................................... Treatment of Diabetes Mellitus - Adequate Compensation ....................................................................... Supportive and Additional Treatment ........................................................................................................ Alpha-lipoic Acid ................................................................................................................................ Symptomatic Treatment ............................................................................................................................. Treatment in Experimental and Clinical Studies ........................................................................................ Anti-inflammatory Drugs ................................................................................................................... Neurotrophic Factors ......................................................................................................................... Angiotensin Converting enzyme (ACE) Inhibitors ............................................................................. Novel Drugs with Antioxidant Functions ...........................................................................................

228 229 233 235 237 241 242 243 245 246 247 247 248 249 249 250 250 250 252 252 263 268 269 273 274 278

Non-pharmacological Therapy .......................................................................................................... inhibitors of Epigenetic Modifications ............................................................................................... CONCLUSION ................................................................................................................................................. CONFLICT OF INTEREST ........................................................................................................................... ACKNOWLEDGEMENT ............................................................................................................................... REFERENCES .................................................................................................................................................

279 280 281 282 282 282

CHAPTER 5 THE NOW AND TOMORROW OF MIGRAINE TREATMENTS ........................................ 297 6HILN(YUHQ(UGHQHUand7XUJD\'DONDUD INTRODUCTION ............................................................................................................................................ PATHOPHYSIOLOGICAL OVERVIEW .................................................................................................... ACUTE ATTACK TREATMENT ................................................................................................................. Currently Available Drugs ......................................................................................................................... Nonsteroidal Anti-inflammatory Drugs (NSAIDs) ............................................................................. Ergot Alkaloids and Triptans ............................................................................................................. Combination Regimens ...................................................................................................................... Narcotic Analgesics ............................................................................................................................ Hyperbaric Oxygen Therapy .............................................................................................................. Medication Overuse Headache .......................................................................................................... Novel Targets for Attack Treatment .......................................................................................................... CGRP ................................................................................................................................................ NOS Inhibition .................................................................................................................................... TRP Channels ..................................................................................................................................... Glutamate Receptors .......................................................................................................................... PACAP-38 .......................................................................................................................................... Cannabinoids ..................................................................................................................................... The Parenchymal Inflammatory Cascade .......................................................................................... PROPHYLACTIC TREATMENT ................................................................................................................. Currently Available Drugs ......................................................................................................................... Antiepileptics ...................................................................................................................................... Antidepressants .................................................................................................................................. Beta Blockers ...................................................................................................................................... Calcium Channel Blockers ................................................................................................................. Glutamate Receptor Antagonists ........................................................................................................ Triptans .............................................................................................................................................. Novel Targets for Prophylactic Treatment ................................................................................................. CGRP ................................................................................................................................................ NOS Inhibition .................................................................................................................................... Gap Junction Blockers ....................................................................................................................... Renin-Angiotensin System .................................................................................................................. Acid Sensing Ion Channels ................................................................................................................. Botulinum Neurotoxin ........................................................................................................................ CONCLUDING REMARKS .......................................................................................................................... CONFLICT OF INTEREST ........................................................................................................................... ACKNOWLEDGEMENTS ............................................................................................................................. REFERENCES .................................................................................................................................................

298 298 301 301 301 303 307 307 308 308 308 308 310 311 311 311 312 312 313 314 314 315 316 316 317 317 318 318 318 319 319 320 320 322 322 322 322

CHAPTER 6 THE NOW AND TOMORROW OF ISCHEMIC STROKE TREATMENT ......................... 346 (WKHP0XUDW$UVDYDand7XUJD\'DONDUD ADVANCES IN TREATMENT OF ACUTE ISCHEMIC STROKE ......................................................... Recanalization/Reperfusion ....................................................................................................................... Neuro-Glial Protection ............................................................................................................................... Prevention of Secondary Complications ....................................................................................................

347 347 354 355

ADVANCES IN SECONDARY PROPHYLAXIS OF ISCHEMIC STROKE .......................................... CONCLUDING REMARKS ........................................................................................................................... CONFLICT OF INTEREST ........................................................................................................................... ACKNOWLEDGEMENTS ............................................................................................................................. REFERENCES .................................................................................................................................................

356 360 361 361 361

SUBJECT INDEX .................................................................................................................................................... 371

i

PREFACE The book series Frontiers in Clinical Drug Research – CNS and Neurological Disorders contains the most noteworthy recent developments for the treatment of several neurological disorders. The volume 4 of this book series is a collection of well written cutting edge reviews contributed by some of the most prominent researchers in the field. Multiple sclerosis (MS) is a potentially disabling disease of the central nervous system in which the immune system attacks the myelin and causes communication complications between the brain and the body. It affects some two million persons across the world. In chapter 1, Rigolio et al. present an excellent overview of the old and new cellular and molecular therapeutic approaches to fight MS neurodegenerative progression. Alzheimer’s disease (AD) is the most common age-related multifactorial neurodegenerative disease which is described as the failure of cognitive performance and behavioral capabilities and there is a desperate need for the treatment to prevent, stop or reverse this devastating disorder. The magnitude of the problem can be judged from the fact that of the 46.8 million people suffering from dementia worldwide, the majority belong to those suffering from AD and this number is expected to triple over the next 30 years. In chapter 2 Villegas et al. discuss several biomarkers for early detection, clinical trials under way on new drugs, and preclinical research involving different approaches to tackle Alzheimer’s disease. In chapter 3 Cavanagh & Krantic discuss two aspects of AD, hyperexcitability and neuroinflammation, which can be used for future therapeutic intervention. They also summarize the studies, which are related to hyperexcitability and neuroinflammation in the early phases of the disease. Diabetic neuropathy (DN) is characterized by neurodegeneration associated with diabetes mellitus which belongs to the earliest and most frequent chronic diabetic complications. It may occur in clinical form or in subclinical form. High blood sugar affects nerve fibers throughout the body, but diabetic neuropathy most often damages nerves in the legs and feet. Vojtková et al. in chapter 4 present a comprehensive review about current possibilities and future perspectives in the management and treatment of diabetic neuropathy. Migraine is a primary headache disorder characterized by moderate to severe recurrent headaches. Erdener & Dalkara in chapter 5 focus on the current and future therapeutic agents for acute and prophylactic migraine treatment and their mechanisms of action. In chapter 6, the Arsava and Dalkara present a review on developments in the treatment of acute ischemic stroke. They also discuss the recent advancements in the secondary prophylaxis of ischemic

ii

stroke. The 4th volume of the book series represents the results of a significant amount of work by eminent researchers in the field. I am grateful to the authors for these valuable contributions. I also wish to thank the excellent team of Bentham Science Publishers, especially Mr. Shehzad Naqvi (Senior Manager Publications), led by Mr. Mahmood Alam (Director Publications), who deserve our appreciation.

Atta-ur-Rahman, FRS Kings College University of Cambridge Cambridge UK

iii

List of Contributors Chelsea Cavanagh

Department of Neuroscience, Douglas Hospital Research Center, Montreal, Quebec

Elisa Ballarini

Experimental Neurology Unit, School of Medicine and Surgery, Università Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy NeuroMI- Milan Center for Neuroscience, San Gerardo Hospital, Via Pergolesi, 33 - 20052, Monza, Italy

Ethem Murat Arsava

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Gabriele Di Sante

Institute of General Pathology, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168, Rome, Italy

Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain Gisela Esquerda-Canals Departament de Biologia Cellular, Fisiologia i Immunologia, Unitat de Citologia i Histologia, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain Jofre Güell-Bosch

Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain

Jarmila Vojtková

Department of Pediatrics, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital Martin, Martin, Slovakia

Laia Montoliu-Gaya

Departament de Biologia Cellular, Fisiologia i Immunologia, Unitat de Citologia i Histologia, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain

Maria Grimoldi

Experimental Neurology Unit, School of Medicine and Surgery, Università Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy

Margherita Gardinetti

Experimental Neurology Unit, School of Medicine and Surgery, Università Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy

Miriam Čiljaková

Department of Pediatrics, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital Martin, Martin, Slovakia

Peter Bánovčin

Department of Pediatrics, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital Martin, Martin, Slovakia

Roberta Rigolio

Experimental Neurology Unit, School of Medicine and Surgery, Università Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy NeuroMI- Milan Center for Neuroscience, San Gerardo Hospital, Via Pergolesi, 33 - 20052, Monza, Italy

iv Sandra Villegas

Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain

Slavica Krantic

Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche, des Cordeliers, F-75006, Paris, France INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France Centre National de la Recherche Scientifique (ou CNRS) ERL 8228, Centre de Recherche des Cordeliers, Paris, France

Sefik Evren Erdener

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Turgay Dalkara

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey

Frontiers in Clinical Drug Research - CNS and Neurological Disorders, 2016, Vol. 4, 3-113

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CHAPTER 1

Multiple Sclerosis Drug Therapy: From the Classical Pharmaceutical Down to Cellular and Molecular Approach Roberta Rigolio1,2,*, Elisa Ballarini1,2, Maria Grimoldi1, Margherita Gardinetti1, Gabriele Di Sante3 Experimental Neurology Unit, School of Medicine and Surgery, Università Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy 1

NeuroMI- Milan Center for Neuroscience, San Gerardo Hospital, Via Pergolesi, 33 - 20052, Monza, Italy 2

Institute of General Pathology, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy 3

Abstract: Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) affecting over 2.000.000 individuals around the world. Although MS etiopathogenesis is still not completely defined environmental factor exposure and genetic background are relevant in disease development. Moreover, MS shows heterogeneous onset and course so that different disease forms can be described which are all characterized by motor and/or sensory and even cognitive impairment. Two steps in the disease progression can be described. First MS lesions are originated by the activated immune system which recognizes CNS myelin as a foreign element thus leading to the formation of demyelinated plaques that evolve into axonal damage and subsequent neurodegeneration over the time. Since the beginning MS therapy has been focused on counteracting immune system action. Nevertheless, besides the immunosuppressive/immunomodulating drugs such as Glatiramer acetate, Beta-interferons and steroids, the advance in the comprehension of the immune-mediated mechanisms has sustained the development and use of molecular * Corresponding author Roberta Rigolio: Experimental Neurology Unit, School of Medicine and Surgery, Università Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy; Tel: +39 (0)2 64488114; Fax: +39 (0)2 64488250; Email: [email protected]

Atta-ur-Rahman (Ed.) All rights reserved-© 2016 Bentham Science Publishers

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and cellular-focused approaches, e.g. monoclonal antibodies and stem cells. At the same time very few weapons are specifically available for fighting MS neurodegenerative progression. We report an overview on MS and both old and new therapeutic approaches to the disease.

Keywords: Alemtuzumab, Anti-Lingo-1 antibody, Daclizumab, Diseasemodifying drugs, Ethiopathology, Helminthes, Histopathology, Immune system, Masinitib mesylate, Monoclonal antibodies, MOR103, Multiple Sclerosis, Ocrelizumab, Ofatumumab, Remyelination strategies, Rituximab, Secukinumab, Stem cells, Tabalumab, Tolerogenic vaccines, Vitamin D. MULTIPLE SCLEROSIS Over the past 100 years the advances in immunology and neurobiology have led us to the current definition of Multiple Sclerosis (MS) as a chronic inflammatory disease of the central nervous system (CNS) primarily triggered by the activation of immune system elements against myelin sheath components, which is subsequently followed by irreversible damage to axons and neurons leading to permanent disability. Until now, no single etiopathogenetic factor has been identified and MS is generally considered to be a complex multifactorial autoimmune disease depending on genetic predisposition and environmental factors. MS is characterized by a dissemination of CNS lesions in time and space with heterogeneous signs and symptoms that usually indicate more than one lesion and that can be due to injury to any part of the neuraxis. Moreover MS clinical presentation and course are highly variable. Several disease types can be recognized: relapsing-remitting MS (RRMS), primary-progressive (PPMS), secondary-progressive (SPMS). Although our current pathogenetic concepts might be too simple to define such a multifaceted disease, our current knowledge of the MS-related immunological mechanisms has made possible the clinical viability of various effective immunomodulating/immunosuppressive strategies. These are mainly aimed at

Multiple Sclerosis Drug Therapy

FCDR - CNS and Neurological Disorders, Vol. 4 5

limiting/modifying the inflammation-related component of the disease so that the main part of the research activity and treatments has been focused on the RRMS form, while the MS symptoms are mainly managed by means of non-specific symptomatic therapies. Epidemiology, Environmental Agents and Genetics MS is the most frequently diagnosed neurological disease leading to nontraumatic disability among young adults, affecting more than 2 million individuals worldwide [1]. As with many other autoimmune diseases, the prevalence of MS is 2-3 times as high in women as in men and this ratio seems to have increased slightly over time, mainly in the polar latitude countries [2]. The incidence of MS has increased in various countries due both to the improvement in diagnostic tools and to the lengthening of patients’ lives together with the improvement in hygiene conditions over the last century [3]. MS can affect individuals at any age with the first clinical signs occurring most frequently between 20 and 40 years of age although the disease can occur even in individuals over 50 years of age; pediatric MS has also been recognized and diagnostic criteria have recently been redefined [4, 5]. The prevalence of the disease has been shown to increase from the equator to the pole with important exceptions such as the Sardinian and the Inuit populations in the Mediterranean and Canada respectively. Moreover, the migration studies which have shown changes in the risk of MS susceptibility in individuals moving to different MSrisk areas before pubescence [6] and the fluctuations in the rates of MS patients in some areas such as the North Atlantic islands have suggested a strong interaction between genetically-based and environmental factors, i.e. viruses, vitamin D deficiency and other factors [1, 7]. Thanks to these epidemiological studies, a hygiene hypothesis has been put forward suggesting that the higher incidence of MS in industrialized countries is due to certain infections or inappropriate responses to certain substances [8]. This notion is supported by analogies of the geographical distribution of certain infections [9], and by the fact that, in developed countries, certain typical childhood diseases, such as measles or mononucleosis, are contracted at later

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ages, and also by a recent study that noted an amelioration of the clinical course of MS in the presence of parasitic infestations [10]. Some scientists have associated MS with the seasons and, consequently, with seasonal infections, such as arbovirus and epidemic influenza, or with zoonoses, such as visna from sheep and the canine distemper virus from dogs. MS and infectious agents, particularly viruses, and more recently the human microbiome have been widely studied in order to investigate the manner in which they interact with each other and with the human genome to influence the risk of MS; however, until now there has been an absence of conclusive data [11]. Furthermore, the different pathological lesions described and classified in MS probably derive from multiple mechanisms of pathogenesis, and this accounts for the fact that there could be multiple causes and mechanisms involved in its etiology. In particular, viruses have been widely detected in MS patients due to their ability to induce demyelination and axonal loss through many different mechanisms, directly and indirectly [12]. Recent reports have focused on herpesviruses, i.e. HHV6 [13], and Chlamydia pneuomoniae [14], all ubiquitous and essentially asymptomatic infections occurring especially in childhood, while Epstein-Barr virus (EBV) antigens such as EBNA-1 and VCA are more frequently present in MS patients [15]. In fact, the presence of EBV latency-associated genes has been demonstrated in inflamed tissue and lesions of MS brains [16], although EBV detection in CSF (cerebrospinal fluid) and CSF-associated cells has shown no variation in results between MS and other neurologically affected patients [17]. The discovery of human endogenous retroviruses (HERVs) has raised other questions regarding their relationship with autoimmune diseases: in particular, MSRV (MS-associated retroviral virus) has been correlated with progression of the disease [18]. At the moment there is no definitive virus identified that triggers MS. The landmark discovery of a group of surface molecules has increased understanding regarding the way in which the host rapidly responds to invading pathogens. This group includes named pattern-recognition receptors (PRRs) such

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as CD14, β2-integrins, CR1/CD35, CR2/CD21, all receptors of conserved structures of bacteria, and also includes Toll-like receptors (TLRs) [19]. Proof that infections might play a role in triggering MS comes from a recent post-infectious model of Experimental Autoimmune Encephalomyelitis (EAE) [20]. Not only pathogenic but also commensal bacteria may be involved in the induction and modulation of CNS autoimmunity. The gut microbiota, for example, influence T-helper cells (Th) polarization and the development of EAE [21] by direct activation of T-cells, influencing the local system of the antigen presenting cells (APC) and releasing immunoactive metabolites, thereby contributing to the generation of a proinflammatory context and the breakdown of tolerance. Accordingly, autoimmune cells seem to be primed in the peripheral tissues before invading the CNS [22]. The encounter of pathogenic and nonpathogenic microbes profoundly modifies the immune system with effects that can range from protection to the induction of autoimmunity [23]. Both types of microbial agents and the individual genetic background modulate the balance between these possible outcomes. Infectious agents may promote autoimmunity of the CNS through distinct mechanisms. On the one hand, they may directly infect the CNS and either alter the blood brain barrier (BBB) or induce the release of sequestered autoantigens. On the other hand, they may prime self-reactive T-cells in the periphery by antigenic cross-reactivity (molecular mimicry), and these may migrate into the CNS and contribute to its damage. Finally, the occurrence and/or severity of MS may be the result of an encounter with several infectious agents, each contributing in different ways to the full-blown disease. The active form of vitamin D, the 1,25-dihydroxyvitamin D3, plays a central role in the modulation of the immune response [24] and its receptor (vitamin D receptor, VDR) levels and kinetics are crucial for T-cell proliferation [25]. A growing body of evidence has connected vitamin D and autoimmune diseases. With regard to the geographical distribution of MS, the sunlight exposure necessary for the production of the active form of vitamin D has been correlated with MS susceptibility [26]. Recent studies have investigated the VDR gene polymorphisms and their relationship with MS [27], illustrating why different patients respond differently to vitamin D administration [28].

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Although MS cannot be strictly included in the category of Mendelian genetic hereditary diseases, both the increased MS risk in monozygotic twins and its prevalence in dizygotic twins (14%-25% and 2-5% respectively) [29] taken together with the family clustering of the disease and the geometrical fall of MS occurrence beyond first-degree relatives, suggest that MS is a polygenetic disease. While the Human Leukocyte Antigen (HLA) DRB1*1501 gene and haplotype association to MS have been clearly recognized, several other genes and genetic traits have been associated with a susceptibility factor for MS as well as affecting the course of the disease [30]. Besides the mere association between MS and genetic traits, the role of the epigenome in MS is gradually gaining ground based on epidemiological studies and increased knowledge of the mechanisms responsible for gene expression control exerted by several environmental factors, i.e. UV light exposure and vitamin D synthesis [31]. While the etiology of the disease has not yet been clarified, on the clinical side, several attempts have been made to define clear criteria both to diagnose MS and to associate different clinical manifestations with particular forms of MS. Pathogenesis Under normal conditions the immune system has the task of defending the body from external agents, mainly viruses and bacteria and exerts this control through lymphocytes, macrophages and other cells that circulate in the blood and that, in case of necessity, attack and destroy the foreign microorganisms, both directly and through the release of antibodies and other chemicals. In MS, the immune system attacks parts of the CNS mistaking them for extraneous agents. This mechanism of damage is defined as “autoimmune” or, more generally, “dysimmune”. One of the main targets of an impaired immune response to myelin is the “myelin basic protein” (MBP), which is one of the constituents of myelin itself. The cells of the immune system overcome the BBB and penetrate the CNS causing inflammation and loss of myelin. The causes of this alteration in the functioning of the immune system are many and are the subject of countless

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research studies. The presence of inflammatory cells in brain lesions reported by several studies, both in MS patients and animal models, contributed to consider MS as a disease mediated by anti-myelin antigen pathogenic CD4+ T-cells with the consequence of a T-cell-monocyte infiltration into the CNS and a wider neurodegenerative process [32, 33]. The auto-reactive T-cells migrate across the BBB and are involved in the damage to neurons, myelin sheaths and axons. Over the past few decades, it has been thought that in the pathogenesis of MS one of the crucial points of the involvement of auto-reactive T-cells is the immune privilege of the CNS: like other “immunological sanctuaries”, e.g. the testes, reinforcing the dogma that any inflammation seen in the CNS must be mitigated by the systemic inhibition of immune cells. The CNS presents a physiological reduction in resident immune cells, except for microglia, but we now know that the immunological processes are complex and that, like any other peripheral organ, the CNS needs circulating immune cells for its repair and control [34]. The balance between immune activity and the risk of an overwhelming response is the key to the relationship between the immune system and the CNS, and the main mechanisms capable of protecting the CNS from immune reactions are the central tolerance, the suppression of the immunological axis and the high BBB controlled cell trafficking. The BBB is a specialized structure with a fence function responsible for protecting the CNS from immune cells and pathogens [35]. It consists of a three layer barrier: endothelial cells, interconnected with each other by tight junctions (tighter than peripheral microvessels), the surrounding lamina propria formed by pericytes, and externally astrocytes associated with perivascular macrophages and mast cells [36]. In MS patients myelin auto-reactive T-cells are not negatively selected in the thymus [37] and the increased avidity and potency of the peripheral myelinspecific T-cells have been demonstrated [38]. Auto-reactive T-cells are activated through the cross-reaction of alloantigens (from microbes, for example) and myelin because of a similar sequence (molecular mimicry hypothesis) [39], or due to a non-specific event (bystander effect hypothesis) such as cytokines, chemokines, superantigens and TLRs [40]. Furthermore, for their activation T-

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cells need a two-step process composed of antigen recognition and costimulation: physiologically in the CNS, the absence of APC such as dendritic cells (DC), and the low expression of MHC molecules protects neurons from T-cell-mediated damage. In addition, it has been demonstrated that the CNS, in its healthy state, is not a good environment for T-cells due to the expression of many pro-apoptotic and immune regulator molecules such as Fas-ligand, B7-H1, TGFβ [41], somatostatin and VIP [42]. During neuroimmune disorders such as MS and EAE, the immune privilege of the CNS is compromised due to an unknown inciting event (discussed above) and auto-reactive effector CD4+ T-cells are ready to access their targets. To delimit the BBB the endothelial adhesion molecules, such as ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) are upregulated by cytokine production (IFNγ and TNFα in particular) [43] and interact with LFA-1 (Lymphocyte function-associated antigen-1) and VLA-4 (very late activation antigen-4) expressed by CD4+ T-cells. An important molecule involved in the VLA-4 binding is osteopontin, particularly expressed during relapse phases [44] and able to exacerbate EAE when administered to mice [45]. The ability to transmigrate via the transcellular and/or paracellular routes [46] is associated with the tight junction modulation through the metalloproteinases (MMPs) [47]. The perivascular phagocytes are able to reactivate CD4+ T-cells provoking their expansion, the invasion of CNS parenchyma and the consequent damage that may be direct (granzyme B-mediated) or through other cells such as CD8+ T-cells, Bcells, macrophages and microglia. The pathological role of these immune cells and each CD4+ T-cell subset is discussed below. The typical morphological aspect of MS is the primary demyelination of nerve axons able to reduce or even to block the signal conduction at the site of damage and the simultaneous involvement of a significant number of fibers results in neurological symptoms [48]. The recovery of the CNS inflammation and oedema and the glial ensheathment and remyelination are thought to coincide with restoration of CNS conduction and consequent clinical remission. In contrast in chronic MS the persistence of neurological dysfunction is related to irreversible axonal loss.

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Animal Models The most commonly used animal model for MS is EAE that, sharing clinical and pathological aspects with MS, provide an appropriate tool in the study of the inflammatory processes throughout the course of the disease and for the development of new treatments, although there are differences in the outcomes between MS and EAE. The induction of EAE in susceptible animals is obtained through the immunization with an emulsion of myelin antigens (one or a number of) or homogenated CNS with mineral oil adjuvant, able to stimulate the immune response directed against CNS antigens [49]. In 1930s Rivers et al. induced EAE for the first time in primates using homogenates of normal rabbit brain tissue. EAE as an autoimmune T-cel-mediated disease was hypothesized in 1947 by Kabat et al. with a model developed in monkeys, using myelin antigens dissolved in the “newly developed” Jules Freund oil and which was initially named Experimental “Allergic” Encephalomyelitis. Then in mice EAE was induced for the first time in 1949 with spinal cord homogenates [50]. Subsequently, the protocols for induction have been refined over the years, not only for the advent of Freund’s adjuvant, but also for the use of the pertussis toxin, specific mice strains and the identification of encephalitogenic myelin antigens such as myelin basic protein (MBP) and proteolipid protein (PLP), and, subsequently, myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG). Moreover, Paterson in 1960 showed that the disease could be transferred from an immunized rat to a naïve syngeneic one through the “lymph node cells”, this constituting the first “passive” or “transfer” model [51]. It was successively refined by Ben Nun in 1981 [52] who showed the possibility to induce the disease by adoptive transfer of in vitro activated myelin-specific CD4+ T-cells from EAE rats into naïve recipient ones [53]; thus the term “allergic” evolved into “autoimmune”. Both protocols of active and passive inductions are based on the same principle: activation of the circulating myelin-specific CD4+ T-cells that infiltrate the CNS

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crossing the BBB [54, 55]. APC (both resident and infiltrating CNS district) reactivate encephalitogenic T-cells, presenting myelin peptides complexed with major histocompatibility complex (MHC) class II and causing a subsequent cascade of events and inflammatory processes, including chemokines secretion involved in the recruitment of macrophages to the sites of T-cell activation.

Fig. (1). CNS infiltrating leucocytes and demyelinated area in EAE. Infiltrating leucocytes can be detected by Hematoxylin and Eosin staining and they are placed around the white matter blood vessels (B and C, white arrows) or even disseminated in the CNS parenchyma (B) compared to the healthy animals (A). Moreover demyelinated areas can be detected by using Luxol Fast blue staining and they appear as pale blue regions (E and F, white arrows) compared to the more colorful stained sections in the healthy animals (D).

Similarly to the pathology of MS, the neuro-inflammation results in cellular infiltrates composed by different leukocyte populations and focally demyelinated plaques in the CNS (Fig. 1). The development of numerous EAE models allowed the study of various clinical and pathological features of MS.

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In the SJL (H-2s) mouse strain, EAE can be actively induced with CNS homogenate, PLP, MOG or epitopes such as PLP139-151, PLP178-191, MOG92-106 or MBP84-104, and that develops a typical relapsing-remitting course of paralysis while other commonly used mouse strains are C57BL/6 (H-2b) mice in which EAE can be induced with MOG35-55 leading to a chronic progressive disease course, B10.PL and PL/J (H-2u) mice with acute disease and more recently NOD mice immunized with MOG35-55 that present a chronic progressive stage after initial relapsingremitting stages [56]. Although less commonly used, the EAE rat models have been principally developed using the Lewis rat strain and obtained after immunization with MBP or MBP peptide in complete Freund’s adjuvant generally leading to an acute and transient paralysis that is reversed after a few days, characterized by poor demyelination and mononuclear cell infiltration into the spinal cord. Both acute and chronic disease can be obtained instead in the Dark Agouti (DA) strain using MOG epitopes or homogenated spinal cord in CFA or IFA, while in the BrownNorway strain EAE can be induced with MOG in CFA. EAE can also be induced in rabbits [57] and guinea-pigs [58] which show inflammation in the spinal cord and brain at the same time, similarly to that which happens in humans with MS, while the marmosets, a non-human primate, are good models for studying the role of demyelinating antibodies in EAE [59, 60]. The limitations of the outbred species, such as the variability in disease induction and the low availability of purchasable reagents, have meant that the most commonly studied EAE models are rats and mice. The analysis of the pathogenic mechanisms in EAE is facilitated by the abundance of genetically engineered rodent models, useful in dissecting the genetic and environmental factors involved in the susceptibility to EAE [61]. Besides EAE, natural animal models of acute and chronic viral demyelinating diseases are good models for MS. They include the previously-mentioned Theiler’s and neurotropic hepatitis viruses in mice, the Visna virus in sheep, the caprine arthritis-encephalitis virus in goat, the SV40 in macaque monkeys and the canine distemper virus in dogs. Other MS models have been obtained in order to mirror the demyelinating processes in vivo, i.e. isoleucine and cuprizone [62], and

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to investigate anti-demyelinating approaches. Different Immune System Players on the MS Stage T-cells The classic textbook perspective regarding MS inflammation considers that peripheral and activated T-cells specific for myelin are able to cross the BBB by means of chemotactic [63] and adhesion molecules [64]. When they reach the CNS, they recognize specific target structures able to restimulate them through local APC [65], causing consequent damage to myelin sheaths and the successive recruitment and transmigration of other immune cells [66], such as B-cells and plasma cells [67], with the final result of demyelination. B-cells play a central role in the pathogenesis of MS, although there have been recent modifications to this classic model of MS as a T-mediated disease. In fact, not only as previously described the main genetic risk factor for MS is HLA-DR2, but also both CD4+ and CD8+ subpopulations have been identified in MS lesions [68]; particularly CD4+ T-cells myelin-specific circulate in periphery and can be revealed in the CSF of MS patients [69]. The relevance of T-cells in the pathogenesis of MS is confirmed by the successful therapies targeting T-cells, such as drugs involved in the block of leukocyte trafficking into the CNS or of the lymphocytes egress from lymph nodes [70]. CD4 - Th1 In 1986 the identification by Mosmann and Coffman of two subpopulations of activated effector CD4+ T-cells, then named Th1 and Th2 cells, because of their distinct pattern of cytokine production and their different involvement in immunity against pathogens and in autoimmunity and allergy. Th1 cells play an important role in the pathogenesis of MS, although in the recent years it has been widely debated. It has been suggested the BBB, initially crossed by only Th1 cells, facilitates a subsequent recruitment of other immune cells [55]. CD4- Th17 IL-17-expressing T-cells were proposed as a new Th lineage. Since their

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discovery, Th17 cells have been associated with autoimmune diseases and, in particular formally Th1 autoimmune responses like EAE have been attributed to the expansion of Th17 cells in the periphery, their CNS infiltration and demyelination [71]. IL-17A, initially cloned as CTLA-8, is the signature cytokine of this subset of T-cells. The reduction of the severity of the EAE treating mice with IL-17 blocking antibodies, confirms the central role of Th17 cells in the development and pathogenesis of EAE, while the block of IFN-γ production exacerbates the disease [72], suggesting that selective elimination of Th17 subset may protect against MS. Clinical trials studying the role of antibodies against IL17, IL-12p40 and IL-23 are currently in progress for a different autoimmune diseases [73 - 75]. Recently other cytokines have been suggested to be involved in EAE and MS. IL22 in particular, initially considered part of the Th17 signature, has now been shown to occur in a unique subset of CD4+ T-cells, termed Th22 cells whose number grows up in the peripheral blood (PB) and the CSF mainly in the active disease phase of remitting relapsing (RRMS) patients [76]. Th17 cells are highly sensitive to the inhibitory effect of IFNbeta because of their high expression of IFNaR1 [77], which may be involved in the IFNbeta effect in MS. By contrast, Th22 cells express low IFNaR1 levels [76] and are more resistant to the inhibitory effect of IFNbeta treatment. Th1/Th17 It has been observed that T-cells producing both IL-17 and IFNγ and expressing transcription factors such as T-bet (T-box expressed in T-cells) and ROR-γ-t (retinoid-related orphan receptors), infiltrate CNS during EAE. Therefore Th17 cells when transferred are able to switch to IFNγ production; these findings suggest a plasticity within these subsets [78, 79]. In particular T-bet expression seems to be involved in the encephalogenicity of T-cells, more than their cytokine profile [80]: in fact inhibiting this transcription factor EAE ameliorates decreasing both Th1 and Th17 cells population [81, 82]. Similar correlations have been found in brain lesions and with disease activity in MS patients through microarray analyses [83]. Interestingly, the enrichment of T-cells producing both IL-17 and IFNγ in active MS brain tissue suggests that both Th17 and Th1 subsets may be

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involved in MS [84]. CD8 Immunohistology of MS lesions shows a prevalence of CD8+ T-cells that display signs of clonality in inflamed plaques, CSF and blood, thereby suggesting an antigen-specific recruitment [85]. This represents a marked difference between MS and the experimental model EAE, where it is widely believed that the pathogenesis is due to a CD4-mediated response. In addition, data derived from the experimental models and from human subjects also indicate that the immune response spreads to other epitopes of the same antigen and to other self-molecules during the course of the disease. Recent literature indicates that CD8+ T-cells are critical in MS pathogenesis: killing of neurons and axonal injury have been correlated with cytotoxic granzyme B mediator and a number of CD8+ T-cells in MS patients [86]; CD4+ T-cell subset target therapies could be ineffective in some patients in clinical trials; the protective role of HLA-A2 (MHC I class gene) against the disease and the association of HLA-A3 and MS both imply an important role for CD8+ Tcells in the etiology of MS. In fact, it has been demonstrated that CD8+ T-cells secrete IL-17 providing evidence of their role in pathogenesis of MS [87]. Treg A growing body of evidence suggests that the overshooting autoimmune reactions are controlled by a subpopulation of T-lymphocytes named regulatory T-cells (Treg) that include both natural (nTreg) and adaptive (also termed inducible) Treg cells (iTreg) [88]. The transcription factor forkhead box P3 (FoxP3) expressed by CD25+/CD127lo+ T-cells (nTreg) is essential for preventing autoimmunity and for maintaining homeostasis [89, 90]. T regulatory-1 (Tr1), Th3 and CD8+ Treg cells usually have an immunosuppressive role through different cytokines such as IL-10, TGF-β or retinoic acid, and are known as adaptive Treg (iTreg) because of their ability to respond to foreign antigens. The discrepancy regarding the number and functions of these recently-discovered T-cell subsets is due to the fact that Treg markers are

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inducible also on effector cells and may confound the phenotypes. Their involvement in MS has been studied with reference to a possible imbalance in the regulation of the normally circulating and potentially auto-reactive T-cells specific for myelin antigens: Tr1 and IL-10 are reduced in MS patients [91] and nTreg have been found in the CSF of MS patients [92] in spite of their similar frequency in the peripheral blood; moreover, a subset of nTreg (CD39+) from RRMS patients had an impaired ability to suppress IL-17 [93]. Therefore, in the animal models of MS, it has been demonstrated that Treg can control the development and severity of the disease [94] by IL-10 [95] and/or TGFβ production [96] and that nTreg cell-based therapies in the mouse model are able to prevent EAE [97]. B-cells Since the 1950s, the presence of oligoclonal bands (OCB) has been considered as pathognomonic of MS and intrathecal IgG production can be detected in early, as well as in chronic, disease. Many efforts have been made to determine their specificities. CSF-derived plasma cells produce autoantibodies specific to myelin [98] and the MOG has been the prime candidate target antigen because of its localization on the outer surface of the myelin sheath. Nevertheless during demyelination MOG-specific autoantibodies can be detected in only a few MS patients [99] and the presence of OCB (mainly immunoglobulins) in the CSF is still unexplained. The role of B-cells in MS is controversial and not limited to autoantibody production. They may promote neuronal protection [100] although it has been clearly observed that their enrichment in the CSF is associated with a more severe course of the disease [67]. Moreover, their proliferation and clonal expansion within the CNS is due to a local environment capable of promoting their transmigration through CCL12 and CXCL13 chemokines [101] and their physiological organization in a pathological area, such as “pseudo-follicular” structures in the meninges of chronic MS patients [89, 102]. The efficacy of the monoclonal anti-CD20 antibody therapy (Rituximab, see below) with a reduction of the lesions without significant alterations of the immunoglobulin levels, suggested other functions of B-cells in MS; their roles in antigen presentation to Th-cells and antigen transport to cervical lymph nodes has been demonstrated in EAE [103] and MS [104]. B-cells are important also for cytokine production; in

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particular the IL-10 secreting B regulatory (Breg) subpopulation or at least the IL-10 levels seem to be reduced in MS patients [105]. A recent study identified deficiency in peripheral B-cell tolerance in patients with MS [106]. Other Immune System Cells and the Innate Immune Response In acute lesions, macrophages and activated microglia are the most important components of the inflammation process, at least in numerical terms. Those cells are responsible for the effector mechanisms of the damage: the release of proteolytic enzymes, such as MMPs, the production of nitric oxide and reactive oxygen species (ROS) and cytotoxicity via the secretion of pro-apoptotic cytokines and antibody and/or complement-dependent [107]. Mast cells were first described in post-mortem brains of MS patient plaques in 1890 [108] and later within demyelinated lesions together with infiltrating leukocytes, and also in the CNS parenchyma [109, 110]; they were claimed to play a potential role in MS pathogenesis and currently their role is under investigation in in vivo EAE studies [111]. The neutrophils are terminally differentiated immune cells generally considered as being the first line of defense against pathogens despite the fact that they have shown their ability in shaping the acquired immune system response [112]. Recent literature has also shown that MS can affect their phenotype and function in patients while their role in EAE development has been more extensively studied in mice rather than in humans [113 - 116]. The immunomodulatory properties of infectious agents are mainly due to their ability to engage a group of highly conserved PRRs that recognize conserved molecular motifs on bacteria and viruses. TLRs comprise a set of these PRRs and are involved in the maintenance of tolerance to commensal microbiota, as well as in the induction of inflammation against pathogens. Triggering TLRs induces distinct signaling pathways, resulting in cytokines and chemokines production and the transcription of genes involved in the control of infections [117]. TLR activation is the hallmark of the innate immune response, but there is also evidence that TLRs are important for adaptive immune cell functions such as the regulation of B-lymphocyte development [118] and T- lymphocyte activities such

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as Treg signaling [119]. It has been demonstrated that TLR2 and TLR4 mRNA are constitutively expressed on T-cells [120]. TLR ligands may represent an additional signal that influences the development of Th-cell responses, supporting T-cell development through innate immune activation, and directly regulating the functions of certain Th-cell subsets. TLRs have been identified in CD4+ T-cells at the mRNA level but their protein expression capability is still being debated [121]. It has been demonstrated that TLRs play a role in EAE [122]. Environmental agents (mainly viruses and bacteria) can influence MS in terms of lesion distribution and of severity of the disease along a pathway that, through the engagement of TLRs, involves innate and adaptive immune cell functions. The damaged BBB is the pathogenic mechanism that allows cells infiltrate and it is evident with Gadolinium (Gd) enhancement at Magnetic Resonance Imaging (MRI). The migration of leukocytes through the wall of cerebral vessels requires three components: the expression of adhesion molecules, chemokines and proteases, all enhanced by the inflammatory process that causes the disorganization of endothelial junction molecules [123, 124]. For example VLA4, expressed on the leukocyte surface and interacting with VCAM1 expressed by microglia and endothelial cells, is an effective target for treatment in MS [125]. Similarly other studies have analyzed the role of other adhesion molecules such as ninjurin-1 [126] and melanoma cell adhesion molecules [127]. Many chemokines and their selective receptors, through which leukocytes are directly attracted on the basis of the concentration gradients, have been found to be linked with MS lesions [128] such as CCR7 that is expressed in central memory T-cells and DC and is important for Fingolimod treatment [129]. Proteolytic enzymes are very important for the cleavage of auto-antigens of the CNS and they are also involved in the induction and propagation of demyelination and axonal damage [130]; it has been observed in EAE that their inhibition can ameliorate the clinical course of the disease [131] and is considered to be one of the mechanisms of action of interferon in the treatment of MS [132]. The BBB dysfunction is the result of acute inflammation with all its components of leukocyte migration, cytokine and chemokine production, and is much more evident during active lesions than inactive; chronic inflammation may be responsible for the BBB disturbance even if it has not been possible to find any correlation between inflammation and BBB

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damage during chronic disease [133]. Histopathology MS can be consider as the prototype of the inflammatory demyelinating diseases of the CNS and, from a pathological point of view, consists of the progressive accumulation of areas of demyelination, particularly in the periventricular white matter. The plaques show different degrees of myelin loss, reactive gliosis and inflammatory infiltrates of mononuclear cells: lymphocytes, macrophages and plasma cells. Demyelination may involve also cortical [134] and grey matter structures [135], even if they are not damaged by a degenerative process related to the subcortical pathology: the severity of axonal loss and the extent of cortical and subpial damage is disproportionate to the number of lesions of the white matter [136]. Axonal injury is generated by the release of free radicals and immune mediators such as cytokines and proteases cytotoxic T-cells [137], activated macrophages and microglia [138], able to cause increased oxidative stress, mitochondrial damage, and energy deficiency [139, 140]. These alterations are associated with disease progression, increasing global neuronal loss and brain atrophy, and it is an open question as to whether these evolutions are part of the degenerative process or a secondary effect after axonal destruction. The axonal damage occurs early in the process of plaque formation following or sometimes preceding demyelination [141]. For this reason it may be both mechanisms that lead to axonal injury: on the one hand the inflammatory reaction and mediators actively damage axons; on the other hand a slowly progression of axonal loss occurs also in inactive lesions. The plaques are well-demarcated brown-grey faded areas, usually in periventricular and subcortical white matter and consist of focal lesions of demyelination that may occur in the form of a large subpial-cortical band [142] or strips encircling the cerebral ventricles. The demyelinated nerve fibers traverse the plaques, from the node of Ranvier, as denuded axons and this process is called “segmental” demyelination. The active lesions can be distinguished from the firm and grey inactive lesions by their pink color and soft-tissue texture at gross

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inspection. During the different phases of the disease, several plaque types and levels of demyelinating activity are evident, and various mechanisms and triggering factors may contribute to the loss of myelin: infiltrates of mononuclear cells, high levels of antibodies, the presence of elevated complement fragments and/or the absence of inflammation, but always accompanied by a massive loss of oligodendrocytes. Further phases could be divided into early and late active plaques; in both a degradation of myelin proteins is evident in macrophages. In early lesions there is partial degradation in particular of minor myelin proteins, persisting for a few days, and showing synchronous myelin destruction with poor or absent remyelination. Late plaques present an inactive center with macrophage vacuoles containing remnants of major myelin proteins and also neutral lipid degradation products, such as cholesterol and triglycerides that may persist for months. Many exceptions depend on the course of the disease and individual peculiarities and, for this reason, post-mortem examinations and brain biopsies from MS patients show a wide variety of pathologies and their lesions show considerable pathological heterogeneity. The complex role of oligodendrocytes in MS lesions was controversial for many years and the presence of mature cells or progenitors could be effective in some lesions or fail in others. The formation of plaques is due to several basic processes: inflammation, myelin breakdown, astrogliosis, axonal loss, oligodendrocyte damage and neuro-degeneration. All the above-described mechanisms are present in PPMS patients, defined as being those with a slow and uninterrupted disease progression [143], even if the inflammation in focal white matter lesions is less severe compared to that in patients with SPMS. The main characteristic of RRMS is the presence of new focal white matter plaques. The nature of inflammatory processes is another difference between RRMS and progressive diseases: new plaques are associated with leukocyte trafficking and profound BBB damage while, in PPMS and SPMS, inflammatory cells are usually trapped within the CNS for the often-repaired BBB [133]. This compartmentalization of the inflammation in progressive MS causes an auto-amplification of the activation of microglia, mitochondrial defects and age-dependent iron accumulation in the CNS and could explain the failure of the

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immunosuppressive treatment that is more effective in RRMS. Active Lesions Inflammation, demyelination and vasogenic oedema are responsible of the acute active lesions, typically of RRMS patients, where demyelinated plaques are massively infiltrated by macrophages and debris from degraded and destroyed myelin sheaths is contained inside phagocytes [144]. Early active demyelination presents the degradation of smaller myelin proteins, while the larger such as PLP and MBP are more slowly digested and may persist in lesions indicating a late active lesion. Inflammatory infiltrates usually accumulate in the connective spaces of the brain and spinal cord, i.e. the meninges and the perivascular Virchow-Robin spaces, and are composed of macrophages/microglia and lymphocytes mainly cytotoxic Tcells [145], and their key role in induction and maintenance is recently confirmed by genome-wide association studies [146]. Few B- cells can be find in early active plaques, but they are the dominant cell type in “lymphatic B-cell follicles”, meningeal aggregates of inflammatory cells important for antigen presentation, cytokine and immunoglobulin production (IgG, IgM and IgA) [147]. Usually abundant macrophages and activated microglia in early MS lesions are engaged in the removal and degradation of tissue debris and in antigen presentation, while some very aggressive and fulminant MS may present granulocytes a massive deposition of complement, perhaps driven by an autoantibody reaction against channel acquaporin-4, Chronic Plaques In chronic plaques demyelination is evident in well-defined areas; several myelinladen macrophages and a slowly expanding rim of activated microglia characterize the borders of the lesions that centrally contain only few cells. Other typical characteristics of chronic lesions are the loss of oligodendrocytes and axons, astrogliosis and minor infiltrations by lymphocytes and macrophages/ microglia. In chronic plaque an intact BBB often occurs with perivascular infiltrates, organized in lymphoid follicular structures full of plasma cells [148].

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The inflammation in MS usually depends on the duration of the disease and the age of the patients; chronic inactive lesions show a decrease in oedema and macrophages/microglia, the appearance of astrocytic fibrillary gliosis, axonal loss, neurodegeneration and brownish discolouration and retraction of the brain. Several mechanisms are involved in chronic neurodegeneration and axonal damage in MS: (1) demyelination of previously remyelinated lesions; (2) lacking trophic support from oligodendrocytes and myelin; (3) oxidative burst and chronic mitochondrial failure; (4) alterations in axonal ion channels; (5) Wallerian degeneration [149, 150]. Remyelinated Plaques Remyelination is the process through which myelin sheaths are newly formed by oligodendrocytes. Remyelinated lesions are frequently found during early MS where the recruitment of oligodendrocyte precursor cells may provide the new formation, usually resulting in thinly myelinated axons with short internodal distances, detectable with the osmic acid impregnation technique [151]. In the later phases of remyelination this reduced myelin density is evident and, for this reason, these lesions are called “shadow plaques”; they are extensive in progressive [152] and relapsing MS patients [153] and more susceptible to a second bout of inflammation/demyelination, possibly because the newly formed myelin in MS lesions is unstable as long as the inflammation is active. Remyelination occurs equally among patients with different clinical courses of the disease, but sometimes this process may fail to take place depending on many variables: genetics, age, disease duration, trophic support of microglia, oligodendrocyte loss, inappropriate interactions between axons and oligodendrocytes, or a barrier constituted by a dense glial scar that may prevent the migration of oligodendrocyte precursor cells into lesions. Unfortunately, no biomarkers are currently available to identify these different groups of patients. That being so, MS is not simply a focal demyelinating disease as Charcot postulated in 1880; we now know that MS is a disease of the whole CNS in which inflammation and degeneration involve myelin, axons and neurons; there is no MS lesion in which axons are completely preserved [154].

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Clinical Features and Diagnostic Criteria MS is characterized by a dissemination of lesions in time and space. Exacerbations and remissions occur frequently. In addition, signs and symptoms usually indicate more than one lesion. Clinical manifestations may be transient and some may seem bizarre. The patient may experience unusual sensations that are difficult to describe and impossible to objectively verify. Since any part of the neuraxis can be injured the symptoms and signs are diverse. The most frequent initial complaints are focal sudden loss or blurring of vision in one eye, diplopia, weakness, numbness, tingling, or unsteadiness in a limb, disequilibrium or a bladder-function disturbance. The symptoms generally appear over a period of hours or days, at times being so trifling that they are ignored and, less often, coming on so acutely and prominently as to bring the patient urgently to the doctor. Several syndromes can be considered as being an initial manifestation of MS: (a) optic neuritis, (b) transverse myelitis, (c) cerebellar ataxia, and (d) various brainstem syndromes (vertigo, facial pain or numbness, dysarthria, diplopia) and they may pose diagnostic questions, since they also certainly occur with numerous diseases other than MS as well also occurring during the course of MS. The clinical tool currently used to evaluate the neurologic impairment, the disease progression and the effectiveness of the therapy is the Expanded Disability Status Scale (EDSS). This is a numerical scale with rankings from 0 (normal neurological examination) to 10 (death due to MS), and its main concern is walking ability, focusing on whether it is possible for the patient to walk without assistance, with monolateral or bilateral assistance or whether deambulation is impossible [155]. EDSS evaluates 8 “functional systems” (piramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral and other), giving each of them a different score on the basis of patient's disability in that functional system. EDSS is easily reproducible and it's used as an outcome measure in most of the clinical trials, even though it tends to focus mostly on the walking ability, not estimating properly, for example, cognitive issues in MS patients.

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Although the clinical presentation and course of the disease are highly variable, several disease types can be recognized, including relapsing-remitting MS (RRMS), primary-progressive (PPMS), secondary-progressive (SPMS) and progressive-relapsing (PRMS): ●

●

●

RRMS is the most common form of MS. A relapse is characterized by recurrent attacks in which neurologic deficits are caused by a lesion in different parts of the nervous system that may last weeks, months, or even longer and it can resolve completely or almost completely, whether treated or not. The incomplete recovery from repeated individual relapses has been usually considered to be responsible for the cumulative deficit which characterizes the general clinical deterioration although increasing evidence indicates that an ongoing neurologic deterioration can be independent of relapses. Approximately 50% of patients with RRMS convert to a SPMS within 10-15 years after disease onset [156]. SPMS starts when RRMS patients begin to experience a worsening of their symptoms and disability, generally without developing new relapses and without modifications of the global burden of disease as seen in MRI. In some cases, individuals with SPMS continue to experience relapses, and it has been shown that about two thirds of patients with RRMS go on to develop SPMS [157]. Since this phase of the disease is probably driven mostly by neurodegeneration, and no more by inflammation, drugs used for RRMS aren't effective and unfortunately no specific markers have been found to identify the phase of transition from RRMS to SPMS [158]. PPMS accounts for approximately 10% of MS cases and is defined as progression without previous relapses. Occasionally relapses are superimposed on progressive disease in the progressive-relapsing MS form (PRMS), which represents 5% of patients with MS [159].

Other two clinical conditions have recently been described and related to the risk of developing MS. These conditions, called “Radiologically Isolated Syndrome (RIS)” and “Clinically Isolated Syndrome (CIS)”, don't satisfy diagnostic criteria for MS, but can be highly suggestive of the risk of developing MS. The main goal is to identify clinical and paraclinical markers that can predict the risk of conversion from CIS/RIS to MS.

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RIS is defined as the “incidental finding of MRI anomalies highly suggestive of demyelinating pathology, not better accounted for by another disease process” [160]. Several studies have recently been carried out to estimate the risk of developing MS in these patients, but larger prospective studies are still needed. However, it seems that Gd enhancement is associated with a greater risk of developing new lesions in a subsequent MRI, and that spinal cord lesions tend to be related to a progression to MS [160, 161]. No specific treatment is nowadays recommended for these patients. CIS is the term used to describe a first episode of neurological symptoms that lasts at least 24 hours and is caused by inflammation and demyelination affecting the optic nerves, brainstem or spinal cord. CIS can be either monofocal or multifocal [162]. To our knowledge, patients tend to spontaneously recover, although a consistent percentage of them will develop MS later, so CIS can be considered as the first manifestation of MS. Recent studies have tried to point out clinical and paraclinical factors that can predict conversion from CIS to MS: particularly, it seems that age of onset of the disease, radiological burden of disease and presence of oligoclonal bands are associated with a consistent risk of conversion from CIS to MS, while the role of vitamin D remains still unclear, and no laboratory markers are still defined [163].

Moreover, it seems that grey matter atrophy relates with the risk of developing MS too [164]. As far as we know, 30-70% of patients diagnosed with CIS will subsequently develop MS [165]. The diagnosis of MS is primarily clinical and relies on the demonstration of symptoms and signs attributable to white matter lesions that are disseminated in time and space, together with the exclusion of other conditions that may mimic MS. Single clinical feature or diagnostic test is not adequate to diagnose MS by itself. Therefore, diagnostic criteria have included a combination of both clinical and paraclinical data such as the detection of intrathecal OCB in the CSF; this is a diagnostically useful laboratory criterion since the OCB response is found in 90-

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95% of MS patients, although it is not an exclusive MS marker [166]. Moreover, other instrumental tests are routinely performed to support the MS diagnosis, at least in its early diagnostic phase, such as the evoked potential tests which measure the electrical activity of certain brain areas in response to the stimulation of specific sensory nerve pathways. In fact demyelination reduces, alters or arrests nerve impulses thus producing the MS symptoms. While Visual Evoked Potentials (VEP), Brainstem Auditory Evoked Potentials (BAEP), Sensory Evoked Potentials (SEP) have been used in the past to support MS diagnosis nowadays only VEP findings are considered in MS diagnosis [167]. As previously stated, the diagnosis of MS requires the demonstration that lesions are disseminated in time and space. MRI helps in demonstrating this dissemination, and is the most sensitive imaging technique in MS diagnosis. In the 2010 revision of the McDonald diagnostic criteria for MS, dissemination in space (DIS) is defined as the presence of at least one T2 lesion in at least two out of four SNC specific areas (periventricular, juxtacortical, infratentorial and spinal cord), according to the MAGNIM DIS criteria [168] (Fig. 2).

Fig. (2). MRI of CNS lesions in MS patients. MS lesion can be located in multiple different regions of the neuraxis. They result in hyperintense foci in FLAIR MRI (A and B, white arrows) while the active lesions are characterized by Gd-enhancement in T1-weighted MRI (C and D, white arrows).

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On the other hand, dissemination in time (DIT) is defined, according to the MAGNIM DIT criteria, as the contemporary presence of Gd-enhancing and nonenhancing lesions in a single MRI or as the presence of a new T2 lesion, enhancing or not, on a follow-up MRI in comparison with baseline MRI [169]. The following table summarizes the revised 2010 McDonald criteria for the diagnosis of MS (Table 1). Table 1. MS diagnosis criteria. MS diagnosis is actually made on the basis of McDonald criteria revised in 2010. RRMS Dissemination in space at least one T2 lesion, as seen on MRI, in at least two of these areas: (DIS) periventricular, juxtacortical, infratentorial and spinal cord. Gd enhancement is not required to demonstrate DIS. Symptomatic lesions are excluded from the lesion count. Dissemination in time if a baseline MRI is available, DIT can be demonstrated as the presence of new (DIT) or enlarging lesion(s), or as the presence of Gd-enhancing lesion(s). More simply, DIT can be demonstrated as the contemporary presence of enhancing and non-enhancing lesions on the same MRI CSF findings

the finding of oligoclonal bands in CSF is not mandatory for MS diagnosis, but it supports the hypothesis of inflammatory pathology of the CNS

PPMS Clinical determination of at least one year of disease progression At least 2 out of 3 of the following criteria: - evidence of DIS as previously described - evidence of DIT as previously described - CSF findings consistent with the hypothesis of inflammatory pathology (elevated IgG index or oligoclonal bands) SUMMARY Clinical history

Other required data

at least 2 clinical attacks with objective evidence of both none at least 2 clinical attacks with objective evidence of one DIT demonstration, or, if possible, to wait for of them another attack with objective clinical evidence of it 1 clinical attack with evidence of at least 2 lesions

DIT demonstration

CIS

DIS demonstration

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On the basis of their location, the cortical demyelinating lesions can be divided into three types: intracortical lesions (small, perivascular and confined within the cortex), subpial lesions (from the pial surface to cortical layer or to the entire width of the cortex) and leukocortical lesions (involving both grey and white matter). The cortical-subcortical lesions are present at all stages of the disease. In some patients extensive cortical demyelination is present in the near absence of focal white matter plaques and particular areas are mainly involved such as the cerebellum and/or insular, cingulated, frontobasal and temporobasal cortex [170]. High-field MRI has improved the sensitivity for in vivo detection of most cortical lesions and this is important, mainly because the cortical involvement is negatively associated with disease progression and disability such as cognitive impairment and epilepsy in RRMS. In MRI widespread cortical atrophy has been observed, even in the absence of demyelinated areas [171]. The explanation for this could be the loss of neurons for anterograde or retrograde degeneration, in particular in the cortical areas anatomically connected to the damaged areas in the white matter or in the deep grey matter [172]. Few pathological studies are available regarding the involvement of deep grey matter in the brain and in the spinal cord: lesions can occur at any site and at any stage of the disease but, in particular, in the hypothalamus and lateral columns of the cervical cord [173] and in early disease [174]. Irreversible disability, progression and cognitive deficits are prominent and extensive in chronic MS patients, at least partly correlating with cortical atrophy, especially when the cortical demyelinating lesions are detected in the frontal, temporal, insular and cerebellar cortices in addition to the cingulate gyrus and hippocampus. DISEASE-MODIFYING DRUGS According to the MS patient post-mortem CNS analysis, genetic and in vivo studies on animal models, MS is a bona fide, defined, inflammatory autoimmune disease affecting the CNS. The disease compromises the myelination status of the axon which can be almost completely recovered or be hindered either by repeated inflammatory attacks or by inadequate remyelination processes determining an irreversible axonopathy, neuronal loss and thus permanent neurological decline.

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The first MS specific modifying drugs were first developed over 20 years ago in the 1990’s with the FDA approval of IFNbeta for RRMS treatment. Thanks to the improved knowledge of the disease’s pathophysiology, more focused therapeutic approaches are now available or under consideration. The landscape of MS treatment has dramatically changed over the past decade and now includes injectable therapies, newer oral options, and targeted monoclonal antibody agents. Efficacy is demonstrated by reducing the number of clinical relapses and the appearance of new lesions on imaging. Although currently available therapeutic approaches are mainly focused on the immune system compartment to slow down the disease’s progression and the neurodegeneration, some encouraging experimental attempts are present in the literature to sustain and improve the myelin regeneration. Steroids The treatment of MS relapses is important as it may help to shorten and lessen the disability associated with their course [175]; historically, it was the first, and for a period of time the only, approach to MS treatment. In 1970, Rose randomized 197 MS patients to 40 units of adrenocorticotropic hormone (ACTH) gel that was given as intramuscular (i.m.) injections, assessing its effect versus similarly administered placebo gel thereby demonstrating the beneficial effects of ACTH so that ACTH had broad regulatory approval by the FDA [176]. Several studies were then performed comparing intravenous (i.v) methylprednisolone to ACTH, which was considered the “gold standard” for MS exacerbation treatment [177, 178], and to placebo [179, 180]. A typical regimen of methylprednisolone is 500-1,000mg once daily for 3-5 days [181]. Steroids in MS treatment are safe when they are used up to 7 days. Bone problems are rare and exceedingly unlikely. When steroids are continually used for a long term, they can cause serious side effects including weight gain, fluid retention, increased risk of infection, risk of stomach ulcers, muscle weakness, behavioural changes (e.g. depression or psychosis), cataracts and osteoporosis [182]. Longterm use is not a good option unless there is no therapeutic alternative [183].

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Injectable Drugs Beta-Interferons Interferon-beta (IFNbeta) precise mechanism of action in MS is still unclear, but it's thought that it can modulate immune responsiveness in an anti-inflammatory way [184]. INFbeta-1b was the first disease-modifying approved drug for MS in 1993. It has to be taken every day by subcutaneous (s.c.) injection and its efficacy was proved in a double-blind placebo-controlled trial which lasted 5 years [185] and demonstrated a dose-dependent reduction in the annualized relapse rate (ARR) together with better disease progression. This IFNbeta-1b formulation proved its effectiveness even in comparison with i.m. IFNbeta-1a (see below) in the INCOMIN study, a 2-year prospective randomized clinical trial, with more marked differences during the second year of treatment [186]. Another i.m. injectable IFNbeta formulation was evaluated. Its effectiveness was proved in a double-blinded, placebo-controlled multicentre phase III trial which reported a lower ARR, a significant delay in time to sustained disability progression and an even lower number of new T2 Gd-enhancing lesions in IFNbeta-1b treated patients. In addition to the i.m. route, IFNbeta-1a is also available for s.c. administration at a dosage of 22mcg or 44mcg three times a week. Its effectiveness was first proved in the PRISMS trial (NCT01034644) which consisted of a first phase, lasting 2 years, in which the patients were assigned either to 22mcg INFbeta-1a three times a week, 44mcg three times a week or placebo. This study showed that both dosages were able to decrease the relapse rate and the disability progression in a statistically significant manner, prolonging the time to first relapse and the relapse-free period [187]. The trial was extended for a further 2 years proving that the beneficial effects of the drug persist even in a longer follow-up period [188]. A long term follow-up study confirmed the results already obtained, highlighting the fact that the sooner the treatment was started the better were the results.

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The higher dose seemed to be the most effective [189] with the evidence for the efficacy of INFbeta-1a in delaying the onset of clinically definite MS in individuals with CIS. The i.m. IFNbeta-1a, showing a higher proportion of relapse-free patients in the former group and less active MRI lesions [190] A subsequent extension of this study, involving patients shifted from i.m. to s.c. INFbeta-1a demonstrated an improvement in clinical and radiological disease features after the therapeutic shift [191]. Pegilated IFNbeta-1a (PEG-interferon) is a modified formulation of IFNbeta-1a which was obtained by attaching a polyethylene-glycol group, thus modifying the pharmacokinetic and pharmacodynamics properties of the drug [192]. PEGinterferon seemed to have the same efficacy as IFNbeta-1a, but a more tolerable regimen, being administered twice a month. Patients with poor or no response to INFbeta treatment should be tested for IFNbeta-neutralizing antibodies which are known to determine a reduction of treatment effectiveness [193]. No significant difference in efficacy or safety has been described between different INFbeta formulations, even though clinical trial outcomes are disomogeneous and phase IV studies lack in duration, aren't blinded and can include selection bias [194]. IFNbeta is currently approved for the treatment of RRMS while it has been showed to improve disease progression and neurological outcome in CIS patients [195]. Moreover, IFNbeta has been investigated for its potential beneficial effects in progressive MS: as underlined in a recent review, the therapeutic action of this drug is limited to the relapsing phase of the disease, where the damage is driven by an inflammatory activity [196]. The most common side effects reported during IFNbeta treatment are flu-like symptoms, cutaneous reactions at the injection site, thyroid dysfunction and asymptomatic liver dysfunction commonly detected by blood exams, while leukopenia, anemia and polyneuropathy have also been reported.

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Glatiramer Acetate Glatiramer acetate (GA) (Copolymer-1) is a pool of synthetic peptides randomly composed of L-tyrosine (Y), L-glutamic acid (E), L-alanine (A), and L-lysine (K) ranging from 40 to 100 residues to mimic the encephalitogenic properties of MBP, one suspected auto-antigen in MS. The mechanism of action of GA in the treatment of MS is not still clear, although it is reported to induce the development of Th2-polarized GA-reactive CD4+ T-cells and to restore the deficiency in T-cells and Treg cells. Furthermore, GA modulates immunomodulatory activity on APC [197]. The first preclinical studies demonstrated the effectiveness of GA in protecting mice from subsequent attempts to induce EAE instead of inducing the disease itself [198]. GA was first approved for the immunomodulatory treatment of relapsing-type MS in 1996 [199]. The BECOME study (NCT00176592) demonstrated that patients with relapsing MS randomized for IFNbeta-1b or GA showed similar MRI and clinical activity [200]. A dose-comparison study has recently been performed: 40mg GA was administered 3 times/week and compared with placebo; the results were a 34.0% reduction in the risk of confirmed relapses compared with placebo, and a highly significant reduction in the cumulative number of Gd-enhancing T1 (44.8%) and new or newly enlarged T2 lesions (34.7%) at months 6 and 12 (GALA, NCT01067521) [201]. FDA approves GA for use in reducing the relapse frequency in RRMS patients, including those who have had a first clinical episode and have MRI features consistent with MS (CIS). GA is for daily s.c. injection only at a dose of 20mg. GA stands out as having excellent long-term safety data [202]. Lipoatrophy is the main side effect in GA treatment and it occurs in approximately 2% of 20mg/mL daily treated patients.

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Oral Drugs Teriflunomide Teriflunomide is the principal active metabolite of lefluoniomide, a pyrimidine synthesis inhibitor. It selectively and reversibly inhibits dihydroorotate dehydrogenase, a mitochondrial enzyme for de-novo pyrimidine synthesis, required by rapidly dividing B and T-cells [203]. A global, phase III, randomized trial involving over 1,000 patients (TEMSONCT00134563) demonstrated that compared with placebo, Teriflunomide significantly reduces relapse rates, disability progression and MRI evidence of disease activity. These results have been replicated by another multicentre, double-blind, parallel-group, placebo-controlled study (TENERE- NCT00883337) with an open-label extension which randomized a total of 1,169 patients to treatment with 7 or 14mg of Teriflunomide or placebo in order to establish the ARR, the number of confirmed relapses per patient-year and the sustained accumulation of disability at 12 weeks. Two other phase III trials are still ongoing: TOPIC (NCT00622700), including early MS or CIS individuals, and TERACLES (NCT01252355), investigating Teriflunomide as an adjunct to therapy with IFNbeta. Teriflunomide is an approved drug for rheumatoid arthritis and an oral, oncedaily, disease-modifying approved therapy for the treatment of RRMS in several countries including the USA (September 2012) and the European Union (August 2013). Animal studies have shown reproductive toxicity, embryo lethality and teratogenicity. To date, controlled data in human pregnancy are still missing. In the TEMSO study the most frequent side effects were diarrhoea, nausea, hair thinning and elevated alanine aminotransferase levels. Serious infections were reported in 1.6%, 2.5%, and 2.2% of patients in the three groups, respectively [204]. Teriflunomide has been detected in human semen [205]. Liver failure is another FDA Black Box Warning for treatment with Teriflunomide. If a drugliver injury is suspected, cholestyramine can be used in order to accelerate drug elimination [206].

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Dimethyl Fumarate (DMF, BG-12) Dimethyl fumarate (DMF), also known as BG-12, is the methyl ester of fumaric acid. Although its exact mechanism of action is not known, DMF is thought to exert both an anti-inflammatory and a neuroprotective action by activating the nuclear factor E2-related factor 2 pathway [207] and by inhibiting the nuclear binding of NF-kappaB1, a key player in inflammation pathways [208]. The efficacy and safety of DMF were evaluated in two large, global phase III clinical studies: DEFINE (NCT00420212) and CONFIRM (NCT00451451). Results from both studies demonstrated that DMF provides clinical and radiological efficacy over 2 years across a range of outcomes: 240mg DMF twice and three times per day reduced ARR by 44% and 51% and the risk of relapse by 49% and 50% respectively compared with placebo. DMF has been introduced in the late 1950’s for the treatment of psoriasis by the German biochemist Schweckendiek who was himself afflicted by the disease [209]. In March 2013, 240mg BG-12 twice a day received FDA approval as an oral treatment for RRMS. DMF can cause flushing and gastrointestinal discomfort (e.g. nausea, vomiting, diarrhoea, abdominal pain, and dyspepsia) within the first month of treatment. An increased incidence of the hepatic transaminase elevations was seen, these being primarily reported during the first six months of treatment. The long-term efficacy and tolerability is currently under investigation in the ENDORSE trial (NCT00835770). DMF for psoriasis have been reported [210] and more recently the FDA warned about a patient with MS who developed progressive multifocal leukoencephalopathy (PML) after DMF administration and later died. Since this patient had an extremely low count of lymphocytes before developing PML, it is currently under debate if PML has emerged by chance or as a direct consequence of DMF action; it is also questioned whether considering low lymphocyte count as a risk factor for PML development in DMF-treated patients [211].

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Fingolimod (FTY720) The mechanism of action of Fingolimod is not fully understood, although it is well known that Fingolimod is metabolized into its active form, Fingolimodphosphate, which resembles the naturally occurring sphingosine-1-phosphate (S1P) [212]. At least 5 receptors have been listed for S1P (S1P receptors 1-5) which are expressed on different cell types in various organs. The S1P1 receptor regulates lymphocyte trafficking in lymphoid organs. Fingolimod-phosphate binds to and activates the S1P1 receptor, initially acting as an agonist. However, subsequently, this interaction induces internalization of the receptor, thus functionally assuming the characteristic of a S1P1 antagonist. Physiologically, the interaction between S1P and S1P1 receptors leads to the egress of lymphocytes from the lymph node. Therefore, Fingolimod, through its active metabolites, leads to a reduction in the number of auto-aggressive circulating lymphocytes (i.e. Th17) that can reach the CNS [213]. Moreover, Fingolimod may exert a direct effect on the CNS because it crosses the BBB due to its lipophilic nature, and it can interact with S1P receptor family members present on several CNS resident cells [214]. Fingolimod has been shown to reduce the disease's severity in chronic MS models reversing the already present clinical features, while its prophylactic administration leads to a complete prevention of the disease features [215]. In RRMS patients it has proved its effectiveness in two large clinical trials named FREEDOMS (NCT00289978) and TRANSFORMS (NCT00340834). The FREEDOMS was a double-blind, randomized trial which lasted 24 months and involved over 1,000 RRMS patients who received either two different (0.5 or 1.25mg) Fingolimod dosages or placebo [216]. In this trial, Fingolimod proved its efficacy in reducing the relapse rate in both naïve patients and those previously treated with other drugs. Moreover, the times to the first relapse and the total relapse-free period were longer in treated patients and there was a reduction in the disability progression and time to disability progression irrespective of the dosage. Moreover, the effectiveness of the Fingolimod treatment was also assessed by MRI with a reduction in the number of Gd-enhancing lesions and the global burden of disease estimated as new or enlarged T2 lesions.

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On the other hand, the double-blind, randomized TRANSFORMS trial compared the efficacy and safety of Fingolimod to intramuscular (i.m.) IFNbeta-1a. It was a study involving more than a thousand patients with active RRMS randomly assigned to two different dosages of Fingolimod 0.5mg and 1.25 mg/day, or to i.m. 30mcg IFNbeta-1a weekly. Fingolimod proved to be more effective than IFNbeta-1a in reducing the relapse rate, prolonging the time to the first relapse and the relapse-free period [217]. Fingolimod proved its major effectiveness in comparison to IFNbeta-1a even on MRI outcomes, while no differences between the groups were observed regarding the disability outcomes. A phase IV study (EPOC; NCT01216072) had been conducted in order to determine the impact of Fingolimod versus active comparators on health-related quality of life. It demonstrated that switching from GA or IFNbeta to Fingolimod therapy may significantly improve several aspects of quality of life [218]. Fingolimod was the first oral treatment approved for MS by the FDA in 2010. Fingolimod 0.5mg/day is approved as first-line medication in the USA and in Australia, while in Europe it is a second-line treatment for patients with high disease activity despite treatment with a first-line modifying therapy [219]; however it can be used even in patients with rapidly evolving severe RRMS without prior treatment. Although the treatment does not seem to carry a significant increase in infectious disease rates, two cases of lethal viral infections and a case of PML have been recently reported [220] inducing FDA to require detailed phase IV studies to explore lower dose and to precisely define the risk for opportunistic infections. Rates of Varicella Zoster Virus (VZV) infections resulted higher in patients treated with Fingolimod although serious or complicated cases of herpes zoster were uncommon; VZV immune status should be established before initiating Fingolimod therapy and immunization for patients susceptible to primary VZV infection should be performed. The most frequently reported side effects are macular oedema, lowering of the heart rate up to atrio-ventricular block and elevation in liver enzymes. Fingolimod cannot be used in patients with a recent history of cardiac disease or severe bradyarrhytmias due to the risk of bradycardia (up to atrio-ventricular block)

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which usually arises within 6 hours of the first dose although it may occur as late as 20 hours after the first dose in some patients. Moreover electrocardiogram prior to dosing and at the end of the observation period and hourly pulse and blood pressure for at least 6 hours after the first dose should be performed. Particular attention must be paid to monitor diabetes, macular oedema, liver enzyme levels, blood cell count and infectious diseases. No clear association between treatment with Fingolimod and the risk of developing neoplasms has been established yet. Fingolimod therapy is also contraindicated in pregnancy since it may exert harmful effects on the foetus. Conventional Immunosuppressants Cyclophosphamide Cyclophosphamide (Cy) is an alkylating agent which interferes with cellular DNA synthesis and the growth of rapidly proliferating cells thus leading to cell death. When the immune system is activated the normally resting state immune cells, such as T-cells, start proliferating and this provide a rationale for the use of Cy in the treatment of autoimmune diseases. Open-label studies dating back to 1966 support the use of Cy in MS [221]. The efficacy results of the studies are often conflicting because of the type of selected patients and the therapeutic protocols [222]. Despite this limitation the studies have also shown that the greatest benefits can be seen in younger patients with an aggressive course that is refractory to treatment [223]. The most widely used therapeutic schedule is monthly pulses of 700-800mg/m2 for 1 year, followed by bimonthly pulses in treatment responders [224]. Even though the main regulatory agencies have not approved Cy in MS it should only be considered when approved first-line and second-line drugs fail or when Fingolimod, Natalizumab, or Methotrexate are contraindicated [225]. The most frequent adverse events include alopecia, nausea/vomiting, transient myelosuppression, amenorrhea, oligo/azoospermia and haemorrhagic cystitis. Increasing risk of bladder cancer occurrence has been reported up to 17 years in

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Cy-treated patients and it is favoured by toxic cystitis, chronic urinary tract infections related to bladder dysfunction, and permanent catheterization. Moreover Cy has been demonstrated to be teratogenic in animals, therefore its administration so should be interrupted before conception [226]. Azathioprine Azathioprine is a purine analogue, that is rapidly metabolized to the cytotoxic and immunosuppressant derivatives 6-mercaptopurine and thioinosine acid [227]. Though its precise mechanism of action in MS is still unclear, a Cochrane metaanalysis stated the effectiveness of azathioprine in reducing relapses and the progression of disability over 3 year follow-up period [227]. A randomized controlled study in 94 patients with RRMS suggested that Azathioprine may be as effective as IFNbeta in reducing relapses and decreasing disability progression as measured by EDSS [228] while more recent multicenter, randomized, controlled, single blinded trial demonstrated that its efficacy was not inferior to that of IFNbeta when ARR and MRI annualized T2 lesion rate were considered (EUDRACT number 2006-004937-13) [229]. Azathioprine is the most widely used immunosuppressant in MS and, although it has not been approved for use in MS by the main regulatory agencies, it has been approved in Germany. It is generally used at a dose of 2.5-3mg/kg/day or lower in the event of haematological (white blood cell count 600g [227]. For all these reasons Azathioprine is reserved for those with an inadequate response to first-line therapy.

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Mitoxantrone Mitoxantrone (MTX) is a synthetic antracenedione derivative which inhibits a specific enzyme involved in DNA repair [232] and which is especially used in the treatment of leukemia and breast cancer. Besides possessing antineoplastic properties, MTX exerts its action also on the immune system, reducing the number of circulating lymphocytes and macrophages with a long-lasting effect [233] and this is the reason why it's used in MS. In preclinical studies, MTX treatment was effective in suppressing the development of acute EAE and suppressing the onset of relapsing EAE [234, 235]; it improved the disability in SPMS and RRMS patients, reducing the number of relapses for at least 12 months after discontinuation of the treatment [236]. MTX is used in PPMS and SPMS and was approved by the FDA in 2000. It is meaningful to underline that nowadays MTX is the only drug approved by the main regulatory agencies for the treatment of progressive MS (both PPMS and SPMS). MTX has serious side effects, including cardiotoxicity (systolic dysfunction) and the risk of developing acute leukemia even a long time after treatment discontinuation. For this reason, MTX is currently indicated only for patients with active MS not responding to other safer drugs, or in patients with progressive MS. Even if it's not approved for MS treatment and doesn't seem to have strong immunomodulatory activity, MTX seems to exert a neuroprotective action that makes it worth to be quoted. Laquinimod Laquinimod is a synthetic derivative of Roquinimex, an oral immunomodulatory compound exerting beneficial effects on both clinical and radiological outcomes in MS patients during clinical trials, despite its toxicity [237]. The effectiveness of Laquinimod was first demonstrated in preclinical studies [238]. In the double-blind, placebo-controlled, multicentre ALLEGRO clinical trial (NCT00509145), versus placebo, lasting 24 months; Laquinimod proved its efficacy in minimally reducing ARR, disease progression and the number of Gd-

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enhancing or new or enlarged T2 lesions as seen on MRI [239], but in the multicentre, double-blind BRAVO clinical trial (NCT00605215), which compared Laquinimod’s effectiveness with IFNbeta-1a, after 24 months Laquinimod showed only a statistically significant reduction in brain atrophy as measured by MRI, with no effect on ARR and disability progression [240]. The CONCERTO clinical trial (NCT01707992), whose primary outcome is to measure the impact of Laquinimod on disability progression in RRMS patients, is still ongoing and the ARPEGGIO (A Randomized Placebo-controlled trial Evaluating Laquinimod in PPMS, Gauging Gradations In MRI and Clinical Outcomes) study, which aims to establish the effectiveness and safety of Laquinimod in PPMS patients, is about to start with completion estimated to be in 2017. Laquinimod is not currently approved for MS treatment. Laquinimod is generally well tolerated despite an elevation in liver enzymes, headache, arthralgia, diarrhoea, an elevated risk of urinary tract infections and sinusitis compared to placebo which disappear with drug discontinuation [239]. Methotrexate Methotrexate (MTX) is a strong immunosuppressive oral drug, traditionally used in the treatment of psoriasis and rheumatoid arthritis. Its mechanism of action is interference with DNA synthesis [241]. Despite its marked immunosuppressive action, few benefits have been demonstrated in MS patients’ treatment. In 1995, a randomized, double-blind, placebo-controlled clinical trial showed that low dose MTX (7.5mg/week) in a small cohort of progressive MS patients was able to reduce disability progression, evaluated by specific tests [242]. This result was not confirmed by other trials, nor when MTX was used as combination therapy with IFNbeta [243]. Due to its significant side effects and poor clinical results, MTX is considered an off-label therapy for MS patients. Biologics (Monoclonal Antibodies) Antibodies are glycoproteins physiologically produced by B-cells with a basic

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structural unit composed of two identical heavy and two identical light chains. The antibodies are classified according to their heavy chain C-regions into different classes termed isotypes. The variable regions of each heavy and light chain combine together to constitute the antigen-binding site, which shows high specificity for the target epitope. On the other hand, the constant part of the heavy chains interacts with other immune system elements such as complement proteins and Fc receptors, determining the immune system reaction. The modern era of therapeutic antibodies dates back to the invention of mouse hybridoma technology by Kolher and Milstein 1975 [244] while various strategies have evolved to improve their therapeutical effectiveness, i.e. increasing the antibody life span and reducing immunogenicity [245]. Based on their structure the antibodies are defined by the suffix: ● ●

●

●

omab: murine-generated antibodies; ximab: chimeric mouse-human antibodies with the entire antigen specific variable domain of murine origin coupled to human constant domains; zumab: humanized antibodies with a murine hypervariable region grafted onto a human framework. umab: completely human monoclonal antibodies.

The clinically used antibodies exert their effect by a) depleting the target cells by an antibody dependent cellular toxicity (ADCC); b) depleting the target cells by a complement dependent cytoxicity (CDC); c) directly inducing intracellular pathways leading the targeted cells to cell death; d) blocking of soluble and membrane-associated cytokines and growth factors; e) blocking of the ligandreceptor interactions not only targeting the receptor but also downregulating the cell surface expression of the targeted receptors [245]. The use of monoclonal antibodies in clinical practice has the advantage of targeting pathogenic cells or molecules with high specificity, hopefully limiting undesired off-target effects, although not eliminating them completely. Several monoclonal antibodies are currently available in clinical practice as a therapeutical approach in various medical disciplines such as rheumatology and oncology.

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Together with increasing knowledge regarding the players on the MS stage, several therapeutic antibody strategies have been developed. While some of them have already reached the bedside others are still being validated. As previously reported, T-cells have been considered as being the main players in sustained CNS lesions. Therefore, limiting their egress into the CNS and the circulation of the auto-aggressive clone was the first goal in anti T-cell antibody development. Natalizumab Natalizumab is a humanized immunoglobulin IgG4 antibody targeting the α4chain present in the α4β1 integrin heterodimer, also termed the VLA-4. VLA-4 is expressed on the surface of T-cells and its interaction with the VCAM-1 enables the leukocytes to firmly adhere to the blood vessel wall and to subsequently migrate through the BBB into the CNS [246]. Natalizumab, therefore, is the first antagonist in the class of selective adhesion molecule inhibitors blocking the immune cell adhesion to the endothelium of the BBB. As demonstrated in animal models, an additional mechanism of Natalizumab action is the modulation of leukocytes trafficking with a reduced migration into the brain's parenchyma. Two large phase III double-blind trials, AFFIRM (Natalizumab versus placebo) and SENTINEL (Natalizumab plus IFNbeta-1a versus placebo plus IFNbeta-1a), assessed Natalizumab’s safety and efficacy over a 2-year period. In the AFFIRM trial (NCT00027300), over 900 patients with relapsing MS were randomly assigned to receive either monotherapy with 300 mg Natalizumab or placebo by intravenous (i.v.) infusion every four weeks for two years. A 68% relative reduction in ARR in the Natalizumab group and a 42% reduction in the risk of sustained progression of disability at 2 years. New or enlarging T2 MRI lesions and the mean number of Gd-enhancing lesions were reduced by 83% and 92% respectively [125]. The SENTINEL trial (NCT00030966) evaluated the effect of the addition of Natalizumab treatment in over 1,100 participants who were receiving IFNbeta-1a.

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This combination therapy resulted in a 24% reduction in the relative risk of sustained progression of disability at 2 years while the ARR was 0.34 compared to 0.75 on IFNbeta-1a alone and new or enlarging T2 MRI lesions were reduced by 83% [235]. Two patients subsequently developed PML and died; consequently, in February 2005 Natalizumab marketing and clinical trials were suspended. In June 2006, Natalizumab was reapproved only for monotherapy [247]. By January 2010, 31 cases of PML were ascribed to Natalizumab. However, the FDA did not withdraw the drug from the market because its clinical benefits overbalanced the risks. Natalizumab has been the most effective therapy against MS since its approval in 2004. It is used to prevent episodes of symptoms and to slow down the worsening of disability in individuals who have relapsing forms of MS while it is also recommended for patients who have had an inadequate response to, or are unable to tolerate, an alternative MS therapy [248]. It is administered by monthly i.v. infusion. The increased risk of PML, an opportunistic viral infection of the brain caused by the JC virus (JCV), is the most serious side effect. JCV is a common virus that is generally harmless in healthy individuals but it can cause PML in immune-compromised patients due to human immunodeficiency virus (HIV) infection, blood dyscrasias such as leukaemia or lymphoma, or medications such as Natalizumab. PML primarily affects oligodendrocytes in the brain causing a progressive demyelinating damage with a mortality rate ranging between 30 and 50% in the first months while surviving people can develop varying degrees of neurological disabilities. PML symptoms depend on location and the degree of damage. The most commonly involved areas are the parietal and occipital lobes, but it is now recognised that all cerebral, cerebellar and brainstem regions can be affected. The most common symptoms at onset are clumsiness, progressive weakness, visual field impairment, cognitive disorders and personality changes [249]. The risk of PML is lowest among the MS patients (a) who had been treated with Natalizumab for the shortest periods, (b) who had used few if any

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immunosuppressant drugs, and (c) who had no anti-JC virus antibodies. Combining these three factors predicts the pre-treatment risk of developing PML as a complication of Natalizumab treatment. Hence, testing for anti-JC virus antibodies through an analytically and clinically validated immunoassay is mandatory in order to assess the risk of subsequent developing PML before starting a treatment with Natalizumab. Patients initially testing negative for antiJCV antibodies are not completely PML risk free because of the potential risk of new JCV infection and, on the other hand, the risk of a false negative test result. Periodic re-testing of previously anti-JCV antibody negative determined patients should be considered. Common Natalizumab adverse effects are fatigue, allergic reactions (with a low risk of anaphylaxis) and elevated blood levels of liver enzymes Alemtuzumab (Campath-1H) CD52-antigen is mainly present on the surface of mature lymphocytes and to a lesser extent on monocytes, DC and granulocytes as well as being produced by the epithelial cells in the epididymis [250 - 254]. CD52 is a small glycoprotein that is linked to the cell membrane by a phosphatidylinositol linker [255, 256] and whose function remains uncertain. It is thought, however, that it may serve to promote cell-cell adhesion or may be involved in T-cell migration and costimulation [257 259]. Alemtuzumab is a humanized IgG1 antibody which specifically recognizes CD52. It was originally developed as a rat monoclonal antibody (Campath-1G (IgG2b)) by researchers of the Department of Pathology at the University of Cambridge (Cam(bridge)path(ology): Campath) and then humanized (Campath-1H) through genetic engineering to limit the anti-globulin response [260]. Alemtuzumab induces both ADCC, CDC and apoptosis; however, the complement-mediated and antibody direct cellular cytotoxicity is presumed to be important in vivo [261]. It should be noted that Alemtuzumab differentially affects circulating T and B-cell depletion and their restoration time, with the B-cell recovery that tends to precede T-cell one while other immune system compartments do not seem to be affected [262]. Moreover alterations in proportions and properties of lymphocyte subsets

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have been observed such as an increase in Treg cell percentage [263, 264]. The efficacy of Alembtuzumab in MS patients has been proved in several clinical studies that demonstrated its greater effectiveness compared to s.c. IFNbeta administration. Nevertheless, the same studies also showed an increased risk of potentially serious side effects in patients treated with Alemtuzumab [262]. In the phase II CAMMS223 clinical trial (NCT00050778), over 300 randomized RRMS patients received either 12mg Alemtuzumab ev/day or 24mg Alemtuzumab ev/day or s.c. IFNbeta 44mcg three times a week. In this study Alemtuzumab was demonstrated to be more effective in reducing ARR, disability progression and brain atrophy as seen on MRI, although it had serious side effects, including immune thrombocytopenic purpura, with a patient dying due to this side effect [265]. A five-year follow-up of this study confirmed the results already obtained [266]. Two other studies, the CARE MS I (NCT00530348) and the CARE MS II (NCT00548405), confirmed that previously reported in the CAMMS223 trial, also in patients previously treated with first-line disease modifying drugs; however, there was no comparison with placebo and with Natalizumab. Given the evidence of its immunomodulatory effect, Alemtuzumab has been proposed as a therapy for RRMS. It was approved in Europe in September 2013, while it has not been approved in the US yet. Alemtuzumab has to be intravenously administered at a dosage of 12mg/day for 5 consecutive days and then at the same dosage for 3 days, one year after the first course of treatment. The main concern regarding the use of Alemtuzumab is related to its side effects, mainly the autoimmune ones, especially on the thyroid gland, the anti-glomerular basement antibody disease (Goodpasture syndrome) and the immune thrombocytopenic purpura [267]. RECENT EMERGING BIOLOGICAL THERAPEUTICAL APPROACHES

AND

EXPERIMENTAL

The advance in the comprehension of the immune-mediated mechanisms has sustained the development of more specific drugs towards specific immune system elements (Table 2) while other cellular and molecular potential approaches in MS therapy are also been developing (Table 3).

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B-Cells Circulating and secondary lymphoid organ B-cells originate mainly from the Bcell precursors in the bone marrow through different development stages. The precursor B-cells go through Pro- and Pre-B-cell stages before reaching the mature inactive form which may then be activated in a T-cell dependent or independent way. Once the naïve B-cells are activated, they clonally expand and differentiate resulting in the generation of plasma cells which can also secrete antibodies other than IgM and persist as memory B-cells [268]. Each developmental step is characterized by the presence of intracellular processes and several surface markers. B-cells have a double function in the well-organized systemic immune defence since they can be activated against pathogens as well as being able to function as APC in the T-cell mediated immune response. The development of monoclonal antibodies for B-cell depletion was driven by their clinical use in B-cell malignancy but their use has overcome the oncological field. Rituximab CD20 is a membrane-bound protein present on the majority of B-cells, but it is not expressed on lymphoid progenitor cells in the bone marrow or on mature plasma cells [269]. Rituximab is a chimeric high affinity IgG1 anti-CD20 which depletes CD20+ Bcells by means of ADCC, CDC and by directly stimulating the intracellular cell death pathway [270, 271]. Since CD20 is not expressed on progenitor cells in bone marrow, this allows for repopulation after Rituximab therapy while Avivi et al. [272] having reviewed the literature, claim that the drug has a further effect also on T-cells. Rituximab was first approved for the therapeutical treatment of non-Hodgkin lymphoma and rheumatoid arthritis patients. Despite the fact that MS is generally considered to be a T-cell-based pathology, the presence of OCB in the CSF and of ectopic lymphoid follicles in the meninges of SPMS patients has led to B-cells being considered as a new target in MS. Therefore Rituximab is currently being used in clinical trial for both RRMS and progressive MS forms.

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At this point in time, 5 clinical trials enrolling MS patients are present on the clinicaltrial.gov web site with the two oldest ones dating back to the 2000s. The OLYMPUS study (NCT00087529) was a double-blind, placebo-controlled study performed on over 400 randomized PPMS patients to assess the efficacy and safety of two 1g i.v. doses of Rituximab every 24 weeks, through 96 weeks (4 courses). Although the time to confirm disease progression did not reach significance, Rituximab was shown to affect disease progression in younger patients, particularly those with inflammatory lesions in a subgroup analysis [273]. Moreover another double-blind, placebo-controlled, and multicentre 48 week study (NCT00097188) was performed on 100 RRMS patients receiving i.v. 1g Rituximab as OLYMPUS study. This clinical trial demonstrated that a single course of Rituximab reduced inflammatory brain lesions and clinical relapses for 48 weeks, suggesting B-cell involvement in the pathophysiology of RRMS [274, 275]. Since 2010, the first trial to be set up has been the RIVITaLISe (NCT01212094) which has been based upon intrathecal drug release. Intrathecal (i.t.) Rituximab administration has already been used to increase the CNS level of the drug in the treatment of CNS lymphoma. For the same reason, this route could result in higher CSF drug levels disrupting the meningeal ectopic lymphoid follicles, thus reducing cortical lesions and possibly disease progression. RIVITaLIse is a double-blind, placebo-controlled trial enrolling 80 SPMS patients with mild to moderate levels of clinical disability. The primary goal of this study is to define the safety and efficacy of combined systemic and i.t. administration and to collect quantitative neuroimaging measures of CNS tissue destruction as well as carrying out clinical and functional evaluations. At this point in time, no published data are available for this study as is the case for the other two studies that are currently ongoing. The first one was initiated in 2012 recruiting 90 CIS and relapsing patients over time with the purpose of determining the superiority of combined therapy vs. GA monotherapy (i.v. 1g Rituximab followed by s.c. 20mg/day GA versus placebo followed by GA alone), of evaluating the number of disease-free patients and also

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the changes in lymphocyte populations in the CNS (NCT01569451). The other study started in October 2014 and deals with the safety of i.t. 25 mg Rituximab infusion over 10 minutes at two time points, two weeks apart in both SPMS and PPMS patients (NCT02253264). No further patients are currently being enrolled in the study and only 12 patients with progressive MS are expected to be included in the study which will evaluate the number of serious adverse events over the course of the study possibly related to i.t. Rituximab administration. While the primary aim is the safety of intrathecal administration, the investigators are also going to evaluate its effect on the quantity of meningeal lesions on MRI, changes in biomarkers of inflammatory activity and neuronal injury in the CSF. Although Rituximab has generally been well tolerated, the patients may experience sustained B-cell depletion and secondary hypogammaglobulinemia while, in rare cases, PML has been reported when Rituximab was co-administered with other chemotherapeutic drugs [270]. PML in these patients can be considered as a potential warning factor for MS patients previously treated with immunosuppressive drugs and could also suggest to carefully select the MS patients for Rituximab after a screening for anti- JCV antibody titer. Recognizing the potential immunogenicity risk in the use of chimeric antibodies in clinical practice, several new generation anti-CD20 antibodies have been developed and, for some of them, clinical trials on MS patients are already ongoing. Ocrelizumab and Ofatumumab Ocrelizumab and Ofatumumab are both humanized IgG1 antibody which differently recognized CD20 [276]. Ocrelizumab binds to a different but overlapping epitope compared to Rituximab and it shows an increased binding affinity for FCγRIIIa receptor together with an enhanced ADCC and reduced CDC effects in vitro [277]. Instead Ofatumumab binds a more membrane proximal epitope, it has a slower dissociation rate compared to Rituximab triggering ADCC and CDC of B-cells overexpressing CD20 while it has been shown to deplete even Rituximab-resistant B-cells in vitro [278, 279]. Both antibodies have been tested in B-cell malignancy and clinical trials have also

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been performed for safety and efficacy in immune mediated disease [278, 280, 281]. Ocrelizumab is currently under investigation for MS patient treatment. The first registered clinical trial (NCT00676715) was a randomised, parallel, double-blind, placebo-controlled trial enrolling over 200 RRMS patients recruited in centres located in both Europe and America. The patients were randomly assigned to 4 parallel arms to receive either placebo, low-dose i.v. (600 mg) or high-dose i.v. (2,000 mg) Ocrelizumab in two doses on days 1 and 15, or i.m. IFNbeta-1a (30mcg) once a week. A fourth group of patients was treated with IFNbeta-1a and it was included as an active, open-label, rater-masked control. The complete scheme of treatment is reported in Kappos’s study [282]. Safety was assessed throughout the clinical trial up to 96 weeks with the primary outcome being the total number of Gd-enhancing lesions and T1-weighted MRI at 12, 16, 20, and 24 weeks. The annualized protocol-defined relapse rate, the proportion of relapsefree patients and changes in the total volume of T2 lesions from baseline to week 24 were the secondary outcomes taken into consideration. Neither opportunist infections nor PML cases were reported during the study, while Ocrelizumab treatment resulted in fewer Gd-enhancing MRI lesions as well as in reduced ARR. Larger and longer-term randomized, double-blind, double-dummy, parallel-group studies (OPERA I, NCT01247324 and OPERA II, NCT01412333) are currently ongoing, formally involving over 1,600 RRMS patients, to evaluate the efficacy and safety of Ocrelizumab in comparison with IFNbeta-1a. The RRMS patients receive i.v. 600 mg Ocrelizumab every 24 weeks plus s.c. IFNbeta-1a placebo three times a week or ascending s.c. doses of IFNbeta-1a three times a week over 5 weeks plus Ocrelizumab placebo i.v. every 24 weeks. The anticipated time for study treatment is 96 weeks. The primary outcome is the ARR while several other parameters concerning the relapse and clinical signs are evaluated, i.e. the proportion of relapsing-free patients, as well as the immunogenicity of Ocrelizumab, i.e. the Human Anti Human Antibody (HAHA) level. One randomized, parallel group, double-blind, placebo-controlled study is also

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ongoing on PPMS patients (ORATORIO, NCT01194570) to evaluate the efficacy and safety of 2 i.v. 300 mg Ocrelizumab infusions separated by 14 days in each treatment cycle. The blinded treatment period lasts at least 120 weeks and it is followed by an open-label treatment for patients enrolled in Ocrelizumab and placebo group who could benefit from further or newly initiated Ocrelizumab treatment according to the investigator evaluation. The anticipated time for study treatment is up to 5.5 years. The study aims first to evaluate the effect of Ocrelizumab on the time to onset of sustained disability progression, defined as an increase in EDSS score that is sustained for at least 12 weeks going up to 24 weeks. In addition, other secondary outcomes related to both walking capability and total volume of T2 lesion changes will be evaluated. An observational study was started in 2012, enrolling the patients who are a subsample of the above-mentioned ORATORIO, OPERA I and OPERA II studies. This study comprises approximately 100 PPMS and 100 RRMS patients and the treatment groups will only be disclosed after completion of the trials. The present study will investigate the effects of Ocrelizumab on multimodal evoked potentials. Furthermore, quantitative electroencephalography will be explored as a potential correlate of cognitive dysfunction and fatigue. The safety and efficacy of three different Ofatumumab doses were also tested in RRMS patients in a double-blind, randomized, placebo-controlled multicentre study performed in Europe (NCT00640328). The trial consisted of two phases: a 48 week treatment period, followed by an individualized treatment period of up to two years. Three dose patient cohorts were set up including 38 RRMS patients who received 100, 300 or 700 mg Ofatumumab infusions separated by 2 weeks during the first 24-week treatment period. The active/placebo group received treatment with Ofatumumab during the first treatment period and placebo during the second treatment period. Participants in the placebo/active group received treatment with placebo during the first treatment period and Ofatumumab during the second one. Adverse effects, HAHA reactions and changes in circulating leukocyte subsets together with other haematological changes induced by the drug treatment were considered as primary outcomes. Results of this first clinical trial demonstrated that Ofatumumab did not increase the number of serious adverse events while decreasing the number of new MRI lesions [283].

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A second double-blind, placebo-controlled, parallel-group, multicentre study is going to be completed soon (NCT01457924). It has evaluated the safety and MRI efficacy of three different doses (3, 30 or 60 mg) of a s.c. Ofatumumab formulation in over 200 RRMS patients over 24 weeks. While no official data have been placed on the clinicaltrial.gov website, some results have been recently presented at an ECTRIMS meeting (European Comitee for the Treatment and Research in Multiple Sclerosis), demonstrating that the most common Ofatumumab adverse effect was injection-related reactions while only a few serious adverse events were observed in the highest dose group. Neither PML nor infections were recorded and Ofatumumab reduced the cumulative number of new T1 Gd-enhancing lesions for each Ofatumumab dose regimen [284]. MEDI-551 Certain B-cell malignancies do not respond to Rituximab because of low levels of CD20 expression [285], while the cancer progression or relapse during Rituximab therapy may be due to the down-regulation in CD20 expression [286, 287] as well as to the establishing of mutation in CD20 which affects the Rituximab-CD20 interaction [288]. To circumvent such drug resistance is of great importance in Bcell malignancies. Therefore, new targets have been identified such as the transmembrane glycoprotein CD19 which is broadly expressed on the B-cell lineage from pro-B-cells to early plasma cells but it is absent on haematopoietic stem cells and plasma cells. CD19 is a transmembrane B-cell Receptor (BCR) co-receptor mainly clustered in a protein complex comprising CD21, CD81 and CD225 [289, 290]. It is essential for intracellular signalling and for amplifying the B-cell response to membranebound ligand stimulation [291]. As with anti-CD20, the anti-CD19 monoclonal antibodies have been tested for the treatment of autoimmune diseases. In fact, anti-CD19 antibodies can remove the potentially auto-reactive B-cell clones as well as reducing the auto-reactive T-cell activation through the elimination of mature B-cells. Despite the fact that several

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anti-CD19 antibodies have been developed, only MEDI-551 is currently under consideration for the treatment of MS. MEDI-551 is a humanized IgG1 monoclonal antibody originated by modification of another fucosylated anti-CD19 antibody. MEDI-551 results in binding characteristics that are favorable for an ADCC-dependent mechanism and demonstrates B-cells depletion at lower concentrations than Rituximab [292]. The only clinical study on MS patients started in 2012 (NCT01585766) and the estimated completion date is early 2015. The trial aims to ascertain the safety and tolerability of ascending doses of i.v. or s.c. administration of MED-551 in 5 MEDI-551 treatment arms plus 2 placebo groups which are expected to enroll more than 25 relapsing MS patients (confirmed RRMS, SPMS, PRMS, or CIS and at least 1 documented relapse within the past 3 years prior to screening). Beside the primary outcomes, the effects of pharmacokinetics, pharmacodynamics and the immunogenicity of MEDI-551 are also being considered. Mast Cells Masitinib mesylate Mast cells (MC) were first described in the post-mortem plaque tissue from brains of MS patient in 1890 [108], and later within demyelinated lesions together with infiltrating leukocytes and also in the CNS parenchyma [109, 110]. Their potential role in disease pathogenesis was supported by in vivo studies in EAE in which they were hypothesized to take part in the modulation of the immune response in the peripheral lymphoid organs as well as supporting the infiltration of immune cells into the CNS [111]. MC are innate immune system cells flowing in the blood stream as precursor cells undergoing maturation in peripheral tissue and phenotypically defined by the expression of high affinity IgE Fc receptor and tyrosine kinase receptor KIT (CD117) [293]. According to the microenvironment they face and as they move downstream, the activation of these and other functional receptors the MC become able to synthesize and release several new mediators such as cytokines, prostaglandins and proteases [110, 111]. The stem cell factor (SCF) is the main survival and cell growth factor for MC, it is the KIT ligand and has been shown to be unregulated in inflammatory conditions [294,

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295]. Masitinib mesylate belongs to the tyrosine kinase inhibitor family targeting KIT with an improved selectivity compared to other molecules of the same family, i.e. Imatinib [296]. Supported by the results obtained in the EAE mouse model and in clinical trials for neurological and inflammatory disorders [297, 298], a first Masitinib multicentre, double-blind, placebo-controlled, exploratory phase IIa clinical trial started in 2005 (NCT01450488). This focused on PPMS and 2 year relapse-free SPMS patients who received 2 different oral doses of 3 or 6mg/kg/day administered in two daily intakes over 12 months. Due to the poor efficacy of a low 3mg/kg/day dose, the investigators placed both MS patient types in a 6mg/kg/day treatment group. Although there was no statistically significant improvement in the efficacy data, an advance in the MS functional composite scale (MSFC) versus baseline was achieved. Masitinib seemed to mainly and positively counteract the degeneration of the lower limbs and to improve hand and arm functionality, although the EDSS scale remained stable. According to the safety analysis data Masitinib had some adverse effects that ranged from asthenia, rash, oedema and diarrhoea commonly related to the TK inhibitor compounds. Starting in 2011, a phase IIb/III multicentre clinical trial has been ongoing on a larger group (450) of MS patients with the same characteristics as the previous one and the collection of the primary outcome data are expected for the end of 2014 (NCT01433497) while in the European clinical trial register (EudraCT number 2010-021219-17) a similar trial is currently being performed treating the same kind of MS patients with a lower Masitinib dose/kg/day (4.5 mg instead of 6 mg). Cytokines and Chemokines Daclizumab IL-2 receptors are defined on the basis of their affinity binding to IL-2. The

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intermediate affinity IL-2 binding receptor is a heterodimer consisting of IL-2beta (ILR2β) and the so-called gamma chain of the cytokine receptor (ILR2). This receptor is present on cells other than T-cells, such as NK cells, where it is constitutively expressed and also on innate lymphoid cells [299]. The high affinity IL-2 binding receptor is a heterotrimer including IL-2Rα (CD25), IL-2Rβ and ILR2γ and it is present on recently activated T-cells and Treg. IL-2 is mainly produced by activated T-cells and plays an important role in T-cell proliferation and differentiation by binding to CD25. The latter is present at low levels in resting cells while it is upregulated in activated T-cells so interfering with the capability of IL-2 /CD25 binding may limit T-cell expansion. The monoclonal IgG1 antibody Daclizumab selectively binds to the ILR2α chain masking the IL-2 binding site without activating ADCC, CDC or modulating the cell survival signal pathway and the IL-2R surface rearrangement. Daclizumab was first developed to block adult T-cell leukemia (ATL) and was subsequently used in transplantation medicine and in the treatment of refractory uveitis [300, 301]. The rationale for Daclizumab as a therapeutic option also in MS originates from the idea that MS is a CD4+ auto-reactive CD25+ T-cell dependent disease and that Daclizumab can block auto-reactive activated T-cell clones. Nevertheless Daclizumab has been shown to increase also the circulating CD56brightNK cell number and to affect NK cell immunoregulatory properties on autologous activated T-cells and microglia. Moreover, Daclizumab modulates both DCsecreted cytokines and functional profile and affects the number and phenotype of circulating innate lymphoid cells [302]. These additional findings suggest that Daclizumab can exert a multifactorial effect in counteracting inflammatory process in MS. Several clinical trials have already been performed to prove Daclizumab’s safety and effectiveness [300, 301] and have shown that it is generally very well tolerated and serious adverse events are similar between Daclizumab and placebotreated patients; however, skin rashes, lymphadenopathy and elevation of liver function tests have been reported [303]. No cases of PML have been reported

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while herpes infection is similarly distributed between placebo- and Daclizumabtreated patients [304, 305]. Up to 10 clinical trials are currently registered on the clinicaltrial.gov web site while a summary of them and their related publications has recently been reviewed by Pfender and Martin [300]. The efficacy of Daclizumab has been assessed in small studies in which Daclizumab was intravenously administered to active RRMS and SPMS patients. The published results have indicated that Daclizumab was effective in reducing contrast enhancing lesions and improving clinical condition as evaluated by EDSS and neurological enhancing scale [306, 307] and these results indicated the potential effectiveness of Daclizumab in RR and SPMS patients paving the way for developing larger study. Thus the efficacy of s.c. Daclizumab has been assessed in an add-on therapy with IFNbeta therapy demonstrating mainly radiological benefits, while in the CHOICE study (NCT00109161) no changes in clinical progression and relapse rate were reported despite the fact that the combined therapy was more effective than IFNbeta alone. Moreover, an increase in CD56brightNK cells was reported in Daclizumab-treated patients and it also seemed to correlate well with the clinical and radiological signs [300, 304, 305]. A new formulation of Daclizumab, Daclizumab high-yield process (DAC-HYP), was also obtained by a different glycosylation pattern of the same amino acid sequence and it was recently used in the SELECT study (NCT00390221), a multicentre, double-blind, placebo-controlled, dose-ranging study to determine DAC-HYP safety and efficacy as a 52 week monotherapy treatment in highly active RRMS patients who were assigned to either s.c. 150 or 300 mg DAC-HYP every 4 weeks for 52 weeks or to the placebo group [308]. The study results have indicated a positive effect on radiological diseases as well as on ARR. Therefore, it was extended (SELECTION study, NCT00870740; EUDRA CT number 2008005559-46) to assess the safety and immunogenicity of extended treatment within the same patients previously enrolled in the SELECT trial. Patients who received the placebo in SELECT were randomly assigned to one of the two treatment

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groups every 4 weeks for 52 weeks (treatment initiation group); those who had received DAC-HYP were randomly assigned to continue their present dose with or without a washout period of 20 weeks. No adverse effects or increase in the treatment immunogenicity have been observed during the second year of DACHYP administration in all the trial arms [169]. A phase III clinical study (DECIDE; NCT01064401) was designed to test the superiority of DAC-HYP treatment in preventing relapse and slowing clinical decline compared to IFNbeta1a and was recently completed. The study compared s.c. 150 mg DAC-HYP which was administered once every 4 weeks to i.m. 30mcg IFNbeta-1a once weekly for 96 to 144 weeks (NCT01064401). Tabalumab (LY2127399) B-cell activating factor (BAFF) is a B-cell survival factor expressed by several cell types such as activated T-cells, monocytes and astrocytes. There are two forms of BAFF: a soluble and membrane-bound one. BAFF plays a central role in B-cell maturation and maintenance so that its deregulated expression can affect several B-cell functions, suggesting a potential role for BAFF in both B-cell malignancy and autoimmune disease pathogenesis due to the therapeutic benefit obtained by BAFF neutralization [309, 310]. Therefore, anti-BAFF monoclonal antibodies were generated and screened for the ability to neutralize both membrane-bound and soluble BAFF binding to BAFF receptor. Tabalumab (LY2127399) is a fully humanized IgG4 monoclonal antibody which binds and neutralizes both soluble and membrane-bound biologically active forms of BAFF, preventing BAFF from binding to its receptor [311, 312]. Tabalumab treatment transiently reduces circulating B-cell frequency mainly naïve B-cells in rheumatoid arthritis patients, it does not seem to affect T-cell dependent antibody response in monkey preclinical studies while some effects have been reported in mouse preclinical studies [311, 313, 314]. Tabalumab was first tested in rheumatoid arthritis patients while, up to now, only one clinical study has been completed testing the ability of different doses of subcutaneously injected Tabalumab to reduce the cumulative total of Gdenhancing MRI lesions. This study evaluated Tabalumab effect in more than 200

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RRMS patients compared to placebo, although other secondary outcomes were also considered, e.g. total number of new or newly enlarged T2-weighted MRI lesions and the proportion of relapse-free subjects (NCT00882999). MOR103 Granulocyte macrophage-colony stimulating factor (GM-CSF) was originally defined by its ability to induce the proliferation and maturation of myeloid progenitor cells in vitro. Nevertheless, it is now evident that GM-CSF also plays a role in regulating the properties of the granulocyte and macrophage lineage cells during host defence and inflammatory reactions; it has also been shown to promote the differentiation and pathogenicity of proinflammatory Th17-cells [315 - 317]. Preclinical findings showed that GM-CSF was essential in EAE development and the initiation of CNS inflammatory processes [318, 319] while both an increase in the frequencies of ​GM-CSF-producing Th-cells and the correlation between GMCSF expression and CSF biomarkers in MS progression support the GM-CSF involvement in the disease [320]. GM-CSF might, therefore, provide a therapeutic approach for the treatment of MS. To this end, an anti-GM-CSF fully humanized IgG1 antibody (MOR103) has recently been tested for safety and preliminary efficacy in a randomized, doubleblind, placebo-controlled study which enrolled 32 RR and SPMS patients with two documented relapses within the past 2 years before the screening was performed (NCT01517282). The patients were randomly allocated into 3 treatment groups and treated with i.v. 0.5, 1 or 2mg/kg MOR103 every 2 weeks for a total of 6 times and a placebo group was also present. The number of new T1 Gd-enhancing lesions and of new or enlarged T2 lesions were evaluated besides treatment-related adverse and serious adverse effects, MOR103 pharmacokinetics and anti-MOR103 immunoreactivity. The results are present on the clinicaltrial.gov website [https://www.clinicaltrials.gov/ct2/show/NCT01517282?term=MOR103&rank=2] and indicate that only 31 out of 32 patients have been considered (NCT01517282) and no full paper has been published yet.

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Secukinumab On activation by pathogens, naïve CD4+ T-cells undergo differentiation into different Th subsets that were initially classified on the basis of their cytokine production and functions into two main groups: Th1 and Th2 [321]. Nevertheless, the discovery of Th17 cells has updated the Th1/Th2 paradigm [322] and Th17cells have been found to play a relevant role in autoimmune disease as demonstrated in MS animal models [72, 323]. The commitment, differentiation and activation of Th17 cells are due to different cytokine sets among which the most important are IL-6, TGFβ and IL-23 [324]. The first attempt to limit Th17 effects involved targeting IL-17A and IL-17R. IL-17 is a six-member family, IL-17A to F and, despite the fact that IL-17A can be produced by other immune cell types, it is generally thought to be the signature cytokine of Th17 cells. IL-17A in humans is associated with the pathology of numerous autoimmune and inflammatory conditions such as rheumatoid arthritis, psoriasis, Crohn's disease and Behçet's disease. Moreover, it has been found to play an important role in both MS and EAE as previously reported in this chapter. Thus, the inhibition of IL-17A has been suggested as a therapeutic target in autoimmune diseases. Secukinumab (AIN 457) is a fully humanized IgG1 that selectively neutralizes IL17A, and was first tested for psoriasis, rheumatoid arthritis and uveitis and then for Crohn’s disease [325 - 327]. In 2013 a multicentre, randomized, double-blind, parallel arm, placebo-controlled study was initiated to evaluate the efficacy and safety of Secukinumab in relapsing MS patients (NCT01874340). The RRMS patients were intravenously administered 3 different Secukinumab doses. The cumulative number of new Gd-enhancing T1-weighted lesions was specified as the first outcome to be evaluated while other parameters referring to disease relapse and progression were also considered. The study has been formally completed; however, up to now, no publications are available. Moreover, another study was performed to assess the effect of multiple i.v. doses of Secukinumab on disease activity by monitoring MRI scans in RRMS patients (NCT01051817). Preliminary data were presented in 2013 at the ECTRIMS

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showing a significant reduction in cumulative new Gd-enhancing lesion number. The raw data of the study have been recently displayed on the clinicaltrila.gov web site (https://clinicaltrials.gov/ct2/show/results/NCT01051817). Table 2. Recent emerging biological approaches. New developed monoclonal antibodies and a kinase inhibitor have been considered in clinical trial to improve the specificity in targeting different immune system elements. MAIN IMMUNE SYSTEM TARGET B-CELLS

DRUG/TREATMENT

Rituximab

Chimeric IgG1

TARGET ADMINISTRATION PATIENTS ROUTE

CD20

i.v.

PPMS

NCT00087529

i.t.

SPMS

NCT01212094

CIS/RRMS

NCT01569451

i.v. i.t. Ocrelizumb

Humanized IgG1

Ofatumumab Humanized IgG1

MAST CELLS

CD20

SPMS/PPMS NCT02253264

i.v.

RRMS

NCT00676715

i.v.

RRMS

NCT01247324

i.v.

RRMS

NCT01412333

i.v.

PPMS

NCT01194570

CD20

i.v.

RRMS

NCT00640328

RRMS

NCT01457924

s.c.

MEDI-551

Humanized IgG1

CD19

i.v. / s.c.

Masitinib mesylate

Tyr kinase inhibitor

KIT

oral

CYTOKINES Daclizumab Recombinant IgG1

CLINICAL TRIAL

RR,SP,PRMS, NCT01585766 CIS PPMS/SPMS NCT01450488

IL-2Rα

s.c.

RRMS

NCT00109161

Daclizumab different glycosylation pattern

IL-2Rα

s.c.

RRMS

NCT00390221

Tabalumab

Humanized IgG4

BAFF

s.c.

MOR103

Humanized IgG1

GM-CSF

i.v.

IL-17

i.v.

RRMS

NCT01874340

i.v.

RRMS

NCT01051817

DAC-HYP

Secukinumab Humanized IgG1

NCT00870740 NCT01064401 RRMS

NCT00882999

RRMS/ SPMS NCT01517282

Other strategies to limit the contribution of Th17 to autoimmune diseases are

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being developed: e.g. targeting IL-23 and IL-6, although they have had poor or as yet unclear results in MS (Ustekinumab, NCT00207727 [328]). Tolerogenic Vaccines for MS The tolerogenic vaccines are a new type of vaccines: which have been developed to induce immunological tolerance and are useful to restrain mainly autoimmune diseases. Immunogenic and tolerogenic vaccinations differ in many aspects [329]. Usually a successful immunogenic vaccination requires strong immunological adjuvants to prime the initial immunization; it needs the activation, expansion and differentiation of B and T-cells and it aims to induce disease prevention rather than therapy. Immunogenic vaccines are largely used for prevention of several disease in clinic. On the other hand, tolerogenic vaccination is based on minimal adjuvant activity; therefore the antigens administered in the vaccines are recognized in nonactivated, non-inflammatory environments so that antigen self-tolerance can be developed. Moreover this kind of vaccination is contingent on the generation of long-lasting memory of antigen-specific Treg that mediates antigen-specific inhibitory activity. Tolerogenic vaccines are intended to be used in the therapy rather than prevention mainly for chronic diseases. Tolerogenic vaccination is currently carried out in pre-clinical settings and its clinical application is limited to early phase clinical trials [329]. The rationale for MS patient vaccination is based on the model of the molecular mimicry of the infectious agent and the different efficacy of T-cells in recognizing epitopes. The molecular mimicry model predicts that chronic or recurring infectious agents etiologically instigate MS, stimulating a strong T-cell-mediated immunity to the endogenous cross-reactive myelin epitopes. Re-emerging from latency, these infectious agents may drive MS relapses causing the RRMS form. Moreover, the infectious agents may persist in the CNS by camouflaging epitopes that resemble the host tissue [329]. Epitope spreading is a mechanism by which myelin-reactive clones initiate CNS inflammation while recruiting new naïve

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myelin reactive clones into the CNS, which in turn differentiate into effector cells and are responsible for additional neurological damage. Thus these heterogeneous anti-myelin T-cell clones may sustain a progressive disease course despite the absence of the infectious agents. Usually as a consequence of the induction of self-tolerance, auto-reactive naïve Tcells recognize self-antigens (i.e. endogenous myelin antigens) with a low efficiency. Moreover myelin auto-antigens stimulate Treg differentiation so that their inhibitory action prevents the activation and differentiation of potentially autoreactive T-cells which drive autoimmune disease development. Despite the development of these control mechanisms, cross-reactive pathogenic antigens (i.e. myelin-mimicking antigens) may induce a high efficiency recognition driving the differentiation of myelin auto-reactive T-cells into effector/memory cells and potentially representing the critical stimulus to initiate an autoimmune reaction. The rationale in tolerogenic vaccines development is that they are able to reinforce myelin-specific Treg cells and, as consequence, to induce anergy of auto-reactive naïve, memory or effector T-cells therefore promoting tolerance rather than autoimmunity. One of the main concerns in the use of a tolerogenic vaccine is the possibility that it results in an immunogenic rather than tolerogenic effect, thus even worsening the disease course. Indeed, tolerogenic vaccines are adjuvant-free so that antigens face the immune system in the absence of co-stimulatory molecules and proinflammatory cytokines and this fact is a critical aspect for the inhibitory properties of the vaccines. However, if a patient has non-symptomatic infections, a tolerogenic vaccine could be administered in a pro-inflammatory environment and it could even worsen the disease’s course [329]. One promising tolerogenic vaccine is the GM-CSF-neurogenic fusion protein (GM-CSF-NAg) obtained by the fusion of GM-CSF to the major encephalitogenic epitopes derived from MBP, MOG and PLP. An important characteristic of GM-

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CSF-Nag is targeting the vaccine to DC to promote an efficient antigen nonactivating presentation thereby promoting tolerance. This vaccine has been found to inhibit both mouse and rat EAE when administered both at disease onset and during established chronic EAE [329]. It was also shown to be effective even when it was administered under pro-inflammatory conditions in vivo [330]. The attempt to use potentially tolerogenic myelin protein vaccines has already been tested in human clinical trials as for ATX-MS-1467 which is a mixture of 4 human MBP peptides selected on the basis of the immunodominant epitopes retrieved in MS patients with HLA haplotypes DRB1*1501 and DQB1*0602. In EAE Lewis rats, ATX-MS-1467 administered before disease induction caused a significant delay in disease onset and a reduction in severity [331]. This was associated with reduced leukocytes infiltrating the CNS together with an increased secretion of anti-inflammatory cytokines, thus suggesting that ATX-MA-1467 could re-establish the self-tolerance to CNS-derived myelin peptides [331]. The same tolerogenic vaccine was then tested in a phase I clinical trial (NCT01097668) in HLA DRB1*15 positive RRMS patients with high baseline levels of T-cell proliferation in response to MBP. The study was based on an open-label upward titration over five dose levels via intradermal (i.d.) or s.c. routes followed by a 22 week follow-up period. The first aim of the study was to assess the safety and biological activity of ATX-MS-1467 and on the other end to follow the course of MS trough brain MRI scans (number of new or persisting Gd-enhancing lesions). Although no results have been published in the literature yet, the companies producing ATX-MS-1467 have announced that no safety issues have emerged and a remarkable improvement in visual-sight has been observed. These positive results have paved the way for a multicentre, open-label, single arm, baseline-controlled phase IIa study (NCT01973491, still ongoing) which aims to evaluate the clinical and biological effects of ATX-MS-1467 (s.c. 800mcg/bi-weekly) in RRMS patients and to assess its effect on immune tolerance according to MRI and MBP peripheral blood leukocyte proliferation. Beside ATX-MS-1467, another tolerogenic vaccine formulated on MBP was MBP8298 (Dirucotide). It is a synthetic MPB peptide that has been shown to be immunodominant in MBP-specific T-cells as well as in auto-antibodies isolated mainly from patients with particular HLAs such as DR2 and DR4 [332 - 334].

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MBP8298 has been intravenously administered and has been tested in a phase II trial for RRMS (MINDSET01, NCT008699866) and in three phase III trials for SPMS (NCT00869726, NCT00870155, NCT00468611). In a phase II clinical trial for RRMS (NCT00869986), MBP8298 failed to improve the clinical course of MS and in a phase III clinical trial on SPMS patients (NCT00468611), an infusion of MBP8298 every six months for 24 months resulted in no significant difference between treated patients and placebo controls. However, a significant delay in clinical progression was found in people with a specific HLA aplotype [335]. Another approach to vaccinate MS patients was made using autologous peripheral mononuclear cells (PBMC) pulsed with different numbers of immunodominant myelin peptides (MOG, MBP and PLP) in the presence of the chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Studies on EAE animals have proved that syngenic splenocytes coupled with encephalitogenic myelin peptides/proteins induce antigen-specific tolerance in vivo, not only preventing the disease’s onset but also affecting its clinical course when administered after the EAE induction [336]. A first clinical trial (ETIMS, NCT01414634) has been performed on 7 RRMS (phase I) and 2 SPMS (phase II) patients with the aim of assessing the feasibility, safety and tolerability of a single infusion of PBMC isolated from MS patients by leukapheresis and chemically coupled with seven myelin peptides. Neurological, laboratory, immunological and MRI examinations demonstrated that the infusions were well-tolerated, with no increase in clinical and MRI parameters of disease activity; moreover patients receiving a high dose of the vaccine (> 1x 109cells) showed a decrease in the antigen specific T-cell response. None of the first six treated patients showed a relapse during the first 3 months after treatment and, as regards the three patients with high disease activity, one showed disease exacerbation and one of the remaining two presented mild fine motor skills dysfunction and dysaesthesia in both upper extremities. To address the treatment’s effect on the frequency of T-cells specific for the antigens used in the tolerization, the antigen-specific T-cell response was measured before and three months after the treatment. Before treatment, one patient showed positive T-cell responses to all seven myelin peptides, which were all reduced three months after the treatment [337]. A second clinical trial was performed to extend the safety analysis and to

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assess the effect on antibody levels of common auto-antigens and myelin peptides. Serum samples were collected from MS patients one month before, 3 and 12 months after the treatment; it was shown that none of the patients had antibodies against native MOG, two patients had anti-MBP antibodies but their levels were reduced after treatment, and no new auto-antibody responses were induced by the treatment. In summary, the extended safety analysis of the ETIMS phase I study did not suggest an increase in auto-antibodies or the induction of new autoantibody reactivity in MS patients treated with ETIMS [338]. Moreover in MS patients the immunoregulatory processes that lead to limit the inflammation have been demonstrated to be less effective than in healthy subjects [339]. Therefore another strategy could be to restore the deficient suppressive mechanisms developing an immune-based vaccine strategy based on the use of a therapeutic TCR Peptide Vaccine. This approach is actually under consideration and two clinical studies not yet recruiting patients are present on clinicaltrial.gov web site dealing with a new trivalent TCR peptide vaccine (IR902) to be tested in SPMS (NCT02057159) and pediatric MS patients (NCT02200718) [339, 340]. Stem Cells Haematopoietic Stem Cells (HSCs) Bone marrow houses two types of stem cells with immunotherapeutic potential: the haematopoietic (HSCs) and the mesenchymal stem cells (MSCs). HSCs are surrounded by stromal cells which form the niche where haematopoiesis takes place and which support HSCs self-renewal and maintenance by preventing their apoptosis [341]. HSCs are characterized by the expression of markers such as CD34 and CD133 and they are characterized by their capacity to form clonogenic colonies in vitro while in vivo they can differentiate into myeloid and lymphoid lineage precursors. HSCs are routinely used in haematology to reconstitute the immune system after myelo- and immune-ablative chemotherapy. They are mobilized from the haematopoietic niche using immunosuppressive agents and collected from the blood stream by leukapheresis, while an alternative source for HSCs is umbilical

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cord blood. When HSCs are collected and reinfused in the same patient the procedure is defined as autologous haematopoietic stem cell transplantation (HSCT) while transplantation from a HLA-matching donor is termed allogenic transplantation. HSCT is mainly indicated to treat different malignant haematological disorders, while a therapeutic potential for autoimmune disorders has also been proved [341]. The most radical approach to eradicate disease-inducing autoimmune cells is immune ablation which eliminates the pathogenic auto-reactive immune cells while the regeneration of a healthy immune system occurs through HSCT. The assumption is that during immune reconstitution, HSCs can regenerate a healthy immune system but autologous HSCs obviously confer no correction of genetic deficits. However, there is strong evidence that MS is not a merely genetic disease and that an environmental component is implicated [341]. During the past 15 years HSCT has become an emerging indication for autoimmune diseases indeed autologous HSCT is performed for patients with MS, systemic sclerosis, systemic lupus, Crohn’s disease, type I diabetes and juvenile idiopathic arthritis, while allogeneic HSCT is still considered too dangerous for autoimmune diseases. Several studies in EAE have revealed that HSCT can induce a remission showing HSCs recruitment to sites of neurological damage [341]. In RRMS patients, pilot studies began in the 1990s and provided proof of the principle that HSCT could induce stabilization in patients with severe MS due to the replacement of the existing immune system by the immature haematopoietic progenitors. Immunodepletion followed by autologous HSCT has been limited to patients with highly active, rapidly degenerating, refractory forms of MS. Numerous patients have undergone autologous HSCT for the treatment of severe MS. Suppression of relapses and of Gd-enhancing MRI lesions after HSCT has been clearly demonstrated [342]. No other treatment has shown such a degree of suppression of new MRI lesions. Because of its greater impact on patients’ quality of life, progression free survival (PFS), which means no increase in EDSS score, is considered a particularly important outcome measure.

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The largest body of data currently available is a retrospective analysis of data from more than 170 patients. This analysis reported a PFS in 60% to 70% of patients after 3 years and 50% to 60% after 6 to 8 years. Twenty-one RRMS patients were treated with a reduced-intensity conditioning HSCT regimen and reported 100% PFS, with 81% showing an improvement in neurological function after a median of 3 years of follow-up [343]. Krasulova et al. described a series of Russian cases and reported a significantly higher rate of PFS in RRMS than in SPMS patients, asserting that there is an association of better clinical outcomes in patients with short disease duration and a younger age [344]. Young patients with highly aggressive, rapidly evolving forms of MS constitute a group that seems to especially benefit from HSCT. Candidate patients for autologous HSCT should have evidence of inflammatory activity. Patients with advanced disabilities, especially those with PPMS or SPMS, without clear inflammatory activity are less likely to benefit from HSCT because the disabilities result from a neurodegenerative process rather than an acute inflammation [342]. Large series of MS patients was evaluated by the European Group for Blood and Marrow Transplantation (EBMT) Working Party on Autoimmune Diseases. The study included PPMS (26%) and SPMS (70%) patients who were subjected to HSCT. After a follow-up period, the chance of PFS was 74% at 3 years. Five patients died of treatment-related complications including infection and cardiac failure [345]. Taken together, these results show that in most cases HSCT is able to improve MS symptoms in the progressive phase of MS. European and North American investigators are currently developing a larger multicentre, randomized, controlled phase III trial which will assess the efficacy of autologous HSCT transplantation versus standard of care in highly active, treatment-refractory RRMS [342]. There have been numerous clinical trials on HSCs used in the treatment of MS and many of them have already been published. Currently, however, there are 4 ongoing active clinical trials as reported on the clinicaltrial.gov web site (NCT00273364; NCT01099930; NCT00497952; NCT00288626).

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Mesenchymal Stem Cells (MSCs) MSCs display adult stem cells’ multipotency by differentiating into bone, adipose tissue and cartilage and they can easily be isolated from bone marrow although they can be also retrieved from placenta, umbilical cord, adipose tissue and teeth [346] and then be expanded in vitro. MSCs are immune privileged cells since they exhibit low levels of MHC class I molecules and rarely express MHC II, thus escaping the T-cell recognition. Moreover they do not express co-stimulatory molecules [346], are able to suppress the T-cell response to mitogens and they can mediate immunosuppression of innate and adaptive immune system cells by affecting Bcells and inhibiting the NK cell proliferation, cytotoxicity and cytokine production. MSCs affect in vitro maturation of DC through the down-regulation of MHC II, CD11c, CD83 and co-stimulatory molecules. MSCs promote the generation of CD4+ Treg and they are able to switch CD4+ T-cell responses from a Th1 to a Th2 polarized phenotype [347]. Furthermore, MSCs are able to sense dangered tissues on receiving chemotactic signals, and they migrate to the site of injury homing in different organs including the CNS. Most in vivo studies have tested the immunomodulatory properties of MSCs in EAE. Intravenously injected MSCs ameliorated EAE by homing in lymphoid organs and reducing T, B-cells and macrophage infiltration in the CNS [348]. They can also decrease inflammation, demyelination and axonal loss by homing in the CNS but without trans-differentiating into neural cells [349]. It is supposed that MSCs’ therapeutic activity depends on the release of anti-apoptotic, antiinflammatory and trophic factors. Moreover it’s also possible that MSCs act recruiting local progenitors and inducing their differentiation into neurons [341]. Currently, there are guidelines for the culture of MSCs and for the treatment of MS patients [350]. Autologous MSCs are mainly chosen for MS patients’ treatment since this minimizes the risk of transmitting infections [351]. Most clinical studies administrate MSCs intrathecally, intravenously or

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intravenously plus intrathecally. A recent in vivo work on EAE has demonstrated that (i.t.) and i.v. administration result in similar clinical effects [352], therefore, since an i.t. injection is invasive, i.v. injection should be considered a safer option [351]. As regards the dose of MSCs, most preclinical studies in murine models have considered to use a single dose of 1x106 MSCs which clinically would amount to a dose of 50x106 MSCs/Kg of body weight. Unfortunately, it is not possible to collect and expand such a quantity of MSCs from BM aspiration and so the common dose of cells used in clinical trials is about 1-2x106 MSCs/Kg of body weight [351]. Finally, no serious adverse events have been reported in clinical studies, except some cases of meningeal irritation after i.t. injection and cutaneous rash, scalp pruritus, respiratory and urinary tract infection which may occur after i.v. injection. Despite these minor limitations, MSCs are considered safe and well tolerated [351]. A phase I clinical trial (NCT00781872) tested the feasibility and safety of i.t. and i.v. injection of autologous MSCs showing no adverse effects and an improvement in EDSS. MRI showed the presence of MSCs in the occipital horns of the ventricles and the proportion of Treg cells was increased with a decrease in lymphocytic proliferation [353]. A pilot study was performed on 10 patients with non-responsive MS and MSCs i.t. injection resulted in no significant adverse events [354] while the i.t. injection of autologous MSCs in patients with advanced MS was safe and it also resulted in clinical but not radiological efficacy [355]. In another study (NCT00395200) autologous i.v. MSCs were used in SPMS showing an improvement in visual acuity, in visual evoked response latency and it increased the optic nerve area of recipients [356]. Even (NCT01364246) umbilical-derived MSCs have been tested in progressive MS patients to evaluate their safety and efficacy, nevertheless the results of this trial have not been reported yet.

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Actually there are 19 registered clinical trials for MSC treatment in MS on the clinicaltrial.gov web site (NCT01377870; NCT01895439; NCT00395200; NCT01730547; NCT01854957; NCT01364246; NCT01056471; NCT02403947; NCT02034188; NCT02326935; NCT01228266; NCT01606215, NCT00813969; NCT01933802; NCT01745783; NCT02239393; NCT00781872; NCT02166021; NCT02249676) among which 4 trials have been completed (NCT01377870; NCT00395200; NCT01228266; NCT00813969). Helminths In the developed world throughout the 20th century, the improvement in general living conditions contributed to reducing the exposure of humans to infections such as viruses, bacteria and worms. Although this may have led to less contact with potential autoimmune antigens, on the other hand, this microbial deprivation may, at least in part, account for the current epidemic of autoimmune diseases [357 - 359]. In particular, there has been a reduction in human-intestinal worm cohabitation: the intestine is the largest compartment of the immune system and the helminths have a particularly strong effect on the immunoregulatory pathways closely associated with the inflammatory response and autoimmunity [360 - 362]. Fleming identified several mechanisms that are involved in the helminths’ escape from the immune attack including the promotion of regulatory lymphocytes and the immunological drift towards an anti-inflammatory response [363]. Thus the decrease in worm infections could account for the increase in the incidence of allergy and autoimmune diseases since the absence of these worm infections influences the pro- and anti-inflammatory T-cell development leading to a loss of self-tolerance [361]. The use of the EAE model together with the first long-term observational studies paved the way for the first clinical safety studies in RRMS patients. Correale and Farez [3, 10] were the first to investigate the influence of parasitic infections on RRMS individuals. They selected two demographically-matched cohorts of patients, one affected by naturally contracted parasitism and the other without worm infection. After an observation period of over 7 years, the infected individuals showed a reduced number of clinical attack and new lesion

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appearance, while the anti-helminthic therapy induced disease exacerbations in such patients. In addition to the clinical results, it was shown that there was an increase in various anti-inflammatory networks in the infected MS patients. On the other hand, in both mouse and rat EAE, the infection with different kinds of helminth eggs and larvae during the preimmunization and preclinical phase was effective in controlling the disease [364]. In spite of their promising relevance in limiting MS clinical impairment, at this point in time, only four clinical studies are formally listed and they are mainly safety and efficacy phase I clinical trials using a porcine whipworm ova, Trichuris suis ova (TSO) (3 out of 4 reported trials) or Hookworm larvae. The first clinical trial (HINT, NCT00645749) aimed at testing the safety and tolerability of TSO in 5 newly-diagnosed, disease-modifying drug-free RRMS patients. As reported by Fleming et al. [364], TSO were used since they are neither pathogenic for humans nor do they entail the risk of transmitting human pathogens; moreover, they can be supplied at an adequate purity and safety standard (GMP, good manufacturing product) to be used in humans [365]. Therefore, after 1 month pre-treatment screening, 2,500 GMP TSO were orally administered every 2 weeks for 3 months and the patients monitored for 2 more months after the treatment ended. TSO was found to be well tolerated with mild gastrointestinal symptoms at most which did not interfere with everyday activities; a reduction in active lesions was reported during the treatment period as well as an increase in the serum levels of anti-inflammatory IL-4 and IL-10. These very preliminary data led to permission being granted for further a clinical step (HINT 2) in which 18 RRMS patients were recruited for a longer period (20 months); the results were expected in late 2013. Another safety study, named TRIMS A (NCT01006941) was conducted on 10 RRMS or SPMS patients with relapse who were orally treated with 2,500 TSO / dose every second week for 12 weeks without showing any of the neurological benefits reported by Fleming et al., 2013, although official results of the studies have not been made available up to now. The study protocol for a randomized control trial on TSO safety/effectiveness in RRSM and CIS patients has recently been published and is referred to as the TRIOMS study (NCT01413243) [366].

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The study started in 2012, enrolling patients with active recurrent remittent MS or CIS; they were patients with clinical activity and with inefficacy or intolerance for therapy with IFNbeta and not undergoing any other standard therapy. Also in this trial TSO were used and 2,500 eggs were given every 2 weeks for 12 months; a placebo control group was also present. While the final data collection for the primary outcome measure is expected in late 2016 and no preliminary results are available, the same research team has recently published the results of the first pilot study to be performed on 4 SPMS patients recruited in Berlin and administered the same TSO schedule over 6 months and then monitored for 4 more months. The TSO treatment was well tolerated also in SPMS patients, while the results dealt mainly with the haematological effect of helminth treatment. A first transient increase in pro-inflammatory cytokine IFNγ was followed by a trend towards a decrease in both IFNγ and IL-2, a slight change in the T-cell main subset and an eosinophil count suggesting a possible systemic influence to be further studied [367]. The WIRMS study (NCT01470521) is a unique study among those presented both for the choice of parasite and for the method of exposure. In fact, the 72 enrolled RRMS patients received a single dose of 25 live hookworm larvae (Necator americanus larvae) that were pipetted onto a plaster dressing which was then placed on the upper forearm for 24 hours; the placebo patient group received pharmacopoeia grade water by the same method. The RRMS patients were clinically, immunologically and radiologically monitored for 1 year with the cumulative number of Gd-enhancing lesions on T2 weighted MRI being the primary clinical outcome, and the immunology regulatory network being the secondary one. Although the use of live helminths seems to be a promising strategy for RRMS patients, the possible interference of worm infection with vaccine efficacy, and the complications arising from co-infections, has to be carefully considered. The increased interest in helminths-based therapy could also lead to the development of an approach based on helminth-derived molecules [368].

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Vitamin D The epidemiologists have observed that MS incidence is strictly connected with the geographical localization and with the latitude. The latitude-gradient of MS prevalence can be explained with sun exposure, in fact UV have immunosuppressive properties which are taught to be due to UVB-mediated transformation of vitamin D into 1,25-dihydroxyvitamin D3 [369, 370]. Vitamin D is a steroid hormone that plays a central role in the control of bone and calcium homeostasis. Dietary intake accounts for only 30% of its physiological need while the main route for obtaining vitamin D is exposure to sunlight. Vitamin D is a modulator of the immune response since monocytes, macrophages, DC, activated T and B-cells all express VDR, so it can play a role in autoimmune diseases [369]. Deficiency in vitamin D or mutations in VDR diminish its function on the immune system and could lead to an increased risk for autoimmunity since the nuclear translocation of VDR targets genes involved in cell proliferation, differentiation and immunomodulation [369]. It has been evidenced that low levels of sun exposure, of vitamin D intake or of vitamin D blood levels are associated with a higher risk of MS. In MS patients high serum level of a biologically active form of vitamin D is associated with a lower frequency of new lesions and fewer Gd-enhancing MRI lesions [371]. Moreover variability in vitamin D-related genes such as vitamin D-activating enzyme locus, VDR and the vitamin breakdown enzyme has been also observed in MS [372]. Moreover it has recently been demonstrated that the average serum levels of 25hydroxyvitamin D3 (25(OH)D) are strongly associated with MS prognosis in patients with CIS. The BENEFIT study enrolled 468 CIS patients treated with IFNB-1b and 25(OH)D serum concentration was measured at baseline, 6, 12 and 24 months after CIS. It was observed that within the first 12 months patients with a 25(OH)D serum level of more than 50 nmol/L had a 25% lower modification in T2 lesion volume, a lower neurological disability, a 57% lower relapse rate, a 57% lower rate of new active lesions and a 2 fold lower grade of brain atrophy then patients who had a serum concentration below 50 nmol/L. So low vitamin D

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level was a strong predictor of the disease course and for CIS patients, in which MRI value is very prognostic for the development of long-term disability, the strict connection between MRI measures and 25(OH)D level had a great clinical relevance [373]. The results of BENEFIT clinical trial support the protective role of vitamin D in MS, previously suggested by epidemiological evidences. In line with the observational studies it was also demonstrated that the administration of a biologically active form of vitamin D suppresses the development of Th1 autoimmune disease and that the vitamin D treatment of EAE blocked the disease’s development [374]. Moreover many in vivo studies have demonstrated that vitamin D administration or UV exposure can improve EAE disease and provides protection from disease development [375]. Some randomized controlled trials of vitamin D supplementation have been conducted, but they obtained controversial results and they were quite small. A recent clinical trial (NCT00644904) has showed that short-term vitamin D supplementation at a high dose (40,000 IU/day) appeared to induce fewer relapse events (from 0.44 to 0.26) and a persistent reduction in T-cell proliferation compared to controls [376]. Indeed, another small trial showed that vitamin D supplementation (6,000 IU/day) was associated with a higher EDSS score and a greater number of relapses in MS patients compared to lower doses (1,000 IU/day) but this study cannot be considered conclusive since it enrolled only 23 patients with RRMS [377]. Moreover vitamin D supplementation in association with IFNbeta was safe for more than 60 MS patients and induced fewer Gdenhancing lesions and a trend towards slower disability progression [378]. Currently, there are 2 clinical trials reported on clinicaltrials.gov (NCT01285401; NCT01440062) which will hopefully provide more definitive results about this issue since they enrolled more MS patients than those reported above. Remyelination Strategies in MS MS patients experience acute episodes of neurological dysfunction associated with inflammatory-related white and grey matter demyelination which usually recovers together with neurological impairment. Attempts to remyelinate the denuded axons have been observed in different MS patients’ CNS locations [379]

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together with the more prominent remyelination process that occurs in the early stages of MS [380]. However, the accumulation of disability reflects chronic demyelination which leads to axon damage and neuronal loss within the CNS. In fact, myelin plays a role not only in axon conduction but also in axon survival and full functionality [381]. Intrinsic remyelination is a complex process which involves the oligodendrocyte progenitor cells (OPCs) and their interaction with other glia cells as well as with neurons and the extracellular tissue environment. The OPCs are present all along the CNS and shift from a quiescent towards a migratory phenotype, moving towards the injured area. Here they differentiate into remyelinating oligodendrocytes by making contact with denuded axons, synthesizing myelin protein and wrapping the axon to create the myelin sheath which in MS remyelinated lesions appears to be thinner than the normal surrounding tissue [379, 381]. Because the OPCs are essentially responsible for myelin formation and have been described as accumulating but not myelinating cells in MS lesions [379], the main strategies for supporting the remyelination process in MS patients have focused on promoting the OPCs’ function. While several different approaches for controlling and limiting inflammation in MS have been developed mainly considering RRMS patients management, remyelinating strategies and approaches are still required for the progressive MS forms. Nowadays only a couple of pharmacological approaches in promoting remyelination are currently under clinical consideration (Table 3). BIIB033 The knowledge of the cellular and molecular mechanisms involved in the myelination process is the first and critical step for developing potentially remyelination-specific therapeutic strategies since developmental myelination and remyelination shares certain common mechanisms. Axon regeneration in the adult CNS is prevented by inhibitors present in myelin

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(i.e. MAG, OMG and Nogo-A) which bind to the receptor complex that comprises the Nogo-66 receptor (NgR1)/LINGO-1 and p75 on neurons, thus inducing intracellular downstream signals that block the axon’s outgrowth [382]. LINGO-1 (leucine-rich repeat and Ig-containing, Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS on oligodendrocytes and neurons, with a higher expression in the OPCs, while no mRNA has been retrieved in astrocytes [383]. LINGO-1 was first identified as the additional functional component of the NgR1/p75 required for signal transduction [383]. It functions as a negative regulator of neuronal survival and axonal regeneration, of oligodendrocyte differentiation and myelination [383], and its expression is developmentally regulated while it is upregulated along with CNS injury. In EAE, LINGO-1 is upregulated in comparison with healthy controls while in MS patients LINGO-1 expression is increased in OPCs from the demyelinated white matter of post-mortem MS patients’ CNS [384]. In MS animal models targeted-LINGO-1 inhibition resulted in improved functional recovery together with enhanced remyelination processes [383, 385 - 387]. Taken together, these results have suggested LINGO-1 as a novel target in MS therapy under the hypothesis that LINGO-1 inhibition could enhance remyelination and reduce axonopathy in MS patients. The anti-LINGO-1 IgG1 antibody Li81, formally BIIB033, is a fully humanized monoclonal antibody that binds to the convex surface of the leucine-rich repeat domain of LINGO-1, inhibiting the oligomerization of LINGO-1 and producing a complex that masks functional epitopes that are important for LINGO-1’s role in oligodendrocyte differentiation [388]. Two randomized, blind, placebo-controlled phase I studies were conducted. The single ascending dose (SAD) study enrolled 64 healthy volunteers administered with i.v. ascending doses of BIIB033 from 0.1 up to 100mg/kg and one group was subcutaneously treated with BIIB033 3mg/kg while 18 healthy individuals received placebo treatment (NCT01052506). In the second study, the multiple ascending dose (MAD) study, 42 RRMS/ SPMS patients were enrolled and two

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identical i.v. doses of BIIB033 were given 12-14 days apart (single dose ranging from 0.3 up to 100mg/kg) (NCT01244139). An additional single dose group was added to the trial plan to assess the tolerability of the faster infusion of the highest tested dose in the multiple infusion group. The results have recently been reported by Tran et al., [389]. All doses in both the SAD and MAD studies were well tolerated with an adverse effect rate near to that reported in the placebo group. Moreover, poor immunoreactivity was elicited by BIIB033 administration, while the BIIB033 CSF/serum concentration ratio was similar to that reported for other IgG monoclonal antibody drugs. This ratio appeared to increase in a dosedependent manner, with the exception of the 10mg/kg dose. Starting from the results reported for BIIB033 distribution to the CNS the authors claimed that the four doses higher than 3 and 10mg/kg could have some potential pharmacological activity [389]. In 2013, a further study (NCT01864148, EudraCT number 2011-006262-40) was started to evaluate the concurrent use of BIIB033 and IFNbeta-1a in a larger cohort of RRMS and SPMS patients experiencing active relapse within 12 months of enrolment; the primary outcome was to be the number of patients with confirmed improvement of neuro-physical and/or cognitive function and/or disability. The first estimated completion date for this study is 2016. rHIgM22 Naturally occurring auto-antibodies (NA) are circulating in each individual [390]. Among these antibodies, CNS protecting and repairing NA have been identified based on the results first obtained in a TMEV-mediated spinal cord demyelination model. In fact, the presence of antibodies able to promote remyelination was first demonstrated in the serum of TMEV-affected mice. These antibodies were IgM and could bind to live oligodendrocytes. Based on these in vivo findings, a screening for oligodendrocyte auto-reactive antibodies was performed. High concentration sera from different individuals were screened for their capability to bind to mouse CNS slides irrespective of the recognized antigen. Six out of 52 investigated IgM were positive for this assay while only 2 of them (sHIgM22 and sHIgM46) could also promote remyelination in vivo.

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To ascertain the unique correlation between IgM NA and remyelination processes, a recombinant IgM was then established (rHIgM22). It showed therapeutic potential in virus and toxin-induced MS animal models as demonstrated by MRI, without affecting the immune system function. The capability of rHIgM to cross the BBB and to accumulate in the CNS lesions suggested the use of rHIgM22 for MS patient treatment [391]. A double-blind, placebo-controlled, single ascending i.v. infusion study of rHIgM22 is currently ongoing (NCT01803867), and it has been designed to evaluate the safety, tolerability, pharmacokinetics, and immunogenicity of a single i.v. administration of rHIgM22 in 71 patients all of whom have clinical presentations of MS; the final data collection for the primary outcome measures is due to take place in late 2014. Table 3. Experimental immunomodulating approaches and remyelination strategies. Emerging strategies other than monoclonal antibodies which can affect the immune system auto-reactivity are actually under investigation for treatment of MS patients. IMMUNOMODULATING DRUG/TREATMENT APPROACHES TOLEROGENIC VACCINES

ATX-MS-1467

MBP8298

CLINICAL TRIAL

Mixture of 4 s.c. human MBP s.c. peptides

RRMS

NCT01097668

RRMS

NCT01973491

Synthetic MBP i.v. peptide i.v.

RRMS

NCT00869986

SPMS

NCT00468611

i.v.

SPMS

NCT00869726

i.v.

SPMS

NCT00870155

i.v.

RRMS

NCT01414634

i.v.

SPMS

NCT02057159

i.v.

Pediatric MS NCT02200718

Immunodominant peptide coupled PBMC IR902

ADMINISTRATION PATIENTS ROUTE

Trivalent TCR peptide vaccine

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(Table ) contd.....

IMMUNOMODULATING DRUG/TREATMENT APPROACHES

ADMINISTRATION PATIENTS ROUTE

STEM CELLS

Autologous

i.v.

RRMS/SPMS NCT01099930

Allogenic

i.v.

RRMS

Autologous

i.v.

RRMS/SPMS NCT00288626

i.v.

RRMS

Autologous

i.v.

RRMS/SPMS NCT00813969 PRMS

Autologous

i.v/ i.t.

No responsive MS

NCT00781872

Autologous

i.v.

SPMS

NCT00395200

Umbilical MSC

i.v.

SPMS/ NCT01364246 neuromyelitis optica

oral

RRMS

oral

RRMS/SPMS NCT01006941

oral

RRMS/CIS

NCT01413243

dermal

RRMS

NCT01470521

oral

Clinically defined MS

NCT00644904

RRMS

NCT01339676

HSCs

MSCs

HELMINTHS

Trichuris suis ova

Necator americanus larvae VITAMIN D

Vitamin D3 dietary supplement

IFNbeta-1b + s.c.+ Cholecalciferol oral capsules

REMYELINATION DRUG/ STRATEGIES TREATMENT BIIB033

Human aglycosyl IgG1

rHIgM22

TARGET ADMINISTRATION PATIENTS ROUTE Lingo-1

NCT00497952 NCT00273364

NCT00645749

CLINICAL TRIAL

i.v. / s.c.

Healthy volunteers

i.v.

RRMS/SPMS NCT01244139

i.v.

RRMS/SPMS NCT01864148

i.v. Recombinant hIgM

CLINICAL TRIAL

NCT01052506

MS NCT01803867

CONFLICT OF INTEREST The authors confirm that they have no conflict of interest to declare for this publication.

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ACKNOWLEDGEMENTS The authors wish to thank Ms Elizabeth Genton for her language assistance. ABBREVIATIONS 25(OH)D

=

25–hydroxyvitamin D

ADCC

=

Antibody dependent cellular toxicity

APC

=

Antigen Presenting cells

ARR

=

Annualized relapse rate

BBB

=

Blood Brain Barrier

BCR

=

B cell receptor

BM

=

Bone marrow

CDC

=

complement dependent cytoxicity

CIS

=

Clinically Isolated Syndrome

CNS

=

Central Nervous System

CSF

=

Cerebrospinal Fluid

DC

=

Dendritic cells

EAE

=

Experimental Autoimmune Encephalomyelitis

EBMT

=

European Group for Blood and Marrow Transplantation

EBV

=

Epstein-Barr virus

ECTRIMS

=

European Comitee for the Treatment and Research in Multiple Sclerosis

Gd

=

Gadolinium

GM-CSF

=

Granulocyte macrophage-colony stimulating factor

GM-CSF-NAg =

GM-CSF-neurogenic fusion protein

GMP

=

Good manufacturing product

HAHA

=

Human Anti Human Antibody

HLA

=

Human Leukocyte Antigen

HSCs

=

Haematopoietic stem cells

HSCT

=

Haematopoietic stem cell transplantation

I.D.

=

Intradermal

I.M.

=

Intramuscular

I.T.

=

Intrathecal

I.V.

=

Intravenous

ICAM-1

=

Intercellular adhesion molecule-1

IFN

=

Interferon

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Ig

=

Immunoglobulin

IL

=

Interleukin

iTreg

=

Adaptive regulatory T-cells

JCV

=

JC virus

LFA-1

=

Lymphocyte function-associated antigen-1

LINGO-1

=

Leucine-rich repeat and Ig-containing, Nogo receptor interacting protein-1

MAG

=

Myelin-associated glycoprotein

MBP

=

Myelin basic protein

MHC

=

Major histocompatibility complex

MMPs

=

Metalloprotinases

MOG

=

Myelin oligodendrocyte glycoprotein

MRI

=

Magnetic Resonance Imaging;

MS

=

Multiple Sclerosis

MSCs

=

Mesenchymal stem cells

NA

=

Naturally occurring auto-antibodies

nTreg

=

Natural regulatory T-cells

OCB

=

Oligoclonal bands

OMG

=

Oligodendrocyte Myelin Glycoprotein

OPCs

=

Oligodendrocyte progenitor cells

PB

=

Peripheral blood

PBMC

=

Peripheral mononuclear cells

PFS

=

Progression free survival

PLP

=

Proteolipid protein

PML

=

Progressive multifocal leukoencephalopathy

PPMS

=

Primary Progressive Multiple Sclerosis

PRR

=

Pattern Recognition Receptor

RIS

=

Radiologically Isolated Syndrome

ROS

=

Reactive oxygen species

RRMS

=

Remitting Relapsing Multiple Sclerosis

S.C.

=

Subcutaneous

SPMS

=

Secondary Progressive Multiple Sclerosis

TNF

=

Tumor necrosis factor

Treg

=

Regulatory T-cells

Tr1

=

T regulatory-1

TSO

=

Trichuris suis ova

82 FCDR - CNS and Neurological Disorders, Vol. 4 VCAM-1

=

Vascular cell adhesion molecule-1

VDR

=

Vitamin D receptor

VLA-4

=

Very late activation antigen-4

Rigolio et al.

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CHAPTER 2

Prospective Therapies for Alzheimer Disease: Biomarkers, Clinical Trials and Preclinical Research Jofre Güell-Bosch1, Gisela Esquerda-Canals1,2, Laia Montoliu-Gaya1, Sandra Villegas1,* Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès. Spain 1

Departament de Biologia Cellular, Fisiologia i Immunologia, Unitat de Citologia i Histologia, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain 2

Abstract: Most of the 46.8 million people estimated in the year 2015 as living with dementia worldwide were attributed to have Alzheimer’s disease (AD), with projections set to be almost tripled by 2050. Current drugs to treat AD are focused on ameliorating symptoms instead of treating its underlying causes, becoming only palliative. Consequently, treatments to prevent, stop or reverse this overwhelming disease are desperately needful. This Chapter takes a tour through clinical and preclinical studies from different approaches, ranging from those based on small molecules to immunotherapy. But first it also addresses the role of biomarkers in early diagnosis, which is necessary not only to properly recruit patients but also for an accurate assessment of efficiency and safety in clinical trials. Among other approaches, Aβ- immunotherapy has emerged as a promising tool for the treatment of AD. Whereas active immunotherapy, namely administering fragments of the Aβ-peptide, is currently in Phase II, passive immunotherapy, specifically administering antibodies against the Aβ-peptide, reached Phase III. Some of these Phase III trials failed probably because they were performed in patients with mild-to-moderate AD, a too advanced stage of the disease. Currently, different cohorts have been recruited for clinical trials: Corresponding author Sandra Villegas: Protein Folding and Stability Group, Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Campus Bellaterra 08193, Cerdanyola del Vallès, Spain; Tel/Fax: 34935814258/1264; Email: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2016 Bentham Science Publishers

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asymptomatic and very-mildly symptomatic carriers of autosomal-dominant AD mutations as well as symptomatic elderly patients with amyloid positive PET. More studies are needed, but we are getting closer to find a disease-modifying drug to cure this devastating disease.

Keywords: AD biomarkers, AD imaging, Alzheimer's disease, Amyloid-β, APOE, APP, Immunotherapy, New therapies, PSEN, Vaccine. I. INTRODUCTION I.1. Background On November 1906, Alois Alzheimer reported an oral communication “On a peculiar severe disease of the cerebral cortex” to the 37th Meeting of South-West German Psychiatrists in Tubingen. He described the pathology of a 50-year-old woman whom he had followed since her admission for memory loss, disorientation, hallucinations, and dementia, until her death 5 years later. His report noted particular features in the brain histology: a dramatic reduction in the number of cortical cells as well as distinctive extracellular plaques and intracellular neurofibrillary tangles (NFTs). Little interest was aroused despite an enthusiastic response from Emil Kraepelin, who included the term “Alzheimer's disease” (AD) in the 8th edition of his text “Psychiatrie” in 1910. The term “amyloid”, stemmed from the Latin word “amylym”, was first referred in the XIX century as the substances featuring a positive staining for starch. Just after cellulose was known to change from brown to blue upon iodine-sulfuric acid reaction, positive small round deposits were described in the nervous system. It was not until 1984 when Glenner and Wong first reported a 4 kDa peptide as the main component of cerebrovascular amyloids associated to AD [1]. Subsequently, the sequencing of the A4 peptide (“A” stands for “amyloid” and “4” refers to the Mw) from the core of amyloid plaques dissected from AD and old-age DS (Down Syndrome) individuals allowed the localization of the gene coding its precursor (APP, Amyloid Precursor Protein), a cell-surface receptor, on human chromosome 21 [2]. Nowadays the A4 peptide is known as the Aβ peptide because it aggregates featuring β-sheet secondary structure [3]. The intracellular NFTs turned out to be constituted by aberrant forms (typically hyperphosphorylated and

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fragmented) of tau, a microtubule-associated protein [4]. In contrast to Aβpeptide, abnormal tau metabolism is also associated to other neurodegenerative diseases, collectively referred to as tauopathies [5]. Both, the Aβ-peptide and abnormal tau, are known to trigger oxidative stress and inflammatory responses during AD progression. I.2. Alzheimer's Disease and the Need for New Drugs Most of the 46.8 million people estimated in the year 2015 as living with dementia worldwide were attributed to have AD, with projections set to 74.7 million by 2030 and 131.5 million by 2050 [6]. AD begins with trouble remembering some daily events and evolves into a progressive dysfunction in orientation in time and space and, finally, into a general deficit in motor functions. Because the neurodegenerative process may extend for over a decade, the associated dementia causes a long suffering to both patients and their caregivers. In consonance, the economic impact is huge, with an US$818 billion worldwide estimated in 2015 [6]. This cost comprises social care provided by community professionals, informal care provided mostly by relatives and medical care. Because there are no drugs that can halt its progression, treatments to prevent, stop or reverse this overwhelming disease are desperately needful. Currently, two types of drugs are approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the palliation of AD symptoms: those acting on the cholinergic system and one acting on the glutamatergic system. Because the death of cholinergic neurons is the main output of the pathology, increasing the levels of acetylcholine partially compensates for the loss of this neurotransmitter [7]. The cholinesterase inhibitors donepezil, galantamine, rivastigmine and tacrine are used for this purpose. On the other hand, it is known that glutamate is increased in AD patients, as well as in other neurodegenerative diseases, and that this unbalance leads to cell death. Memantine, an antagonist of N-methyl-D-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors, is thought to have a neuroprotective effect [8]. However, these drugs only have palliative benefit during some stages and are very individual dependent.

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I.3. The Amyloid Cascade Hypothesis (ACH) The main reason why no disease-modifying treatment for AD has been achieved up to now is that the exact mechanisms leading to the disease are still not well understood. The first attempt to propose a theory that could sequentially explain how AD is developed was done by Hardy and Higgins in 1992, who formulated the amyloid cascade hypothesis (ACH) [9]. The ACH asserts that the aggregation of the Aβ peptide is the cause triggering AD pathology, followed by the downstream formation of NFTs, cell loss, vascular damage and, ultimately, dementia (Fig. 1, top). ACH was well accepted and supported by the discovery of dominant mutations in the APP gene, as well as in some genes for APPprocessing enzymes (presenilins) although it has been modulated over time (Fig. 1, bottom) [10]. Original Calcium disruption

APP mutations Aβ deposition

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Inflammatory response Oxidative stress

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Fig. (1). Overview of the amyloid cascade hypothesis (ACH) evolution. On the top, the ACH in its original form, which situated amyloid β (Aβ) aggregation in the beginning of the cascade triggering the rest of events until the final dementia occurs. On the bottom, the current vision of ACH, with Aβ oligomers having a central role in the disease triggering. It is not clear how this is correlated with tau hyperphosphorilation and how this downstream event leads to neuronal loss. The current vision, although remaining Aβ- centric, is much more complete than the original one and incorporates the main events in AD pathology. AD: Alzheimer’s disease, FAD: Familial Alzheimer’s disease, SAD: Sporadic Alzheimer’s disease, ApoE: apolipoprotein E, APP: Amyloid precursor protein, PS1/PS2: Presenilin 1 and 2, NFTs: Neurofibrillary tangles, PHFs: Paired helical filaments. [Figure adapted from [10]].

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DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVML 39/40 42/43

Fig. (2). The Amyloid Precursor Protein (APP) processing. On the top, in the nonamyloidogenic pathway APP is sequentially processed by α-secretase and γ-secretase. The α-secretase cleavage occurs within the Aβ sequence. On the bottom, in the amyloidogenic pathway APP is cleaved by β-site APP cleaving enzyme 1 (BACE1), also known as β-secretase, instead of by α-secretase and, finally, γ-secretase cleavage generates the Aβ-peptide. The γ-secretase cleavage can occur at different sites resulting in the generation of different size Aβ peptides (from 37 to 43 residues), being the Aβ 1-40 the most abundant one, and the Aβ 1-42 the most amyloidogenic one. AICD: APP intracellular C-terminal domain. [Reviewed in [11]].

The current vision, although remaining Aβ-centric, is much more complete than the original one and incorporates the main events in AD pathology. It must be mentioned, however, that some authors claim that factors downstream Aβ oligomers depicted in (Fig. 1) (bottom panel), such as calcium disruption and/or oxidative stress, could have a more important role. These factors have even been postulated as the ones triggering the disease, what have given rise to the nonamyloidogenic hypothesis of AD (see next section). APP is a type-1 transmembrane protein mainly located in neurons’ synapses where it acts regulating synapse formation, among other functions [12]. Within the cell, APP can also be found at endosomal, lysosomal and mitochondrial membranes, in the trans-Golgi network or in the endoplasmic reticulum [13]. APP is normally cleaved by α-secretase within the Aβ sequence (Fig. 2, top), releasing the extracellular fragment sAPPα, related to neuronal plasticity, and leaving the

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C83 carboxyl-terminal (C-terminal) fragment anchored within the cellular membrane. Then C83 can be either degraded in lysosomes or cleaved by γsecretase releasing the extracellular small fragment known as p3 and the APP intracellular C-terminal domain (AICD), both related to neuron cholesterol homeostasis [11]. However, if instead of α-secretase is β-secretase, also known as β-site APP cleaving enzyme 1 (BACE1), the one cleaving APP, the amyloidogenic pathway is taken (Fig. 2, bottom). The generated soluble form of APP, sAPPβ, is shorter, and the C-terminal domain anchored within the cellular membrane, C99, is longer. The latter fragment can be processed by γ-secretase, resulting in the AICD and, importantly, the extracellular Aβ peptide. It is likely that γ-secretase cleaves the substrate several times leading to the generation of multiple lengths Aβ peptides, from Aβ1-37 to Aβ1-43, being the Aβ1-40 species the most abundant one in the human brain. The site where the Aβ peptide is cleaved is not trivial, as it has implications in the aggregation potential of the peptide and, thus, in its pathogenicity. Aβ1-42, although commonly found in a ratio around 1:10 with respect to Aβ1-40, has been shown to be more hydrophobic and prone to aggregation, and constitutes the predominant form in amyloid plaques, whereas Aβ1-40 tends to aggregate in the vasculature. Indeed, the variation in the Aβ1-42/Aβ1-40 ratio is thought to be a major determinant in the pathogenicity of the peptide [14]. The difference in size between p3 and Aβ, the final products of the nonamyloidogenic and amyloidogenic pathways, respectively, is due to the longer sequence of the Aβ peptide at the amino-terminus (N-terminus). This sequence is involved, from residue 6, in the β-hairpin in which the trimeric Aβ1-40 threedimensional structure is based (Fig. 3) [15]. It is known that Aβ1-40 peptide monomers tend to aggregate in oligomers multiple of three units (trimers, hexamers, nonamers and dodecamers), where the N-termini (DAEFR, residues 15) within the trimers are exposed to the solvent, while the C-termini, highly hydrophobic, are buried in the trimer core (Fig. 3). Likewise, 0.5nm-twisted hydrophobic package of the trimers leads to the formation of amyloid fibrils, in such a way that all of the N-termini within the fibril are solvent-exposed. Actually, Aβ aggregation is known to occur in a nucleated polymerization manner, so that the Aβ monomers would associate into soluble oligomers which,

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acting as seeds, would be able to subsequently form insoluble oligomers, protofibrils, long amyloid fibrils and finally amyloid (or senile) plaques [16].

Fig. (3). Three-dimensional structure of Aβ1-40 trimers. The structure obtained by Nuclear Magnetic Resonance (NMR) (pdb 2M4J [15]) shows the side-chain of the five N-terminal residues (DAEFR) exposed to the solvent, whereas those in the C-termini (V40 is shown) are buried in the hydrophobic core. β-sheet spans residues L17-A21. Each monomer is arbitrarily colored.

Among all Aβ species, soluble oligomers are the ones which occurrence better correlates with cognitive decline, thus contradicting the earlier hypothesis claiming that amyloid plaques were the main toxic Aβ species [17]. However, the mechanisms by which these oligomers induce toxicity and how they are correlated with other hallmarks such as neuronal loss and, specially, NFTs formation, remain unclear. Different theories arising from a wide range of in vitro, in vivo and even clinical studies have been proposed. I.4. Correlation among Aβ and other AD Hallmarks NFTs, composed by hyperphosphorylated tau and fragmented tau, are one of the main hallmarks of AD. Tau is a highly soluble cytoplasmic protein with a length of 352-441 amino acid residues and six different isoforms in the human brain. It participates in functions such as neurite outgrowth, axonal transport and, mainly, in microtubule assembly and stability (reviewed in [18]). Phosphorylation in

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specific sites of tau protein modulates its function and its localization. However, tau hyperphosphorylation leads to its dissociation from microtubules (preventing axonal transport) and sequential aggregation into paired helical filaments (PHFs), which subsequently form NFTs. Similarly to Aβ oligomers, tau oligomeric forms are cytotoxic and can cause cognitive deficits [19], which correlate well with disease progression but not with neuronal loss [20]. Furthermore, tau alone is able to cause cell loss and dementia in other diseases such as frontotemporal dementia (FTD). Taking this into account, tau aggregation may not be a mere consequence of Aβ formation, as it was initially postulated by the ACH, but rather it is likely that some kind of feedback exists between Aβ and tau (reviewed in [21]). Besides the two traditional hallmarks, many others have been added to the AD phenotype such as synaptic and neuron loss, oxidative stress and metabolism alterations, vascular injury or disruption of calcium regulation (see Fig. 1, bottom) (reviewed in [22]). A relationship among them, especially with Aβ aggregation, has been partially demonstrated although the clue for understanding the whole picture is still missing and the non-amyloidogenic hypothesis of AD is under consideration. Synaptic loss is a consequence of aging itself but, in AD brains, synapses are disproportionately reduced in relation to neural loss and better correlated to cognitive decline [23]. Aβ oligomers may impair synaptic plasticity facilitating the endocytosis by N-methyl-D-aspartate (NMDA) and α-amino-3-hidroxl-5-metil-4-isoxazol-propionate (AMPA) receptors, altering the balance between long-term potentiation (LTP) and long-term depression (LTD) [24,25]. In addition, Aβ would also bind to α-7 nicotinic acetylcholine receptors (nAChR) impairing the release of acetylcholine and dysregulating the whole cholinergic system [26]. Indeed, neurotrophin receptors in cholinergic neurons and neurotrophines such as brain-derived neurotrophic factor (BDNF) are found to be reduced in AD brains, preventing neuron proliferation and differentiation [27]. Mitochondrial functions are also altered in AD. Thus, affected mitochondria release oxidizing free radicals that cause a considerable oxidative stress [28]. Aβ,

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which is a potent generator of reactive oxygen species (ROS), could trigger this damage by producing elevated levels of free divalent transition metal ions, which cause neurodegeneration and tau aggregation. In addition, Aβ increases the membrane permeability to calcium ions. The calcium balance is modulated by presenilins, as AD-associated mutations cause Ca2+ overload of endoplasmic reticulum stores, which results in instability of mature synaptic spines and subsequent memory loss [29]. In turn, elevated concentrations of cytosolic calcium stimulate Aβ aggregation and amyloidogenesis [30]. Mitochondrial dysfunction also affects the metabolism of different substances involved in AD. Thus, glucose intolerance, as that in type 2 diabetes, is considered to be a risk factor for dementia, and has even been postulated as the cause of AD. The idea of oxidative stress as a link between insulin resistance and AD is currently under evaluation by several research groups, since the brain of AD subjects shows both insulin resistance and increased heme oxygenase-1 protein levels (for a review see [31]). Actually, heme oxygenase-1 (HO-1) and inducible nitric oxide synthase were found to be up-regulated by biliverdin reductase-A (BVR-A) in the aged beagle model of AD treated with atorvastatin [32]. In addition, nitrosative stress-induced modifications on hippocampal BVR-A [33], as well as oxidative stress-induced modifications on HO-1 [34], are an early event in the pathogenesis of AD since they appear also in MCI subjects (see biomarkers section). The induction of the heme HO-1/BVR-A system in the brain represents one of the earliest mechanisms activated by cells to counteract the noxious effects of increased reactive oxygen species and reactive nitrogen species, and thus it plays a pivotal role in AD pathogenesis. On the other hand, glucose transporter proteins, insulin receptors and other components in the insulin signaling pathway in the brain have been found to be reduced in some AD patients [35]. Moreover, high serum glucose levels are likely to up-regulate glycogen synthase kinase 3β (GSK-3β), which is a tau kinase, and to reduce the levels of the insulin-degrading enzyme (IDE), which is also able to degrade the Aβ peptide [22]. Finally, AD also implicates vascular damage and inflammation. Capillary abnormalities, disruption of the blood-brain barrier (BBB) and especially cerebral

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amyloid angiopathy (CAA), affecting more than 90% of AD patients, have been described [36]. It is likely that the Aβ transport across the BBB (mostly Aβ1-40, as is the one forming vascular deposits) is impaired because there is an imbalanced expression of the molecules responsible for the Aβ efflux (the low-density lipoprotein receptor-related protein 1, LRP1) and Aβ influx (receptors for advanced glycation end products, RAGE), thus, affecting Aβ clearance [37]. Other clearance mechanisms also involve activated microglia and reactive astrocytes, whose biochemical markers are elevated in AD and which co-localize with amyloid plaques [38]. The activation of microglia, which also induces the expression of Aβ binding RAGE receptors, could trigger the generation of cytokines and the classic complement pathway activation, leading to inflammatory responses [39]. I.5. Genetics of AD Depending on the age at which clinical symptoms appear two main forms of AD are classified: the early-onset AD (EOAD) and the late-onset AD (LOAD), being the threshold around 60-65 years old. EOAD represents only 1-2% of the AD cases, and a minor part of these is considered to be familial AD (FAD) because hereditary autosomal dominant mutations are found. Thus, the rest of EOAD cases are usually classified together with the LOAD cases as the sporadic form of the disease (SAD). The finding of autosomal dominant mutations causing AD has been of capital importance to focus AD research. I.5.1. APP The APP gene is located on chromosome 21, causing a dose-effect in DS patients that leads to the development EOAD. In 1991, Goate and colleagues found the first FAD mutation in the APP gene, an amino acid substitution close to the Aβ Cterminus [40]. Since then 49 mutations in the APP gene have been described, 42 of which have already been demonstrated as pathological [41]. In general, APP mutations can be divided in three categories depending on the site they occur: the ones at the β-secretase cleavage site, the ones at the γ-secretase cleavage site and, finally, the ones within the Aβ region (so, close to the α-cleavage site) (Fig. 4). Therefore, they have different effects on APP processing and, consequently, on

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AD development [14]. These mutations are usually named according to the geographic origin of the first identified carrier family, and the residue position for each mutation always refers to the longest APP isoform (APP770) (Fig. 4). NH2NH2NH2-

KPI KPI OX2

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-COOH

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-COOH

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V K M DAE F R H D S GYE V H H Q K LV F FAE D V G S N K GAI I G LM V G G VV IATV I V I

K670N/M671L (Swedish)

E693G (Arctic) E693Q (Dutch) A692G (Flemish)

V717F (Indiana) V717I (London) I716V (Florida) V715M (French) V715A (German) V714I (Austrian)

Fig. (4). Most common isoforms and mutations in APP. A) The alternative splicing of exons 7 (encoding a Kunitz protease inhibitor, KPI) and 8 (encoding an OX2 domain, OX2) results in three APP isoforms: APP695, APP751 and APP770. B). Some representative APP mutations immediately flanking or within the Aβ region. The given residue position for each mutation is referred to the longest APP isoform (APP770). [Figure adapted from [42]].

Swedish double mutation (K670N/M671L, known as APPSwe) is located at the βcleavage site. Because the mutation favors β-secretase activity, an increment in Aβ production occurs. In contrast, the variety of mutations described close to the γ-cleavage site do not increase the total Aβ, but promote the more toxic Aβ1-42 species relative to Aβ1-40. London (V717I) and Indiana (V717F) are the most prevalent ones, although many others have been reported such as Florida (I716V), French (V715M), German (V715A) and Austrian (T714I). Moreover, mutations within the Aβ region are reported to be highly amyloidogenic, triggering an

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increase in Aβ accumulation within neural tissue and/or in the blood vessels walls: Flemish (A692G) and Dutch (E693Q) mutations are involved in CAA, whereas Arctic mutation (E693G) causes an aggressive Aβ aggregation despite the lower Aβ1-42/Aβ1-40 ratio exhibited. I.5.2. PSEN1/PSEN2 Presenilin 1 (PS1) and 2 (PS2) proteins form the catalytic core of the γ-secretase complex, which also contains the anterior pharynx-defective 1 protein (APH-1), nicastrin and presenilin enhance protein 2 (PEN2) involved in the maturation and stability of the complex [43]. In 1995 several mutations were described in the PSEN1 gene coding for PS1, and located on chromosome 14 [44]. Few months later, the homologous protein PS2 was described. Presenilin 2 gene (PSEN2) was found on chromosome 1 and reported to carry a FAD mutation [45]. Since then 25 mutations have been described in the PSEN2 gene, 13 of which already demonstrated as pathological, and 203 in the PSEN1 gene, most of them pathological (192) and some being the most common cause of FAD, i.e. M146V, M146L, L286V and ΔE9 [41]. Typically, PS mutations have been evidenced to alter the γ-secretase activity by triggering a higher Aβ1-42 production relative to other species. Nevertheless, recent approaches suggest that in some cases the loss of other essential functions of PS1, instead of the altered processing of APP, could better explain neurodegeneration and dementia in AD [46]. In addition to APP, PSEN1 and PSEN2, which as mentioned before account for less than 1-2% of the AD cases, there are many others genes considered to confer different extent of risk to develop the disease; being the apolipoprotein E gene (APOE) the most significant one [47]. I.5.3. APOE APOE is the main known locus increasing LOAD risk, with the APOE Ɛ4 allele as the major risk factor for its development. Recently, high-throughput genotyping analyses also pointed to the APOJ, CR1, PICALM, BIN1, ABCA7 and CD33 loci as new genetic determinants of AD risk [48], with at least one independent

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genome-wide association study (GWAS) replication for each candidate [49]. What is most striking from this list is the predominance of lipid-related genes. Although the exact mechanisms by which any of these lipid-related risk factors affect the pathophysiology of AD remain to be clarified, it is well stablished that there is a relation between cholesterol metabolism and AD. I.5.3.1. The Relationship between Cholesterol Metabolism and AD Brain contains approximately 30% of the total body cholesterol, which means it is the cholesterol-richest organ. Cholesterol is an essential component of cell membranes and plays a crucial role in the development and maintenance of neuronal plasticity and function, which are deeply compromised in AD [50]. Several studies have demonstrated that brain cholesterol levels (total, free and esterified cholesterol) are significantly reduced in hippocampal and cortical areas of AD patients compared to age-matched controls, but no differences are detected in the pathology-free cerebellum. On the other hand, experiments performed in cell cultures and animal models have consistently demonstrated that hypercholesterolemia is associated with increased deposition of cerebral Aβ peptides [51]. This apparent contradiction leads to the following question: if increasing cholesterol levels promotes Aβ generation, how can AD be simultaneously associated with decreased total cholesterol levels and increased Aβ levels in the brain? [49]. The amyloidogenic processing of APP (Fig. 1) is believed to occur in, or in close proximity to, lipid rafts, cholesterol-rich membrane microdomains where α-secretase activity is negatively regulated, but both β and γ-secretase activities are stimulated, resulting in increased Aβ production. Taking this into account, it is easy to understand that it is not the total amount of cholesterol what affects Aβ production, but rather its distribution. Because of the low permeability of the BBB to peripheral cholesterol, most of the cholesterol present in the brain derives from de novo synthesis. In the central nervous system (CNS) apoE, in partnership with apoJ and apoC1, plays a pivotal role in cholesterol delivery during the membrane modeling associated with synaptic turnover and dendritic organization [52]. ApoE is normally synthesized and secreted by astrocytes and microglia in the brain and binds to high-density lipoproteins (HDLs) to facilitate cholesterol and phospholipids mobilization and

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transport between glia and neurons [48]. ApoE interacts with a variety of cellsurface receptors of the high-density lipoproteins receptor (LDLR) family to allow particle uptake, including low-density lipoproteins receptor (LDLR), LDL-related protein receptor 1 (LRP1), very-low-density lipoproteins receptor (VLDLR) and apoEr2 [53]. Internalization of the apoE-HDL particles by members of the LDL receptor family occurs primarily in specific clathrin-coated pit structures in the plasma membrane, where both BIN1 and PICALM gene products were shown to facilitate endocytosis of large complexes. Once internalized via endocytic processes, the HDL complex is degraded releasing cholesterol that can be used for synapse formation, terminal proliferation or for medium-term storage purposes [48]. ApoE is synthesized with an 18 amino acid N-terminal peptide that undergoes intracellular processing before secretion of a mature 35kDa glycoprotein containing 299 residues. Human apoE has two separate N-terminal and C-terminal domains joined by a flexible hinge region. The N-terminal domain constitutes the receptor-binding region and the C-terminal domain the lipid-binding region. The three isoforms apoE2, apoE3, and apoE4, differ only at positions 112 and/or 158. ApoE3 contains a cysteine at position 112 and an arginine at 158, while apoE2 contains a cysteine at both positions and apoE4 contains arginine at both sites [54]. Although differences of just one or two amino acids are located in the receptor-binding domain they significantly alter the apoE’s folding structure and change its ability to bind both lipids and receptors. For instance, apoE3 and apoE2 display preference for HDLs, whereas apoE4 is frequently associated with VLDLs and LDLs [55]. The main consequence of the differential lipoprotein preference by apoE4 is the increase in plasma cholesterol levels. Apart from increasing the risk for cardiovascular diseases, poor synaptic remodeling capacity results in increasing the risk for the development of AD [51]. Another remarkable structural difference among apoE isoforms is the distinctive stability of their N-terminal domains, with apoE4 being the least resistant to thermal and chemical denaturation, apoE2 the most and apoE3 showing an intermediate resistance [56].

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I.5.3.2. The Relationship between apoE and Aβ Histological analyses of AD brain reveal that apoE is co-deposited with Aβ amyloid plaques in an isoform dependent manner (apoE4> apoE3> apoE2) [57], suggesting a relevant role for apoE in modulating Aβ metabolism, aggregation and deposition. Epitope mapping reveals that Aβ can interact with both the lipidbinding site and the receptor-binding site within apoE. ApoE’s lipidation status may dictate both the Aβ-binding and the receptors’ affinities [58]. Although their interaction is demonstrated, the effect of apoE on Aβ aggregation, whether it is facilitated or inhibited, is controversial [59]. On one hand, high concentrations of apoE form high molecular weight co-aggregates with Aβ, in which apoE4 is likely to promote Aβ aggregation to a higher extent than apoE3 [60]. As it occurs with co-deposition in amyloid plaques, apoE increases the level of Aβ oligomers in an isoform dependent manner, apoE4 stabilizing Aβ oligomers more efficiently than apoE3 [61]. ApoE is also known to self-aggregate with irregular protofilament-like morphology, in which the aggregates form at substantially different rates depending on the isoform [62]. Thus, it is likely that apoE may be able to produce co-aggregates with Aβ through itself aggregation propensity. In contrast, it has been reported that apoE decreases Aβ fibrillogenesis as it prefers to interact with Aβ peptides that show β-sheet conformation, probably by capturing Aβ nuclei and preventing its seeding effects [63]. In this case, apoE3 appears to interact with Aβ better than apoE4, so it is also possible that apoE4 is less effective in the inhibition of Aβ fibril formation [62]. Even though the apparent contradiction, in both cases (aggregation and binding) it is evident that it is the apoE4 isoform the one precipitating the pathological hallmarks of AD. On the other hand, it is also unclear what influence apoE has on Aβ clearance. Because Aβ is generated by secretases cleavage, it is released to the interstitial space fluid (ISF) where it is cleared by a variety of mechanisms, in which the presence of one or another apoE isoform has a great importance. One clearance mechanism is the efflux across the BBB to the periphery where Aβ is rapidly degraded [59]. Not only apoE and Aβ can interact with each other, but they also

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share common receptors including LRP1, LDLR and HSPG (heparan sulfate proteoglycans) on cell surface [64]. ApoE likely competes with Aβ for their receptor binding but can also facilitate cellular Aβ uptake by forming apoE/Aβ complexes depending on several parameters as their concentrations, involved apoE isoform, lipidation status, Aβ aggregation extent and receptor distributionpatterns [48, 62]. Receptor-mediated clearance at the BBB is also affected by the apoE isoform. While apoE2 and apoE3 use both LRP1 and VLDLR to cross the BBB, apoE4 shifts Aβ transport from LRP1 to the VLDL receptor [65], resulting in a slower rate of Aβ clearance, as it has been described that LRP1 has the higher endocytic rate for apoE [64]. Aβ can also be cleared by degradation by Aβ proteases: intracellularly by neprilysin and extracellularly by IDE. While apoE2 and apoE3 isoforms enhance Aβ degradation by neprilysin, apoE4 appears to have the weakest effect in vitro. [66]. Endocytic trafficking to lysosomes is also altered by apoE isoform. ApoE3 promotes Aβ uptake and degradation by lysosomes in vitro, while apoE4 is less effective [67]. In addition, the aggregative nature of both Aβ and apoE4 in the acidic lysosomes suggest that these AD pathogenic molecules might co-aggregate within these organelles [62]. Another way by which apoE probably facilitates Aβ clearance is by activating phagocytosis by the microglia in an isoform dependent-manner, with apoE4 showing reduced capacity to induce uptake when compared to apoE3 [62]. As a conclusion, it could be said that although apoE role in AD is unclear, it has been proven that there are several ways by which apoE could affect AD pathology, whether it is inhibiting or favoring Aβ neurotoxicity. More studies are required to clarify this issue, but it is likely that it is not apoE as a molecule what is positive or negative in the development of AD, but its isoforms seem to be the key that makes the difference in terms of disease.

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I.5.4 . APOJ/CLU As mentioned, GWAS have determined APOJ as an important risk factor for developing AD. Surprisingly, and although it has been reported to be expressed abundantly in the CNS (at similar concentrations to apoE), very little is known about the role this apolipoprotein plays in AD pathogenesis. Apolipoprotein J, also called clusterin, is thought to act as an extracellular chaperone with the capacity to interact with a wide range of molecules [68], modulating the membrane-attack complex and preventing the inflammatory response associated with complement activation after protein aggregation. Clusterin binds to LRP2, apoEr2 and VLDLr, and is internalized by cells expressing either one of these receptors [68]. It has been proven that clusterin expression is increased in AD, where it is found associated with Aβ plaques. Although some authors have pointed out that clusterin expression facilitates amyloid formation [69], others have shown that apoJ inhibits Aβ fibril formation in vitro and promotes the clearance of protein aggregates via endocytosis [70]. As a consequence of such opposite observations, the role of apoJ is undetermined and further research is needed to find out the precise mechanism this apparently important molecule plays in AD pathogenesis. I.5.5. The Role of Genetic Testing in Clinical Care Genetic testing of patients with EOAD and a positive family history can be a valuable tool for choosing the correct diagnosis among some differential ones, and offering at-risk relatives the option of predictive testing [71]. However, in the case of asymptomatic relatives of a FAD case the availability of genetic testing creates both advances and dilemmas. Advances are mainly the chance of early intervention and opportunity to join clinical trials for groups at genetic risk, such as the Alzheimer's Prevention Initiative and Dominantly Inherited Alzheimer Network initiatives (see later). On the other hand, the main dilemma is the potential risk of emotional stress. However, it has been reported in two independent studies that the use of a standardized counseling protocol was beneficial [72, 73]. Although genetic variations other than that in APP and PSEN genes also contribute to AD risk, among which variants in APOE gene have the

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greatest effect, its genetic testing is not recommended for asymptomatic individuals [74]. Guidelines and recommendations on genetic counseling procedures in asymptomatic AD have been published [71]. Genetic testing in the characterization of participants for clinical trials consists of the testing of, a part of the well-established APP and PSEN loci, the strongest risk APOE allele (see later). However, testing of other genes is not being used because, as mentioned before, they have been related to the disease by GWAS. Since there is not a direct cause/effect relationship between the presence of these mutations and the development of the disease, their inclusion in clinical trials will, at the moment, complicate the studies. However, the future study of both the physiopathological mechanism behind the already GWSA AD-related genes and the ongoing and next-generation GWAS should lead to the identification of novel targets for genetic testing, as well as developing curative therapies for AD [75]. As a general conclusion, Alzheimer’s disease is a very complex disease with multiple agents involved, affecting many systems and with a great amount of mechanisms behind its pathogenicity still unknown or not well understood (summarized in Fig. 1, bottom). Adding to these factors the urgency to find an effective treatment to cure the disease before it becomes a worldwide pandemic, it is understandable that a lot of different therapies are being tested in both preclinical and clinical trials. II. THE ROLE OF BIOMARKERS IN EARLY DIAGNOSIS AND CLINICAL TRIALS ASSESSMENT Two main things learned from clinical trials will be deeply discussed in the next Section. The first one is that patients recruited for these studies must be at early stages of the disease progression to appraisal effectiveness of the treatment. This concurs with the fact that neuropathological changes of AD are thought to start more than a decade before the onset of clinical symptomatology. Second, not only effectiveness but also safety must be carefully monitored during clinical trials. According to the Consensus Report of the Working Group on Molecular and Biochemical Markers of AD, a biomarker must reflect AD pathology and discriminate it from other dementia with a high sensitivity, be reliable and

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reproducible, be easy to perform and analyze, and remain relatively inexpensive [76]. Four main categories of AD markers have been widely considered: cognitive and behavioral assessments, genetic procedures to determine the risk of AD, biochemical markers in both blood (plasma and serum) and CSF, and neuroimaging approaches including structural, functional and metabolic studies. However, only the last two categories (CSF analysis and neuroimaging) are currently ascertained to be effective in early diagnosis [77]. Whereas genetic susceptibility may become extremely useful in prevention, well established cognitive tests (such as mini-mental state examination, MMSE) will continue to be useful in the appraisal of the mild-to-late stages of the disease. In view of this, exhaustive studies are being carried out to define accurate biomarkers for biochemical (CSF- and blood-based) and neuroimaging characterization. II.1. Biochemical Markers II.1.1. Cerebrospinal Fluid Biomarkers Because the CSF straightly contacts with the brain parenchyma, its composition reflects those changes occurring in the neural tissue, becoming a magnificent tool to identify neuropathological fluctuations. For this reason, many molecules from the CSF have been proposed as putative AD biomarkers (Table 1) [78]. The most commonly used are Aβ1-42 peptide, total tau protein (t-tau) and its phosphorylated form (p-tau) [79]. Specifically, CSF levels of Aβ1-42 diminish in AD because of the accumulation of amyloid species at cortical regions; whereas, the increase in t-tau concentration indicates axonal damage and neuronal degeneration, and p-tau correlates with NFTs pathology. Interestingly, better diagnosis efficiency is obtained when biomarker ratios are considered instead of single biomarkers determinations. For instance, the Aβ1-42/Aβ1-40 ratio has been positively valued as a biomarker in several studies [80]. Several other Aβ species in CSF are also suggested to become useful biomarkers for AD diagnosis and prognosis. Different C-t and N-t truncated and posttranscriptionally modified Aβ peptides found in CSF have been suggested to prognosticate AD [81]. Interestingly, a very recent publication proposed the measurement of Aβ oligomers in CSF as a potential biomarker, since its

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accumulation is directly related to AD progression [82]. In addition, other potential biomarkers for primary alterations have been proposed, as increased BACE1 levels and activity [83, 84] or increased apoE levels [85]. According to proteomic and metabolomic assessments, there are many other potential molecules which concentration statistically differ between CSF samples from AD and control subjects [86]; for instance, a list of 8 amino acids and metabolic compounds (threonine, glutamate, arginine, ornithine, citrulline, alphaaminobutyric acid, ammonia and urea) or some metabolite profiles such as cortisol, cysteine and uridine. Also, the YKL-40 glycoprotein has been recently considered as an inflammatory marker for AD and other neurodegenerative diseases as FTD [87]. Furthermore, the levels of cell-free mitochondrial DNA (mtDNA) has been shown to be decreased in CSF at a very-early preclinical phase of AD [88] and the levels of some circulating miRNAs change in CSF and blood as AD progresses [89]. Subsequently, next steps must be based in defining which of these candidate biomarkers contribute with high accuracy to diagnosis, prognosis and/or therapeutic monitoring [78]. Moreover, further efforts are focused on the standardization of CSF biomarkers measurements in order to improve analytical precision and facilitate comparisons among laboratories, which will drive to the establishment of pathological values ranges (see below) [90]. Table 1. Potential biomarkers for AD. Principal characteristics of CSF- and blood-based biochemical and neuroimaging potential biomarkers for AD diagnosis and prognosis. Current diagnosis recommendations only include CSF assays for Aβ and tau, brain volumetry and glucose metabolism and amyloid deposition detection by PET, in addition to cognitive tests.

Biomarkers

Characteristics

Biochemical biomarkers CSF-based Aβ1-42

Levels of Aβ1-42 decrease in CSF because of the amyloid accumulation at cortical regions in AD.

t-tau

Increased levels of t-tau indicate axonal damage and/or neurodegeneration.

p-tau

Neurofibrillary pathology causes an increase in p-tau.

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(Table ) contd.....

Biomarkers

Characteristics

Aβ1-42/Aβ1-40

Decreased Aβ1-42/Aβ1-40 ratio in AD. Prediction of the progression risk from MCI to AD dementia.

C-/N-terminal truncated Aβ

Prediction of the progression risk from MCI to AD dementia.

Aβ oligomers

Oligomeric amyloid accumulation and toxicity.

BACE1 activity

Increased in MCI and AD.

ApoE

Elevated levels of apoE in CSF from AD patients.

Metabolic compounds

Elevated levels of urea, threonine, glutamate, citrulline, alpha-aminobutyric acid, ornithine, ammonia and arginine in AD. Cortisol, cysteine and uridine profiles inform about AD progression.

Inflammatory markers

E.g. Levels of YKL-40 glycoprotein reports about inflammation. It does not discriminate AD from other neurodegenerative diseases as FTD.

mtDNA

Decrease in cell-free mtDNA at very early preclinical stages of AD.

miRNAs

Levels of circulating miRNA change as AD progresses.

Blood-based Aβ1-42/Aβ1-40

Decrease in plasmatic Aβ1-42/Aβ1-40 ratio indicates a higher risk for imminent progression.

tau

Plasmatic tau is increased in AD dementia.

tau-A

Tau-A indicates neurodegeneration. Its concentration in serum inversely correlates with cognitive functions.

C-/N-terminal truncated Aβ

Various Aβ species are altered in preclinical AD. E.g. the free-/cell-bound Aβ1-17 ratio varies as AD progresses.

QC

Its mRNA and protein levels correlate with AD progression.

miRNAs

Levels of circulating miRNA change as AD progresses.

Inflammatory markers

E.g. sTNF-R indicates pre-demential stages of AD. Combinatorial algorithm of plasmatic neuroinflammatory proteins for an accurate diagnosis of AD.

Serum ceramide

Altered levels of ceramide indicate neuronal dysfunction.

VCAM-1

Marker for microvascular injury, common in AD.

HO-1/BVR-A

Marker for oxidative and nitrosative stress at early stages of AD.

Imaging biomarkers sMRI Volumetry

Reduction of hippocampal or brain volume indicates neuronal loss. It cannot discriminate AD from other neuropathologies.

Gray matter measure

Local measures of gray matter concentration in specific regions may support the typical atrophy pattern of AD.

Cortical thickness

Cortical thickness changes reflect neuronal and dendritic architecture alterations.

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(Table ) contd.....

Characteristics

Biomarkers DTI Water diffusion

Informs about tissue composition, physical properties of its constituents, architectural organization and microstructure. Useful to detect neuronal damage in AD.

Deoxyhemoglobin

Deoxyhemoglobin concentration correlates with cognitive impairment. It can be detected at very early stages of AD.

fMRI

MRS

Brain metabolites

E.g. myo-inositol, choline-containing compounds and N-acetyl aspartate, glutamate, taurine, present a varying profile through AD progression. Increased levels of myo-inositol indicate astrogliosis at early stages of AD, suggesting brain injury. Increased levels of choline are also detected in MCI phase. N-acetyl aspartate profile declines as AD progresses.

Glucose metabolism

Measures of the glucose analogue FDG determine the metabolic pattern in the brain. It allows the detection of very early stages of preclinical phases of AD.

Amyloid deposition

E.g. 11C-PiB or 18F-AV-45 tracers indicate amyloid deposition.

PET

II.1.2. Blood Biomarkers Since blood is a fluid much more easily obtained and manageable than CSF, the discovery of a consistent blood-based biomarker becomes a priority. However, the strong partitioning between blood and brain parenchyma by the BBB, as well as the cellular diversity and the dynamic environment of blood, strongly impact on its composition [91]. Controversial results in Aβ quantification performance in blood have been published [92]: some studies reported a decrease in plasmatic Aβ1-42/Aβ1-40 ratio in those cases at risk of imminent progression, whereas some others detected a very modest correspondence between plasmatic Aβ levels and clinical progression. These confusing results are attributable to the variability through studies, timing of sample collection, different antibodies used against Aβ, among others. What is a fact, though, is that both pre-analytic and analytic processes require of standardization protocols in order to improve the reproducibility and diminish variability among studies. Recently, some modified Aβ species have been proposed to be useful for AD diagnosis. In particular, Aβ1-17 has been shown to be a major Aβ fragment in CSF

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and blood, becoming a potential biomarker [93]. The ratio of free/cell-bound Aβ1-17 in blood seems to significantly differ among preclinical, early clinical stages and dementia AD. Similarly, other truncated and/or modified Aβ species have been suggested as blood-based biomarkers. An interesting promise arises from the relevant role of the pyroglutamate-modified Aβ peptide (pE-Aβ) in the development of AD. Specifically, mRNA and protein levels of glutaminyl cyclase (QC), the enzyme that catalyzes the N-terminal addition of pyroglutamate, correlate with AD cognitive decline [94]. Taking into account the drawbacks of using different Aβ determinations in blood as biomarkers, the scientific community is also focusing in downstream biomarkers that reflect secondary pathological phenomena such as axonal degeneration or neuroinflammation [95]. For instance, plasmatic tau concentrations increases in dementia patients of AD relative to previous stages [96]. In addition, tau is cleaved by ADAM10 (a disintegrin and metalloproteinase domain-containing protein), interestingly an enzyme also showing α-secretase activity. The concentrations in serum of the produced fragment, named tau-A, inversely correlate with cognitive functions [97]. Besides, because of the contribution of systemic inflammation over neurodegeneration, the serum soluble form of the tumor necrosis factor receptor (sTNF-R) has been suggested as a potential biomarker enabling identification of pre-demential stages of the disease [98]. Moreover, an algorithm based on a set of 30 neuroinflammatory proteins and which combines demographic and clinical data has been developed for diagnosis prediction [99]. Regarding neuronal and vascular injuries, which are also pathophysiological features of the AD condition, ceramide levels in serum have been proposed as indicators of neuronal dysfunction [100], whereas plasmatic VCAM-1 (vascular cell-adhesion molecule) has been suggested as an indicator of microvascular injury [101]. On the other hand, and because an increase of oxidative and nitrosative stress occurs in AD, the activation of heme oxygenase-1/biliverdin reductase-A (HO1/BVR-A) system, one of the earlier events in the adaptive response to stress, has

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been suggested as a putative blood biomarker for the prediction of AD at the earliest stages of the disease [102]. Further research is needed for defining the more appropriate biomarkers in blood and their pathological value ranges. II.2. Imaging-based Biomarkers Imaging techniques provide information about regional and temporal distribution of those pathological changes occurring in AD brain, through both macroscopic and mesoscopic analysis by using structural, functional and metabolic approaches [77]. II.2.1. Structural Magnetic Resonance Imaging Structural Magnetic Resonance Imaging (sMRI) is employed to test brain atrophy in AD, either by volumetric assessment or, in specific cases, local measures of gray matter concentration or cortical thickness. Reduction of hippocampal volume evidences the neuronal loss occurring in AD [103], but it is not a specific AD feature as it also occurs in other pathologies such as FTD, vascular dementia, Lewi-body disease or depression. Total brain volumetry is used to determine the disease progression in such conditions. Local measures of cortical thickness or gray matter concentration in specific neuroanatomic regions allow obtaining an estimate of the atrophy pattern, which facilitates discrimination among different types of dementia [104]. Noteworthy, cortical thinning does not reflect neuronal loss since this parameter is not associated with regional neuron amount neither density, but rather reflects neuronal and dendritic architecture changes [105]. Taking into account all of the parameters measured by structural MRI, a temporal evolution of neurodegeneration has been proposed [106]. II.2.2. Diffusion Tensor Imaging Diffusion tensor imaging (DTI) is another variant of MRI and measures the random thermal motion of water molecules within the neuronal tissue, providing

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unique information about tissue composition, physical properties of its constituents, architectural organization and microstructure, while avoiding exogenous contrast agents [107]. In normal conditions, cell membranes increase the hindrance of water diffusion [108], so mean water diffusion increases by neuronal damage and axonal bundle disruption as evidenced by DTI analyses in AD patients [109]. It is also a valuable approach to detect one of the most severe adverse effects that has appeared in some clinical trials, the occurrence of cerebral edema. Edema is referred as ARIA (Amyloid-Related Imaging Abnormalities) and the technique is able to distinguish between vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H). The main difference is that vasogenic edema refers to an increase in extracellular fluid volume related to increased permeability of brain capillary endothelial cells to serum proteins while microhemorrhages generate small deposits of iron in tissues, formed by the blood leaked from a vessel. Precisely the appearance of ARIA-E was the reason to stop a Phase III clinical study in which bapineuzumab was being tested (see below). The pathophysiological mechanisms causing vasogenic edema are not clear but clinical studies are providing some information about risk factors, such as the presence of the APOE Ɛ4 allele [110]. II.2.3. Functional Magnetic Resonance Imaging Functional MRI (fMRI) assesses neuronal activity by measuring neuronal metabolic demand. In particular, high deoxyhemoglobin concentration produces an increase in the intensity of local signal due to its paramagnetic properties [111]. For instance, cognitive procedures promote blood flow and oxygenation, while cognitive impairment reduces the metabolic demand from neurons. Thus, neuronal dysfunction can be identified prior to neuronal loss. Functional MRI is widely employed because of its accurate spatial and temporal resolution, and numerous studies have reported abnormal patterns of memoryrelated activity in mild cognitive stages of AD and dementia [112]. II.2.4. Proton Magnetic Resonance Spectroscopy Proton Magnetic Resonance Spectroscopy (1H-MRS) permits to assess variations

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in the biochemical composition of tissues, as those associated with neurodegeneration [77]. It allows regional measurements of different metabolites such as N-acetyl aspartate (NAA), myo-inositol (mI), choline (Cho), glutamate or taurine in a single session. Quantitative results are usually given under an internal standard correction, for instance ratios with other metabolites, being creatinine (Cr) commonly used as an internal reference. NAA is generated in mitochondria and located intraneuronally, so its measurements provide information about mitochondrial integrity and function, as well as neuronal density and viability [113]. Otherwise, mI is mainly located within glial cells, so it becomes a marker for neuroinflammation or gliosis [114]. For instance, AD patients present reduced NAA/mI ratios in parietal cortex compared with frontal cortex [115]. Although the combination of 1H-MRS with other biomarkers increases the diagnostic accuracy, this technique per se enables preclinical identification of AD and permits discrimination among other neurodegenerative disorders [116]. II.2.5. Fluorodeoxyglucose-Positron Emission Tomography Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET) allows determining the glucose metabolism in the brain. The glucose analogue 18F-FDG contains a radioactive isotope, leading to distinguish those activated neurons when the requirements of glucose shoot up [117]. In the case of AD, synaptic dysfunction and neurodegeneration result in a hypometabolic pattern. Abnormalities in the metabolic pattern of pre-dementia AD patients may predict a higher risk to develop AD dementia [118]. Interestingly, the high sensitivity of this technique permits distinguishing even preclinical asymptomatic APOE Ɛ4 carriers from non-carriers [119]. FDG-PET also discriminates among heterogeneous groups of AD patients in terms of amyloid deposition [120]. II.2.6. Amyloid-Positron Emission Tomography Positron Emission Tomography (PET) is the main neuroimaging tool for detecting and quantifying amyloid deposition. The major advantages are the possible in vivo

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diagnoses and its efficiency in early stages of the disease. Measurements are obtained by PET specific radiotracers. The 11C-Pittsburg-Compund B (PiB) is the most widely utilized [121], although its short 20-minutes half-life makes its use somehow restrictive. Thus, a variety of tracers containing 18F -with a half-life of 110 minutes- have been developed: 18F-AV-45 (florbetapir) was approved by FDA in 2012, whereas flutemetamol, florbetaben, and AZD4694 continue being improved because they have shown non-specific binding to white matter [122]. Several convergent works have been performed in order to standardize this diagnostic technique, focusing in the acquisition of images and measurements, identification of tracer-binding AD patterns discriminating from other dementias, and the correlation between amyloid deposition and cognitive decline [123]. In any case, amyloid-PET is currently the most widely used technique in clinical practice [124] and also the best tool to detect AD before cognitive decline starts, and therefore is also currently being used to recruit patients for clinical trials (see below). II.3. The Continuum of AD: Diagnosis and Prognosis Several opinions diverge when establishing the onset of AD, whether it begins when the neuropathologic changes occur or once clinical symptoms appear [125]. According to the International Working Group (IWG), the pathology starts with the clinical evidences of early but significant episodic memory impairment, known as the prodromal phase, which would evolve towards dementia. Preclinical presence of the core AD features is an indicative of the risk to develop the disease, but it is not considered as pathological [126]. By contrast, the National Institute on Aging and Alzheimer’s Association (NIA-AA), establishes the neuropathology onset long time before the emergence of clinical symptoms (Fig. 5): Preclinical AD is considered to start with asymptomatic amyloidosis (stage 1), which may give rise to neurodegeneration (stage 2) followed by subtle cognitive decline (stage 3); such an asymptomatic phase triggers to mild cognitive impairment (MCI) and AD dementia at last [127 - 129]. Additionally, the preclinical phase also covers those individuals genetically predisposed to develop the disease, as the autosomal dominant mutations or APOE Ɛ4 carriers. Staging references in the

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following text relate to the NIA-AA consideration, which includes the preclinical phase, MCI and AD dementia. Cognitive function

Asymptomatic amyloidosis Aβ (PET or CSF)

Aging

Amyloidosis + Neurodegeneration Aβ (PET or CSF) Neuronal injury (tau, FDG, sMRI) Alzheimer’s disease Amyloidosis + Neurodegeneration + SCD Aβ (PET or CSF) Neuronal injury (tau, FDG, sMRI) Subtle cognitive/behavioral decline

Preclinical

Years

MCI

Dementia

Fig. (5). The continuum of Alzheimer’s disease. The pathological development model proposed by the National Institute on Aging and Alzheimer’s Association (NIA-AA) includes a preclinical phase that may progress to mild cognitive impairment (MCI) and dementia at last. Preclinical staging is detailed with those specific diagnostic tips. Aβ: amyloid-beta, PET: Positron Emission Tomography, CSF: Cerebrospinal Fluid, FDG: Fluorodeoxyglucose, sMRI: structural Magnetic Resonance Imaging, SCD: Subtle Cognitive Decline, MCI: Mild Cognitive Impairment. [Figure adapted from [129]].

In 2010, Jack et al. described a hypothetical model of the evolution of biological markers during early AD progression [130]. First, it includes those biomarkers related to APP abnormal processing, CSF Aβ1-42 measurement and PET-Aβ imaging, which point out to the accumulation and deposition of the Aβ peptide. Second, CSF tau, FDG-PET and sMRI assessments reveal neurodegeneration and synaptic dysfunction. Finally, more severe changes in biomarkers, in addition to cognitive and functional tests, would correlate with more advanced stages of the disease. Of course, this general model would be affected by genetic factors and lifestyle.

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Further longitudinal projects are required to elucidate the evolution pattern of biomarkers throughout the disease progression, so that the diagnostic and prognostic standards will be defined. For the time being, several studies have quantified the Aβ peptide and the tau protein in CSF at all 3 phases of AD. In the dementia phase, a reduction of 50% of the Aβ1-42 levels and an increase in t- and ptau to approximately 300 and 200%, respectively, have been described [131]. As expected, biomarkers alterations are not so pronounced in the MCI phase as in dementia [132]. In the preclinical phase, a reduction in CSF Aβ1-42 but normal t- or p-tau have been detected [133 - 135]. Interestingly, subjects carrying FAD mutations already exhibit both Aβ1-42 and tau levels altered in the preclinical phase, evidencing that the pathological development starts long before the symptoms onset [136]. Furthermore, an increase in tau/Aβ42 ratio has been associated to the progression from preclinical phase to MCI, especially in APOE Ɛ4 carriers [137]. In turn, rapid progression from MCI to AD dementia is prognosticated in subjects with high levels of tau [138], being enhanced in those individuals with the APOE Ɛ4 homozygous genotype [139]. Finally, it is important to emphasize that the use of biomarkers in drug discovery plays a double role by monitoring the efficacy of therapies and easing individual selection for clinical trials [140]. This latter application of biomarkers allows a reduction of sample size and, what is more important, leads to much more precise results [141]. This is so because variability among individuals in the same cohort is reduced as they have been assigned to the same AD stage or determined to present identical genetic risk factors. II.4. Regulatory Framework Given the apparent controversy among some biomarkers analysis, several international working groups are currently focused on the exact relation between results obtained from biomarkers and their pathophysiological meaning [142]. It is of great importance to define standard criteria to avoid controversial conclusions and to accurately diagnose. This standardization refers to the whole study process: since the experimental design, with the individual selection as an example, to pre-

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analytical and analytical procedures such as sampling, treatment of the sample and measurements. Numerous regulatory criteria, recommendations, quality tools and protocols bring out the comparison of data coming from worldwide studies for validating specific biomarkers for AD and, eventually, defining the cutoff values for diagnosis [79]. In 1984, the National Institute on Neurologic and Communicative Disorders and Stroke (NINCDS) and the Alzheimer’s disease and Related Disorders Association (ADRDA), now called the Alzheimer’s Association (AA), published a review of clinical criteria for the diagnosis of AD [143]. It was revised by the International Working Group (IWG) in 2007, and updated in 2010 [144]. Next, the National Institute on Aging and the Alzheimer’s Association (NIA-AA) suggested an introductory guideline including recommendations for the diagnosis of AD [145], which also propose the 3-phase criterion for the progression of the pathology. At the same time, three working groups derived from the NIA-AA discussed the most appropriate biomarkers for the diagnosis of each AD phase, preclinical [129], MCI [127] and dementia [128]. Because of the exponential growth of AD research, several meetings are recurrently convened in order to compile novel discoveries and to standardize the diagnostic tools [146,147]. Furthermore, the Global Biomarker Standardization Consortium (GBSC), which contains representation from researchers, clinicians, industry and regulatory agencies, intends to standardize and validate biomarkers-based tests for clinical practices [148]. Two projects are currently being performed: CSF Biomarkers Standardization, in which Alzheimer’s Association CSF Quality Control Program is working since 2009, and Hippocampal Volumetry Harmonization, in which European Alzheimer’s disease Consortium (EADC) [149] and Alzheimer’s disease Neuroimaging Initiative (ADNI) [150] are joining efforts. EADC is a European network of research centers focused on AD, which develops various projects in collaboration with other entities, from neuroimaging investigations to collection of biochemical biomarkers information. ADNI is a longitudinal multicenter initiative working on the validation of AD biomarkers for diagnosis and clinical trials, involving blood, CSF and neuroimaging (MRI and PET). A guideline for the standardization of pre-analytic variables for blood-based biomarkers studies in AD research is available [151].

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In addition, the regulatory entities as the European Medicines Agency [152] and the Food and Drug Administration [153] publish numerous guidelines not only for biomarkers standardization and AD diagnosis, but also for drug discovery, quality assessment and criteria for preclinical and clinical trials. III. PROSPECTIVE THERAPIES As mentioned in the introduction Section, there are currently only two types of drugs to palliatively treat AD, cholinesterase inhibitors (donepezil, galantamine, rivastigmine and tacrine) and an NMDA receptor antagonist (memantine). Prospective therapies being tested in clinical trials, as well as some preclinical studies, are reviewed. The material on to be reviewed and discussed is current and accurate as of December 15 2015, and updated in Alzforum and ClinicalTrials databases [154, 155]. III.1. Non-immunologic Therapies In this category very diverse strategies that intend to stop the disease progression by many different methods are found. Clinical studies have been grouped on 7 sections according to their approach: neuronal receptors modifiers, Aβ aggregation inhibitors, secretases modifiers, metabolic targeting strategies, tau targeting therapies and others. A summary is found in Table 2. Next, a selection of the most interesting preclinical studies is also shown. Table 2. Non-immunotherapeutic approaches that are currently under clinical trials. Phase stage is indicated in brackets, and in Arabic numbers for the sake of clarity. Information extracted from Alzforum and Clinical Trials databases and updated on December 15, 2015 [154, 155]. Therapeutic approach

Neuronal receptors modifiers

Secretases inhibitors / modulators

Mechanism of action

Drug (company)

Identifier (Phase)

α4β2/α2β2 receptors agonist

AZD3480 (Targacept Inc.)

NCT01466088 (2)

5-HT6 receptor antagonist

Lu AE58054 (H. Lundbeck A/S)

NCT02006641 (3) NCT01955161 (3) NCT02006654 (3) NCT02079246 (3)

MK-8931 (Merk Sharp and Dohme Corp.)

NCT01739348 (2/3) NCT01953601 (3)

JNJ-54861911 (Janssen)

NCT02406027 (2) NCT02260674 (2) NCT02569398 (2/3)

β-secretase inhibitor

γ-secretase modulator

E2609 (Eisai Inc.)

NCT02322021 (2)

AZD3293 (LY3314814) (Eli Lilly and Co./ AstraZeneca)

NCT02245737 (2)

EVP-0962 (FRM-0962), (Forum Pharma. Inc)

NCT01661673 (2)

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Therapeutic approach

Mechanism of action

Drug (company)

Identifier (Phase)

Statin/HMG-CoA reductase inhibitor

Simvastatin

NCT00303277 (4) NCT00842920 (4)

Metformin

NCT00620191 (2) NCT02409238 (4)

MSDC-0160 (Metabolic Solution Develop. Co.)

NCT01374438 (2)

Tau aggregation inhibitor

Trx0237 (TauRx Therapeutics Ltd.),

NCT01689246 (3) NCT01689233 (3) NCT02245568 (3)

GSK-3β inhibitor

Lithium

NCT02129348 (2)

Anti-inflammatory drugs

Microglial modulator

CHF-5074 (CERESPIR)

NCT01303744 (2) NCT01723670 (2)

Calcium related molecules

Calcium influx modulator

Piracetam (Janssen-Cilag G.m.b.H)

NCT01009476 (4)

Aβ clearance enhancer

Nilvadipine

NCT02017340 (3)

Sirtuin activator

Resveratrol

NCT01504854 (2)

Aβ oligomer binding molecule

Curcumin

NCT01811381 (2)

Metabolic related molecules

Tau targeting drugs

Insulin sensitizer

Natural compounds

Phase stage is indicated in brackets, and in Arabic numbers for the sake of clarity. Information extracted from Alzforum and ClinicalTrials databases and updated on December 15 2015 [154,155]. III.1.1. Clinical Trials III.1.1.1. Neuronal Receptors Modifiers Following the strategy of drugs currently in the market, there are some drugs that intend to potentiate the cholinergic system. In these cases, however, drugs are agonists of nicotinic acetylcholine receptors. AZD3480 (Targacept Inc.), which acts as an α4β2 and α2β2 receptors agonist, has completed a Phase II clinical trial without positive results (NCT01466088) [156,157]. Lu AE58054 (H. Lundbeck A/S) is a 5-hydroxytryptamine 6 (5-HT6) receptor antagonist that affects the serotonergic system. Preclinical results showed an

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improvement in cognitive tasks in a rat model with cognitive impairment [158]. Currently, many Phase III clinical trials are recruiting participants to test Lu AE58054 together with the already accepted drug donepezil (NCT02006641; NCT01955161; NCT02006654; NCT02079246). Other studies with neuronal receptors modifiers are no longer under development. These include the α7 receptor agonists GTS-21 and MEM3454, the AMPA receptor enhancer LY451395, and the muscarinic acetylcholine receptor antagonist ACI-91. In brief, among new neuronal receptors modifiers the 5-HT6 receptor antagonist Lu AE58054 is the most advanced one, but as an add-on to donepezil treatment. III.1.1.2. Aβ Aggregation Inhibitors ELND005, also known as scyllo-inositol (Transition Therapeutics Ireland Limited) is the only drug intended to inhibit Aβ aggregation currently under development. However, its mechanism of action seems to be lowering brain levels of its endogenous isoform myo-inositol (mI), and exerting effects similar to lithium in the treatment of bipolar disorder. Thus, it has been repurposed to treat severe agitation and aggression in AD patients rather than to improve cognitive impairment [154]. Other studies with Aβ aggregation modifiers to treat AD are no longer under development. These include the mimetic glycosaminoglycan tramiprosate and the copper and zinc ionophore PBT2. Thus, the only drug for inhibiting Aβ aggregation under development has been repurposed to treat severe agitation and aggression. III.1.1.3. Secretases Inhibitors/Activators There is a large group of approaches trying to divert the APP amyloidogenic pathway to the non-amyloidogenic one by modulating the secretases enzymes activity, either by potentiating α-secretase or by inhibiting BACE1 or γ-secretase.

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III.1.1.3.1. α-secretase Activators The only tested drug following this approach is etazolate hydrochloride or EHT0202, which was assayed as adjunctive therapy to acetylcholinesterase inhibitor in a Phase II clinical trial completed in 2009. However, dose-dependent numbers of early withdrawal and CNS related adverse events were observed [159]. III.1.1.3.2. β-secretase Inhibitors Since the first potent BACE1 inhibitor, OM99-2, was published by Ghosh and colleagues in 2001 [160] many others have been developed, being verubecestat, also known as MK-8931 (Merk Sharp and Dohme Corp.), the one that has gone further [161]. Currently, and after significantly reducing Aβ concentrations in cerebrospinal fluid (CSF) in Phase I clinical trials, one combined Phase II/III is ongoing (NCT01739348) and one Phase III is recruiting participants (NCT01953601). Similarly, JNJ-54861911 (Janssen) has been tested in multiple Phase I trials, and two Phase II (NCT02406027, NCT02260674) and one II/III (NCT02569398) trials are recruiting. This inhibitor was safe and well-tolerated, entered the blood and CSF with favorable pharmacokinetics and pharmacodynamics, and dosedependently reduced different size Aβ peptides (37, 38, 40, 42) and BACE concentrations, whereas levels of sAPPα rose. On the other hand, a Phase II clinical trial (NCT02322021) is currently recruiting participants to test E2609 (Eisai Inc.) after Phase I clinical studies showed marked reductions of Aβ concentrations in plasma and CSF [161]. Finally, Eli Lilly and Co. is recruiting participants for a Phase II clinical trial with AZD3293 (LY3314814) (NCT02245737). Although the results have not yet been published, AstraZeneca, the company handling manufacturing, reported that the inhibitor appeared safe and strongly reduced CSF Aβ levels. Other BACE1 inhibitors that have been tested in clinical trials but are no longer under development are LY2886721, CTS21166 and AZD3839.

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III.1.1.3.3. γ-secretase Inhibitors and Modulators Trying to inhibit or modulate the γ-secretase enzyme is the most difficult approach among strategies focused on secretases in terms of side effects, as it has multiple cleavage actions on physiologically important substrates like the Notch receptor [162]. Thus, several compounds have been tested but discontinued, as the non-selective γ-secretase inhibitors semagacestat (LY450139) and MK0752, the selective thiophene sulfonamide inhibitor begacestat (GSI-953), the sulphonamide pharmacophore avagacestat (BMS-708163), and the non-steroidal antiinflammatory drug flurizan (tarenflurbil, R-flurbiprofen or MPC-7869). In contrast, EVP-0962 (or FRM-0962, Forum Pharmaceuticals Inc) is a γsecretase modulator that affects the enzyme complex differently than inhibitors, and a Phase II has been completed (NCT01661673) although not published yet. In brief, the only activator of α-secretase tested was discontinued, as well as happened with several inhibitors of γ-secretase. Results of a Phase II trial with a modulator of γ-secretase activity are not published. In contrast, four β-secretase inhibitors are currently in Phase II or III, and therefore are the ones that hold promise among secretase modifiers. III.1.1.4. Metabolism Targeting Strategies III.1.1.4.1. Statins As discussed above, many associations have been done between lowering plasma cholesterol and reducing CNS amyloid deposition so 3-hidroxi-3-metilglutail-coenzyme A (HMG-CoA) reductase inhibitors, commonly known as statins, could decrease the risk of AD [163]. Two of these inhibitors, simvastatin and lovastatin have been tested with such a purpose but no clear benefits have been demonstrated. Simvastatin showed no benefits on the progression of AD despite lowering plasmatic cholesterol levels in a Phase III clinical trial, and ended Phase IV (NCT00303277) with negative results. Currently, there is another Phase IV clinical study recruiting participants (NCT00842920). Lovastatin also completed a Phase IV long ago, but it was not published. In addition to these unclear results, Wong and colleagues were not very optimistic after performing a meta-analysis of

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overall results on statins [164]. III.1.1.4.2. Insulin Signaling Modulation Many studies have demonstrated the correlation between the regulation of both insulin and Aβ peptide [165]. Thus, some drugs approved for diabetes treatment have been tested in AD patients. That is the case for metformin, a peripheral insulin sensitizer drug that has completed a Phase II clinical trial for AD condition (NCT00620191) and is recruiting patients to start a Phase IV one (NCT02409238). Another insulin sensitizer, MSDC-0160 (Metabolic Solution Development Company) has also completed a Phase II trial (NCT01374438) and reported some differences in glucose metabolism in the brain when referred to cerebellum [166]. III.1.1.4.3. Mitochondria-targeting Approaches Mitochondrion is one of the most affected organelles in AD and accumulation of Aβ amyloid within the mitochondria has been demonstrated [167]. Thus, mitochondrial recovery/rejuvenation drugs have been investigated as AD therapies. Dimebon or PF-01913539 (Pfizer), after obtaining promising results in a Phase II trial for Huntington disease (HD) (NCT00497159), failed to show significant improvements in a Phase III study for AD condition (NCT00838110) [168] and investigations were halted. Thus, metabolism targeting strategies have not succeeded for statins neither for the mitochondria-targeted drug dimebon, whereas insulin sensitizers are currently being tested in clinical trials. III.1.1.5. Tau Targeting Therapies In addition to the approaches mentioned above, substances targeting tau protein rather than Aβ effects are also been tested. Methylthioninium chloride (MTC) was the first identified tau aggregation inhibitor and a Phase II clinical trial showed significant results as assessed by PET [169]. A modified version, TRx0237 (TauRx Therapeutics Ltd.), has already entered Phase III , with two trials currently ongoing and a follow-up trial

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recruiting participants (NCT01689246; NCT01689233; NCT02245568) [169]. Tideglusib or NP-12 (Noscira SA) is a kinase inhibitor that inhibits GSK-3β. After showing promising results in a Phase I trial, completed a Phase II without conclusive results and was discontinued [170]. Finally, lithium, a FDA approved mood stabilizing drug, is been tested in AD patients as it has been reported to inhibit GSK-3β in bipolar disorder. A Phase II trial is currently recruiting participants (NCT02129348). Thus, among molecules targeting tau pathology (reviewed in [171]), one out of two inhibitors of the GSK-3β kinase gave inconclusive results and the other is just recruiting patients for a Phase II, whereas an inhibitor of tau aggregation is currently under Phase III. III.1.1.6. Other Strategies In this section several different strategies that have reached clinical trials but that cannot be grouped in any of the previous sections are reviewed. CHF-5074 (CERESPIR) is a microglial modulator that reached Phase II clinical trials for AD. The most recent one, showed no significant differences between groups even though a positive tendency was found on executive function in APOE Ɛ4 carriers (NCT01303744) [172] and another Phase II trial was withdrawn prior to enrollment. Piracetam and its derivative levetiracetam are nootropic drugs whose mechanism of action is not very well known but they affect glutamate receptors and calcium influx of neuronal cells [173]. Piracetam administration combined with other substances has completed a Phase IV clinical study (NCT01009476) and a Phase II is currently recruiting patients with EOAD to test efficacy of levetiracetam on AD-associated epileptiform activity (LEV-AD) (NCT02002819). Nilvadipine, a licensed calcium channel blocker, has shown to enhance Aβ clearance from brain in an AD mice model. After being shown safe and well tolerated, a Phase III clinical trial is ongoing (NCT02017340).

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Resveratrol, a grape-derived polyphenol that is the proposed ingredient responsible for the benefits of the red wine, acts mimicking the beneficial effects of dietary restriction [174]. A Phase II clinical study (NCT01504854) showing biomarker changes associated with treatment has just been published, although further studies are required to interpret them [175]. Curcumin is a neuroprotective compound isolated from turmeric root that inhibits the formation of Aβ oligomers and fibrils and binds to Aβ plaques. Preclinical studies showed that curcumin was able to cross the BBB, bind to Aβ plaques and reduce both Aβ levels and plaques after a peripherally injection in Tg2576 mice [176]. Few clinical trials have been done and, currently, a Phase II clinical trial is recruiting participants (NCT01811381). In brief, a microglial modulator failed in Phase II, the nootropic drug piracetam combined with other substances just completed Phase IV, a licensed calcium channel blocker is in Phase III, and natural products such as resveratrol or cucurmin are also being tested. III.1.2. Preclinical Studies Several preclinical studies are being carried out following either the same approaches that have reached clinical trials or different strategies to improve the already tested drugs. Anyway, in this section studies showing promising potential to enter clinical trials are reviewed. The ones modulating secretases activity are grouped in the same section. III.1.2.1. Secretases Activity Modulators NbutGT is a specific inhibitor of the O-linked β-N-acetylglucosaminase (OGLcNAcase), an enzyme that carries out this post-transcriptional modification depending on glucose metabolism and that it is known to act on nicastrin. Thus, inhibition of O-GLcNAcase diminishes γ-secretase activity and, as a consequence, attenuates the accumulation of Aβ, neuroinflammation and memory impairment in 5XFAD mice [177]. LY379268 is a dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonist that incremented sAPPα release in cultured astrocytes by inducing α-secretase

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expression and reducing β-secretase level. mGlu2/3 receptor was found to be decreased in PDAPP-J20 transgenic mice, suggesting a specific role in the disease [178]. However, further studies are needed before it could enter clinical trials. MicroRNAs (miRNAs) are small regulatory RNAs that participate in posttranscriptional gene regulation in a sequence-specific manner. A microarray study performed by Wang and colleagues found that miRNA-107 was already reduced in AD early stages, accelerating the disease progression by increasing β-secretase level [179]. A Phase 0 clinical study is about to recruit participants to test Gemfibrozil, a compound that modulates miRNA-107 level (NCT02045056). Other investigations focusing on RNA are being carried out, such as the reduction in the APP mRNA translation by controlling its 5’UTR secondary structure [180] and even histone-modifications are being proposed to modulate APP transcription [181]. III.1.2.2. Others Agonists of cannabinoid receptors CB1 and CB2 have been shown to reduce the Aβ peptide action and tau phosphorylation in different AD transgenic mice models. In addition, the fact that endocannabinoid signaling modulates many processes such as neuroinflammation, mitochondria dysfunction or oxidative stress makes cannabinoid compounds interesting drugs for AD treatment research (reviewed in [182]). At this moment, the ∆9-tetrahydrocannabinol (THC) analogue nabilone is being tested in a pilot study as a pharmacological option for managing agitation and pain in patients with moderate to severe AD (NCT02351882). Erythropoietin (EPO), besides promoting erythrocytes formation, acts as a neuronal protector. This compound and the carbamylated-erythropoietin (CEPO) were administrated to APP/PS1 transgenic mice, resulting in memory improvement. Moreover, EPO decreased soluble Aβ1-40 and Aβ plaques amount [183]. The AchE18ANH2 is an apoE mimetic peptide that was supposed to exert protective effects on neuroinflammation due to the apoE anti-inflammatory capacity. Preclinical trials results showed a cognitive improvement, a decrease in Aβ plaque deposition, a reduction of activated microglia and astrocytes and an

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increase in apoE brain levels after administration in APP/PS1 transgenic mice [184]. EP2 is a prostaglandin E2 receptor that has been implicated in the microglia dysfunction found in AD. Studies with C57BL/6 Ep2fl/+ mice showed that deleting the gene encoding for EP2 resulted in the restoration of microglial chemotaxis and Aβ clearance, as well as in the prevention of memory deficits and synaptic injuries [185]. Glutamyl cyclase (QC) is, as already mentioned in the biomarkers’ Section, the enzyme that catalyzes the formation of pE-Aβ. A QC inhibitor has shown to reduce both Aβ levels and plaques and to improve cognitive performance after oral administration in Tg2576 and TASD-41 mice models [186]. On the other hand, stem cells therapies are being investigated to treat AD. A recent study showed that transplantation of human neural stem cells (HuCNS-SC) improved cognition and increased synaptic and growth-associated markers in the 3xTg-AD and CaM/Tet-DTA mice. It was also demonstrated that these cells can migrate and differentiate into immature neurons and glia [187]. Together, these results open a great potential pathway for stem-cell therapies in AD. III.2. Immunotherapy Among all different strategies developed to fight Alzheimer’s disease, immunotherapy is the one in which more hope is deposited as reflected in the large amount of clinical trials carried out until now [156]. The majority of those trials have the Aβ peptide as the target molecule but there are also recent strategies on tau protein, and even preclinical DNA strategies recombinantly expressing a fragment of the Aβ peptide or Aβ-directed antibody fragments. Immunotherapeutic approaches can be generally classified as either active or passive and are summarized in Table 3. Passive immunotherapy drugs that have been discontinued development or repurposed are shown in italics. Phase stage is indicated in brackets, and in Arabic numbers for the sake of clarity. Information extracted from Alzforum and ClinicalTrials databases and updated on December 15 2015 [154, 155].

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Table 3. Immunotherapeutic approaches that are currently under clinical trials. Passive immunotherapy drugs that have been discontinued development or repurposed are shown in italics. Phase stage is indicated in brackets, and in Arabic numbers for the sake of clarity. Information extracted from Alzforum and ClinicalTrials databases and updated on December 15 2015 [154, 155] Therapeutic approach

Active immunotherapy

Mechanism of action

Drug (company)

Identifier (Phase)

N-t (1-6) Aβ + bacteriophage Qβ

CAD106 (Novartis Pharmaceuticals)

NCT02565511 (2/3)

Tetra-palmitoylated N-t (1-15) Aβ liposome-based vaccine

ACI-24 (AC Immune)

EudraCT 2008006257-40 (1/2)

UBITh® helper T-cell epitopes +Aβ1-14

UB-311 (United Biochemical Inc.)

NCT02551809 (2)

Tau peptide + KLH

AADvac1 (Axon Neuroscience SE)

NCT01850238 (1) NCT02031198 (1) NCT02579252 (2)

mAb anti-N-t (1-5) Aβ Binds monomeric, oligomeric and fibrillar

Bapineuzumab (AAB-001 & AAB-003) (Janssen / Pfizer)

 

mAb anti-C-t (30-40) Aβ Binds plasmatic Aβ

Ponezumab (Pfizer)

 

mAb anti-mid-region (16-24) Aβ Binds monomers

Solanezumab (Eli Lilly and Co./ Hoffmann–La Roche)

NCT01127633 (3) NCT01900665 (3) NCT01760005 (2/3) NCT02008357 (3)

mAb anti-N-t (1-10) and midregion (23-25) Aβ Binds fibrils Passive immunotherapy

NCT01224106 (3) Gantenerumab (Hoffmann-La NCT01760005 (2/3) Roche) NCT02051608 (3)

mAb anti-mid-region (12-23) Aβ Binds protofibrils

Crenezumab (Genentech)

NCT01723826 (2) NCT01998841 (2)

mAb anti-N-t (3-6) and midregion Aβ Binds aggregated forms

Aducamab (BIIB037) (Biogen Idec)

NCT02484547 (3) NCT02477800 (3)

mAb anti-Aβ E22G Binds protofibrils

BAN2401 (Eisai Inc.)

NCT01767311 (2)

mAb anti- N-t (4-12) and midregion (9-20) Aβ Binds prefibrillar aggregates

SAR228810 (Sanofi)

NCT01485302 (1)

Gammagard (Baxalta)

 

Octagam (Octapharma)

 

IVIG (Grifols Biol. Inc.)

NCT01561053 (3)

IVIG (Sutter Health NI)

NCT01300728 (2)

Intravenous IgG Binds different linear and conformational epitopes

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III.2.1. Active Immunotherapy Active immunotherapy consists of administering an antigen (or a molecule that mimics the antigen) to activate the immunologic response of the organism, and thus corresponds to the classical concept of vaccination. Active immunotherapy first demonstrated its effectiveness in Alzheimer’s research in 1999, when Schenk and colleagues reached to prevent amyloid accumulation after injecting the Aβ1-42 peptide in 6-weeks old PDAPP transgenic mice [188]. The first active Aβ immunotherapy tested in clinic trials was AN1792 (Janssen & Wyeth), a synthetic Aβ1-42 peptide co-administered with the QS-21 adjuvant [189]. Although it was halted in 2002 because of the appearance of meningoencephalitis in 6% of the immunized patients, almost 20% of the participants developed the predetermined antibody response. Moreover, a follow-up study with those patients demonstrated a persistent anti-Aβ antibody titer and a reduced functional decline more than 4 years after the last injection [190]. Thus, although halted in Phase II, the AN1792 clinical trial paved the way for a new generation of anti-Aβ vaccines, which are currently being tested. The main difference between these second generation vaccines and the AN1792 is that they do not use the entire Aβ1-42 peptide, which is thought to be the cause of meningoencephalitis appearance as it contains the Tcell epitopes (in the C-terminus) that trigger the T-cell response [191]. In this way, different strategies have been followed to avoid this response such as the administration of N-terminal short Aβ peptides, fragmented peptides corresponding to the central sequence or mimetic peptides [192]. The active immunotherapy approaches currently being tested in clinical or preclinical trials are shown next. III.2.1.1. Clinical Trials III.2.1.1.1. Discontinued Trials Because it is also important to know which drugs have unsuccessfully been tested, a brief description of their mechanism of action and the results of the performed clinical trials is shown. ACC-001, also called vanutide cridificar (Janssen and Pfizer), is a conjugate of multiple copies of Aβ1-7 peptide linked to a nontoxic variant of diphtheria toxin

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(CRM197), which was administered intramuscularly together with QS-21 adjuvant. Preclinical data showed that ACC-001 generates anti- N-terminal-Aβ antibodies without inducing T-cell response [192]. Many Phase II clinical trials have been completed, but a recent paper reports a variety of treatment-emergent adverse events (TEAEs) from most subjects during the studies [193]. All doses of ACC-001 + QS-21 elicited high, sustained anti-Aβ antibody titers, although QS21 was necessary for this effect. Affitope AD01 and AD02 (Affiris AG) are composed of a 6-amino acid peptide that mimics the N-terminus of Aβ [194]. They are non-endogenous compounds so they avoid breaking the tolerance normally established against self-proteins. In addition, their N-terminal sequence and their controlled specificity prevent both autoreactive T-cell activation and cross-reactivity with APP. Although Phase I clinical trials showed a favorable safety profile with both compounds, Phase II clinical trials with AD02 did not reach either primary or secondary outcome results but rather found a new formulation with a greater benefit on the primary outcome. V950 (Merck Sharp & Dohme Corp.) is a multivalent Aβ peptide vaccine. Preclinical studies showed that administration of V950 results in Aβ antibodies in the serum and in the CSF that recognize different pE-modified and other Nterminally truncated Aβ fragments [192]. A Phase I trial was completed and results about safety and tolerability are available online (NCT00464334). The company discontinued V950 development. III.2.1.1.2. CAD106 CAD106 (Novartis Pharmaceuticals) consists of the Aβ1-6 peptide coupled to the Qβ coat protein, which is present in 180 copies in the virus-like Qβ particle [195]. CAD106 stimulates B-cell response and carrier-induced T-cell help but avoids activating an Aβ-specific T-cell response [196]. After completing different Phase II studies, the main conclusions were that CAD106 showed a favorable tolerability profile and that long-term treatment induced Aβ-specific antibody titers [197], and therefore a Phase II/III clinical trial (NCT02565511) has just been registered [155].

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III.2.1.1.3. ACI 24 ACI-24 (AC Immune) is a tetra-palmitoylated Aβ1-15 peptide liposome-based vaccine. Preclinical results with double-transgenic APP/PS1 mice showed induction of significant levels of systemic Aβ antibodies, a complete restoration of cognitive non-spatial memory and plaque reduction after two intraperitoneal inoculations of ACI-24 [198]. Nowadays, a Phase I/II clinical trial testing its safety and efficacy is ongoing (EudraCT 2008-006257-40). III.2.1.1.4. UB-311 The UB-311 vaccine, which consists of two synthetic peptides, the UBITh® helper T-cell epitope and the Aβ1-14, is designed to minimize inflammatory reactivity through the use of a proprietary vaccine delivery system that biases Thelper type 2 (Tht 2) regulatory response in preference to T-helper type 1 (Tht 1) proinflammatory response [199]. Phase I studies demonstrated its safety and tolerability and a Phase II study is recruiting patients (NCT02551809). III.2.1.1.5. AADvac1 AADvac1 (Axon Neuroscience SE) consists of a tau peptide (tau294-305) conjugated to a keyhole limpet hemocyanin (KLH) and is administered with aluminum hydroxide adjuvant. Preclinical data shown that besides the reduction of neurofibrillar pathology, vaccination with AADvac1 improved behavioral deficits [200]. A Phase I study is completed (NCT01850238), another is ongoing (NCT02031198), and a Phase II study has just been registered (NCT02579252). As a summary, the active immunotherapy approach is currently being assayed with four drugs: the Aβ1-6 peptide coupled to the Qβ coat protein (CAD106), a tetra-palmitoylated Aβ1-15 peptide liposome-based vaccine (ACI-24), a combination of UBITh® helper T-cell epitope and Aβ1-14 (UB-311) and a KLHconjugated tau294-305 peptide (AADvac1). III.2.1.2. Preclinical Studies Besides vaccines that have reached clinical trials, there are many other substances that are being investigated in preclinical studies and that could eventually result in

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successful active immunization strategies. Lu AF20513 (Lundbeck and Otsuka) is an Aβ1-12 peptide with two foreign Thelper epitopes from tetanus toxin, instead of the T-helper cell epitopes of the Aβ1-42 peptide, which stimulates existing memory T-helper cells to promote Aβ antibodies production from B cells [201]. Lu AF20513 induced robust non-self Tcell responses and production of anti-Aβ antibodies, resulting in the reduction of AD-like pathology without inducing microglial activation and enhancing astrocytosis in Tg2576 mice. MER5101 (Mercia Pharmaceutical) is an Aβ1-15 peptide conjugated to a diphtheria toxin and administered with a nanoparticle emulsion-based adjuvant [202]. Results showed high anti-Aβ levels, reduction in cerebral Aβ plaque burden and improvement of cognitive deficits in APP/PS1 transgenic mice. Immunization of tau/PS1 transgenic mice with Tau379-408[P-Ser396, 404] induced a good IgG antibody response, reduced soluble and insoluble tau peptide in the brain and improved behavior [203]. These results postulate immunotherapy against tau peptide as a possible alternative/complementary therapy to Aβtargeting immunotherapy. ACI 35 (AC Immune) is a liposome-based vaccine which uses the same principles that ACI 24 (explained above). In this case, the 16 amino acid synthetic peptide does not correspond to the Aβ peptide but to Tau393-408[P-Ser396,404]. The vaccine provoked fast and robust specific antibody responses for p-tau in tau.P301L mice [204]. Similarly, two KLH (keyhole limpet hemocyanin)-conjugated peptides containing the phosphorylated Serine 422 tau epitope were demonstrated to be effective in THY-tau22 transgenic mice [205]. A decrease in insoluble tau species in the brain and an increase in tau concentrations in the blood were observed, suggesting clearance from the brain to the periphery. Ankyrin G (ankG) is a neuronal cytoskeletal protein the expression of which was shown to be higher in AD brains. After administration of ankG protein with the Freund’s adjuvant in ArcAβ or Tg2576 mice there was a reduction in Aβ-related

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pathology and an increment in soluble Aβ1-42 levels [206]. In DNA immunizations, the DNA encoding the Aβ1-42 peptide is injected into the skin or muscle using a gene gun. Then, the DNA is transcribed and translated to the Aβ peptide causing a general immune response [18]. Preclinical studies with AD transgenic mice showed a reduction of plaques and Aβ1-42 levels in the brain. They also proved the effectiveness and safety of the approach by the absence of T-cell proliferation in full-length Aβ1-42 DNA-immunized mice compared to those directly immunized with Aβ1-42 peptide [207]. III.2.2. Passive Immunotherapy Passive immunotherapy consists of the administration of a monoclonal antibody (mAb) against a specific target which, in Alzheimer’s disease, usually is the full Aβ1-42 or a part of it. These antibodies can be obtained in a murine model and then humanized (the humanization consists of the mice framework regions (FR) substitution for the human corresponding ones, maintaining the complementary determining regions (CDR), and takes the form -zumab), or they can be obtained directly from human cellular lines (-umab). As opposed to active immunotherapy, passive immunotherapy requires frequent re-administrations and can be quite expensive. However, it has multiple advantages including the possibility to immediately stop administration whenever an adverse effect is observed, the avoidance of host’s immune system activation and the targeting of specific protein forms or epitopes. Different passive immunotherapy strategies that are being investigated in clinical and preclinical trials are shown below. III.2.2.1. Clinical Trials III.2.2.1.1. Discontinued or Repurposed Drugs This section is dedicated to discontinued or repurposed developments of anti-Aβ mAbs, not only because much effort has been paid to them but also because they have generated wealth of knowledge. Bapineuzumab

In 2000 it was demonstrated that peripheral injection of a monoclonal antibody

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(mAb) specific for the 1-5 N-terminal residues of the Aβ peptide and which recognizes all soluble forms of Aβ (monomeric, oligomeric and fibrillar), 3D6mAb, generated the antibody transfer to the brain, the antibody binding to plaques and the induction of fragment-crystallizable region (Fc) receptor-mediated microglial phagocytosis of Aβ deposits in PDAPP mice [208]. Bapineuzumab or AAB-001 (Janssen & Pfizer) is the humanized successor of this antibody and has reached many Phase III clinical trials. In a Phase I study bapineuzumab was welltolerated by patients with mild-to-moderate AD, but DTI monitoring showed microhemorrhage in three out of ten patients on the highest dose. This finding led to the new terms ARIA-E and ARIA-H described above. ARIAs are thought to result from transient leakiness of cerebral vessels following vascular amyloid clearance induced by the mAb (see the biomarkers Section). In the subsequent Phase II trial the highest dose was lowered. Despite no significant difference was seen by two different cognitive assessments, reduced ptau in CSF and a considerable reduction in amyloid burden were shown [209]. However, the occurrence of ARIA-E in APOE Ɛ4 allele carriers led to recruiting APOE Ɛ4 (NCT00575055) and non-APOE Ɛ4 carriers (NCT00574132; EudraCT 2009-012748-17) separately for Phase III. CSF analyses showed a reduction in ptau with treatment only in the APOE Ɛ4 group. sMRI did not show any differences between treated and untreated groups, but amyloid-PET scans demonstrated the clearance of some fibrillar cerebral Aβ. On the other hand, DTI showed that ARIA-E occurred in at least 5% of the bapineuzumab-treated patients, especially in the APOE Ɛ4 carrier cohort (ten out of twelve cases analyzed), and led to the discontinuation of the study at the highest dose. These facts were attributed to the increased Aβ burden, including vascular amyloid, in mild-to-moderate AD APOE Ɛ4 carriers. Because bapineuzumab did not improve clinical outcomes in patients with AD, despite treatment differences in CSF p-tau observed in APOE Ɛ4 carriers, other Phase III clinical trials with bapineuzumab were halted in August 2012 [210]. However, the main hypothesis for the absence of clinical efficacy is that the application of the treatment was done in too advanced stages of the disease. Pfizer and Janssen in 2014 completed a couple of Phase I trials with AAB-003 (NCT01193608; NCT01369225), a redesigned mAb intended to minimize the risk

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of vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H). The redesign, consisting of the modification of the Fc portion of the antibody, did not achieve its goal and the company discontinued its development. However, other strategies trying to avoid these adverse side effects are being developed in preclinical studies (see below). Ponezumab

Ponezumab, also known as PF-04360365RN1219 (Pfizer), is an IgG2a humanized mAb designed to recognize the C-terminus Aβ30-40 peptide region. This allows a major efficiency in binding the Aβ peptide in plasma, where this epitope is solvent-exposed, increasing the amount of the peptide there and diminishing the hippocampal amyloid burden [211]. In addition, Ponezumab contains two mutations (A33S, P331S) that eliminate effector function, and thus it is immunologically inert. Early clinical data pointed to good safety profile, although no improvement in cognitive impairment was observed in two completed Phase II trials. Because it is more appropriate to recognize Aβ1-40 than Aβ1-42, it was repurposed for treating CAA and a Phase II clinical trial is ongoing (NCT01821118). III.2.2.1.2. Solanezumab Solanezumab or LY206243 (Eli Lilly and Co./Hoffmann-LaRoche) is a humanized mAb based on the m266 described by DeMattos in 2001, which was directed to the Aβ16-24 mid-region and able to bind and sequester plasma Aβ in PDAPP mice [212]. Thus, the peripheral administration of m266 reduced Aβ deposition without binding Aβ deposits in the brain, meaning that m266 seems to promote Aβ efflux from CNS towards plasma. In Phase I, solanezumab was well tolerated in healthy volunteers and patients with mild-to-moderate AD. No evidence of inflammation, vasogenic edema, or microhemorrhage were found by MRI. Biochemical marker studies found changes in plasma and CSF Aβ1-40, Aβ1-42 and N-terminally truncated Aβ, and plasma pEAβ, but not total tau and p-tau in CSF. In the subsequent Phase II trial doses were increased and the antibody's safety and tolerability were confirmed. While dosedependent increases of various Aβ species in plasma and CSF were found, no

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cognitive improvement was achieved. In Phase III, carried out with mild AD patients, 33% reduction in the rate of decline was shown (NCT00905372; NCT00904683) [213]. The benefit appeared after some months of treatment, grew over time, and is consistent with a disease-modifying effect. However, this benefit was not better than that of the palliative acetylcholinesterase inhibitor drugs. Two Phase III and one Phase II/III trials are ongoing (NCT01900665; NCT01127633; NCT01760005) and another one is recruiting patients (NCT02008357). The two last trials deserve a special mention. The Dominantly Inherited Alzheimer Network (DIAN) recruited individuals with FAD but still cognitively normal or with MCI (DIAN-TU; NCT01760005). Solanezumab (also gantenerumab, see next section) is the main treatment being tested based on good safety, some clinical benefit, and a similar efficacy in patients with or without the APOE Ɛ4 allele [214]. Since this trial began in 2012, some results with untreated patients have been published [215]. Cross-sectional measurements by imaging (PET and MRI) and CSF and plasma biomarkers levels assessed from non-treated 54 PS1E280A kindred members (age range 20-59 years) were performed. Compared with non-carriers, PS1E280A carriers had significantly lower precuneus cerebral metabolic rates for glucose, a smaller hippocampal volume, lower CSF Aβ1-42, higher CSF t-tau and p-tau181, and higher plasma Aβ1-42 levels. Another prevention study in which solanezumab will be tested is the Alzheimer's disease Cooperative study A4, in which asymptomatic or very mildly symptomatic people aged 65-85 years and who have evidence of brain amyloid deposition by PET are being recruited (NCT02008357). III.2.2.1.3. Gantenerumab Gantenerumab (Hoffmann-La Roche) is the first fully human IgG1 for Aβimmunotherapy. It was optimized by HuCAL(®) phage display technologies for binding to a conformational epitope shown by amyloid-β fibrils. This conformational epitope is made by both the N-terminal and the mid-region of Aβ. Epitope mapping using overlapping decameric Aβ peptides revealed peptides spanning residues 3-12 and 18-27 as those showing the strongest binding. It is quite interesting that the crystal structure of the Fab-Aβ1-11 complex exhibited a novel orientation of the N-terminal Aβ bound to the CDRs. This makes sense

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because, in contrast to what happened with bapineuzumab, the first two residues of the Aβ peptide are not included within the epitope. Gantenerumab preferentially interacts with aggregated brain Aβ, both parenchymal and vascular. In a mouse model of AD, this mAb showed sustained binding to cerebral amyloid and reduced amyloid plaques by recruiting microglia and preventing new plaque formation, while plasma Aβ remained the same [216]. Phase I of clinical trials in mild-to-moderate AD patients treated with gantenerumab reduced the brain amyloid load, but two patients in the high dose group experienced ARIA-E at sites with the highest level of amyloid reduction, as seen by MRI [217]. The Phase II study enrolled people aged 50 and older whose memory function tests were below normal and who were positive for amyloid on PET assessments. Although this phase was extended to Phase III, an interim futility analysis made Roche discontinue this trial in December 2014. At this moment, and based on inclusion criteria, two trials are currently on going: a Phase III trial with patients in the prodromal phase (NCT01224106) and a Phase II/III with those FAD mutations carriers in the DIAN-TU study mentioned above (NCT01760005). In addition, another Phase III trial is recruiting patients with mild AD (NCT02051608). III.2.2.1.4. Crenezumab Crenezumab, also known as MABT5102A (Genentech), is a humanized mAb against the mid-region of the Aβ peptide (12-23) that was designed on an IgG4 scaffold, instead of the IgG1 one for bapineuzumab, solanezumab and gantenerumab, to avoid inflammatory effects as microglia activation, edema and microhemorrhages [218]. When compared in a mouse model with a human IgG1 with the same antigen-binding variable domains, crenezumab showed reduced activation of stress-activated p38MAPK in microglia and induced less release of the pro-inflammatory cytokine TNFα. This antibody binds to protofibrillar species, including oligomers, resulting in the prevention of aggregation and also in the disaggregation of amyloid plaques. Remarkably, the central Aβ residues mentioned above as recognized by solanezumab are almost the same recognized by crenezumab, explaining the observed shared cross-reactivity of these mAbs [219]. However, amino acidic changes in positions 33 and 36 are responsible for

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the recognition of different species, i.e. monomers by solanezumab and protofobrilar species by crenezumab. Phase I trials showed good safety data for mild-to-moderate AD patients, and Phase II testing with higher doses was completed in spring 2014 and continued into an open-label-extension trial that will run until February 2017 (NCT01723826). It is also being evaluated as a part of the Alzheimer Prevention Initiative (API), in which pre-symptomatic PS1E280A carriers were recruited for a Phase II trial set to run until 2020 (NCT01998841). III.2.2.1.5. Aducanumab Aducanumab (Biogen Idec), also known as BIIB037, is a human IgG1 mAb derived from an AD patient with an unusual stable clinical course. Reverse translational medicine was used to isolate, clone and recombinantly express the anti-Aβ antibody, which showed a high affinity for insoluble fibrillar Aβ and reduced amyloid burden in Tg2576 mice [220]. Similarly to bapineuzumab, aducanumab recognizes Aβ’s N-terminal residues 3 to 6 but also a structural epitope that is present on aggregated Aβ but absent from monomers. In a Phase I clinical trial, patients with mild AD who took a 10-mg dose of the drug showed reduced cognitive decline and decreased brain levels of amyloid, but microhemorrhages occurred in some cases and much smaller doses had statistically insignificant benefits. So, an intermediate dose of 6 mg was tested and decreased levels of amyloid were found. Although mental decline was not reduced, it showed glimmers of promise and two Phase III trials are currently recruiting participants (NCT02484547; NCT02477800). III.2.2.1.6. BAN2401 BAN2401 (Eisai Inc.) is a humanized mAb, mAb158, that is conformationdependent. Because it was directed against the Arctic APP mutation (E22G in Aβ), which generates a form of AD characterized by particularly high levels of Aβ protofibrils, it shows the ability to recognize a unique conformation in the Aβ protofibril [221]. It has recently been demonstrated that it selectively reduces amyloid-β protofibrils in the brain and CSF of APPArcSwe mice [222].

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Two Phase I clinical safety studies were completed and no serious adverse events were observed. Phase II was started in January 2013 in patients with MCI and early AD (NCT01767311). III.2.2.1.7. SAR228810 SAR228810 (Sanofi) is a humanized mAb, mAb13C3, that recognizes a repeating conformational epitope of prefibrillar Aβ aggregates organized by residues 4-12 and 9-20 [223]. Similarly to crenezumab, it was derived form an IgG4 subclass in order to prevent ARIA. A Phase I clinical trial in 48 patients with mild-t-moderate AD has just being completed (NCT01485302). III.2.2.1.8. IVIG Intravenous immunoglobulins (IVIGs) are a mixture of human IgG antibodies derived from the plasma of healthy young volunteers that naturally occurs. Interestingly, IVIGs contain elevated levels of antibodies against different conformation of Aβ monomers and aggregates. Even though Phase II and III trials of octagam (Octapharma) and gammagard (Baxalta US Inc.), respectively, had shown favorable results to some extent, they had failed to achieve primary endpoints such as the slowing of functional and cognitive decline (reviewed in [224]). Although there is not any trial active for octagam, last pipeline page of Octapharma Canada still mentions its octagam program; In contrast, gammagard has been discontinued. However, research in IVIG has not been stopped and, currently, there is a Phase II trial (NCT01300728) and a Phase II/III trial (NCT01561053) being carried out by Sutter Health Neuroscience Institute and Grifols Biologicals Inc., respectively. It is noteworthy that apart from recognizing plasma Aβ, other interesting properties of IVIG are related to its immunomodulatory and anti-inflammatory effects. IVIG might influence modulation of microglial activation and increase microglial phagocytosis of fibrillar Aβ. In addition, IVIG can inhibit complement activation, neutralize inflammatory cytokines, and modulate chemokine expression and regulatory T-cell subsets. Moreover, IVIG has plenty of components that could be helpful in AD treatment. For example anti-RAGE

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antibodies have been reported in IVIG preparations, so Aβ influx into the brain could be reduced. Likewise, soluble LRP (sLRP) is present and could prevent plasma Aβ from entering the brain [225]. As a summary, bapineuzumap development has been discontinued and ponezumab repurposed for treating CAA, whereas solanezumab, gantenerumab and crenezumab are being tested in prevention studies. Aducamab, BAN2401 and SAR228810 are in Phase III, II, I clinical trials, respectively; and several IVIG preparations are in different stages of development. III.2.2.2. Preclinical Studies Apart from the clinical trials ongoing at this moment, several different preclinical studies are being carried out in order to improve these therapies or to search new molecules that could avoid the reported adverse effects. Despite some of them have a promising potential, more investigation is needed before reaching the desired clinical trials. A-887755 is a highly Aβ oligomer-specific mAb generated from Aβ20-42 oligomers (named globulomers). The affinity for oligomeric Aβ species and not monomeric or fibrillar Aβ demonstrated to alleviate synapse loss in Tg2576 mice, suggesting that lowering the fibrillary amyloid load is not necessary to improve cognitive function [226]. mAb07/1 (Probiodrug AG) is a mAb targeting pE-Aβ, present in almost all cerebral plaques and vascular amyloid deposits. Chronic passive immunization with this mAb significantly reduced plaque deposition in APP/PS1 transgenic mice without producing microhemorrhages, postulating it as a putative Aβ clearance enhancer [227]. A F(ab’)2 consists of two antigen-binding fragments (Fabs), each one composed of one constant and one variable domain of each of the antibody heavy and light chain. Aβ1-13-F(ab’)2 derives from an IgG1 mAb and targets the N-terminal Aβ1-13. The lack of the Fc fragment is supposed to avoid the activation of microglia and thus the main adverse effects reported until now such as vasogenic edema and microhemorrhages. Preclinical results showed a reduction in plaque formation and

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in phagocytic infiltration in the CNS (compared to full mAb) after it was intraperitoneally or intracranially injected in Tg2576 mice [228]. More recent investigations have been carried out using Fabs but they have not yet been tried in animal models. ScFv-h3D6 is a single chain variable fragment (scFv) derived from bapineuzumab. ScFvs consist of the antibody heavy chain variable domain (VH) joint to the light chain variable domain (VL) by a polypeptide linker [229, 230]. Similarly to Fabs, they are supposed to avoid microglia activation and thus the vasogenic edema that halted bapineuzumab clinical trials. Preclinical results showed a significant effect at the behavioral and molecular levels in the 3xTg-AD mouse model [231]. After a single intraperitoneal dose of scFv-h3D6, the accelerated swimming speed characteristic of this model was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease in Aβ oligomers in the cortex and in the olfactory bulb. In addition, the increased concentrations of apoJ and apoE in the brain of the 3xTg-AD mouse model were diminished to non-pathological levels after treatment. This concurs with the involvement of these lipoproteins in the pathology, as mentioned above. Also, a preliminary study with this mouse model showed the ability of scFv-h3D6 to protect deep cerebellar nuclei neurons from death [232]. ScFv59 is a single chain variable fragment derived from solanezumab (currently in the DIAN-TU and A4 studies, see above). Preclinical results showed that both Aβ brain deposits and Aβ CSF levels decreased while serum Aβ increased after muscularly injecting an AD mice model (B6.Cg-Tg 85Dbo/J) with a recombinant adeno-associated virus (rAAV1) encoding scFv59 [233]. These results support the peripheral sink hypothesis and postulate the scFv59 delivery via rAAV injection as a prophylactic option for AD. W20 is a conformation-dependent oligomer specific scFv that binds to many oligomeric assemblies in vitro, including those formed by Aβ1-40 and Aβ1-42. Preclinical results showed a cognitive impairment rescue and an interference with Aβ levels and deposits in APP/PS1 transgenic mice [234].

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The discovery of a novel type of antibodies termed heavy-chain antibodies (hcAbs) in the immune system of camelid species (camels and llamas) and cartilaginous fish (wobbegong and nurse sharks), lead to the engineering of stable single-domain antibodies (sdAb), also called nanobodies, for applications such as immunotherapy. Preclinical results with an anti-Aβ1-42 VH that targets Aβ mid-/Cterminal region showed a reduction of amyloid deposits in Tg2576 mice after intracranial administration [235]. Designed ankyrin repeat proteins (DARPins) are small and highly stable proteins ideal for high affinity protein-protein interactions. Apart from other applications, they have been used as a scaffold for CDRs targeting the Aβ peptide [236]. After the prevention of Aβ aggregation and the reduction of Aβ-mediated neurotoxicity in cell culture, results in Tg2576 mice showed an improved cognitive performance and reduced soluble Aβ levels when mono- and trivalent Aβ-specific DARPins (D23 and 3xD23) were intracerebroventricularly infused. PHF1 is a mAb which targets p-tau by reacting with phosphorylated Ser396 and Ser404. Reduction of tau pathology and improvement in behavioral tasks have been shown in JNPL/3 mice [237]. Anti-BACE1 is a high-affinity phage-derived human antibody against the βsecretase enzyme. Results of systemic dosing in mice and in nonhuman primates showed a reduction of CNS and peripheral Aβ. However, the success of this approach will depend on improving the capacity of the antibody to cross the BBB [238]. CONCLUSION AND FUTURE PERSPECTIVES Alzheimer’s disease is a complex neurodegenerative disease which involves and affects many different biological mechanisms. As compiled in this Chapter, many therapies are being developed trying to modify different targets that are thought to participate in the disease progression but, until now, none of them has been able to succeed. On the other hand, epidemiological research evidenced modifiable risk and protective factors for the sporadic form of the disease, from vascular and metabolic issues to lifestyle, diet, and psychosocial factors, among others. Such is the importance of trying to prevent the disease, that the World Alzheimer Report

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2014 is entirely dedicated to this issue [239]. On the other hand, the World Alzheimer Report 2015 updates the global impact of dementia [6]. Since the vast majority of approaches searching for an efficient and safe AD therapy are Aβ-centric, their failures have led to question the amyloid cascade hypothesis (ACH). Thus, there is a stream of scientists claiming a new standpoint for the sporadic form of the disease. A modified scheme of the ACH claims that aging and the increase in allostatic load would be the primary factors and both Aβ peptide and tau protein would appear later as a consequence of synaptic loss, triggering a secondary degeneration [10]. Other visions point to calcium or glucose dysregulation as upstream events in the disease development [29,31]. Although it is true that many questions remain unsolved, ACH has provided many evidences that have pushed AD research to a huge evolution in the last two decades [14]. Before accepting a secondary role for Aβ, it is worthy to analyze the reasons why Aβ-centric clinical trials have failed. In an extensive work, Karran and Hardy reviewed the studies made with 6 compounds that have failed in Phase 3 clinical trials. In general terms, they conclude that the desperation for an effective treatment leads, too often, to start clinical trials without enough verified preclinical information, and that the problem lies in the way the studies were done, instead of the target itself [240]. Special emphasis is made in passive immunotherapy failures as, in those cases, preclinical data were robust and optimistic. In the specific case of immunotherapy, two hypotheses exist about how antibodies are able to reduce Aβ burden: the microglia-mediated hypothesis and the peripheral sink one [241]. The first one suggests that anti-Aβ antibodies bind to amyloid plaques in the brain and the complex activates local microglia that clears them by Fc receptor-mediated phagocytosis. Although it has been demonstrated that Fc receptor-mediated phagocytosis is unnecessary for the clearance of the antigen-antibody complex [242], and that the proportion of antibodies crossing the BBB is lesser than 0.1% [243], the antibodies that are able to bind amyloid plaques are thought to trigger some other clearance mechanisms. Conversely, the peripheral sink hypothesis suggests that anti-Aβ antibodies in blood that bind to Aβ in serum trigger an efflux of Aβ from CNS through the BBB [241].

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While it is true that the lack of a conclusive hypothesis explaining the pathomechanism of Alzheimer's is a drawback in terms of achieving clinical effectiveness, it is not the only one. Regarding immunotherapy again, one of the main concerns is the stage of the disease at which the treatment is applied. Patients engaging AD clinical trials have often clear symptoms of AD or MCI, stages in which the disease is too advanced to be effectively stopped or even reversed [240]. In this sense, three consortia are working to test different drugs in younger patients in order to prevent the disease development: the Alzheimer’s Prevention Initiative (API), the Dominantly Inherited Alzheimer’s Network (DIAN) and the Anti-Amyloid treatment in Asymptomatic Alzheimer’s (A4). API tests crenezumab in individuals from a large Colombian family which carries the PS1E280A mutation; DIAN tests immunotherapy with solanezumab or gantenerumab in adult children of a parent with the same mutation; and, finally, A4 tests solanezumab in asymptomatic elderly patients with amyloid positive PET. Therefore, in order to facilitate early intervention and an appropriate design of clinical trials biological markers improvement is mandatory. Thus, they are considered one of the most promising working lines in AD research. Given the exponential growth of prospective biomarkers, standardization and harmonization of protocols become extremely required to facilitate reliable diagnosis. In turn, accurate diagnosis does not only allow early intervention, but it also enables suitable criteria for selection or inclusion of individuals for clinical trials and the achievement of real assessments on efficiency and safety of the assayed treatments. Moreover, it is highly important to ease and refine biomarkers detection. Despite the great recent development on neuroimaging and CSF analyses, these procedures are invasive and expensive. Consequently, many efforts are focused in discovering potential peripheral biomarkers, such as bloodbased biomarkers. Such a kind of biomarkers would extraordinary improve AD pandemic by regular testing of the population at risk [79]. So, drug development and early detection must evolve together. As a general conclusion, and even though there are many issues that need to be addressed, AD research is on the right path to find a disease-modifying drug that could stop this devastating pandemic.

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CONFLICT OF INTEREST The authors confirm that they have no conflict of interest to declare for this publication. FUNDING Instituto de Salud Carlos III/FEDER [FIS-PI113-01330], Generalitat de Catalunya [SGR-GRC-2014-00885] & Generalitat de Catalunya/FEDER [2014PROD00032]. PIF-UAB student grants (JGB, GEC & LMG). ACKNOWLEDGMENTS Declared none. REFERENCES [1]

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CHAPTER 3

At the Crossroad between Neuronal Hyperexcitability and Neuroinflammation: New Therapeutic Opportunities for Alzheimer’s Disease? Chelsea Cavanagh1, Slavica Krantic2,3,4,5,* 1

Douglas Hospital Research Center, Department of Neuroscience, Montreal, Quebec, Canada

Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche, des Cordeliers, F-75006, Paris, France 2

3

INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France

Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France 4

Centre National de la Recherche Scientifique (ou CNRS) ERL 8228, Centre de Recherche des Cordeliers, Paris, France 5

Abstract: Alzheimer’s disease (AD) is a multi-faceted neurodegenerative disease. Clinically available treatments, such as cholinesterase inhibitors, are based mainly on the cholinergic hypothesis of AD. These treatments, as well as those targeting the NMDA type of glutamate receptors all provide only a limited therapeutic benefit. The field of AD research has also shifted focus to develop intervention strategies that prevent overt symptoms, such as amyloid plaque deposition and memory loss, in agreement with the more recent amyloid hypothesis of AD. However, to date, all amyloid-directed therapeutics for the treatment of AD have failed, suggesting that additional factors may be involved in the etiology of the disease and mobilizing the Corresponding author Slavica Krantic: Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche, des Cordeliers, F-75006, Paris, France; INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France; Centre National de la Recherche Scientifique (ou CNRS) ERL 8228, Centre de Recherche des Cordeliers, Paris, France; Email: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2016 Bentham Science Publishers

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search for additional drug targets. By studying the early stages of AD, candidate drug targets (e.g. cytokines or neuronal network activity) have been identified and are now at advanced stages of preclinical development. Throughout this chapter we will focus on two aspects of AD that have garnered widespread attention with respect to future therapeutic intervention strategies. First, a common feature of both mouse models of AD and patients with the disease is hyperexcitability at the level of the synapse as well as neuronal networks. New research is starting to uncover the causes of this hyperexcitability and which cell types are vulnerable, thus, providing attractive therapeutic targets. Second, AD brains are affected by neuroinflammation-like alterations at early stages, which turn into overt neuroinflammation at the late stages. Reducing this activity by targeting the proinflammatory cytokine, tumor necrosis factor-α (TNFα), is thought to be a promising strategy to treat AD. Furthermore, given the cross-talk between the nervous system and the immune system, we hypothesize that the hyperexcitability and progressive induction of neuroinflammation may be related. Here, we summarize studies in both animal models of AD and AD patients related to hyperexcitability and neuroinflammation in the early stages of the disease. Finally, we propose that a combination treatment targeting these factors in addition to the amyloid burden would be a possible way to target more facets of AD.

Keywords: Alzheimer’s disease, Amyloid, Hyperexcitability, Inflammation, Intervention, Prodromal, Synaptic, TNFα. INTRODUCTION Alzheimer’s disease (AD) and related dementias affect an estimated 44 million people worldwide [1], a number that will only grow with the aging population unless new therapies are developed. AD develops insidiously over decades and can remain undiagnosed for years until the manifestation of overt symptoms. These overt symptoms include loss of cognitive functions that interfere with the individuals’ ability to perform daily tasks, trouble remembering recent events and eventually total memory loss, as well as a host of other symptoms such as agitation, paranoia, sleep disturbances and aggression. The hallmark histopathological features of AD include extracellular, senile plaques composed of amyloidbeta (Aβ), intracellular tangles composed of hyperphosphorylated tau and neuronal loss [2]. However, these features represent only a fraction of this multifaceted disease and research in animal models is uncovering additional hypotheses for possible causes of AD.

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The purpose of this chapter is to summarize and provide new insights into the treatment of AD. We will focus on two aspects of AD that research suggests are particularly important to the pathogenesis of the disease namely, neuroinflammation and synaptic function, as well as how they are related. We propose that the treatment of AD will ultimately require a multi-targeted, stagespecific approach to address multiple facets of the disease. 1. BACKGROUND ON AD The prevailing theory on the cause of AD is the amyloid cascade hypothesis, which states that the overproduction of Aβ from the amyloid precursor protein (APP) initiates a series of events, including synaptic dysfunction, microglial and astrocytic activation and hyperphosphorylation of tau, which culminates in widespread neuronal death [2]. Although this hypothesis is supported by strong genetic evidence, which will be described below, all amyloid-directed therapeutics tested to date have failed in clinical trials. In addition, this hypothesis has been challenged by findings of amyloid plaques in cognitively healthy individuals upon post-mortem analysis [3]. However, these findings may reflect the ability of some to cope better with AD pathology than others [3]. Although it is hard to discount the contribution of Aβ to the disease, the search is underway for additional drug targets and contributors to AD pathology. 1.1. APP Processing Aβ is a 4kDa protein produced through the sequential cleavage of APP, which is a transmembrane protein. APP can be processed in one of two ways, either along the amyloidogenic pathway, which generates Aβ or the non-amyloidogenic pathway. The amyloidogenic pathway involves an initial cleavage step by βsecretase or β-site APP cleaving enzyme 1 (BACE1) to produce the β-C-terminal fragment (βCTF), which is retained in the membrane and the secreted APP-β fragment (sAPPβ). βCTF then undergoes a second cleavage step by γ-secretase to liberate Aβ and the APP intracellular domain (AICD) peptide. Along the nonamyloidogenic pathway, the first cleavage step is by α-secretase, which produces the α-C-terminal fragment (αCTF) and the secreted APP-α (sAPPα) fragment. The second cleavage step along this pathway is also by γ-secretase, which cleaves α-

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CTF to produce AICD and the P3 peptide. [4]. Under physiological conditions, it is thought that APP cleavage occurs primarily along the non-amyloidogenic pathway. However, rare genetic mutations and unknown, disease-prone conditions can shift APP processing primarily along the amyloidogenic pathway and thereby increase the production of Aβ to toxic amounts [5, 6]. Aβ is a ‘sticky’ protein that easily aggregates to form various oligomeric forms, fibrils and plaques. Although there is much debate in the field over which aggregate of Aβ is the most toxic variant, a top contender are soluble, oligomeric species of Aβ, such as dimers, which can negatively impact synaptic transmission [7]. However, the precise oligomeric species involved and the mechanism of action remain unknown. In addition to Aβ, other APP fragments have been considered for their toxicity. The βCTF fragment, which is rate-limiting to the production of Aβ, has shown neurotoxicity in PC12 cells and rat cortical neurons in culture [8]. βCTF can also lead to endosomal dysfunction in fibroblasts even when Aβ production is reduced by inhibiting γ-secretase [9], induce the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1β) [10] and disrupt calcium homeostasis [11]. Furthermore, when expressed in vivo, βCTF impaired learning and long-term potentiation (LTP) in mice [12]. These studies are just a few examples from a body of evidence clearly showing that there is more to the story than just Aβ. Even when narrowing potential culprits to APP fragments, there is incriminating evidence against βCTF in addition to a host of Aβ aggregates. Targeting APP processing by inhibiting β-secretase cleavage may be a viable strategy to reduce the production of these toxic fragments altogether and inhibit the production of toxic βCTF [13]. Issues facing this approach will be described in section 1.3. 1.2. Genetics of AD As it stands, AD is classified as one of two forms, both with the same hallmark pathological features albeit differing etiologies; a familial autosomal dominant form that accounts for approximately 0.5% of cases [14] or a sporadic form with an unknown etiology accounting for more than 99% of cases. The autosomal dominant form of AD is caused by genetic mutations that affect the processing of APP. These mutations can be found in the gene for APP itself, APP, or in the

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genes for presenilin 1 and 2, PSEN1 and PSEN2, respectively. Both presenilin 1 and 2 are part of the γ-secretase protein complex. In addition, the APP gene is located on chromosome 21 and virtually all individuals with Down’s syndrome (trisomy 21) develop AD by middle age [15]. The evidence is clear that AD can be caused by a mutation in one of these genes, which alters this pathway such that Aβ is overproduced. Moreover, a polymorphism in APP was recently discovered that decreases the chance of developing AD by preferentially processing APP along the non-amyloidogenic pathway and reducing the production of Aβ [16]. These mutations provide solid support for the amyloid cascade hypothesis and furthermore, were used as the basis for the majority of transgenic mouse models of AD. Notably, despite the growing body of data on the role of the APP processing pathway under pathological conditions, the physiological roles of APP and Aβ are unknown. Some studies suggest that at physiological levels, Aβ is actually required for efficient synaptic plasticity and memory [17]. In contrast, the γ-secretase protein complex, which includes presenilins 1 and 2 has well-known physiological substrates, including the notch receptor, which is required for normal development. Despite this solid genetic evidence for the cause of AD with a familial autosomal dominant mutation, the cause of the more prevalent, sporadic form has yet to be elucidated. In addition to these autosomal dominant mutations, the major genetic risk factor for AD is the apolipoprotein E (apoE) genotype. In comparison to APP and Aβ, the contribution of the apoE genotype to AD pathology is under-represented, especially considering that the presence of one apoE ε4 allele confers a 29% risk [18] of developing the sporadic form of AD, which accounts for the vast majority of cases. The ε4 allele is also associated with an increased rate of cognitive decline in patients with AD and cognitive symptoms that emerge 16 years earlier than non-carriers [19, 20]. These associations provide a strong case for the involvement of apoE4 in the pathogenesis of AD. However, it is important to note that half of homozygous ε4 carriers do not develop AD by the age of 90 [21]. Furthermore, although ε4-negative individuals do have a lower associated risk of 9%, [18, 20, 21], they are not protected from the disease. Understanding the precise contribution of apoE genotype to AD pathogenesis would help explain these findings and develop therapeutic strategies.

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There are three isoforms of the apoE protein commonly found in humans, apoE2, apoE3 and apoE4 resulting from the APOE ε2, ε3, and ε4 alleles, respectively. Lipoproteins are involved in transporting and clearing lipids, such as cholesterol and triglycerides [22]. ApoE is expressed primarily by astrocytes but expression can also be induced in neurons under conditions of insult or stress [23, 24]. ApoE is involved in several neurobiological functions that vary with each isoform. For example, apoE isoforms are involved in remodelling neurons by redistributing lipids from degenerating neurons to proliferating neurons as well as for membrane repair and myelin formation for new axons. However, apoE3 increases growth whereas apoE4 decreases growth [24, 25]. The same differential effect of apoE isoforms is present for the intracellular trafficking of glutamate receptors, where apoE4, but not E2 or E3, decreases cell surface levels of glutmatergic transmission, thereby impairing glutamatergic neurotransmission and plasticity [26]. ApoE4 is also more susceptible to proteolytic cleavage than apoE2 or apoE3 [27, 28], which produces neurotoxic C-terminal truncated fragments. These toxic fragments have been found in AD brains at high levels [29] and can lead to learning and memory deficits and the loss of hilar GABAergic interneurons when expressed in transgenic mice [30]. C-terminal truncated apoE4 fragments also clear Aβ inefficiently [31] and lead to tau hyperphosphorylation [32]. It is hypothesized that in response to stress or injury that would occur in AD, apoE is induced to remodel and repair neurons. However, since apoE4 is more susceptible to proteolytic cleavage, its induction leads to the production of neurotoxic fragments that can actually exacerbate AD pathology [24, 25]. Aside from the APOE gene, the search is on for additional genes that may be risk factors for AD. Genome-wide association studies (GWAS) have been used to screen up to a million genetic markers and assess them for correlations with a disease. In the context of AD, a number of independent GWAS studies have confirmed that the genes CD33 (CD33), CLU (clusterin), CR1 (complement receptor 1), BIN1 (bridging integrator 1), PICALM (phosphatidylinositol binding clathrin assembly protein) [33] and TREM2 (triggering receptor expressed on myeloid cells 2) [34, 35] are associated with the sporadic form of AD albeit at to a lower degree than APOE. These genes are primarily associated with the immune system and inflammation or with lipoprotein vesicles. For example, CD33, is part

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of a family of receptors called sialic acid binding immunoglobulin-like lectins (Siglecs) and is involved in regulating monocyte activation [36]. Clusterin is a widely expressed chaperone molecule that is involved in inflammation, lipid transport and inhibition of apoptosis [37]. CR1 is a receptor for the C3b complement protein, which is activated in AD [38]. Bin1 is a nucleocytoplasmic adaptor protein involved in receptor-mediated endocytosis [33] and PICALM is involved in clathrin-mediated endocytosis [39]. Finally, the innate immune receptor, TREM2, is expressed at high levels on myeloid cells surrounding amyloid plaques and is associated with inflammation and phagocytosis [40, 41]. There are many theories on how these genes may contribute to AD pathogenesis. However, GWAS studies have several limitations. For example, the only loci that reach genome-wide significance occur with a frequency greater than 1%, which restricts detection to relatively common polymorphisms [33]. In addition, reproducibility and effect sizes remain low [42]. The advent of whole genome sequencing will be able to detect rare variants that may confer a significant susceptibility to AD. 1.3. Clinical Trials & Treatment Strategies There is no cure for AD and currently available disease-modifying agents offer limited benefit. Three out of the four drugs currently available for the treatment of AD target the cholinergic system, rivastigmine, galantamine and donepezil. The fourth drug currently approved for AD, memantine, was approved for use in 2003 and targets the glutamatergic system by blocking NMDA receptors. However, neither of these drug targets is currently thought to be causally involved in the development of AD and may only be secondary targets. In order to get closer insight into the primary targets of AD, the focus has shifted to the amyloid cascade hypothesis. In recent years a number of clinical trials have been performed targeting amyloid. One strategy to combat Aβ production was to inhibit γ-secretase, the second cleavage step in the processing of APP to liberate Aβ. Initial γ-secretase inhibitors (GSIs) failed in clinical trials due to the worsening of cognitive performance and an increased incidence of skin cancer [43, 44]. Since γ-secretase has many substrates besides APP, including the notch receptor, a second generation of γ-secretase-targeting drugs was developed that spared notch signaling, called γ-secretase modulators (GSMs). Although initial GSMs failed in

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clinical trials, studies are ongoing to test new GSMs [44]. Importantly, GSMs do not inhibit the production of βCTF, which is produced from APP by β-secretase cleavage, and has its own toxic effects as discussed in section 1.1. Targeting βsecretase is another strategy being studied to attenuate production of Aβ. A number of BACE1 inhibitors have already failed in clinical trials due to safety concerns. One compound, MK-8931, has made it to phase 3, which is currently underway [44]. Notably, both GSMs and BACE1 inhibitors target the production of Aβ. However, it has been hypothesized that the sporadic form of AD may be caused by reduced clearance of Aβ [45]. In order to target clearance, antibodies against Aβ are thought to be a possible strategy to treat AD. Both active and passive immunization approaches have been investigated to decrease the levels of Aβ. Active immunization is done by administering an antigen in the form of a vaccine so that the immune system can develop its own antibodies against Aβ. Passive immunization is done by administering antibodies against Aβ that are not derived from the patient’s own immune system [45]. Both techniques have advantages and disadvantages. For example, active immunization leads to a prolonged immune response, however, there may be great inter-patient variability, especially in older patients with a weakened immune system. In contrast, passive immunization would require ongoing treatments, yet the dosing would be controlled [45]. In addition, there are no known active transport mechanisms for antibodies to get into the central nervous system (CNS), however, it is thought that anti-Aβ antibodies in the peripheral circulation can still lower Aβ in the CNS by affecting the equilibrium, a phenomenon known as the ‘peripheral sink hypothesis’. Both immunization techniques have been tested in clinical trials with some studies still ongoing. An early active immunization trial with the compound, AN-1792, had to be terminated due to an increased risk of meningoencephalitis. However, postmortem studies revealed a reduced number of amyloid plaques as well as a decrease in plaque-associated dystrophic neurites in the absence of documented cognitive improvement [46]. This was a surprising finding and supported the notion that amyloid plaques are not directly related to cognitive status. Nevertheless, additional immunizations are currently being tested and a number have moved on to phase 2 and 3 trials.

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Experts speculate that a possible reason for the failure of these trials is that they were started at a stage when the disease was already too far along and the neurodegeneration far too advanced for cognitive function to be rescued. Due to the long latency before symptoms manifest and the extensive neurodegeneration present in AD brains, intervention strategies to prevent the development of the full-blown pathology may stand a better chance at success. Therefore, the focus has been to shift to studying earlier stages of the disease to intervene as early as possible. In order to do so, biomarkers must be ascertained so that individuals at risk can be identified. Several biomarkers have been proposed with help from the Dominantly Inherited Alzheimer Network (DIAN), a registry of individuals at risk of developing AD due to autosomal dominant mutations. Researchers can estimate the expected age of onset based on the age at which the parents began experiencing symptoms. In addition, researchers can study these individuals longitudinally to document changes in various parameters related to AD as they occur. Alarmingly, changes in CSF Aβ levels have been detected as early as 25 years before the expected age of onset [47]. Various other biomarkers are also being studied for their potential to identify people at risk of AD. For example the GWAS studies (section 1.2) have pointed to a number of genes that may serve as potential biomarkers. Using biomarkers we may be able to intervene before extensive neurodegeneration occurs. Indeed, the DIAN-Trials Unit currently has a trial underway to test solanezumab and gantenerumab, humanized monoclonal Aβ antibodies, in individuals from the DIAN cohort at an early, presymptomatic stage with the aim to attenuate Aβ production before the damage is done to ward off the disease [48, 49]. Whether it is to attenuate amyloid production or intervene on another aspect of the disease or both, early intervention and prevention are promising strategies to treat AD. 2. PRECLINICAL AVENUES AD develops decades before clinical symptoms arise and can remain undiagnosed for years, eventually leading to complete loss of cognitive abilities and widespread neurodegeneration. For this reason, there has been a shift of focus in the field to study the earliest stages of the disease and identify additional culprits to intervene and halt the progression of AD before the development of overt pathology. In the following section we will provide an overview of various facets

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of AD that, importantly, are altered at early stages of the disease. Although there are dozens of plausible suspects that may be contributing to AD, we will focus on those that have garnered widespread attention and are the most promising targets for clinical applications. Notably, these potential targets are not mutually exclusive, competing effectors, but rather, are connected and dependent on one another via common signaling pathways. 2.1. Neuroinflammation & Tumor Necrosis Factor-α Neuroinflammation is often associated with a heavy plaque burden and accumulation of neurofibrillary tangles, however, emerging evidence suggests that neuroinflammatory processes may also be involved in the prodromal, pre-plaque stages of AD [50], thereby providing a possible upstream mediator in the pathogenesis of the disease. As mentioned in section 1.2, various GWAS studies have implicated a number of genes related to the immune system in the sporadic form of AD, including TREM2, CR1, CD33 and CLU [51]. Moreover, CLU, is an acute phase protein and therefore is a marker of an increased inflammatory response [52]. Finally, the APOE gene is associated with the immune response and apoE4 has been linked to a greater inflammatory response [53]. Although these genetic findings indicate that neuroinflammation clearly plays a role in AD, whether it is a cause or a consequence of the disease is still unknown. Curbing the inflammatory response to treat AD is not a new idea. Initial studies investigating this approach revealed a lower prevalence of AD among rheumatoid arthritis patients taking non-steroidal anti-inflammatory drugs (NSAIDs) [54]. Subsequent epidemiological studies have supported the association between longterm NSAID use and decreased risk of AD [55]. Additional findings in mouse models have shown that treatment with the NSAID, ibuprofen, led to both reduced amyloid pathology and activated microglia [56, 57]. However, other studies have shown conflicting results [58]. To further complicate the story, extended results from the Alzheimer’s disease anti-inflammatory prevention trial (ADAPT) suggest that NSAIDs may be used to reduce the risk of AD in asymptomatic individuals as NSAID treatment actually had an adverse effect in patients with advanced AD [59]. In addition, the preclinical phase can be further divided based on the rate of cognitive decline [60]. Within these phases, the NSAID, naproxen,

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attenuated cognitive decline in slow decliners, however worsened cognitive decline in fast decliners [61]. These findings are consistent with the notion that neuroinflammation can be a “double-edged sword” [62] and may be deleterious in early stages of AD, yet beneficial at later stages. Thus, targeting the immune system via NSAID treatment or manipulating distinct aspects of the system with specific pro-inflammatory inhibitors warrants further attention. Activation of the immune system involves a complex cascade of cytokines, chemokines, complement factors and acute phase proteins that has been likened to a web, whereby one set of these mediators can likely induce activation of many others [63]. Activation cascades in the innate immune system are thought to actively contribute to AD pathology [64]. For instance, activation of the NLRP3 inflammasome controls caspase-1 activation, which in turn cleaves and activates IL-1β from its pro-form. Caspase-1 is also activated in MCI brains [65], which further points to an upstream role of inflammasomes. In APP/PS1/NLRP3 -/- mice this cascade is attenuated, such that caspase-1 is not activated and IL-1β levels are similar to controls, leading to enhanced Aβ clearance and protecting spatial memory [65]. Moreover, immune mediators are not limited to classical immune functions and there is significant crossover between systems. For example, the mitochondrial translocator protein (TSPO) was initially characterized for its role in steroid synthesis by translocating cholesterol from the outer to the inner mitochondrial membrane [66]. However, recently TSPO has been used as a marker of reactive gliosis and brain inflammation in AD patients [67]. In fact, TSPO may even be more than a marker of inflammation since intervention and treatment with TSPO ligands demonstrated reduced neuropathology and behavioral impairments in a mouse model of AD [68]. This positive effect may be due to the pleiotropic functions attributed to TSPO including, nerve regeneration, steroidogenesis and reducing oxidative damage [69] and is a testament to the crossover between systems. In addition to crossover with other systems, many inflammatory mediators, such as cytokines and chemokines, are thought to have overlapping functions. Importantly, many inflammatory mediators have been detected at early stages of amyloid pathology. For example, in APP V717I mice IL-1β, interleukin-6 and major histocompatibility complex-ΙΙ (MHC-II) mRNA levels were significantly

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increased at the early pre-plaque stage [70]. Other markers of microglial activation such as MHC-II, inducible nitric oxide synthase (iNOS) and cluster of differentiation 40 (CD40) are increased in the pre-plaque stage of the McGillThy1-APP model, which suggests that microglia may be the source of the immune mediators [71]. Interferon-γ mRNA expression is elevated as early as 3 months in the Tg2576 model [72]. In addition, TNFα was also shown to be increased along with monocyte chemoattractant protein (MCP-1) in the entorhinal cortex of 3xTgAD mice before plaque deposition [73]. These findings indicate that the inflammatory response is well underway even at the pre-plaque stage. Nonetheless, the pro-inflammatory cytokine, TNFα has been referred to as the “master cytokine” that initiates the inflammatory process [52]. However, this distinction may be related to its popularity in research studies compared to other inflammatory mediators. This popularity of TNFα is further reinforced by that fact that TNFα inhibitors, such as infliximab and etanercept, were among the top ten selling drugs in 2013. For these reasons it is important to remember that additional inflammatory mediators may play equally important or overlapping roles. Despite this possibility, it remains that TNFα is a relatively well-characterized and promising therapeutic target for the treatment of AD. Genomic studies have revealed a significant association between TNFα polymorphisms and AD [74] and TNF signaling has been associated with conversion to dementia in patients with mild cognitive impairment (MCI) [75]. Furthermore, TNFα expression is increased at the prodromal stages of AD-like pathology in mouse models of the disease [71, 76] and correlates with the expression of βCTF, which has its own toxic properties, as described above [76]. This increase in TNFα at early stages supports a possible causal role in the pathogenesis of AD (Fig. 1). TNFα comes in two forms, a soluble form (solTNFα) and also a transmembrane form (tmTNFα). Both forms bind TNF receptor 1 (TNFR1), although solTNFα does so preferentially. TNFR1 is expressed in the majority of cell types, including neurons and mediates signaling that is often pathogenic and leads to cell death [77]. TNF receptor 2 (TNFR2) binds with greatest affinity to tmTNFα and is expressed principally by microglia and endothelial cells. Signaling through TNFR2 is involved in the innate immune system and promotes cell survival [78].

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Drugs that target TNFR1 signaling specifically may have fewer adverse side effects, such as congestive heart failure, de-myelination and risk of infection, since the immunomodulatory role of TNFR2 would be spared [79]. Indeed, specific inhibition of solTNFα has been studied in a 3xTgAD mouse model of AD challenged with lipopolysaccharide. In this paradigm, long-term treatment with a dominant negative solTNFα inhibitor prevented the accumulation of βCTF and Aβ [80]. Additional studies using mouse models of AD have also reported the positive effects of chronic interruption of TNFα signaling on behavior, markers of inflammation and amyloid pathology [80 - 83]. Moreover, cognitive improvement was witnessed upon perispinal injection of a TNFα inhibitor in a human case of AD [84]. These preclinical studies provide support to move on to testing TNFα inhibitors in the clinical setting. The positive effects of TNFα inhibition in AD may be attributable to the interruption of its effect on APP processing [85 - 87]. In APP expressing astrocytes and cortical neurons, TNFα stimulation can increase BACE1 expression as well as Aβ production in a dose-dependent manner, an effect that was blocked by a TNFα-neutralizing antibody [85]. In addition, TNFα signaling through TNFR1 can activate NF-κB, which has binding sites near the BACE1 promoter [88, 89]. Treating cells with an NF-κB inhibitor significantly decreased TNFα-induced BACE1 promoter activity, indicating that TNFR1-mediated activation of NF-κB is responsible for the increase in BACE1 expression and APP processing [87]. Thus, TNFα inhibitors may improve AD pathology by reducing APP processing [90]. In addition to the effect of TNFα on APP processing, this cytokine can also affect cerebral insulin resistance. Insulin resistance is characterized by chronic high levels of insulin in the periphery, a decreased ability of insulin to mediate glucose uptake, and importantly, the decreased transport of insulin to the brain [91]. Insulin in the brain is known to modulate neurotransmitters, membrane potentials, and synaptic plasticity [91]. Furthermore, intranasal insulin administration has also been tested in the clinical setting and improved cognition in patients with MCI and early-stage AD [92]. With respect to inflammation, it is clear that insulin resistance and inflammatory mediators are dependent on one another [52]. Peripherally induced hyperinsulinemia can increase cytokines, including TNFα, in

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the cerebrospinal fluid [93]. In turn, pro-inflammatory cytokines can potentiate insulin resistance [94]. Accordingly, the TNFα inhibitor, infliximab, improved insulin signaling in obese diabetic mice [95]. Similarly, mice with a targeted null mutation in the gene for TNFα were protected from obesity-induced insulin resistance [96]. In a complementary approach, intranasal insulin administration reduced microglial activation and Aβ levels in the 3xTgAD mouse model of AD [97]. These studies highlight the interconnected nature of the signaling pathways involved and how inhibiting TNFα signaling may be beneficial because it attenuates cerebral insulin resistance. Alternatively, as discussed in section 3, the therapeutic effect of inhibiting TNFα signaling may be related to the direct impact of this cytokine on synaptic function. 2.2. Network Hyperexcitability Synaptic loss is the best correlate of cognitive decline in AD [98]. Alterations at the synapse, and specifically at dendritic spines, are thought to be among the first pathological events in early AD. Since proper synaptic function occurs within a range of activity that is neither too high nor too low, maintaining synaptic function within this optimal range allows for stable neuronal and network function, which underlies healthy cognitive processing. Network dysfunctions are often reported in AD patients and hyperactivity is a common, yet paradoxical feature of the prodromal stage [99]. Increased activity at the early stages of AD has been studied in various different contexts, which will be described further below, including using functional magnetic resonance imaging (fMRI) to measure blood flow as well as studying seizure susceptibility and the molecular mechanisms that may contribute to hyperexcitability. Although these phenotypes may arise via entirely different mechanisms, these phenomena represent a similar shift towards increased activation. A number of fMRI studies have demonstrated increases in blood-oxygen leveldependent signals in multiple brain regions of patients with MCI [99]. Hyperactivation in the hippocampus was associated with greater cortical thinning in patients with MCI and even in cognitively normal elderly individuals [100]. The associated cortical thinning was present in regions susceptible to AD pathology that are functionally connected to the hippocampus, such as the medial

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temporal lobe [100]. Furthermore, hyperactivation may not be limited to the hippocampus since amnestic MCI (aMCI) patients displayed higher activation in parietal and temporal lobes during the maintenance phase of a verbal working memory task [101]. Importantly, studies at different stages of disease, even within the MCI stage, can yield opposite results on activation [99, 102]. Nonetheless, increased fMRI activation during a cognitive task is thought to be related to neural compensation since hyperactivation was associated with recruitment of additional brain regions and more detailed memories in Aβ-positive, cognitively normal older individuals [103]. In addition to hyperactivation in fMRI studies, evidence of increased activity has also been documented in the context of intrinsic network activity. There is a clear association between seizures and AD such that seizure prevalence is increased in sporadic as well as in familial AD cases [104]. Moreover, the APOE ε4 allele is associated with epileptiform activity [104]. Alterations in network activity due to increased seizures are also apparent in various mouse models of AD. For example, pre-plaque TgCRND8 mice display an increased susceptibility to pentylenetetrazole-induced seizures in comparison to control mice [105]. Accordingly, APdE9 mice have an increased incidence of unprovoked seizures at the onset of Aβ deposition [106]. Indeed, Aβ production is thought to be dependent on neuronal activity [107] and inducing neuronal activity with glycine lead to the convergence of APP and BACE1 to the same endocytic vesicles, which increases APP processing and amyloidogenesis [108]. Moreover, the level of lactate, a marker of neuronal activity, in the interstitial fluid of Tg2576 mice was predictive of Aβ levels in the interstitial fluid for a given brain region [109]. Further evidence suggests that the accumulation of soluble Aβ leads to spontaneous, non-convulsive seizure activity, compensatory GABAergic remodeling and deficits in synaptic plasticity [110]. Recent evidence from the hAPP-J20 mouse model of AD as well as in AD patients suggests that this hyperactivity may be due to decreased GABAergic transmission resulting from decreased expression of the voltage-gated sodium channel subunit Nav1.1 [111] or GABAergic loss [112 - 115]. Increasing inhibition of neuronal networks by rescuing Nav1.1 expression using a bacterial artificial chromosome reduced memory deficits and premature mortality in this mouse model of AD [111].

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Similarly, inhibiting hippocampal hyperactivity with the anti-epileptic drug, levetiracetam, reduced cognitive impairments in patients with aMCI [116] and also reversed synaptic and cognitive deficits in a mouse model of the disease [117]. As mentioned, the early paradoxical increase in activity, arising from either a task-dependent increase in fMRI activation or via an increase in the intrinsic excitability of neurons, may be an effort to compensate for early stage AD-related pathology, such as amyloid overproduction or inflammation. However, as the pathology progresses, the nervous system becomes overwhelmed and cannot compensate further and suffers from low activity and degeneration. As AD pathology progresses, low synaptic function and hypoactivity prevail, which are more characteristic of the disease. This dichotomy highlights the idea that treatments for AD should be stage-specific. What is beneficial at one stage may not be recommended at another. At early stages, rescuing hyperexcitability and restoring stable network function may be a valid treatment strategy for AD patients. Since brain networks are thought to require highly coordinated, synchronous activity of multiple neuronal assemblies for higher order cognitive processing, alterations in excitatory and inhibitory activity may lead to the cognitive deficits, characteristic of AD. Indeed, using in vivo two-photon calcium imaging in APP23xPS145 mice, a redistribution of neuronal activity in the frontal cortex was observed that showed hyperactive neurons in proximity to amyloid plaques that were related to a decrease in synaptic inhibition [118]. A similar phenomenon was also documented in the hippocampus before plaque deposition [119]. The rhythmic activity of neuronal populations is produced by local field potentials oscillating at different frequencies. In the hippocampus, oscillations in the theta [120] and gamma [121] ranges are thought to play an important role in memory consolidation and executive functioning. Accumulating evidence from electroencephalogram (EEG) studies suggests that cortical theta [122 - 124] and gamma [125, 126] activity are altered in AD patients. In addition, the crossfrequency coupling (CFC) between theta phase and gamma amplitude in both hippocampal and cortical areas is also thought to be an important component of learning and memory [127]. Furthermore, the strength of CFC correlates with

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performance in learning tasks [127]. Using a complete hippocampal preparation [128], CFC was shown to be altered in a prodromal mouse model of AD, such that CFC was specifically impaired in the high-gamma range, before the occurrence of cognitive deficits and concurrent with increased APP and βCTF levels [129]. This finding is consistent with in vivo studies showing that the shift in theta phase at the highest gamma power during REM sleep was attenuated in APP/PS1 mice [130]. These alterations in CFC may be among the earliest pathological deviations that contribute to cognitive impairment in AD and may provide a novel target for AD detection and prevention. Although, EEG recordings have the potential to detect alterations in the synchronization of neuronal network activities [131], such as CFC, these recordings would only be able to detect changes in cortical oscillation networks since intrinsic hippocampal oscillatory activity is not readily detectable via scalp EEG. Therefore, alterations in hippocampal EEG activity detected in mouse models of AD are not directly translatable to cortical EEG activity in humans. Detecting changes in CFC in the hippocampus is challenging since the hippocampus is a relatively deep brain structure. One option would be to transpose the search for diagnostic markers from the brain to the retina, which is a part of the central nervous system and is amenable to non-invasive study [132]. Furthermore, specific network signatures in the retina can easily be compared across rodents and humans. Visual disturbances represent some of the earliest complaints of AD patients [133, 134]. Moreover, Aβ plaques are detectable in the retina [135] as well as functional changes in electroretinogram recordings [136] and visual evoked potentials [137] at later stages. Importantly, changes in the retina are also detectable at early stages. For example, imaging the retinal nerve fiber layer (RNFL) using optical coherence tomography showed that RNFL thickness was significantly decreased in patients with MCI compared to controls [138]. These findings support the possibility of using the retina as a surrogate for early pathological changes occurring in the brain that may be harder to detect. Moreover, prodromal changes in neuronal excitability may also be detectable in the retina, which could serve as a biomarker for early intervention [132]. This is particularly relevant in the light of recent data using hyperspectral analysis, which demonstrated the presence of soluble Aβ aggregates in human retinal tissue during

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the preclinical stage as well as in the retina of a mouse model of AD up to 2 months earlier than in the hippocampus [139]. 3. CROSS-TALK BETWEEN SYNAPTIC HYPEREXCITABILITY & TNF A growing body of evidence supports a role for cytokines and other inflammatory mediators in neuronal activities, including learning, memory and neural plasticity [140]. Although TNFα is a well-known immune mediator, increasing data indicates that TNFα is an important regulator of synaptic function as well. Since synaptic dysfunction is ultimately responsible for cognitive impairments in AD, the effect TNFα has on synaptic integrity is crucial to understanding disease pathogenesis. The roles of inflammatory mediators can shift from regulatory or neuroprotective under physiological conditions to neurotoxic under pathological conditions. Physiologically, TNFα can act on neuronal TNFR1 to increase the surface expression of glutamate receptor 2 (GluA2) subunit-lacking, calcium-permeable, AMPA receptors and decrease the surface expression of GABAA receptors [141, 142]. The effect of TNFα on ionic homeostasis and on the balance between excitatory and inhibitory synaptic inputs can be detrimental to neurons as chronic up-regulation of extrasynaptic GluA2-lacking AMPA receptors can potentiate glutamate-induced excitotoxicity [143]. Conversely, TNFα can prevent kainatemediated excitotoxicity in motor neurons under physiological conditions, by increasing the expression of GluA2-containing, calcium-impermeable AMPA receptors, [144], suggesting that the effect of TNFα may be region-specific. In addition to its effects on receptor trafficking, TNFα can decrease glutamate uptake by astrocytes [145]. These effects on neuronal excitability may contribute to an increased susceptibility to seizures; a symptom that has been associated with altered TNFα expression and activation of TNFR1 [146]. As discussed in section 2.2, aberrant excitatory neuronal activity has been postulated as one of the primary upstream mechanisms contributing to cognitive impairments in animal models of AD [104]. Several lines of epidemiological data also underline the association between epileptic seizure activity and AD in humans [104]. Since TNFα has been linked to increased excitatory activity, pathological TNFα

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signaling may provide a mechanistic link to the synaptic hyperexcitability observed in AD.

Fig. (1). Hypothetical cascade induced by neuroimmune activity in early AD. The increased accumulation of Aβ induces a classical immune response, glial activation and increased production of TNFα, which creates a positive feedback loop (red arrows) leading to increased neuroinflammation. TNFα can also potentiate this loop through its effects on APP processing and lead to increased levels of βCTF and Aβ. At pathological levels these toxic APP cleavage products interfere with synaptic transmission (green arrows), which initiates a homeostatic glial response mediated by TNFα to scale the synaptic activity by increasing the surface expression of AMPA receptors. These collateral synaptic effects of TNFα can lead to hyperexcitability and affect synaptic plasticity, if the cascade is not stopped.

In keeping with the theme of this chapter, whereby one alteration does not occur without collateral effects, it is important to recognize the impact that synaptic hyperexcitability can have on activity-dependent plasticity. Plastic processes that are associated with learning and memory, such as LTP or long-term depression (LTD), can only occur in the presence of a functional baseline set point. Several homeostatic mechanisms have emerged that actively maintain these functional neural set points, including intrinsic excitability, receptor trafficking and presynaptic neurotransmitter release [147]. Aberrant synaptic excitability outside the functional range is not as amenable to plastic changes and may therefore contribute to learning and memory defects in AD. Notably, TNFα is involved in the homeostatic response to prolonged inactivity, through its role in AMPA and GABA receptor trafficking, which occurs under physiological conditions as

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described above and helps maintain the functional neural set point [142, 148]. Consequently, under pathological conditions when TNFα is chronically expressed, this role of TNFα in the trafficking of receptors suggests that it may also affect synaptic plasticity. In fact, treatment of rat hippocampal slices with TNFα increases basal synaptic transmission, but inhibits LTP in a dose-dependent manner (1-100 nM) [149]. Other studies suggest that Aβ may inhibit LTP via TNFα. Wang et al. (2005) found that Aβ-induced LTP inhibition could be blocked via the interruption of TNFα signaling through the use of TNFα receptor antagonists or use of TNFR1 null mice, as well as through the blockage of TNFα biosynthesis [150]. TNFα antagonists were also able to prevent Aβ-mediated inhibition of LTP in vivo, an effect that was mirrored by antagonists selective for GluN2B-containing NMDA receptors [151]. Impairments in synaptic plasticity, and specifically in LTP, may underlie the cognitive deficits that are so typical of AD and pathological TNFα signaling may therefore contribute to such impairments (Fig. 1). In addition to its effects on receptor trafficking and LTP, TNFα can also affect calcium homeostasis through multiple mechanisms [152]. Considering that calcium is arguably the most important signaling messenger in a cell, this effect is rather significant. Increased intracellular calcium has been associated with increased Aβ accumulation, tau hyperphosphorylation and neuronal death [153], not to mention disrupted synaptic function, which requires acute calcium regulation. TNFα can potentiate neuronal intracellular calcium levels by increasing expression of the type-1 inositol 1,4,5-trisphosphate receptor, which regulates calcium release from the endoplasmic reticulum [154]. Moreover, TNFα can increase calcium currents through L-type voltage-sensitive calcium channels (L-VSCC) [155], an increase that was rescued in aged rates by blocking TNFα signaling [156]. As previously described, TNFα can also increase the surface expression of GluA2-lacking, calcium-permeable AMPA receptors, which can lead to increased influx of calcium across the plasma membrane [142]. By increasing intracellular calcium, TNFα can negatively impact synaptic function and potentially precipitate network dysfunctions documented in AD, including hyperexcitability.

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CONCLUSION The studies described in this chapter review promising therapeutic avenues for the prevention and treatment of AD. A common theme throughout this chapter is the inherent crossover among pathways. It is hard to target one pathway without affecting another, which can be seen as a benefit as well as a hindrance. In the case of GSIs, the crossover with the notch signaling pathway led to negative outcomes in clinical trials. However, in the case of TNFα, the soluble form can be targeted specifically to spare the immunomodulatory roles of tmTNFα and hopefully produce fewer negative side effects. The second key theme of the chapter is timing. AD develops over decades so it is not surprising that disease stage is an important consideration when it comes to treatment. Findings from clinical trials also support this notion, such that NSAID treatment was preventative at early presymptomatic stages of AD, yet actually worsened disease condition when given at later stages. A corollary to these results is that intervening early to prevent the full-blown pathology would be ideal. It is often hypothesized that the reason for the failure of many amyloid-directed therapeutics, such as antibodies and BACE1 inhibitors, is that treatment was given too late. In order to intervene at an early time point, years before symptoms arise, it is necessary to establish biomarkers that can be used to identify individuals with a high risk of developing AD. Possible biomarkers include subtle changes in neuronal networks, which may be detected in the retina better than in traditional EEG recordings. Due to the complexity of this disease, a multi-targeted and disease stage-specific approach will ultimately be necessary for treatment. We propose that targeting inflammation and synaptic dysfunction in addition to amyloid is a promising strategy and will target the main contributors to AD. CONFLICT OF INTEREST The authors confirm that they have no conflict of interest to declare for this publication. ACKNOWLEDGEMENTS We would like to thank Profs Tak Pan Wong and John Breitner, all the members of their labs, as well as the members of the previous Quirion lab for exciting

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scientific discussions on many of the topics covered in the Chapter. Our profound gratitude goes to Prof Rémi Quirion for his constant support, enlightened criticism, constructive suggestions and unconditional encouragement. We are greatly indebted for all these years of science we had a chance to share with him. FUNDING CC is a FRQS fellow. SK was a CIHR scholar as a Visiting Scientist at McGill University. REFERENCES [1]

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glutamate receptor 2-lacking AMPA receptors to extrasynaptic plasma membrane potentiates excitotoxicity. J Neurosci 2008; 28(9): 2119-30. [http://dx.doi.org/10.1523/JNEUROSCI.5159-07.2008] [PMID: 18305246] [144] Rainey-Smith SR, Andersson DA, Williams RJ, Rattray M. Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration. J Neurochem 2010; 113(3): 692-703. [http://dx.doi.org/10.1111/j.1471-4159.2010.06634.x] [PMID: 20132465] [145] Santello M, Bezzi P, Volterra A. TNFα controls glutamatergic gliotransmission in the hippocampal dentate gyrus. Neuron 2011; 69(5): 988-1001. [http://dx.doi.org/10.1016/j.neuron.2011.02.003] [PMID: 21382557] [146] Li G, Bauer S, Nowak M, et al. Cytokines and epilepsy. Seizure 2011; 20(3): 249-56. [http://dx.doi.org/10.1016/j.seizure.2010.12.005] [PMID: 21216630] [147] Davis GW. Homeostatic signaling and the stabilization of neural function. Neuron 2013; 80(3): 71828. [http://dx.doi.org/10.1016/j.neuron.2013.09.044] [PMID: 24183022] [148] Pribiag H, Stellwagen D. Neuroimmune regulation of homeostatic synaptic plasticity. Neuropharmacology 2014; 78: 13-22. [http://dx.doi.org/10.1016/j.neuropharm.2013.06.008] [PMID: 23774138] [149] Tancredi V, D’Arcangelo G, Grassi F, et al. Tumor necrosis factor alters synaptic transmission in rat hippocampal slices. Neurosci Lett 1992; 146(2): 176-8. [http://dx.doi.org/10.1016/0304-3940(92)90071-E] [PMID: 1337194] [150] Wang Q, Wu J, Rowan MJ, Anwyl R. Beta-amyloid inhibition of long-term potentiation is mediated via tumor necrosis factor. Eur J Neurosci 2005; 22(11): 2827-32. [http://dx.doi.org/10.1111/j.1460-9568.2005.04457.x] [PMID: 16324117] [151] Hu NW, Klyubin I, Anwyl R, Rowan MJ. GluN2B subunit-containing NMDA receptor antagonists prevent Abeta-mediated synaptic plasticity disruption in vivo. Proc Natl Acad Sci USA 2009; 106(48): 20504-9. [http://dx.doi.org/10.1073/pnas.0908083106] [PMID: 19918059] [152] Sama DM, Norris CM. Calcium dysregulation and neuroinflammation: discrete and integrated mechanisms for age-related synaptic dysfunction. Ageing Res Rev 2013; 12(4): 982-95. [http://dx.doi.org/10.1016/j.arr.2013.05.008] [PMID: 23751484] [153] LaFerla FM. Calcium dyshomeostasis and intracellular signalling in Alzheimer’s disease. Nat Rev Neurosci 2002; 3(11): 862-72. [http://dx.doi.org/10.1038/nrn960] [PMID: 12415294] [154] Park KM, Yule DI, Bowers WJ. Tumor necrosis factor-alpha potentiates intraneuronal Ca2+ signaling via regulation of the inositol 1,4,5-trisphosphate receptor. J Biol Chem 2008; 283(48): 33069-79. [http://dx.doi.org/10.1074/jbc.M802209200] [PMID: 18838384] [155] Furukawa K, Mattson MP. The transcription factor NF-kappaB mediates increases in calcium currents and decreases in NMDA- and AMPA/kainate-induced currents induced by tumor necrosis factor-alpha

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Treatment of Diabetic Neuropathy – Current Possibilities and Perspectives Jarmila Vojtková*, Miriam Čiljaková, Peter Bánovčin Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital Martin, Department of Pediatrics, Martin, Slovakia Abstract: Diabetic neuropathy (DN), characterized by nerve damage associated with diabetes mellitus, belongs among the earliest and most frequent chronic diabetic complications. It may occur in clinical form (as peripheral sensory/motor, autonomic, proximal, painful or focal) or in subclinical form detectable just by sensitive diagnostic methods. The etiology of DN is complex and not fully understood, untill now. Longterm hyperglycemia triggers a variety of interacting pathways such as production of advanced glycation end products (AGEs), products of oxidative stress and polyol pathway, protein kinase C activation, decrease activity of Na+K+ATP-ase, changed concentration of neural growth factor and production of proinflammatory cytokines. These pathomechanisms may target directly on nerve cells or on endothelial cells causing the microangiopathy of vasa nervorum. According to multicentric studies, duration and poor compensation of diabetes are the principal risk factors associated with the development of chronic diabetic complications, so the basis for the management is to maintain adequate metabolic compensation. Intensified insulin regimen is the most effective in the treatment of patients with type 1 diabetes. In patients with type 2 diabetes, administration of selected peroral antidiabetics or insulin therapy is considered. Physical activity, lifestyle and dietary management also contribute to euglycemia. Currently used management of DN includes supportive (alpha-lipoic acid, vitamins, antioxidants) and symptomatic treatment (painkillers, beta blockers, magnetotherapy). Other therapeutic possibilities are experimental so far. These drugs interfere with the pathophysiological processes and few of them have been shown to be beneficial in clinical studies Corresponding author Jarmila Vojtková: Department of Pediatrics, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital, Kollarova 2, 036 01 Martin, Slovakia; Tel: +421 43 4203 254; Fax: +421 4222 678; Email: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2016 Bentham Science Publishers

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(inhibitors of aldose reductase, selective inhibitor of protein kinase C beta, C peptide substitution), however, the effect of other medicines seems to be controversial (vascular endothelial growth factor, erythropoietin). This chapter brings comprehensive review about current possibilities and future perspectives in the management of diabetic neuropathy.

Keywords: Actovegin, Aldose-reductase inhibitors, Alpha-lipoic acid, Angiotensin converting enzyme inhibitors, Anti-inflammatory drugs, Antioxidants, Chronic complications, C-peptide, Diabetes compensation, Diabetes mellitus, Diabetic neuropathy, Electrical nerve stimulation, Epigenetic modifications, Erythropoietin, Experimental studies, Growth factors, Kinin B1 receptor, Management, Magnetic field therapy, Neurotrophic factors, Pain relief, Ruboxistaurin, Spinal cord stimulation, Vascular endothelial growth factor, Vitamins. INTRODUCTION Diabetes mellitus represents a chronic metabolic disease of heterogeneous etiology with hyperglycemia as the main symptom caused by insufficient action of insulin because of its absolute or relative deficiency and is accompanied by the disorder of metabolism of sugars, lipids, proteins, water and minerals. Considering dramatically the increasing incidence of diabetes worldwide, it is becoming a pandemic with a huge impact on the individual and on the whole society, as well. Diabetes can negatively influence the quality of life, especially by the presence of chronic diabetic complications. Among the earliest diabetic complications belongs diabetic neuropathy, characterized as a disorder of the peripheral nervous system associated with diabetes mellitus. According to the type of affected nerves, it can be divided into sensory, motor, autonomic or mixed neuropathy. Concerning the still earlier onset of diabetes, diabetic neuropathy can be found also in childhood and adolescence, even just few years after type 1 diabetes onset. In its complex etiopathogenesis, the main risk factor is long-lasting hyperglycemia, the impact of which is enhanced by poor compensation and longer duration of diabetes, together with genetic predisposition and probably also immunologic, epigenetic or other factors. Hyperglycemia may trigger a variety of processes, such as non-enzymatic glycation, oxidative stress, polyol pathway,

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activation of protein kinase C, proinflammatory cytokines, changed concentration of neural growth factor or decrease activity of Na+K+ATP-ase. These processes may either directly alter the structure and function of neurocytes or may indirectly damage the blood supply of neurons through disorder of endothelial cells. The management of diabetic neuropathy is complex. The first step involves the maintenance of adequate diabetes compensation – to control glycemia, lipid profile and blood pressure. In addition to insulin therapy (or per oral antidiabetic drugs), lifestyle modification, dietary management and adequate physical activity significantly contribute to euglycemia. Current treatment of diabetic neuropathy includes alpha-lipoic acid and vitamins (C, E and group B). Symptomatic treatment is aimed to relieve pain and to relieve possible symptoms of autonomic neuropathy (such as tachycardia, hypotension, gastroparesis and bladder atonia). Other possibilities are mostly experimental, and interfere with the mentioned pathogenic pathways. Results of some clinical studies are very promising and may represent possible options in complex management of diabetic neuropathy. DIABETES MELLITUS Diabetes mellitus represents a chronic metabolic disease of heterogeneous etiology with hyperglycemia as the main symptom caused by insufficient action of insulin because of its absolute or relative deficiency. Diabetes is accompanied by disorders of metabolism of sugars, lipids, proteins, water and minerals. According to American Diabetes Association [1] the classification of diabetes mellitus is: I. Type 1 diabetes mellitus (T1D) II. Type 2 diabetes mellitus (T2D) III. Specific types of diabetes Genetic defects of beta cells, Genetic defects of insulin action, Diseases of exocrine pancreas, Endocrinopathies, Diabetes induced by drugs and chemicals, Infections, Uncommon forms of immune-mediated diabetes, Other genetic syndromes sometimes associated with diabetes IV. Gestational diabetes mellitus

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Type 1 diabetes (T1D) affects less than 10 % of subjects with diabetes in the whole population; however, it represents the most common form of diabetes in childhood. Main characteristic of T1D is unclear quickly ongoing inflammation of beta cells of pancreas leading to absolute lack of insulin that determines the need of its substitution. Based on the etiopathogenesis, there are two types of T1D – type 1a diabetes and type 1b diabetes. In the majority of cases, type 1a diabetes is found, which is characterized by an autoimmune inflammation of beta cells with the destruction by T lymphocytes and auto-antibodies produced by B lymphocytes. Type 1b diabetes, found in minority of cases, is characterized by idiopathic inflammation without autoimmunity, but with tendency to ketoacidosis and absolute lack of insulin. Important risk factors for T1D are genetic predisposition of an individual (HLA alleles DQ2, DQ8, DR3, DR4), other endogenous factors (immune system, hormonal status) [2] and exogenous environmental factors (viral infection, radiation, nutrition, chemicals, drugs, stress). T1D has typical phases: preclinical phase, phase of manifestation of diabetes, partial remission (“honeymoon period”) and chronic phase with lifetime necessity of exogenous insulin. Preclinical phase precedes the manifestation of T1D for several months or years. In this period of autoimmune inflammation several antibodies can be demonstrated (GADA – glutamic acid decarboxylase antibodies, IA-2 – antibodies against tyrosine phosphatase, ICA – islet cell antibodies, IAA – insulin autoantibodies and ZnT8A – zinc transporter 8 autoantibodies) that are positive at the time of T1D diagnosis in 85 – 90% patients. Nowadays, no possible intervention exists to prevent or delay the manifestation of T1D. Clinical symptoms appear at destruction of approximately 90% pancreatic beta cells. In some children the manifestation is gradual; in constrast, in other children the development of symptoms is rapid with diabetic ketoacidosis. Non-urgent symptoms of T1D include polyuria, polydipsia, blurred vision, loss of weight, despite increased appetite or stagnation of weight in growing children, abdominal pain, frequent urination – also during night or nocturnal enuresis, vaginal candidiasis, emesis, recurrent skin, urinary or other infections, higher excitability and deterioration of results in school. Urgent symptoms of diabetic ketoacidosis include dehydration, recurrent vomiting, acetone odor of breath, hyperventilation (Kussmaul breathing), disorientation, unconsciousness or shock [3]. In laboratory findings, the diagnosis is confirmed

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by hyperglycemia, increased concentration of glycated hemoglobin (A1c) (it may not be present in the case of rapid onset of diabetes), metabolic disruption of water, electrolyte and acid-base balance and low even missing concentration of Cpeptide (except honeymoon period in T1D). Diabetic ketoacidosis may be the first manifested sign in about 30 % children with T1D. Its incidence is higher in patients younger than five years and in children with poor social economic status. In newly diagnosed T1D the incidence of ketoacidosis is higher in countries with lower incidence of diabetes [4]. T1D can manifest at any age. However, more than half of patients with T1D are diagnosed at the age of less than 15 years. Recently, significant increase in the incidence of T1D has been noted in children and youth [5]. Manifestation of T1D in adulthood is referred as LADA (latent autoimmune diabetes in adults). Incidence of T1D is very variable between various countries and ethnic groups (0.1 – 37.4/100 000 people) [6]. Approximately in 80 % children after initiation of therapy by exogenous insulin, the need of insulin transiently decreases. Post-initial remission is defined as the need of exogenous insulin less than 0.5 IU/kg/day while maintenance of A1c less than 7 % (DCCT norm). Ketoacidosis and younger age at diagnosis of T1D decrease the probability of remission onset. Its duration is very variable (few months even few years) and it is followed by the chronic phase of lifetime dependence on exogenous insulin substitution. Type 2 diabetes (T2D), the most frequent form of diabetes in adulthood and in the whole population, is characterized by insulin resistance (impaired insulin action in target tissues) and impaired insulin secretion [1]. In the etiopathogenesis, combination of lifestyle (obesity, lack of physical activity, improper diet, smoking, stress, lack of sleep) and genetic factors play a role. Insulin resistance, which occurs primarily in the muscles, fat tissue and liver, gradually increases demands on insulin secretion, thus leads to compensatory hyperinsulinism, later to disruption of glucose homeostasis and eventually to manifestation of T2D. Insulin resistance, caused by structural or functional change of insulin receptor and/or by the failure of post-receptor mechanisms, causes the disturbances in glucose metabolism. Lack of insulin inhibition on glucose production leads to increased liver gluconeogenesis, increased lipolysis in the liver and fat tissue with elevated concentration of free fatty acids. Glucose influx to the tissues, especially to the

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muscles, is insufficient, so the production of glycogen is decreased. At the beginning of T2D, basal secretion of insulin may be increased even with fasting hyperinsulinism. With continuation of the disorder, insulin secretion decreases due to secondary defect of pancreatic beta cells probably as the consequence of long lasting hyperglycemia (glucose toxicity), chronic increase of free fatty acids, lipid toxicity or storage of amylin. T2D can manifest at any age, but most commonly after 40 years, however, diabetes can run discreetly for a long time before its clinical manifestation. Therefore, T2D can be diagnosed accidentally and some patients can have symptoms of microangiopathic (even macroangiopathic) complications at the time of diagnosis or T2D can manifest as hyperosmolar hyperglycemic (non-ketogenic) coma. Clinical symptoms are the same as those above described non-urgent symptoms of T1D and some patients can have other signs of metabolic syndrome (central obesity, hypertension and dyslipidemia). Monogenic diabetes is the group of hereditary forms of diabetes caused by mutations of certain autosomal dominant gene leading to disruption in the production or action of insulin. It has some typical symptoms: diabetes diagnosed in the first six months after the birth, positive family history of diabetes, mild fasting hyperglycemia (5.6 – 8.5 mmol/l) or diabetes associated with extrapancreatic manifestation. In the majority of cases the diagnosis can be set based on DNA testing. These clinical units include neonatal diabetes (permanent and transient) and MODY diabetes (maturity onset diabetes of the young) that are caused by gene mutations of hepatocyte nuclear factor (HNF) 4α, glucokinase, HNF 1α, insulin promoter-1, HNF 1β and some others. Some types of monogenic diabetes are possibly treated by peroral antidiabetics. More rare causes of diabetes include genetic syndromes associated with diabetes, cystic fibrosis related diabetes, diabetes induced by drugs, pancreatopathy or endocrinopathies. As recommended by American Diabetes Association, diabetes mellitus can be diagnosed based on one of three criteria [1]: 1. Glycated hemoglobin (A1c) ≥ 6.5 % (DCCT method)

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2. Fasting plasma glucose (at least 8 hours without energetic intake) ≥ 7.0 mmol/l 3. Glycemia in the 120th minute of oral glucose-tolerance test ≥ 11.1 mmol/l (examined with the dose of 75g glucose dissolved in water by standard condition according to World Health Organization) 4. In an individual with typical symptoms of hyperglycemia, such as polyuria, polydipsia and weight loss, glycemia at any time during the day (disregarding the time after last meal) ≥ 11.1 mmol/l If unequivocal hyperglycemia is absent, criteria 1 – 3 are recommended to examine repeatedly. CHRONIC COMPLICATIONS OF DIABETES MELLITUS The quality of life in diabetic patients may worsen with chronic diabetic complications, which may even lead to life-threatening situations with their progression [7]. Chronic complications can be grouped into microvascular (neuropathy, nephropathy and retinopathy) and macrovascular complications (ischemic disease of heart, brain and limbs), and can be found in children and adolescents due to early diabetes onset, as well as adults. The presence of chronic complications increases with diabetes duration, however, several years before clinical manifestation functional and structural changes are notable by some specific examinations. Patients with diabetes should be regularly screened for chronic diabetic complications. The aim of the screening is to note the early signs of complications and to begin appropriate therapy as soon as possible. All diabetic patients are recommended to be screened for diabetic peripheral neuropathy – patients with T2D starting at the time of diagnosis and patients with T1D five years after the diagnosis. Then, the screening should be repeated annually, using simple clinical tests (such as a 10 g monofilament test). In children, annual comprehensive neurological examination is recommended to be performed at the start of puberty or at the age of 10 years or in the subject with diabetes duration for five years. The examination includes inspection of feet, palpation of pulses (on a.dorsalis pedis and a.tibialis posterior), assessment of tendon reflexes (such as patellar and Achilles), and investigation of proprioception, vibration and monofilament sensation. It should be performed

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annually together with assessment of possible symptoms of neuropathic pain [1]. According to the American Diabetes Association, annual foot examination is recommended to define risk factors predictive of foot ulcers and amputation. The comprehensive foot examination includes inspection and foot pulse assessment. It is recommended to examine the feet at every visit in patients with disorders of feet sensitivity, foot deformities or ulcers. Similarly, children and adolescents should have foot inspection at each visit as education concerning the importance of foot care [1]. Table 1. The recommendation for screening of diabetic retinopathy [1]. Adults with T1D

Adults with T2D

Children with T1D

Screening at the Within 5 years after Shortly after the At the onset of puberty or at the age of beginning diabetes onset diagnosis of diabetes 10 years or if the child has diabetes for 3 - 5 years Further intervals If retinopathy is not present - every 2 years; Annually (every 2 years may be of screening if retinopathy is present - annually acceptable only on the advice of an eye care professional).

At least annually, quantitative assessment of urinary albumin/creatinine ratio and estimated glomerular filtration rate should be performed in patients (including children) with T1D duration of five years and in all patients with T2D. Blood pressure should be measured at each common visit. If high-normal pressure or hypertension have been found, blood pressure should be confirmed in three separate days. Lipid profile should be examined at the time of diabetes diagnosis and periodically thereafter (for example every 1 – 2 years). In children ≥ 2 years of age, a fasting lipid profile should be examined soon after the diagnosis and after the stabilization of glucose control. In case of abnormal lipid profile, annual monitoring is rational. If values of LDL cholesterol are within the accepted risk concentration (˂ 2.6 mmol/l), the examination of lipid profile is appropriate to repeat every five years. The recommended intervals for screening of diabetic retinopathy (by comprehensive dilated eye examination by an ophthalmologist) [1] are shown in Table 1.

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DIABETIC NEUROPATHY Diabetic neuropathy is a term for nerve disorders associated with diabetes mellitus. It is one of the most frequent chronic diabetic complications and a common reason to visit a doctor. It can be present even at the time of diagnosis of T1D or T2D and it occurs in about 40 – 90% of diabetic patients after ten years of diabetes duration. Several distinctive clinical syndromes of diabetic neuropathies exist, which differ in clinical manifestation, clinical courses and anatomic distribution. Especially children can be affected by its sub-clinical form – without visible symptoms, but detectable by special diagnostic methods. Clinical diabetic neuropathy is characterized by a variety of symptoms (paresthesia, dysesthesia, fault of vibration or thermal sensitivity, impaired proprioception, pain); and diffuse or focal disorders of peripheral somatic or autonomic nerve fibers can be present. Diabetic neuropathy may lead to invalidism due to severe pain, muscle weakness or nerve paresis. Diabetic cardiovascular autonomic neuropathy (CAN) is found to be associated with increased mortality and higher frequency of major cardiovascular events and patients with CAN are at risk of sudden, unexpected death. CAN has been linked to complex cardiovascular changes, such as resting tachycardia, orthostatic hypotension, orthostatic tachycardia and bradycardia, intolerance of physical exercise, loss of baroreceptor sensitivity, intra- and peri-operative cardiovascular instability, decreased hypoxia-induced respiratory drive, asymptomatic ischemia, silent myocardial ischemia and infarction, prolonged QT interval and congestive heart failure [8]. Diabetic foot is the foot of diabetic patients with infection, ulceration and / or destruction of the deep tissues. It can be associated with neurological abnormalities and varying degrees of peripheral vascular disease. Diabetic foot is the main cause of limb amputations and the risk of amputation is 10 – 15 times higher in subjects with diabetes compared to non-diabetic patients [9]. A significant risk factor in etiology of diabetic foot is diabetic neuropathy together with diabetic angiopathy and infection. Diabetic neuropathy contributes to the development of diabetic foot by several mechanisms. Disorder of the innervations of the intrinsic foot muscles causes an imbalance between extension and flexion

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of the foot and anatomical foot deformities. This creates abnormal bone protuberances and pressure points, which progressively lead to skin breakdown and ulcerations. Autonomic neuropathy contributes to diminution in function of sweat and oil glands. As a result, the skin is prone to the development of tears and infections. Peripheral neuropathy may lead to the loss of sensation, so diabetic patients are often unable to identify any insult to their lower limbs and many wounds go unrecognized and may progressively worsen [10]. Regarding the variability of manifestation of diabetic neuropathy several classifications exist, which are based on clinical manifestations. A lot of patients do not suffer from a single type of diabetic neuropathy, but a mixture of neuropathic features often with a dominance of one subtype. Table 2. Diabetic autonomic neuropathy. Organ system Symptoms Cardiovascular rest tachycardia, postural hypotension, orthostatic tachycardia and bradycardia, intolerance of physical exercise, loss of baroreceptor sensitivity, decreased hypoxia-induced respiratory drive, increased arrhythmogenesis of myocardium, cardiomyopathy, cardiovascular instability, silent myocardial ischemia and infarction, cardiovascular events, sudden death Gastrointestinal esophageal enteropathy, nausea, gastroparesis, constipation, diarrhea, fecal incontinence Urogenital

bladder atonia, urine retention, incontinence, frequent infections of urinary tract, erectile dysfunction, retrograde ejaculation

Respiratory

decreased lung functions, decreased cough reflex sensitivity, decreased basal bronchial tone, decreased ventilatory response to hypercapnia, frequent respiratory infections

Sudomotor

feet anhidrosis, sweating disorders (increased sweating of upper body, after food and at night)

Endocrine

asymptomatic hypoglycemia, hormonal contra-regulation disorder

Other

disorders of pupillary reflex

According to Freeman, 2005 [11], the classification of diabetic neuropathy is following: 1. Symmetrical neuropathy a. Distal symmetric sensorimotor polyneuropathy b. Autonomic neuropathy c. Acute painful neuropathy

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d. Hyperglycemic neuropathy e. Treatment-induced neuropathy f. Symmetric proximal lower extremity neuropathy 2. Focal and multifocal neuropathy a. Cranial neuropathy b. Thoraco-abdominal neuropathy c. Focal limb d. Diabetic amyotrophy (proximal motor neuropathy) Diabetic autonomic neuropathy includes the possible disorders of many organ systems, which is shown in Table 2. Diagnostic possibilities of diabetic neuropathies include patient´s history, physical and neurological examination (tactile, vibration, pain and thermal sensitivity and tendon reflexes), which could verify clinical forms of neuropathies. Currently, the emphasis is placed on early diagnosis of sub-clinical forms, therefore several new diagnostic possibilities have been developed such as electrophysiological examinations (nerve conduction, electromyography), quantitative examination of sensitive functions, heart rate variability (the gold standard in diagnostics of cardiovascular autonomic neuropathy) [12] or electrodermal activity. Specific organ systems may be examined by electrogastrography, spirometry or diffuse lung capacity for carbon monoxide. Electrocardiogram can be useful to recognize myocardial ischemia, which may run silently in patients with diabetic autonomic neuropathy. Color skin tests with changing colors depending on sweat amount may be used to diagnose sudomotor neuropathy. Corneal confocal microscopy is a new non-invasive examination which can quantify the pathology of small nerve fibers (their density, morphology or branching) in cornea, the most densely innervated part of body. The future in the diagnosis of diabetic neuropathy may represent genetic analysis of certain gene polymorphisms, which allows personalized medicine and individually tailored approach. ETIOPATHOGENESIS OF DIABETIC NEUROPATHY The etiopathogenesis of chronic diabetic complications is complex and not fully

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understood, up to now. Chronic hyperglycemia (whose impact is enhanced by diabetes duration and poor compensation) triggers the variety of processes, such as non-enzymatic glycation, oxidative stress, polyol pathway, activation of the transcriptional factor nuclear factor-kappa B, increased production of protein kinase C, increased generation of proinflammatory cytokines, decrease production of vasodilatation products (prostaglandins, nitric oxide), decreased origin of myoinositol, change in Na+K+ATP-ase activity or in concentration of neurotrophic factors. These pathomechanisms may lead to diabetic neuropathy either directly through the disorder of nerve cells or indirectly through endothelial damage causing microangiopathy of capillaries supplying the nerves (Fig. 1). Described pathways act in connected interactions. Because of common clinical experience that compensation and duration of diabetes do not always correlate with onset of chronic diabetic complications, some other factors are considered relevant in pathogenesis – genetic, immunologic, environmental or epigenetic factors [13]. Regarding microvascular complications, risk factors at early stages of diabetes include genetic susceptibility to hypertension and cigarette smoking, while at later stages inadequate glycemic control, unfavorable lipid profile and higher blood pressure [14].

Fig. (1). Risk factors and pathogenic pathways in etiology of diabetic neuropathy.

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Diabetes compensation has been shown as significant risk factor in the development of chronic diabetic complications. Multicenter randomized study Diabetes Control and Complication Trial (DCCT) that ran between 1983 and 1993, enrolled 1,441 patients with T1D (duration of diabetes 1 – 15 years). These patients were divided into two subgroups according to the type of the treatment (conventional or intensified insulin regimen). The frequency of microvascular and macrovascular complications was significantly less common in the group treated by intensified regimen in comparison with the conventionally treated group. Intensive therapy reduced the occurrence of clinical neuropathy by 60 % [14]. Most of the patients were subsequently enrolled to the further study Epidemiology of Diabetes Interventions and Complications (EDIC) in which all of the subjects were treated intensively. The risk for confirmed clinical neuropathy reduced from 64 % to 30 % after 14 years of duration of the study [15]. After four years of the following in the EDIC study, the presence of chronic diabetic complications remained still higher in the patients initially treated by conventional regimen, although their actual compensation was satisfactory. In patients treated from the beginning by intensified insulin regimen, lower prevalence of chronic complications was found compared to the subgroup of patients treated conventionally at the beginning. This study demonstrated 'the effect of metabolic memory', in which each period of poor compensation may negatively influence the long-term prognosis of patient with diabetes. Thus, chronic hyperglycemia is the main modifiable factor influencing the onset and progression of chronic diabetic complications in type 1 diabetes. As intensive insulin therapy can effectively slow the progression of chronic diabetic complications, it is necessary to intervene with it immediately after diagnosis. Among the potential mechanisms for the persisting effects of metabolic memory belong epigenetic changes, genetic factors and glycation of proteins. It is generally observed that the prevalence of chronic diabetic complications increases with diabetesduration. The significant association between duration of diabetes and cardiovascular autonomic neuropathy was confirmed in EURODIAB IDDM Complication Study, the biggest study focusing on chronic diabetic complications [16] where 3,250 patients with T1D at the age 15 – 60 years were enrolled. Although similar correlation between diabetes duration and chronic

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diabetic complications was not confirmed in some other studies [17], it was probably due to different study methods and lower number of subjects. In addition to chronic hyperglycemia, also glycemic variability may contribute to the development of diabetic neuropathy and other chronic complications. Acute hyperglycemia may increase circulating cytokines more than continuous hyperglycemia [18]. Acute blood glucose fluctuations can induce chronic inflammation, increase oxidative stress markers [19] and decrease blood antioxidant glutathione [20]. According to the Italian study (35 adults with T1D, 33 adults with T2D), diabetes duration most significantly correlates with diabetic retinopathy, but also glucose variability may have a role as a risk factor of diabetic retinopathy, particularly in the case of acute fluctuations and acute hyperglycemia [21]. Further identified risk factors include higher body mass index, increased diastolic blood pressure ≥ 90 mmHg, increasedtriglycerides > 1,7 mmol/l, decreased serum HDL cholesterol < 1,0 mmol/l, microalbuminuria > 20 µg/min, smoking, presence of retinopathy and the period of puberty [22]. The prepubertal metabolic compensation has minor impact on possible development of chronic complications in comparison with the period after gonadarche [23]. Inadequate compensation within adolescence may be related to endocrine changes in puberty associated with higher insulin resistance (insulin like growth factor 1, sex hormones) [24], redundant food intake, lack of physical activity, neglect of insulin treatment and specific behavior (smoking, alcohol, contraceptives, drugs). Despite undeniable contribution of chronic hyperglycemia on development of chronic complications, another significant risk factor is genetic predisposition, which may modify the rate of onset of chronic complications. Many gene polymorphisms have been identified, which may contribute to the pathogenesis of the development of chronic complications. There are also other factors – immunological, environmental and epigenetic, which are currently not completely clear. According to recent evidence, epigenetic mechanisms play a key role in the complex interaction between genetic predisposition and the environment. Epigenetic mechanisms include covalent modifications of DNA (e.g. cytosine methylation), histone modifications (e.g. lysine acetylation, lysine and arginine

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methylation) and non-coding RNAs. These mechanisms can changes the expression of certain genes in target cells, while preserving DNA sequence. As these alterations may persist for a long time, they are believed to be a key mechanism of ˈmetabolic memoryˈ despite achieving good metabolic control [25]. Advanced Glycation End Products Non-enzymatic glycation includes the condensation reaction (Maillard reaction), in which the reactive carbonyl group of sugar aldehydes reacts with the protein Nterminus or free amino-groups by a nucleophilic addition. The initial product, called a Schiff base, spontaneously rearranges itself into more stable Amadori product. Amadori products stay in equilibrium with glucose and their level depends on the glucose concentration. According to some studies, the Amadori product of albumin is not inert and may be involved in the pathogenesis of diabetic vascular complications [26]. A small part of Amadori products go through further oxidative reactions and may stimulate formation of irreversible advanced glycation end products (AGEs). A great amount of AGEs in the body comes from exogenous sources, such as smoking, meals prepared at high temperature (fried, grilled), or high-fat foods [27]. In contrast, fruit, vegetable and boiled, steamed or braised meals have proven to have a lower content of AGEs. The rate of glycation is directly associated with the proportion of sugar in the open-chain form, so the dicarbonyl compounds (methylglyoxal, glyoxal and 3deoxyglucosone) and glycolytic intermediates (such as dihydroxyacetonephosphate, glyceraldehyde-3-phosphate) form more glycated proteins than do equimolar quantity of glucose [27]. Moreover, reactive intermediates can be generated through the polyol pathway (such as glyceraldehyde-3-phosphate, fructose, fructose-3-phosphate and 3-deoxyglucosone) and can contribute to intracellular formation of AGEs. Similarly, lipid peroxidation can lead to the generation of reactive carbonyl compounds reacting with proteins and giving rise to advanced lipoxidation end products. Glycated proteins have altered their structures and biological functions and the formation of AGEs has been found to have pathophysiological consequences in several conditions, such as ageing, inflammation, atherosclerosis, neurodegenerative disorders and diabetic complications. Some of the biological

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outcomes of AGEs are mediated via the interaction with the receptor for AGEs (RAGE). Advanced glycation contributes to the pathogenesis of diabetic neuropathy by several possible mechanisms. Deposition of AGEs has been found in the axoplasms of nerve fibers and Schwann cells, in the stromal collagens and also in endoneurial vessels [28]. The intensity of AGEs deposition correlates with lower density of nerve fiber myelination [29]. In vitro, after exposition of high AGEs environment, Schwann cells undergo processes of apoptosis with releasing the tumor necrosis factor alpha (TNF-α) and other proinflammatory cytokines. Myelin of peripheral nerves modified by AGEs is susceptible to macrophage phagocytosis and gives rise to segmental demyelination. Glycation of main axonal cytoskeletal proteins (such as tubulin, actin and neurofilament) leads to axonal atrophy and degeneration and impaired axonal transport. Modification of extracellular matrix protein laminin by AGEs results in impaired regenerative activity [29]. RAGE have been identified on vascular endothelial cells, peripheral nerve cells, mesangial cells, macrophages or smooth muscle cells. Consequences of the interaction between AGEs and RAGE include raising oxidative stress, induction of transcriptional factor nuclear factor kappa B (NFκB) and increase of expression of proinflammatory genes for cytokines or growth factor inducing nitric oxide synthase. Binding of AGEs to neural RAGE results in the stimulation of oxidative stress, NFκB and protein kinase C that all contribute to inflammation and apoptosis of nerve cells [28]. AGEs contribute to diabetic neuropathy through direct toxicity to nerve tissues and also indirectly through endoneurial microangiopathy. Oxidative Stress Oxidative stress, as an imbalance between increased production of reactive oxygen species (ROS) and reduced capacity of antioxidant systems, may accompany several pathological conditions including diabetes mellitus and chronic diabetic complications. Hyperglycemia can lead to an excessive formation of reactive oxygen species from the mitochondrial electron transport chain. Condition of hyperglycemia causes elevated mitochondrial membrane potential, changed protein structure of mitochondrial respiratory chain and increased

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production of peroxides. Under hyperglycemia, the activity of mitochondrial antioxidants is inhibited and mitochondrial DNA is damaged, which enhances oxidative stress. Hyperglycemia induces changes in mitochondria including the release of cytochrome C, activation of caspase 3, defective biogenesis and fission, leading to apoptosis of the cell [28]. ROS can directly activate other pathogenic pathways (advanced glycation, polyol pathway, protein kinase C and hexosamine pathway). Moreover, the stimulation of these pathways can further promote generation of ROS, intensify oxidative stress and generate a vicious cycle. Excessive generation of oxygen free radicals causes the reduction of antioxidant systems such as glutathione and superoxide dismutase meantime causing the overproduction of ROS. As a signaling molecule, ROS can affect several intracellular and extracellular pathways, such as poly ADP-ribose transferase (PARP) pathway and mitogen-activated protein kinases (MAPKs) pathways. PARP activation can lead to a disorder in the energy metabolism of nerve cells, inactivation of the sodium channel and the possible change of the membranes that may result in the decline of neuronal excitability and disorders in conduction velocity. Activation of PARP may affect the transcription of several genes, modify the neuronal function and may also lead to dysfunction of endothelial cells. Activation of MAPKs can enhance the expression of pro-apoptotic proteins, leading to apoptosis of nerve cells and impairment of neuronal regeneration [30]. Gene Polymorphisms Of Antioxidant Enzymes And Chronic Diabetic Complications Superoxide dismutase (SOD) is an enzyme catalyzing the transformation of superoxide into oxygen and hydrogen peroxide that is further converted into water and oxygen by catalase. MnSOD2 gene T→C substitution results in a substitution of valine for alanine at position nine (Val9Ala). Variant Ala MnSOD2 has alpha helical structure that is more easily imported into mitochondria and reaches higher mitochondrial activity. Val MnSOD2 variant has partially beta-sheet secondary structure, it is partly retained in the inner mitochondrial membrane and partly degraded in proteasomes. In addition, its mRNA is rapidly degraded, so Val

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MnSOD2 variant has reduced enzyme activity [31]. According to Egyptian and Russian authors [32, 33] the presence of MnSOD2 Ala/Ala genotype is significantly less frequent in patients with diabetic neuropathy and MnSOD2 Val/Val genotype is significantly more frequent in diabetic subjects without neuropathy. A meta-analysis involving 17 studies shows that C allele of C47T polymorphism (Val16Ala) in SOD2 gene is protective against diabetic microvascular complications, diabetic nephropathy and diabetic retinopathy [34]. Regarding gene SOD3, Arg/Arg genotype is significantly more frequent in patients with diabetic neuropathy [35]. Catalase is an enzyme localized in peroxisomes. Its function is to split the hydrogen peroxide into oxygen and water. Regarding C1167T catalase genotype, the prevalence of C allele is higher and the prevalence of T allele is lower in subjects with diabetic neuropathy as compared with diabetic patients without neuropathy [36]. According to another in vitro study in erythrocytes [37], 262TT catalase genotype is associated with increased activity of this antioxidant enzyme compared to 262CC genotype. This finding assumes the protective role of 262TT catalase genotype against rapid development of diabetic neuropathy. However, no significant correlation has been detected between C262T polymorphisms of catalase and the risk of diabetic retinopathy [38]. Glutathione peroxidase (Gpx) is the family of enzymes with peroxidase activity that can reduce lipid peroxides into corresponding alcohols and reduce hydrogen peroxide into water and oxygen. Of the eight previously described Gpx, ubiquitous intracellular enzyme Gpx-1 is the most studied. In codon 198 (rs1050450), T allele is associated with reduced enzyme activity compared to C allele. According to British authors T allele of Gpx-1 represents an increased risk for development of diabetic neuropathy [39]. Uncoupling proteins (UCP1, 2, 3) have a role in thermogenesis, oxidative phosphorylation and in defense against reactive oxygen and nitrogen species. Regarding diabetic complications, G866A UCP2 gene polymorphisms and C55T UCP3 gene polymorphisms seem to be related to lower risk of development of neuropathy in T1D patients [40]. In healthy young Japanese men, the association between heart rate variability and polymorphisms UCP2 (Ins/Del in exon8) and

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UCP3 (C55T) has been confirmed [41]. Metallothionein (MT) is a family of enzymes with function in energy metabolism and with antioxidant activity. Chinese authors studied seven single nucleotide polymorphisms in MT genes. According to their results, polymorphisms of rs10636 in MT2A gene and rs11076161 in MT1A gene are positively associated with neuropathy within type 2 diabetes [42]. Clarifying the association between glutathione S-transferase gene polymorphisms and diabetic complications has been the objective of many already published studies. No significant correlation has been found between GST M1/T1 gene polymorphisms and diabetic sensory-motor neuropathy in adult Russian patients with T1D (216 with diabetic neuropathy and 250 without neuropathy) [37]. In our already published pilot study in Slovak adolescents with T1D (19 with cardiovascular autonomic neuropathy and 27 without neuropathy), GST T1 present and combination GST T1 present/M1 null genotype can be considered as risk factor for development of cardiovascular autonomic neuropathy [43]. Polyol Pathway The polyol pathway is a two-step metabolic pathway. In the first step, aldose reductase catalyzes the reduction of glucose into sorbitol. The second step is oxidation of sorbitol to fructose by the action of sorbitol dehydrogenase (Fig. 2). Aldose reductase is a rate limiting enzyme, which requires a cofactor – nicotinamide adenine dinucleotide phosphate in its reduced form (NADPH).

Fig. (2). Polyol pathway and its contribution to increased oxidative stress [According to 44].

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Increased concentration of intracellular glucose leads to an increase in activity of aldose reductase, increased formation of sorbitol and fructose, which may be deleterious for several reasons. First, aldose reductase competes with glutathione reductase for their cofactor NADPH, leading to a decrease in reduced glutathione. Increased NADH enhances NADH oxidase to produce ROS and metabolites of fructose (fructose-3-phosphate and 3-deoxyglucosone) increase formation of AGEs. All these factors lead to raising oxidative stress [44], which is involved in the etiopathogenesis of diabetic neuropathy. Second, the osmotic theory suggests that increased accumulation of impermeable sorbitol in the cytoplasm causes intracellular hyperosmolarity, cell expansion and lysis of the cells. This theory may be applied in the etiology of diabetic cataract; however, it is not completely clear in the context of diabetic neuropathy. Nevertheless, an accumulation of sorbitol leads to depletion of other osmolytes, such as taurine, myoinositol and adenosine in the cytoplasm. Deficiency of myoinositol leads to phosphatidylinositol depletion and to reduced production of adenosine triphosphate (ATP). The reduction of ATP results in decreased activity of Na+K+ATP-ase and protein kinase C [28]. Pathophysiological consequences are the slowdown of axonal transport, neuronal apoptosis and also microvascular dysfunction. Protein Kinase C Protein kinase C (PKC) is a family of serine-threonine-related protein kinases involved in several intracellular signal transduction cascades. Several isoenzymes of PKC have been identified which differ in their structure and cofactor requirements (diacylglycerol, calcium ions or phosphatidylserine). The production of diacylglycerol is stimulated in condition of increased concentration of intracellular glucose and also within oxidative stress (as ROS inhibits the activity of glyceraldehyde 3-phosphate dehydrogenase, so dihydrogen acetone phosphate is converted to diacylglycerol). Thus, these conditions as well as some growth factors and AGEs can increase the activation of PKC. PKC pathway is implicated in the pathogenesis of diabetic neuropathy especially by affecting the neuronal microvascular circulation. PKC mediates the phosphorylation of target proteins and triggers a cascade of biological responses, such as the generation of transforming growth factor and endothelial cell adhesion molecules, increase of the level of endothelin-1, plasminogen activator inhibitor-1 and vascular

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endothelial growth factor (VEGF), and reduction of activity of NO synthase. These processes result in the increase of endothelial cell permeability (and capillary leak), microvascular occlusion and tissue damage [30]. Stimulation of PKC causes the inhibition of activities of membrane pumps (Na+K+ATP-ase and Ca2+ATP-ase) that contributes to diminution in membrane transport activity. Therefore, activation of PKC results in changes in nerve conduction, capillary pressure and microvascular circulation [45]. PKC activation increases the activity of NF-κB and NADPH oxidase, which leads to higher expression of the proinflammatory genes and oxidative stress. Hexosamine Pathway Hexosamines are amino-sugars arisen by adding an amine group to a hexose. Under normal circumstances, only 1 – 3 % of the glucose enters this pathway. Within the hyperglycemia condition, metabolic intermediate of glycolysis (fructose-6 phosphate) is shifted from the metabolic pathway of glycolysis to the hexosamine pathway, where it is transformed into uridine diphosphate-Nacetylglucosamine (UDP-GlcNAc). Afterwards, UDP-GlcNAc binds to the threonine and serine residues of transcription factors leading to their increased activation. Elevated activation of Sp1, a transcription factor involved in the development of chronic diabetic complications, causes the overexpression of plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1). Hexosamine pathway is increased also by ROS because of inhibition of activity of glyceraldehyde 3-phosphate dehydrogenase and further accumulating of fructose-6 phosphate, which overactivates hexosamine pathway. Collectively, stimulation of hexosamine pathway contributes to multiple metabolic disorders in diabetes [30]. Low C-peptide Concentration C-peptide (connecting peptide) is a 31-amino-acid protein connecting insulin´s αchain to its β-chain (proinsulin molecule). Proinsulin is packaged into secretory granules, where it is processed to C-peptide and insulin. Concentration of serum C-peptide is used to measure the endogenous secretion of insulin (to distinguish between type 1 diabetes and type 2 diabetes or MODY); however, it exerts also

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several biological effects. According to recent information, it can bind to the surface of neuronal, endothelial, fibroblast and renal tubular cells to a receptor (likely G-protein-coupled). This signal activates calcium-dependent intracellular signaling pathways, increases the activity of protein kinase C, MAP-kinase, nuclear receptor PPARγ and some transcriptional factors (NFκB, c-Fos). Cpeptide can improve neuronal function and erythrocyte deformability through increasing of Na+K+ATP-ase activity. C-peptide improves the function of endothelial cells and protects from microvascular damage by induction of nitric oxide synthase. It has also anti-thrombotic effect as it can inhibit the expression of adhesive molecules (P-selectin, ICAM1). It also inhibits apoptosis by increasing the concentration of antiapoptotic protein Bc12. In type 1 diabetes, low concentration of C-peptide may represent a risk factor for diabetic neuropathy [46]. The role of C-peptide in subjects with type 2 diabetes is controversial. In a retrospective cohort study of 2,113 T2D patients, higher baseline concentrations of C-peptide is associated with lower risk of diabetic microvascular complications (neuropathy, retinopathy and nephropathy) but imparts no survival benefit [47]. Cross-sectional study involving 1,410 patients with T2D has shown, that reduced C-peptide concentration is related to diabetic microvascular but not macrovascular complications [48]. Proinflammatory Cytokines According to recent information, proinflammatory processes take place in the development of diabetic neuropathy. Within diabetes, microglia surrounding nerves contains macrophages, sporadically lymphocytes and releases increased levels of TNF-α and interleukins. In experimental diabetic neuropathy in mice, inactivation of cyclooxygenase-2 (COX-2) is protective against diabetes-induced slowing of sensory and motor nerve conduction. These data support the importance of COX-2 activation in the pathogenesis of deficits in motor and sensory nerve conduction velocity in experimental diabetic neuropathy [49]. The proinflammatory condition activates the mitogen-activated protein kinase (MAPK), stress-kinase and NF-κB. Activation of NF-κB leads either to apoptosis of the cell or to its proliferation. Proinflammatory status is stimulated also by polyol pathway and by increased generation of AGEs.

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Neurotrophic Factors The role of neurotrophic factors in the development of diabetic neuropathy is complex [28]. On one hand, the lack of neurotrophins has been found in diabetic neuropathy, especially in the early stages of neuropathy. Nerve growth factor (NGF), the first discovered neurotrophic factor, has selectively trophic function for small fiber sympathetic and sensory neurons. On the other hand, increased concentration of neurotrophins may contribute to pathogenesis of neuroaxonal dystrophy. Increased level of NGF reflects the nerve injury and contributes to neuropathic pain. Concentration of neurotrophin-3 (NT-3) is significantly elevated in skin of person with diabetic neuropathy. This increase of NT-3 may reflect the stage of skin denervation and seems to signify a compensatory mechanism [50]. Angiotensin-converting Enzyme Angiotensin-converting enzyme (ACE) is a circulating exopeptidase, which takes part in the renin-angiotensin-aldosterone system (RAAS). RAAS has a role in retention of sodium and water, vasoconstriction mainly in arterioles, increase of glomerular circulation, increase in sympathetic tone and stimulation of growth of smooth muscle cells and collagen. ACE is secreted by pulmonary and kidney endothelial cells and catalyzes the conversion of angiotensin I (decapeptide) into angiotensin II (Ang II, octapeptide) that mediates the majority of effects of RAAS and acts like a strong vasoconstrictor. ACE also degrades vasoactive peptides such as bradykinin (vasodilator). In addition to this, RAAS plays a role in immune system; it controls leukocyte extravasation and chemotaxis, stimulates the expression of chemokines, chemokine receptors and adhesive molecules. Releasing of renin and Ang II is increased also by macrophages, T lymphocytes and dendritic cells though their production of TNF and IL-1. Recent studies indicate that RAAS contributes to the development of autoimmune diseases [51]. Ang II can enhance the growth of cells and tissues possibly leading to cell hypertrophy and tissue fibrosis. Hyperglycemia stimulates tissue Ang II that results in higher oxidative stress, inflammation, vasoconstriction, thrombosis, endothelial damage and vascular remodeling [52]. Despite many studies describing the association between ACE concentration or gene polymorphisms with diabetic nephropathy, similar information considering diabetic neuropathy

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are not so numerous. According to the meta-analysis of seven studies (including 1,316 cases with diabetic peripheral neuropathy and 1,617 controls), DD + DI genotype of ACE I/D gene polymorphism is associated with a significantly higher risk of neuropathy in T2D patients. According to an ethnicity, significantly increased risk has been found in Caucasians [53]. Kinin B1 Receptor According to recent research, kinins have a role in the regulation of pain and hyperalgesia after tissue injury and inflammation through the activation of the kinin B1 and B2 receptors. B2 receptor is constitutively expressed. Expression of B1 receptor in just low in physiological conditions and is induced by miscellaneous pathological stimuli such as proinflammatory cytokines, bacterial endotoxins, diabetes, ischemia-reperfusion injury, nuclear factor kappa B or angiotensin II. This suggests an important role of B1 receptor in various pathological situations [54]. Binding of the active kinins to B1 and B2 receptors activates intracellular signaling pathways and results in the formation of nitric oxide and prostaglandins. On one hand, both B1 and B2 receptors exert very probably protective effects on diabetic nephropathy [54]. On the other hand, the up-regulation of kinin B1 receptors in spinal dorsal horn microglia seems to be a significant mechanism in early neuropathic pain in streptozotocin-diabetic rats [55]. TREATMENT OF DIABETIC NEUROPATHY Treatment of diabetic neuropathy is complex; however, despite various experimental and clinical studies, it remains insufficient. The management can be divided into the following steps: 1. 2. 3. 4.

treatment of diabetes mellitus, supportive and additional treatment, symptomatic treatment, experimental treatment (mostly interfering with pathophysiological pathways).

Treatment of Diabetes Mellitus - Adequate Compensation The first and the most important step in the management is to maintain the sufficient compensation of diabetes – tight control of glycemia, lipidemia and

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blood pressure [1]. The treatment of type 1 diabetes patients is with intensified insulin regimen and in patients with type 2 diabetes, administration of selected peroral anti-diabetics or insulin therapy is considered. Physical activity, lifestyle modification, smoking cessation and dietary management also contribute to adequate compensation and to a delay of the onset of chronic complications including diabetic neuropathy. Intensified insulin regimen has been shown to prevent the development of chronic complications [14, 15]. The principle of treatment is the application of shortacting insulin (bolus) just before the main meals and long-acting insulin (basal) in the evening (or if necessary, twice daily). During the last decades, insulin analogues are used, which have better pharmacokinetic and pharmacodynamic facilities such as more quickly onset of the effect (ultra-fast analogues) and longer duration of effect (protracted analogues). Patients with insufficient compensation are best treated by an insulin pump that best mimics the natural insulin production by the pancreas. The diet of patients with diabetes should be regular and individual with counting carbohydrates and with sufficient amount of various nutrients, fiber, vitamins and probiotic cultures. Patients with diabetes should not be exempt from physical activity, in contrast, it is desirable to promote physical activity. The management of T2D focuses on lifestyle intervention – physical activity, proper diet, smoking cessation and later on antidiabetic drugs or insulin therapy. Several classes of anti-diabetic medications exist – metformin (biguanide class) as the first choice, then sulfonylureas, nonsulfonylurea secretagogues, thiazolidinediones, alpha glucosidase inhibitors, dipeptidyl peptidase-4 inhibitors and glucagon-like peptide-1 agonists. Oral medication can be used alone or insulin can be administered. If necessary, long-acting insulin is usually given at night (or twice daily) and in case of insufficiency also postprandial short-acting insulin can be added. Self-monitoring of blood glucose (SMBG) by glucometer a few times daily and monitoring of glycosuria is important for the control of the treatment. SMBG enables patients to assess their individual response to treatment and estimate achieving of glycemic targets. Lowering A1c to around 7% or less (DCCT norm) has been observed to decrease the risk of microvascular complications [1].

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Supportive and Additional Treatment The group of supportive and additional treatment is quite wide. However, the mostly used and the most effective medicament is alpha-lipoic acid. Other possibilities include freely available medicines such as vitamins of group B, antioxidants (vitamin C, E, coenzyme Q10, carotene and flavonoids), L-carnitine and poly-unsaturated fatty acids. The clinical benefit of many of these supplements is not completely clear and generally recommended management is to have a varied diet with adequate amount of vegetables, fruit, fish, meat and dairy products [56], as the natural source of active substances is superior to artificial sources. Alpha-lipoic Acid Diabetic subjects with diabetic neuropathy are usually treated by medicaments with the active ingredient alpha-lipoic acid (thioctic acid). Alpha-lipoic acid is an antioxidant which acts like a coenzyme in oxidative decarboxylation of alpha-keto acids in the Krebs cycle. In the metabolism, it changes itself from oxidized form into reduced dihydro-form and it can regenerate by the reduction of antioxidant radicals such as vitamin C or vitamin E. In an experimental environment, alphalipoic acid can stimulate the intake of glucose into the neurons, muscles and adipose cells through the activation of phosphatidyl inositol-3-kinase. The mechanisms of action of alpha-lipoic acid in experimental diabetic neuropathy include also improvement of nerve conduction velocity, nerve blood flow, and several other measures of nerve function [57]. Experimental models have shown that alpha-lipoic acid significantly reduces the expression of neuronal and glial markers, decreases lipid peroxidation, helps correct decline in nerve microcirculation and distal motor and sensory nerve conduction [58]. According to recent evidence, alpha-lipoic acid can decrease nerve sensitivity to pain through selectively blocking T-type calcium channels of nerve cells [59], so it may act also as analgesic treatment. In males with type 1 diabetes, treatment with benfotiamine together with alphalipoic acid has been shown to normalize increased AGEs formation, reduce higher monocyte hexosamine-modified proteins and normalize the decreased activity of

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prostacyclin synthase [60]. According to the meta-analysis of fifteen randomized controlled trials, administration of alpha-lipoic acid 300 – 600 mg intravenously daily for 2 – 4 weeks is related to significant increase in nerve conduction velocity and improvement of neuropathic symptoms in the treatment group with diabetic peripheral neuropathy without serious adverse events [61]. Some studies have not found any significant effect with oral administration of alpha-lipoc acid. In 40 adolescents with T1D, no significant change has been found in markers of oxidative damage, total antioxidant status, A1c and microalbuminuria after oral administration of alpha-lipoic acid for three months [62]. However, according to meta-analysis, a peroral dose of 600 mg alpha-lipoic acid daily up to five weeks leads to improve the symptoms of distal sensory-motor polyneuropathy [63]. According to the meta-analysis of seventeen studies, combination therapy with lipoic acid (300 – 600 mg i.v.) plus methylcobalamin (500 – 1000 mg iv or im) is significantly superior to monotherapy with methylcobalamin alone on diabetic peripheral neuropathy [64]. The recommended length of oral treatment with alpha-lipoic acid is not clear, untill now. A multicenter randomized double-blind placebo-controlled trial involving 460 diabetic subjects with mild to moderate sensory-motor neuropathy has shown that the treatment with alpha-lipoic acid at dose of 600 mg once daily for 4 years results in a clinically significant improvement and prevents the progression of impairments of neuropathy. The treatment is well tolerated; however no significant difference has been found in nerve conduction compared to placebo group [65]. L-carnitine is a quaternary ammonium compound, primarily biosynthesized in the liver and kidneys. L-carnitine acts in transport of long chain acyl groups of fatty acids from the cytoplasm into the mitochondrial matrix. Thus, fatty acids can be utilized in beta-oxidation to acquire available energy. It has also antioxidant effects as it can scavenge hydrogen peroxide or superoxide radical and it can even enhance the activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and the total antioxidant capacity [66]. L-carnitine can prevent lipooxidation of free fatty acids. In T1D children with early stages of diabetic neuropathy (based on electromyographic abnormalities), the treatment with L-carnitine (2 g / m2 of body surface daily) for two months has led to a 44% improvement in all pathologic nerve conduction velocity parameters [67]. An

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analysis of 2 randomized placebo-controlled trials has shown substantial improvements in numbers of sural nerve fibers and regenerating clusters of nerve cells after administration of acetyl-L-carnitine 500 and 1000 mg daily. Despite no increase in nerve conduction velocities and amplitudes, perception of vibration has refined in both studies and pain as most troublesome sign has shown significant release in the cohort taking 1000 mg L-carnitine [68]. Polyunsaturated fatty acids (PUFA) are fatty acids that contain more than one double bond in their backbone. According to their chemical structure, they can be divided into omega-3 fatty acids (such as alpha-linolenic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid/clupadonic acid), dosocahexaenoic acid), omega-6 fatty acids (such as gamma-linolenic acid, linoleic acid, docosapentaenoic acid/osbond acid), omega-9 fatty acids, conjugated fatty acids and other polyunsaturated fatty acids. For humans, two fatty acids – alpha-linolenic acid (omega-3) and linoleic acid (omega-6) –.are essential. Some other can be classified as “conditionally essential” – they can become essential in some developmental or disease conditions, such as gammalinolenic acid (omega-6) and docosahexaenoic acid (omega-3). Essential fatty acids have several biological effects. They are modified to form eicosanoids, endocannabinoids, lipoxins, they can affect cell signaling and regulate transcription factors such as NF-κB. Moreover, polyunsaturated fatty acid may serve as antioxidants. The fatty acids are susceptible to oxidation in relation to their level of unsaturation. Fatty acid micelles are able to directly scavenge superoxide anion in an unsaturation – dependent behavior, while the most effective fatty acid is eicosapentaenoic acid. Dietary sourced rich in PUFA are plant-based oils (sunflower oil, corn oil, soybean oil) and fish (trout, salmon, herring, mackerel). Information from the National Health Nutrition Examination Survey 1999 – 2004 for adults ≥ 40 years of age (a total of 1,062 subjects) with diagnosed diabetes have shown that dietary intake of linolenic acid is significantly reduced in adults with diabetic peripheral neuropathy compared to those without neuropathy. Dietary intake of linolenic acid positively correlates with lower odds ratio for diabetic peripheral neuropathy [69]. Model of diabetic endothelial dysfunction has been studied in high-fat diet fed streptozotocin rats. Supplementation by alpha linolenic acid leads to reduced formation of superoxide

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and peroxynitrite in diabetic vascular segments. Intake of alpha linolenic acid prevents diabetes-induced endothelial dysfunction by enhancing endothelial nitric oxide synthase (eNOS) but attenuates oxidative/nitrative stress by inhibiting inducible nitric oxide synthase (iNOS) [70]. In diabetic rats, diets containing gamma-linolenic acid may prevent the reduction in nerve conduction velocity caused by diabetes and this result does not depend on the nerve phospholipid fatty acid profile, polyol and sugar contents, activity of Na+K+ATP-ase and ouabain binding. Probably, this effect of gamma linolenic is indirect through its function as a precursor of prostaglandins with vasodilator effects [71]. Vitamin E (chemically methylated phenols) exists in eight distinct forms: αtocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol and four types of tocotrienols (α, β, γ, δ), which differ in chemical structure (quantity and position of methyl groups on the chromanol ring) and biological activity. Alpha-tocopherol represents the form of vitamin E preferably absorbed in humans. Vitamin E has positive effects on the formation of sex organs, increases fertility and protects cell membranes (especially in nervous, respiratory system and in erythrocytes) against peroxidation. Tocopherol, as a donor of hydrogen atom of the phenolic group, can intercept the peroxyl radical (ROO.) more quickly than polyunsaturated fatty acid thus preventing lipid peroxidation. Formed phenoxy radical may react with vitamin C, coenzyme Q, reduced glutathione or other free radical with consequent formed oxidized tocopherol that is eliminated in the bile. Vitamin E also acts as an anticoagulant, contributes to membrane stability and inhibits protein kinase C. The main source of vitamin E is represented by vegetable oils (wheat germ, sunflower, almond, olive and peanut oils), nuts (almond, hazelnut, peanut), seeds, whole grains, then butter, milk, tomatoes, asparagus, greens and carrots. Although the effect of vitamin E to decrease the products of oxidative stress is well known, its effect on diabetic neuropathy is not completely clear. In Nigerian adults with type 2 diabetes with painful neuropathy, treatment with vitamin E in a dose of 400 mg in combination with Eve Primrose (in doses 500 – 1000 mg/day) has resulted in a relief from neuropathic pain [72]. In streptozotocin-diabetic rats, high doses of vitamin E (12 g/kg), but not standard vitamin E supplementation (70 mg/kg) partially prevents nerve dysfunction [73]. According to a meta-analysis of randomized controlled trials, long-term antioxidant supplementation with vitamin

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E and/or vitamin C might be efficient to optimize endothelial function in nonobese T2D subjects [74]. Vitamin C (L-ascorbic acid) is an essential nutrient, which acts as a cofactor for several enzymatic reactions. It is necessary in the metabolism of hydroxylysine and hydroxyprolin, amino acids important for the synthesis of collagen that is needed in wound-healing and in prevention of capillary bleeding. It is involved in the synthesis of neurotransmitters, carnitine, in the biosynthesis and catabolism of tyrosine. Vitamin C promotes iron absorption, acts as natural antihistamine, and stimulates the leukocytes formation, development of bones, teeth and cartilage. It acts as reducing agent that donates electrons to miscellaneous enzymatic and also non-enzymatic reactions. Ability to reduce tocopheryl radical is involved in the antioxidant defense of the cells. Oxidized forms of vitamin C may be reduced by glutathione and enzymatic reactions dependent from NADPH. Thus, the presence of glutathione helps maintain vitamin C in a reduced form. Dietary sources of vitamin C include a variety of fruits (citruses, berries), vegetables (chili pepper, tomatoes, potatoes, greens) and plants (rose hip). The effect of vitamin C supplementation to prevent or improve diabetic neuropathy is controversial. Some studies have not confirmed any significant changes in lipids, markers of oxidative stress, inflammation and hypercoagulation in T2D adults after supplementation of vitamin C (doses 250 mg/500 mg/1000 mg per day) for two weeks [75]. On the other hand, infusion of ascorbic acid (3 mg/min) can block acute hyperglycemic impairment of endothelial function (measured by the forearm blood flow reactive hyperemic response to five minutes of upper arm occlusion) in eight adolescents with type 1 diabetes [76]. In Japanese study analyzing the association between fruit intake (rich for carotene, vitamin E, C, retinol equivalent, dietary fiber, sodium and potassium) and incident diabetic retinopathy in 978 patients with T2D, increased intake of fruit and vegetables, vitamin C and carotene is associated with lower risk for diabetic retinopathy [77]. In streptozotocin-induced diabetic rats, oral supplementation of ascorbic acid protects endothelial function against the aggravated ischemic oxidative damage and attenuates the exacerbation of cerebral ischemic injury that can be referred to anti-inflammatory and antiapoptotic effects in the brain [78]. Although the effect of vitamin C supplementation to prevent diabetic neuropathy is not clear, it is generally

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recommended eating meals naturally rich in vitamins, especially vegetables and fruits. Coenzyme Q10 (ubiquinone) is a vitamin-like lipophilic substance present primarily in the inner membrane of mitochondria and also in the membranes of lysosomes, peroxisomes, endoplasmic reticulum and vesicles. As it is a component of the electron transport chain, it participates in aerobic respiration producing cell energy – adenosine triphosphate (ATP). Three redox states of coenzyme Q10 exist – ubiquinone (fully oxidized), ubisemiquinone (semiquinone) and ubiquinol (fully reduced). Fully oxidized form has the function in electron transport chain while fully reduced form plays role as antioxidant. Coenzyme Q10 inhibits the initiation and the propagation of lipid peroxidation as well as protein oxidation. It regenerates vitamin E and prevents the oxidation of DNA bases mainly in mitochondrial DNA. Circulating coenzyme Q10 in LDL cholesterol prevents the oxidation of LDL. There are two sources of coenzyme Q10 – endogenous and exogenous. In the multistep biosynthesis also HMG Co-A reductase plays role, so the treatment with statins leads to decreased production of coenzyme Q10 (causing myopathy). Among rich sources of dietary coenzyme Q10 belong meat and fish, less amounts are found in vegetables (parsley, broccoli) and some fruit (avocado). In mouse model of T1D, low dose and long-term administration of coenzyme Q10 can prevent the development of neuropathic pain, reduce lipid peroxidation and inflammation by down-regulating proinflammatory factors [79]. According to double-blind placebo-controlled clinical trial in T2D adults (24 patients in experimental group versus 25 in control group), treatment with ubiquinone (400 mg) for twelve weeks results in improvement of clinical outcomes and parameters of nerve conduction in diabetic polyneuropathy. Moreover, it also reduces parameters of lipid peroxidation without significant adverse effects [80]. Carotenoids are fat soluble yellow, orange and red pigments necessary for photosynthesis, found in carrot, many other fruits, plants and vegetables (e.g. sweet potatoes, tomatoes, pepper, pumpkin, chanterelle, strawberries, apricots, oranges, apples, orange cantaloupe melon, spinach, broccoli, greens) and also in lower concentration in milk-fat, eggs and butter. Among the most common carotenoids belong alpha-carotene, beta-carotene, lycopene, lutein, beta-

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cryptoxanthin and zeaxanthin. The first three mentioned are precursors for vitamin A through the enzyme carotene-15, 15´-monooxygenase found in the liver and small intestine. This reaction is regulated by the status of vitamin A so carotenoids are very safe sources of vitamin A and increased intake of carotenoids does not lead to hypervitaminosis A. Excessive consumption of beta-carotene can lead to benign carotenodermia (orange skin). However, chronic high intake of synthetic beta-carotene supplements has been related to elevated risk of development of lung cancer in smokers, prostate cancer or intracerebral hemorrhage. In plants, carotenoids act like antioxidants as they deactivate singlet oxygen generated in the process of photosynthesis; however, relevance of this action in humans is less clear. Some studies in vitro indicate that carotenoids can inhibit lipid peroxidation. However, it is still not clear, if the biological functions of carotenoids in humans result from their antioxidant effect or other nonantioxidant behavior. They might modulate cell proliferation as they act like tyrosine kinases, mitogen-activated protein kinase (MAPK) as well as growth factor signaling cascades [81]. In rats with streptozotocin-induced diabetes, oral treatment with lycopene (2.5 mg/kg/day) for seven days after onset of diabetes reduces serum concentration of glucose, total cholesterol, triglyceride, liver aminotransferases (AST, ALT) and increases serum insulin levels, what suggests the possibility that lycopene may be useful for the management of diabetes [82]. In a similar animal model, the therapy with beta-carotene (10 mg/kg/day), vitamin E (40 mg/kg/day) and vitamin C (10 mg/kg/day) has shown significant improvement of cold allodynia and thermal hyperalgesia. Furthermore, treatment with beta-carotene evidently prevents cold allodynia and thermal hyperalgesia [83]. Also, treatment with lycopene for four weeks significantly attenuates thermal hyperalgesia and cold allodynia in streptozotocin-induced diabetic rats [84]. Flavonoids are polyphenolic plant secondary metabolites, sometimes referred as vitamin P. They can be divided into several groups, such as flavones, flavonols, flavanones, isoflavones, anthocyanidins, catechins and chalcones. Several hundreds of flavonoids have been identified untill now and among the most known are resveratrol, silymarine, luteolin, baicalein, and scutellarein. Many of the flavonoids have antioxidant function as they can inhibit reactive oxygen and

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nitrogen species. Flavonoids are also supposed to reduce activity of proinflammatory of enzymes contributing to the generation of reactive species such as inducible nitric oxide synthase, cyclooxygenase or lipoxygenase. They can modify intracellular signaling in immune cells and also seem to have antimicrobial (antibacterial, antiviral and antifungal), anti-cancer, anti-platelet and anti-allergic activity. Several studies have observed that flavonoids can decrease the risk of atherosclerosis and hypertension [85], improve capillary and endothelial function, regulate carbohydrate metabolism, modify concentrations of blood lipid and reduce the risk of cancer [86]. Sources rich in flavonoids include parsley, onions, berries, bananas, citrus fruit, aronia, sea-buckthorns, dark chocolate and beverages (red wine, black and green tea, beer, coffee). In rats with streptozotocin-induced diabetes, administration of diosmin (flavone) at doses of 50 and 100 mg/kg for four weeks significantly restores the decreased body weight and activity of antioxidant enzymes and also restores elevated blood sugar, lipid profile, levels of nitric oxide and malondialdehyde. Dose-dependent improvement has been found in cold allodynia, thermal hyperalgesia and walking function so diosmin seems to be effective in preventing development of early diabetic neuropathy [87]. According to meta-analysis of twenty-two randomized controlled trials involving 1664 participants, injections of puerarin (isoflavone) is effective for the treatment of diabetic polyneuropathy, as it improves the total effective rate and corrects nerve conduction velocity [88]. B vitamins represent a class of water-soluble vitamins playing significant roles in the cellular metabolism. The functions of the main B vitamins are shown in Table 3. Regarding possible management of diabetic neuropathy, the most studied are vitamin B1, B6 and B12. Table 3. Function of B vitamins. Vitamin Name

Function

B1

Thiamin

Generation energy from carbohydrates; RNA and DNA expression; neurotransmitter synthesis; nerve function.

B2

Riboflavin

Production of energy in the electron transport chain, Krebs cycle, beta oxidation of fatty acids.

B3

Niacin, niacinamide

Energy metabolism of glucose, lipids and alcohol; synthesis of lipids and nucleic acid.

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(Table ) contd.....

Vitamin Name

Function

B5

Pantothenic acid

Carbohydrates and fatty acids oxidation; synthesis of coenzyme A that is important for the synthesis of cholesterol, steroid hormones, fatty acids, phospholipids, amino acids, ketones, neurotransmitters and antibodies.

B6

Pyridoxine, pyridoxal, pyridoxamine

Metabolism of amino acids, carbohydrates and lipids; biosynthesis of hemoglobin neurotransmitters,; gene expression; antioxidant function.

B7

Biotin

Metabolism of lipids, proteins and carbohydrates

B9

Folic acid

DNA and RNA synthesis; erythropoiesis; coenzyme in the synthesis of tetrahydrofolate that is involved in the metabolism of amino acids and nucleic acids.

B12

Cobalamin

Metabolism of carbohydrates, proteins and lipids; hematopoiesis; protecting against damage of myelin sheats

Thiamin (vitamin B1) is a vitamin soluble in water, a coenzyme of the enzymes in the catabolism of sugars and amino acids, used in the biosynthesis of the neurotransmitter acetylcholine and gamma-aminobutyric acid (GABA). Active forms of vitamin B1 are phosphorylated derivatives of thiamine. One of the enzymes, where thiamine acts as a cofactor, is transketolatase. It is an enzyme of the pentose phosphate pathway that is important for the generation of reducing equivalents (such as NADPH) and for degradation of some products of glycolysis thus preventing activation of the metabolic pathways such as formation of AGEs, polyol pathway or protein kinase C activation. According to recent in vitro experiments, vitamin B1 plays role as radical scavenger because thiamin and thiamin diphosphate causes considerable suppressive effects on the generation of superoxide in xanthine and hypoxanthine oxidase system and significant suppression against formation of hydrogen peroxide derived from oxidized linoleic acid [89]. To improve pharmacokinetics, new thiamine derivatives have been discovered such as benfothiamine, allithiamine, prosuthiamine, fursuthiamine or sulbuthiamine. Deficiency of thiamin results in disorders of all organ systems, but the nervous system is notably sensitive. It may manifest by peripheral neuropathy (tingling, numbness in the extremities), axonal neuropathy (partial paralysis, sensory loss), muscular atrophy, ataxia or mimicking GuillainBarré syndrome. Syndromes resulted from vitamin B1 deficiency are beri-beri, Wernicke-Korsakoff syndrome and optic neuropathy. Food sources of thiamin include yeast, pork, cereal grains, sunflower seeds, brown rice, oatmeal, flax,

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potatoes, asparagus, liver and eggs. Vitamin B6 represents a group of compounds necessary in biological systems. Its active form, pyridoxal 5-phosphate, plays a role as a cofactor in several enzymes involved in metabolism of amino acids (cofactor of cystathionine-synthase catalyzing the condensation of serine and homocysteine to give cystathionine and next cysteine, conversion of tryptophane to niacin), sugars (glycogen phosphorylase in glycogenolysis) and lipids (biosynthesis of sphingolipids). It is involved in the synthesis of several neurotransmitters (epinephrine, norepinephrine, dopamine, serotonin, and gamma-aminobutyric acid), histamine and hemoglobin and in gene expression. Recently, antioxidant properties of vitamin B6 have been discovered – pyridoxine and pyridoxamine have suppressive effects on glucose-induced lipid peroxidation and superoxide generation in diabetic model experiments [90]. Known forms of vitamin B6 are pyridoxine, pyridoxal, pyridoxamine, pyridoxine 5-phosphate, pyridoxal 5phosphate, pyridoxamine 5-phosphate and 4-pyridoxic acid (catabolite excreted in the urine). Among food sources of vitamin B6 belong whole-grain products, meats, vegetables, nuts and bananas. Deficiency of vitamin B6 leads to symptoms such as angular cheilitis, atrophic glossitis with ulceration, seborrheic dermatitislike eruption, intertrigo, conjunctivitis, neurologic symptoms (neuropathy, confusion, somnolence) and sideropenic anemia. Food sources contain watersoluble form of vitamin B6, so excessive amount of this vitamin is excreted into the urine without adverse toxic effects. However, some supplements contain fatsoluble (or fat and water soluble) derivatives of vitamin B6 which may lead to overdose with symptoms such as motor or sensory neuropathy. Vitamin B12 (cobalamin) consists of a corrin ring made up of four pyrrole subunits with cobalt at the center of the ring. It has an essential role in the physiologic function of the nervous system and in erythropoiesis. It is implicated in the synthesis and regulation of DNA, metabolism of fatty acids and amino acids. It is necessary as a cofactor in homocysteine recycling into methionine. Derivatives of cobalamine are hydroxycobalamin (produced by bacteria), cyanocobalamin (semi-synthetic form), methylcobalamin and 5deoxyadenosylcobalamin (human physiologic forms). Deficiency of vitamin B12 leads to disorders of the nervous system (neuropathies, fatigue, depression, poor

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memory, psychosis), hyperhomocysteinemia and to macrocytic anemia (well known in pernicious anemia with autoimmune damage of gastric parietal cells leading to lack of intrinsic factor crucial for the normal absorption of vitamin B12). Food sources rich in vitamin B12 are fish, meat, liver, poultry, eggs and milk products. The human small intestine may contain bacteria producing vitamin B12; however, it is not known if this amount is sufficient to meet nutritional needs. The question, if patients with diabetic neuropathy should be supplemented by vitamins B even if their serum concentration is normal, is not yet sufficiently answered. According to Serbian authors, treatment with the combination of benfotiamine and vitamin B6 for 45 days leads to significant subjective and objective improvement (motor and sensory conduction velocity) of the symptoms of diabetic polyneuropathy [91]. On the other hand, randomized double-blind placebo-controlled trial has not shown the same results with benfotiamine treatment (900 mg per day) for six weeks. In 31 T2D patients with evident impairment of fasting flow-mediated dilatation, feeding with mixed test meal does not lead to further worsening of flow-mediated dilatation or parameters of microvascular and autonomic nervous function. Deterioration of postprandial parameters occurs not significantly after placebo treatment, so the prevention with benfotiamine is not feasible [92]. A randomized placebo-controlled trial, the Diabetic Intervention with Vitamins to Improve Nephropathy (DIVINe) study, enrolled 238 patients with T1D or T2D and advanced diabetic nephropathy. Subjects received either supplements of pyridoxine (25 mg), folic acid (2.5 mg) and vitamin B12 (1 mg) or placebo daily and were followed-up for a mean period of 32 months. Vitamin supplementation at high doses led to expected decrease in plasma concentration of homocysteine. However, in comparison with placebo, high doses of B vitamins led to more significant reduction of glomerular filtration rate and a greater prevalence of vascular events [93]. Despite decrease in homocysteine level is useful for prevention of vascular events, high-dose of vitamin B supplementation is not recommended in patients with advanced stages of diabetic nephropathy. Vitamin D represents a group of steroid hormonal precursors necessary for the

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synthesis of calcitriol – the hormone with important role in the calcium-phosphate metabolism and right bone formation. According to recent information vitamin D also regulates cell growth and differentiation, induces apoptosis of neoplastic cells, and has immunomodulatory effect. In relation to carbohydrate metabolism, it can increase the pancreatic insulin secretion [94], peripheral insulin sensitivity and its deficit is associated with impaired glucose tolerance. Our already published study in 58 children (aged 9 – 19 years) with type 1 diabetes has shown that nearly two thirds of children have insufficient concentration of vitamin D (< 30 ng/ml). Children with vitamin D deficiency have significantly lower magnesium concentration and lower Z score of lumbar spine compared to diabetic patients with sufficient vitamin D concentration [95]. Recent data have shown that vitamin D deficiency (probably due to inflammatory and angiogenic effects) correlates with an increased frequency of microvascular complications even in young subjects with T1D [96]. According to the National Health and Nutrition Examination Survey (2001-2004), 81 % of adults with diagnosed diabetes (older than 40 years) have vitamin D insufficiency. Insufficiency of vitamin D is associated with the adjusted numbness measure and with the adjusted composite paresthesia measure [97]. According to a metaanalysis of six studies (involving 1,484 T2D patients), concentration of vitamin D is significantly decreased in diabetic patients with peripheral neuropathy and deficiency of vitamin D 2.88-fold increases the risk of diabetic neuropathy [98]. Untill now, just one case report in a single T1D patient has described the improvement of serious symptoms of diabetic neuropathy after adequate supplementation with vitamin D leading to the correction of its deficiency [99]. Symptomatic Treatment Pain is the most bothering symptom accompanying diabetic neuropathy and it is the most common cause for visit a doctor. Information about the presence of pain with peripheral neuropathy vary between 10% up to 50%. Pain within diabetic neuropathy is usually chronic, caused by damage of nerve tissue and it arises without the stimulation of the nociceptors. The neuropathic pain is characteristically worse at night, so regular dosing of the drugs in the early evening and before sleep may improve sleep [11]. The neuropathic pain usually

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retreats with progressive, significant damage of thin nerve fibers. The tricyclic antidepressants and anticonvulsants are the first-line therapy used in the therapy of neuropathic pain. The best studied drug from tricyclic antidepressants is amitriptyline, the tertiary amine. It has been observed to significantly relieve the pain within diabetic neuropathy in several randomized double-blind placebo-controlled trials [100, 101]. Among multiple side effects belong drowsiness, orthostatic hypotension, dry mouth, constipation and weight gain, so the long-term treatment is not well tolerated in many patients. Nortriptyline and desipramine, the secondary amines, have less uncomfortable side effects. The selective serotonin and norepinephrine reuptake inhibitors (SNRI) belong among antidepressants and may be beneficial in the treatment of diabetic neuropathic pain. Because of elevation of serotonin and norepinephrine levels, SNRI has muscarinic, adrenergic and histaminic side effects such as drowsiness, dizziness, fatigue, headache, nausea/vomiting, urinary retention, sexual dysfunction, anxiety, elevated blood pressure and loss of appetite. Duloxetine demonstrates similar efficacy in the treatment of neuropathic pain as amitriptyline and more patients prefer duloxetine. When comparing side effects, patients treated with duloxetine have significantly less frequent dry mouth, more frequent constipation and significantly less frequent moderate to severe adverse events compared to patients treated with amitriptyline [100]. Another drug within SNRI is venlafaxine, which may be used in the treatment of pain within peripheral diabetic neuropathy with minimal adverse effects [102]. Gabapentin is a medication used as an anticonvulsant, originally developed for epilepsy treatment; however, currently it is used also as an analgesic in the management of neuropathic pain. The mechanism of its action is complex and not completely understood. Although it has similar structure to gamma-aminobutyric acid (GABA), gabapentin does not bind to GABA receptors in clinically relevant concentrations. The binding site for gabapentin is α-2-δ subunit of voltagedependent calcium channels and unlike some other anticonvulsants, gabapentin does not bind to sodium channels. Gabapentin modulates the action of two enzymes (glutamate decarboxylase, branched chain aminotransferase) involved in

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GABA biosynthesis. Another possible mechanism of action is that gabapentin blocks the formation of new synapses [103]. In clinical practice, this drug is well tolerated. However, some side effects may occur (somnolence, dizziness). According to the meta-analysis of 21 published trials, gabapentin has most favorable balance between efficacy and safety [101]. The treatment usually begins with the dose of 100 – 300 mg once daily in the evening with a gradual dose escalation by 100 – 200 mg every 3 – 5 days to achieve clinical effect. Maximum dose is 3600 mg, clear efficiency is mostly observable at doses of 1800 mg (300 – 1800 mg). Pregabalin is a higher potency gabapentinoid with a similar mechanism of action to gabapentin. The randomized double-blind trial shows that pregabalin is effective in pain reduction in patients with diabetic neuropathy, which is superior to venlafaxine and carbamazepine [104]. A daily dose of 600 mg is associated with the greatest effect. Several side effects including mood disturbance, ankle edema and sedation have been reported. Abrupt discontinuation of pregabalin can lead to cerebral edema and encephalopathy [105]. Other anticonvulsants possibly effective in the therapy of diabetic neuropathic pain include carbamazepine, phenytoin and topiramate [11]. The use of opiates in the management of diabetic painful neuropathy remains controversial. Opiates are very effective in pain relief; however, their long-term use can be addictive and they have several side effects (sedation, dizziness, nausea, vomiting, constipation, respiratory depression, physical dependence). The meta-analysis of randomized placebo-controlled studies shows that short term opioid therapy (4 – 12 weeks) may be considered in patients with chronic neuropathic pain [106]. Unlike morphine, which acts upon µ-opioid receptors, oxycodone – a semisynthetic opioid – acts on κ-opioid receptors. A multicenter randomized open-labeled study has shown that sustained-release tablets of oxycodone are effective in the treatment of moderate and severe painful diabetic neuropathy. Adverse reactions, but none serious, have occurred in about half of enrolled subjects [107]. In addition to binding to the µ-opioid receptor, tramadol also inhibits the reuptake of norepinephrine and serotonin and provides long-term relief of the neuropathic pain. The most common adverse events include

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constipation, nausea and headache [108]. Similarly, tapentadol has a dual mode action as an agonist of the µ-opioid receptor and as a norepinephrine reuptake inhibitor. Tapentadol extended release seems to be efficient and well tolerated in the treatment of moderate and severe chronic pain related to diabetic polyneuropathy [109]. Topical agents in the management of diabetic painful neuropathy offers several advantages – no drug interactions, very little systemic side effects and direct application to the site of pain generation (in the case of distal pain). The most widely used topical agent is capsaicin, an extract from chili peppers, which leads to activation and subsequent depletion of substance P from the nerve endings of unmyelinated C fibers [11]. Among side effects belong erythema, skin burning and sneezing. Various local anesthetic creams (such as lidocain) are available and can be useful in the management of neuropathic pain. To sum up, in the treatment of painful diabetic neuropathy, in addition to adequate metabolic control and intravenous alpha-lipoic acid (as mentioned above), the first-line pharmacologic therapies for painful diabetic polyneuropathy are tricyclic antidepressants, SNRI (duloxetine, venlafaxine), gabapentin or pregabalin, taking into account patient´s comorbidities and cost. If the pain persists, combination of first-line therapies may be considered, despite a change in first-line monotherapy. In the case of persistent inadequately controlled pain, opiates (such as tramadol) can be added in a combination treatment [110] (Table 4). Table 4. Management of painful diabetic neuropathy. 1. Adequate metabolic control of diabetes mellitus 2. Alpha-lipoic acid 3. First-line monotherapy: tricyclic antidepressants or gabapentin / pregabalin or SNRI (duloxetine, venlafaxine) 4. Combination therapy of first-line drugs 5. Combination therapy - adding a second-line drugs (opioids) 6. Alternative management: topical agents or non-pharmacological treatment

Diabetic autonomic neuropathy, the disorder of autonomic nervous system associated with diabetes, belongs among severe chronic diabetic complications.

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Clinical symptoms of autonomic neuropathy are very heterogeneous, which creates the need for interdisciplinary cooperation within the diagnostics and treatment of its individual manifestations. Cardiovascular autonomic neuropathy, the most serious manifestation of autonomic neuropathy, represents the increased risk of cardiovascular events, silent myocardial ischemia, malignant arrhythmia and sudden death [111]. Basic preventive interventions are regimen recommendations. Rest tachycardia can be treated by cardio-selective beta blockers, orthostatic hypotension might be managed by sufficient intake of fluids and salt, compression bandaging of legs or by pharmacotherapy with fludrocortisone or midodrine. Other symptoms of diabetic autonomic neuropathy such as gastroparesis, diabetic cystopathy, overactive bladder, can be managed in close cooperation with concrete specialists. Current possibilities in symptomatic treatment of diabetic autonomic neuropathy is shown in Table 5. Table 5. Possibilities of the symptomatic treatment of diabetic neuropathy. Symptoms

Possible management

Tachycardia

Cardio-selective beta blockers, verapamil

Orthostatic hypotension

Sufficient intake of fluids, compression bandaging, salt intake, physical maneuvers (leg crossing, sleep in semi sitting position), fludrocortisone, midodrine

Gastroparesis

Prokinetics (metoclopramide, domperidone, itopride)

Diabetic constipation

Diet modification, sufficient hydration, exercise, lactulose, prokinetics

Diabetic diarrhea

Loperamide, codeine, diphenoxylate, clonidine, ondasetron

Excessive sweating

Oxybutynin, glycolpyrolate, local antiperspirants

Anhidrosis

Fatting creams

Overactive incontinence Diabetic residuum

bladder,

cystopathy,

urge Anticholinergics - oxybutynin, solifenacin, darifenacin urine Parasympathomimetics - bethanechol, alpha-adrenergic blockers doxazosin

Erectile dysfunction, impotency

Vardenafil, tadalafil, intrauretral injection

sildenafil,

alprostadil

intracavernosal

Hypoglycemia unawareness

Education, release of tight glycemic control, glucagon

and

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Treatment in Experimental and Clinical Studies Some of the therapeutic possibilities are experimental, untill now. Many of them interfere with the pathophysiological processes within diabetic neuropathy such as oxidative stress, increased products of polyol pathway, formation of AGEs, protein kinase C activation or imbalance in growth factors. A lot of drugs efficient in the animal models have not yet been studied in clinical studies in humans and so their therapeutic potential has not been clearly proved. Aminoguanidine (also known as Pimagedine) is a hydrazine derivative able to reduce concentration of AGEs through its interaction with 3-deoxyglucosone (precursor for the formation of AGEs). It is available on the US market as an antiaging drug possibly alleviating the symptoms associated with diabetes. In experimental animals (rats), administration of aminoguanidine at dose of 100 mg/kg daily for three months shows a significant antioxidant effect and preventive effect against diabetic retinopathy and nephropathy [112]. According to a randomized double-blind placebo-controlled study including 690 T1D patients with nephropathy and retinopathy, treatment with aminoguanidine for 2 – 4 years leads to a more slowly decrement in the estimated glomerular filtration rate and to reduction in the 24-hour total urinary proteinuria. The progression of retinopathy has been less common in aminoguanidine-treated subjects compared to patients treated with placebo [113]. The effect of aminoguanidine on diabetic neuropathy has been studied mostly in animal models. In plexus myentericus in rat ileum, treatment with aminoguanidine completely or partially prevents the diabetesinduced increase in activity of nitric oxide synthase (NOS), in vasoactive intestinal polypeptide (VIP)–containing varicosity size and in fiber width in the circular muscle. However, no effect is shown regarding the diabetes-induced elevation of NOS-containing nerve size as well as proportion of myenteric VIPcontaining neurons. Also, this treatment fails to prevent the decrement in noradrenaline concentrations in nerves resulted from diabetes. Aminoguanidine in the treatment of neuropathy is not equally beneficial for each type of autonomic nerves supplying the ileum [114]. In streptozotocin diabetic rats, treatment with aminoguanidine (25 mg/kg) for sixteen weeks partially but significantly improves the endoneurial microcirculation [115]. Some studies do not confirm its effect. In a primate model of diabetic neuropathy, treatment with aminoguanidine (10

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mg/kg) for three years had no effect on glycemic compensation and did not recover nerve conduction velocity or prevent autonomic dysfunction [116]. Another finding in diabetic rats indicate that the pyridoxal-aminoguanidine adduct is more effective than aminoguanidine in preventing cataracts and diabetic neuropathy [117]. Alagebrium chloride (ALT-711) is a drug able to break the covalent crosslinks between glucose and proteins caused by advanced glycation, whereby supporting the degradation of AGEs. The beneficial effects of alagebrium has been studied mainly in the management of experimental diabetic nephropathy. Administration of alagebrium (2 mg/kg/day) to diabetic mice significantly reduces urinary albumin excretion, pathological changes in kidneys, concentration of nitrotyrosine (marker of oxidative stress) and pentosidine (marker of AGEs), and expression of NADPH oxidase subunits regardless of treatment protocol. Alagebrium also directly decreases hydrogen peroxide in a test tube [118]. In diabetic rats, treatment with insulin together with alagebrium restores erectile dysfunction to similar level as can be seen in normal controls [119]. The influence of alagebrium on diabetic neuropathy has not been extensively investigated. In diabetic mice, treatment with alagebrium restores endothelium-dependent vasodilatation after iontophoretic delivery of acetylcholine; however it does not have significant effect on motor nerve conduction velocity and does not restore C-fiber-mediated nociception threshold [120]. Anti-inflammatory Drugs As proinflammatory cytokines play a role in the development of diabetic neuropathy, anti-inflammatory drugs belong to the possible management not only of neuropathic pain but also of neuropathy itself. Sulphoraphane, a naturally occurring sulfur-containing isothiocyanate, has been identified in broccoli sprouts, cabbage, cauliflower, kale or mustard. It has dual antioxidant and antiinflammatory activities. In rats with streptozotocin-induced diabetes and diabetic neuropathy, administration of sulphoraphane for six weeks leads to amelioration of motor nerve conduction velocity, neuronal microcirculation and pain behavior. Biochemical effects of sulphoraphane includes decreased malondialdehyde, inhibition of NF-κB expression, abrogation of expression of cyclooxygenase-2

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(COX-2) and inducible nitric oxide synthase and reduction of concentration of TNF-α and interleukin-6 [121]. Piroxicam, a non-steroidal anti-inflammatory drug (NSAID) and inhibitor of platelet aggregation, significantly improves decrement in sensory neuron action potential amplitude in streptozotocin-induced diabetic rats [122]. Diabetic mice with cyclooxygenase-2 gene inactivation are protected against nerve fiber loss and biochemical deficits of experimental diabetic peripheral neuropathy. Selective COX-2 inhibition (celecoxib) replicates this protective effect in diabetic rats [123]. An important question remains about possible side effects of these drugs. As the anti-inflammatory mechanisms of NSAIDs are considered to be mediated via COX-2 inhibition and the side effects (especially of gastrointestinal system) via the inhibition of cyclooxygenase-1 (COX-1), selective COX-2 inhibitors may offer a lower incidence of gastrointestinal complications. However, renal and cardiovascular effects limit their application. According to a meta-analysis of 138 randomized trials, administration of selective COX-2 inhibitors is related to a moderately increased risk of vascular events (twofold increase in the risk of myocardial infarction) [124]. Acute interstitial nephritis have been reported in a few case reports after treatment with COX-2 inhibitors and seems to be reversible after withdrawal of these drugs and the application of corticosteroid therapy [125]. Thus, traditional NSAIDs and COX-2 inhibitors are not suitable drugs for long-term treatment of diabetic neuropathy. C-peptide (connecting peptide) is 31-amino-acid protein, released from the pancreatic beta cells during cleavage of insulin from proinsulin. It is not just a byproduct, because it has several biological effects. In animal models (diabetic rats) it has been found that substitution of C-peptide repairs the metabolic abnormalities ameliorating the acute disorder of nerve conduction, corrects imbalance in neurotrophic factors as well as the expression of neuroskeletal proteins. Subsequently, improvements of axonal function and size occurs. Regarding nodal degeneration, C-peptide can prevent and repair it through the modification of the expression of nodal adhesive molecules. Moreover, it can prevent hippocampal apoptosis and cognitive dysfunction. Thus, C-peptide replacement seems to have multiple beneficial effects on diabetic neuropathy and cognitive deficits in type 1 diabetes [126]. C-peptide substitution is useful also in

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the treatment of diabetic nociceptive neuropathy in diabetic rats [127]. According to randomized double-blinded placebo-controlled study including 139 adults with type 1 diabetes, treatment with C-peptide for six months leads to an improvement of sensory nerve conduction velocity and clinical neurological impairment of the lower extremities [46]. Aldose reductase inhibitors (ARIs) represent the group of drugs that interfere with pathophysiological formation of sorbitol and fructose from glucose within the polyol pathway by inhibiting the rate limiting enzyme – aldose reductase. Examples of these drugs are epalrestat, fidarestat, ranirestat, zenarestat, tolrestat and sorbinil. Natural sources with ability to inhibit aldose reductase are spinach, Indian gooseberry, lemon, orange, cumin and fennel seeds, basil leaves, black pepper, curry leaves, cinnamon and lichen. Treatment with epalrestat (150 mg/day) for two years in 38 T2D patients with diabetic neuropathy significantly supresses the worsening of sensory nerve conduction velocity in nervus suralis and motor nerve conduction velocity in nervus tibialis compared to control diabetic group. Serum N-carboxymethyl lysine (marker of AGEs) is significantly decreased in the subjects treated with epalrestat [128]. According to the metaanalysis of ten trials, ARIs can ameliorate cardiac autonomic neuropathy, especially mild or asymptomatic form [129]. Data from the Aldose Reductase Inhibitor-Diabetes Complications Trial has shown that epalrestat prevents progression of diabetic neuropathy (prevention of deterioration of vibration perception threshold, minimum F-wave latency and median motor nerve conduction velocity) and retinopathy/nephropathy [130]. According to the study involving 549 diabetic subjects with diabetic sensorimotor neuropathy, treatment with ranirestat (20 and 40 mg/day) significantly improves the motor nerve conduction velocity in mild to moderate neuropathy, but no significant difference has been found in sensory nerve function compared to placebo [131]. This group of drugs is very perspective and untill now, epalrestat is approved in Japan for the therapy of diabetic neuropathy. It seems to be effective and well tolerated longterm medicament. Ruboxistaurin is an orally active selective inhibitor of protein kinase C beta (PKC beta). It effectively improves parameters of blood flow in the retina, decreases microalbuminuria, reduces macular edema and leads to improvement of the

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symptoms of mild diabetic neuropathy. In randomized double-blind placebocontrolled trial involving 40 subjects with diabetic peripheral neuropathy, administration of ruboxistaurin (32 mg/day) for six months reduces sensory symptoms, enhances skin microvascular blood flow at the distal calf and improves the quality of life (measured by Norfolk Quality-of-Life Questionnaire for Diabetic Neuropathy). No significant differences have been found in morphometry of nerve fibers, nerve conduction studies and quantitative autonomic and sensory function testing between ruboxistaurin- and placebotreated groups. Ruboxistaurin treatment is well tolerated [45]. In Japan, ruboxistaurin is pharmaceutically manufactured for the treatment of macular edema, diabetic retinopathy and neuropathy. In Europe and US it is tested in clinical studies, untill now, and the approval for its use as a medicament is still awaited. Vascular endothelial growth factor (VEGF) is a signal protein stimulating vasculogenesis and angiogenesis and its pathogenic role has been demonstrated in diabetic retinopathy and nephropathy. Conversely, just a little is known regarding diabetic neuropathy. The most significant member of the VEGF family is VEGFA, other members are VEGF-B, VEGF-C, VEGF-D and placenta growth factor. Among the functions of VEGF-A belong the stimulation of endothelial cell mitogenesis and cell migration, and increase in matrix metalloproteinase activity. It acts as chemotactic factor for monocytes, macrophages and granulocytes; it is also a vasodilatator and increases microvascular permeability. According to recent information, VEGF-A is generated as two isoforms, represented by VEGF-A165a and VEGF-A165b [132], which exert opposing actions on vascular permeability, angiogenesis and vasodilatation. Although, VEGF-A165a has neuroprotective effect in hippocampal, dorsal root ganglia, and retinal neurons, its pro-angiogenic, pro-permeability and vasodilatatory effects limit its therapeutic usefulness. To the contrary, neuroprotective effects of endogenous VEGF-A165b, which expression has been detected in hippocampal and cortical neurons, might be advantageous for therapeutic use. Recombinant human VEGF-A165b is a neuroprotective agent that effectively protects both peripheral and central neurons in vivo and in vitro. It may be therapeutically useful for pathologies that involve neuronal damage, including hippocampal neurodegeneration, glaucoma diabetic retinopathy, and peripheral

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neuropathy. The endogenous nature of VEGF-A165b expression suggests that non-isoform-specific inhibition of VEGF-A (for antiangiogenic reasons) may be damaging to retinal and sensory neurons [132]. On the other hand, opposite information exists. As VEGF is implicated in the development of diabetic retinopathy, anti-VEGF drugs are widely used in the management of diabetic retinopathy. However, their effect on nerve cells is not completely clear. In diabetic rats, hyperglycemia early affects neurite outgrowth through the altered regulation of VEGF receptors. Neutralization of VEGF with bevacizumab is protective in vitro as well as in vivo models of diabetic neuropathy [133]. Neurotrophic Factors Administration of all-trans retinoic acid, which supports the endogenous expression of both retinoic acid receptor beta and neural growth factor (NGF), partially reverts the disorders resulted from diabetic neuropathy in diabetic rats and the same therapeutic effect has been shown also in treatment with vitamin A [134]. Treatment with recombinant human NGF for six months in 250 subjects with diabetic polyneuropathy significantly improves two quantitative sensory tests and the sensory part of the neurological examination. The most common side effect is only injection site discomfort [135]. On the other hand, a large-scale phase III clinical trial involving 1,019 diabetic subjects has failed to confirm the former assumptions of effectiveness, when comparing between administration of recombinant human NGF and placebo for 48 weeks. This discrepancy between the two sets of trials can be explained by several factors such as different study populations, inadequate dosage, a robust placebo effect and changes to the formulation of NGF for the phase III trial [136]. Increased concentration of neurotrophins may contribute to pathogenesis of neuroaxonal dystrophy. As shown in some studies, NGF treatment of experimental rats with diabetic autonomic neuropathy increases the occurrence of neuroaxonal dystrophy in ganglion mesentericum superius [137]. Increased levels of NGF reflects the nerve injury that contributes to neuropathic pain, so one of the perspectives in the management of diabetic neuropathic pain may be an antibody against NGF. Fulranumab is a human monoclonal antibody against NGF. According to phase II

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double-blind placebo-controlled trial involving diabetic subjects with moderate to severe peripheral neuropathic pain (randomized to subgroups receiving subcutaneously fulranumab 1, 3 or 10 mg, or placebo every four weeks), administration of 10 mg fulranumab has led to reduction of average daily pain at the 12th week compared to placebo. Treatment is generally well tolerated and the most common adverse events are arthralgia, peripheral edema and diarrhea [138]. In models of diabetic rats with tibial nerve entrapment neuropathy, treatment with prostaglandin E1 (PGE1) alleviates neuropathic pain and results in higher motor conduction velocity compared with the untreated group. PGE1 significantly decreases nerve growth factor mRNA and increases VEGF mRNA in the tibial nerve [139]. Increased levels of NGF in injured tissues contribute to hyperalgesia through upregulation of voltage-gated sodium channels in sensory neurons. Nociceptors are easily sensitized also by agents such as bradykinin, serotonin, prostaglandin E2 and adenosine which are released after nerve or tissue injury. Nav 1.7 is a voltagegated sodium channel (encoded by the gene SCN9A) which is expressed especially in sympathetic ganglion neurons and in the nociceptive neurons at trigeminal ganglion and dorsal root ganglion. Nav 1.8 is a sodium ion channel (encoded by the SCN10A gene), specifically expressed in the dorsal root ganglion and in C-fibers (small-diameter sensory neurons). Considering that these sodium channels are present at the terminals of sensory nociceptive nerves, they play a critical role in nociception and pain. A number of selective Nav 1.7 (and/or Nav 1.8) blockers are in clinical development [105]. For example, administration of a803467, a highly selective blocker of Nav 1.8 channels, to diabetic rats with painful neuropathy leads to 6-fold greater reduction of hyperalgesia and 2-fold greater reduction of allodynia than does lidocaine [140]. Angiotensin Converting enzyme (ACE) Inhibitors The beneficial effect of ACE inhibitors is well known in respect to microalbuminuria/proteinuria, diabetic nephropathy and cardiovascular diseases. However, their use in the treatment of diabetic neuropathy is not so clear. In rats with experimental diabetic peripheral neuropathy, treatment with lisinopril for eight weeks results in higher capillary density of sciatic nerve, better nerve

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conduction velocity, higher level of superoxide dismutase and Na+K+ATP-ase activity [141]. In another study in diabetic rats, administration of enalapril or L158809 (an angiotensin II receptor blocker) reduces the superoxide concentration in the aorta, but seems to be less effective in epineurial arterioles. Treatment with enalapril has greater effect in reversing the impairment of endoneurial microcirculation and motor nerve conduction velocity compared to L-158809. Treatment with enalapril or L-158809 entirely prevents/reverses the diabetesinduced deterioration in calcitonin gene-related peptide (CGRP)-mediated vascular relaxation and this treatment has also beneficial effects to improve impaired acetylcholine-mediated vasodilatation. However, the efficacy is declined depending on the later time of administration [142]. The majority of the effects of ACE inhibitors are ascribed to the inhibition of angiotensin converting enzyme (decrease formation of angiotensin I, II, aldosterone and antidiuretic hormone, increased natriuresis, lower arteriolar resistance, increase of venous capacity, depression of sympathetic activity, lower resistance of renal blood vessels). In addition to this, ACE inhibitors lead to increase concentration of components of kallikrein-kinin system (like bradykinin). Bradykinin exerts vasodilatatory effect via the stimulation of specific endothelial B2 receptors leading to the liberation of nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor (EDHF). According to recent information, bradykinin seems to have a complex role in microvascular endothelial protection, which may contribute to long-term effects of ACE inhibitors in maintaining vascular integrity [143]. While the role of constitutively expressed B2 receptors is considered mostly beneficial, expression of B1 receptors is stimulated in several pathological conditions such as inflammation and ischemia [54], and their role in the pathogenesis of diabetic neuropathy is controversial. In Akita diabetic mice, the absence of both bradykinin B1 and B2 receptors augments neuropathy, nephropathy and loss of bone mineral density, so kallikrein-kinin system is probably protective in chronic diabetic complications. Thus, vasopeptidase inhibitors and kinins agonists, such as ACE inhibitors, might be beneficial in the prevention or treatment of chronic diabetic complications [144]. Moreover, treatment with ACE inhibitors via bradykinin may result in improvement of glucose tolerance and insulin resistance through increase in

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glucose uptake, particularly in skeletal muscle. Possible mechanism is at least partially via the stimulation of bradykinin-NO system and further GLUT4 translocation [145]. On the other hand, inflammation-induced stimulation of kinin B1 receptors may lead to reduction of Na+K+ATP-ase activity and increased oxidative stress resulting in disorders of neuronal activity. Kinin B1 receptors are involved also in the development of diabetic painful neuropathy. In animal models, bradykinin B1 receptor is overexpressed in sciatic nerve during the streptozotocin-induced diabetes and administration of bradykinin B1 receptor antagonist R-954 restores the neuronal activity and attenuates the oxidative stress, suggesting its potential therapeutic effect in the treatment of diabetic neuropathy [146]. Novel mechanisms of action of ACE inhibitors are also the increase of the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-prolin (Ac-SDKP) that participates in the anti-inflammatory and anti-fibrotic effect. Administration of ACE inhibitors results in increased concentration of some other substrates, such as substance P, neurotensin, chemotactic protein and luteinizing hormone-releasing hormone. However, their specific role in the possible therapeutic or adverse effects of ACE inhibitors is not completely clear [147]. Erythropoietin, originally identified as hematopoietic factor stimulating erythropoiesis, is expressed likewise in the nervous system and plays a crucial role in its development and maintenance. Moreover, erythropoietin protects from and enhances the repairment after the injury of neural structures. Regarding peripheral neuropathy associated with diabetes, HIV, cisplatinum, uremia and ageing, erythropoietin has protective effect against its development or it can improve the symptoms of neuropathy [148]. In diabetic mice, HSV-mediated gene transfer of erythropoietin to dorsal root ganglia prevents the decrement of nerve fibers in the skin, prevents the reduction in sensory nerve amplitude characteristic and preserves autonomic function examined by pilocarpine-induce sweating [149]. A single treatment with erythropoietin is sufficiently protective over a period of four weeks without any adverse effects. However, continuous expression of erythropoietin may lead to the development of abnormalities in the vascular supply in the nerve. Thus, regulatable expression of gene transfer of erythropoietin is necessary to control the level and duration of gene expression. In

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diabetic mice, the progression of diabetic neuropathy can be protected by regulatable expression of HSV-mediated erythropoietin in dorsal root ganglion [150]. Actovegin is a highly filtered deproteinized extract manufactured from calf blood, which contains compounds of low-molecular weight up to 5 kDa. It enhances aerobic oxidation, cellular energy metabolism, improves glucose absorption and oxygen uptake in tissues. Actovegin performs insulin-like activity, as it stimulates the glucose transport, activity of pyruvate dehydrogenase and oxidation of glucose. According to animal models (rats), Actovegin improves several parameters of experimental diabetic neuropathy via mechanisms involving suppression of poly-(ADP-ribose) polymerase (PPAR) [151]. In the multicenter randomized double-blind trial, 567 subjects with T2D were randomized to receive either twenty intravenous actovegin infusions (2,000 mg/day) once daily or placebo. Subsequently, the patients were treated perorally either with actovegin (1,800 mg/day) or placebo three times per day for 140 days. Compared to placebo, treatment with actovegin significantly improved the symptoms and sensory function of the lower limbs, while vibration perception threshold decreased by 5 %. No differences were found in the frequency of side effects between the groups [152]. Melatonin is both fat- and water-soluble hormone produced mainly by the pineal gland (also by the retina and gastrointestinal system). It is involved in the circadian rhythms of several biological functions, especially sleep and its production is inhibited by the light. Melatonin is also a powerful antioxidant as it can directly scavenge reactive oxygen species (e.g. hydroxyl radical and superoxide) and reactive nitrogen species (e.g. peroxynitrite), it stimulates the action of other antioxidants, enhances the effectiveness of mitochondrial oxidative phosphorylation, decreases electron leakage (thus reducing generation of superoxide) and it can protect nuclear and mitochondrial DNA [153]. Melatonin has also immunomodulatory effect, anti-aging properties, it influences intestinal motility and acts as an antagonist of pituitary gonadotropins. In many countries it is available in tablet form (on the prescription or as dietary supplement) and it seems to be effective in the treatment of delayed sleep phase disorder. In thirty-six type 2 diabetes patients, significantly lower concentrations of melatonin and also

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the nocturnal melatonin surge have been proved when compared to the healthy controls. Decreased levels of both daily and nocturnal melatonin have been revealed in subjects with autonomic neuropathy [154]. Melatonin administered at doses of 3 and 10 mg/kg per day in rats with streptozotocin-induced diabetic neuropathy leads to improvement in nerve blood flow and motor nerve conduction velocity compared to the treatment with placebo. Melatonin also reduces the increased expression of NF-κB and reduces the elevated levels of inducible nitric oxide synthase (iNOS) , proinflammatory cytokines (IL-6 and TNF-α) and COX-2 in sciatic nerves of animals. Thus, melatonin can mitigate neuroinflammation and exert neuroprotective effect presumably through decreasing oxidative stress and NF-κB [155]. Novel Drugs with Antioxidant Functions Current medical research focus also on novel compounds with antioxidant function and many of them have been shown to be beneficial in animal models. Cardio-protective effect of sodium ferulate (ROS scavenger) has been shown in streptozotocin-induced diabetic rats [156]. Antioxidant mimetics with seleniummanganese complexes can possess catalase, glutathione peroxidase and superoxide dismutase activity and decrease the level of lipid peroxidation products [157]. Selenium-organic glutathione peroxidase mimetic, M-hydroxy ebselen, can diminish diabetes-associated atherosclerosis and diabetic nephropathy in mice [158]. Superoxide dismutase mimetic, tempol, ameliorates the early retinal changes in diabetic rats [159]. Novel copper-zinc superoxide dismutase mimetic D34 seems to have antihyperglycemic and neuroprotective effects [160]. An important role of protein nitration and peroxynitrite in the development of diabetic neuropathy has been shown in experimental study in mice. Treatment with the peroxynitrite decomposition catalyst and the inhibitor of protein nitration for four weeks has led to partial correction of small sensory nerve fiber dysfunction and sensory nerve conduction slowing. Only treatment with peroxynitrite decomposition catalyst has led to an increase in intra-epidermal nerve fiber density and to correction of motor nerve conduction deficit [161]. According to Italian non-randomized open-label study with 50 diabetic patients with an impairment in both sensory and motor nerve conduction, administration of a combination of superoxide dismutase (ALA600SOD®) and alpha-lipoic acid for

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four months significantly improves the perception of pain and electroneurographic parameters [162]. Non-pharmacological Therapy Neuropathic pain may be alleviated by magnetic field therapy. In 375 subjects with diabetic neuropathy, randomized to wear constantly either magnetized insoles or similar placebo unmagnetized device for four months, statistically significant reductions in burning, numbness, tingling and pain have been found in the third and the fourth months of wearing. Static magnetic fields may penetrate up to high of 20 mm and can target the ectopic firing nociceptors situated in the epidermis and dermis [163]. Multicenter randomized double-blind placebocontrolled clinical trial involving 110 subjects with diabetic neuropathy has shown that frequency-modulated electromagnetic neural stimulation (frequency rhythmic electrical modulation system, FREMS) significantly reduces the daily and nocturnal pain as examined by a visual analogue scale promptly after each treatment. However, this useful effect is not measurable three months after the treatment. Treatment with FREMS significanly improves the cold sensation threshold compared to placebo, although not significant differences is found in warm sensation and vibration thresholds. FREMS has no effect on nerve conduction velocity. It seems to be safe treatment for immediate reduction in pain in symptomatic diabetic neuropathy, although this pain releasing effect is only transient [164]. Among the modern electrotherapy of neuromuscular pain belong percutaneous electrical nerve stimulation (PENS or electro-acupuncture), transcutaneous electrical nerve stimulation (TENS) and high-tone external muscle stimulation (HTEMS) [165]. In addition to pain-relieving effect, electrical stimulation can prevent muscle dysfunction and sarcopenia. According to the meta-analysis of twelve studies comparing electromagnetic fields or electrical stimulation versus placebo in patients with painful diabetic neuropathy, treatment with TENS led to significantly greater decrease in the mean pain score compared to placebo, while no significant pain release was found comparing electromagnetic fields with placebo [166]. HTEMS can attenuate the pain and discomfort associated with diabetic peripheral polyneuropathy and also with uremic peripheral poly-

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neuropathy in end-stage renal disease [167]. Alternative treatment approaches include spinal cord stimulation (SCS), in which an electrode is situated in the epidural space to the posterior column at the position of the nerve roots, which transfer the nociceptive information. Patients can modulate the electric current intensity through a device using radio frequency transmission. Multicenter randomized clinical trial involving 36 subjects with severe painful diabetic peripheral neuropathy not responding to conventional treatment shows, that treatment success (≥50% pain release during day or night or improvement in sleep and pain of ≥6 in the score of the Patientˈs Global Impression of Change scale) is found in 59 % patients treated with combination of SCS together with the best medical treatment and in 7 % of patients treated with best medical treatment alone. SCS seems to be effective in pain relief, however it is not without risk, as severe adverse events have been observed in two patients. One patient with SCS died due to a subdural hematoma and another patient acquired an infection of the implantation of SCS system. Despite removal of SCS system and antibiotic treatment, the patient has recovered slowly and not completely and has developed autonomic neuropathy [168]. Inhibitors of Epigenetic Modifications Current research of diabetic complications focuses on epigenetic modifications of genome, such as DNA methylation, posttranslational histone modification, chromatin remodeling and deployment of noncoding RNA. Epigenetic alterations participate in pathologic responses such as inflammation and neurodegeneration, which contribute to the progression of diabetic neuropathy. Whereas Epigenetics is a relatively young scientific areas, inhibitors of epigenetic mechanisms are being studied for a short time. Valproic acid (a drug used for the treatment of epilepsy and migraine) has been established also as a histone deacetylases inhibitor. According to the study in diabetic rats, valproic acid ameliorates the renal injury and fibrosis by preventing the myofibroblast activation and fibrogenesis by inhibition of histone deacetylases [169]. Similarly, fidarestat (an aldose reductase inhibitor) may improve contractile function of cardiomyocytes in diabetic obese mice by a mechanism dependent on histone deacetylase Sir2 [170]. In rats with model of type 2 diabetes, selective inhibitor of histone deacetylase 3,

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BRD3308, reduces hyperglycemia without affecting weight gain and increases insulin secretion [171]. Despite the lack of similar information related to diabetic neuropathy, it is very probably that in the near future inhibitors of epigenetic modifications will be developed and studied in relation to the treatment of diabetic neuropathy. CONCLUSION Diabetic neuropathy represents one of the most common and most bothering chronic complications of diabetes mellitus. In its complex etiopathogenesis, advanced glycation, products of the polyol pathway, oxidative stress, proinflammatory cytokines and dysregulation in neurotrophic factors all play a role. The deep knowledge about the risk factors and pathogenesis may allow a better understanding the principles of possible treatment. According to current results of large trials, the most important aspect in the management of diabetic neuropathy is to maintain adequate compensation of diabetes mellitus (tight control of glycemia, lipidemia and blood pressure) through insulin therapy or/and peroral antidiabetics, physical activity and dietary management. The variety of possible supplements used in the management of diabetic neuropathy refers to incomplete knowledge about the adequate treatment. Alpha-lipoic acid, with antioxidant function, is the most widely used medicament and has been shown to be beneficial in the therapy of neuropathy. Diabetic patients are usually recommended to receive vitamins (group B, C and E), which are considered to have antioxidant and neuroprotective effect. However, these should be preferably received in their natural forms. Patients with diabetes are recommended to eat a varied diet (vegetables, fruits, meat, cereals, milk products) containing all necessary nutrients. The advantage of supplementation of water-soluble vitamins and antioxidants is lack of overdose as their excessive amount is excreted into the urine; however care should be exercised in the taking of fat-soluble supplements because of the possibility of overdose. High-dose supplementation by vitamins of group B (B6, B9 and B12) should be avoided in advanced stages of diabetic nephropathy because of evidenced adverse effects. Symptomatic treatment is aimed to relieve symptoms such as pain, tachycardia, hypotension, constipation or bladder atonia. Current experimental and clinical studies focus on possible influence of the pathologic pathways. Some studies are controversial or done just

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on animal models, while some other are very promising and represent perspective modality in the treatment of diabetic neuropathy. CONFLICT OF INTEREST The authors confirm that they have no conflict of interest to declare for this publication. ACKNOWLEDGEMENTS Declared none. REFERENCES [1]

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CHAPTER 5

The Now and Tomorrow of Migraine Treatments Sefik Evren Erdener1, Turgay Dalkara1,2,* 1

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

2

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey Abstract: The last few decades have witnessed a major progress in migraine treatment based on novel clinical findings as well as advanced pathophysiological understanding of the disease. Studies focusing on the activation of trigeminovascular system during migraine attacks complemented by identification of several genes related to migraine susceptibility have elaborated the role of dural neurogenic inflammation, nociceptive sensitization mechanisms and cortical spreading depression in migraine pathophysiology. Triptans and CGRP antagonists have emerged as novel migrainespecific agents for acute attack treatment although clinical use of CGRP antagonists is hampered by their side effects. Several unrelated classes of drugs ranging from betablockers to antiepileptics have been identified to be effective for migraine prophylaxis. A wide variety of novel targets including CGRP, glutamate receptors, nitric oxide synthase are in drug development pipeline for both acute as well as prophylactic treatment. Availability of a wide range of experimental and human models of migraine is promising in facilitating this progress. This chapter will focus on the current and future therapeutic agents for acute and prophylactic migraine treatment and their mechanisms of action.

Keywords: Antiepileptics, CGRP antagonists, Drug treatment, Headache, Migraine, Neurogenic inflammation, NSAIDs, Spreading depression, Trigeminovascular system, Triptans. Corresponding author Turgay Dalkara: Department of Neurology, Faculty of Medicine and Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey; Tel: +90 312 3052130; Fax: +90 312 3117908; Email: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2016 Bentham Science Publishers

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INTRODUCTION Headache is a common symptom confronted not only by neurologists but by all physicians. Migraine is a common form of primary headaches, affecting about 15% of the World population [1]. Migraine attacks are typically characterized with unilateral, throbbing and disabling headache episodes lasting for 4-72 hours and, are generally associated with nausea, vomiting, photophobia and phonophobia. In 20-30% of migraineurs, transient neurological symptoms, called aura gradually spread over 5 minutes and are accompanied or followed by headache within typically 15-60 minutes. Aura symptoms include visual disturbances (scintillating scotoma, blurring, homonymous visual field defects), migrating paresthesia and numbness in the face or extremities and, occasionally, speech disturbances as well as motor or brain stem dysfunctions. Today’s physicians are fortunate with many therapeutic options available for treating migraine headaches or reducing the attack frequency compared to 30-40 years ago. However, success rate is still not satisfactory and drugs used have unwanted side effects. Overcoming these limitations has been the subject of many recent preclinical and clinical research efforts. Here, we will review the currently used and emerging migraine therapies. PATHOPHYSIOLOGICAL OVERVIEW It is generally agreed that migraine attacks are initiated by transient disturbances in the brain parenchyma and that the first division of the trigeminal cranial nerve along with the upper cervical nerves, which innervate the meninges and cerebral blood vessels mediate the nociceptive impulses to generate headache. Consequently, a detailed understanding of the migraine pathophysiology encompassing multifaceted events in the brain parenchyma, meninges and trigeminovascular system (TVS) is needed for developing better therapies (Fig. 1). Fortunately, research on a wide range of animal models as well as surgical, physiological and neuroimaging studies on human subjects have proved that they could be instrumental in identification of the pathophysiological pathways and novel drug targets.

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Electrical stimulation

Somatosensory perception cortex

Inflammatory soup

MMA Thalamus

Vasodilation

Dura

Pinprick PPE

Subarachnoid space SPG

Pial artery

Mast cell

degranulation

KCI

Electrical stimulation

Pia

SSN

Glia limitans astrocyte

Activated Panx1

CSD

TG Cortex TNC

Fos

expression

Neuron Electrical stimulation

Recording

Recording

HMGB1, IL-1b CGRP, Substance P, NKA ACh, VIP, NO

Trigeminal peripheral projections

Mast cell-released mediators (histamine)

Parasympathetic efferents

Inflammatory mediators released from glia limitans

Central trigeminal projections

Fig. (1). Activation of trigeminovascular system by a parenchymal inflammatory signaling pathway leads to headache. Trigeminal ganglion (TG) neuron processes peripherally terminate over the pial and dural vessels, whereas central processes synapse in TNC. Second-order TNC neurons project to thalamus. Meanwhile, collaterals activate the parasympathetic superior salivatory nucleus (SSN), which innervate dural vessels via spehenopalatine ganglion (SPG) route. TVS activation leads to release of several mediators from perivascular nerve endings, hence, induce dural vasodilatation, mast cell degranulation and extravasation of plasma proteins. The TVS can experimentally be activated by chemical or electrical stimulation of the dura or trigeminal ganglion as well as by CSD. CSD initiates a parenchymal inflammatory signaling cascade by opening of neuronal pannexin-1 (Panx1) channels and release of HMGB1 and IL-1β. This response leads to activation of trigeminal nerve fibers around pial blood vessels. Experimentally, nociceptor activation can be demonstrated by electrical recordings from TG or TNC or by immunolabeling of TNC for Fos. Dural vessel dilatation, mast cell degranulation and protein extravasation can also mark TVS activation. [Reproduced, with permission from [12]].

Activation of the TVS causes release of several vasodilatory peptides like calcitonin gene related peptide (CGRP), pituitary adenylate cyclase-activating peptide (PACAP), substance-P and vasoactive intestinal polypeptide (VIP) from nociceptive nerve endings innervating the dura [2]. These mediators lead to discharge of additional vasodilators like histamine from dural mast cells. The vasoactive mediators induce plasma protein extravasation from dural vessels in addition to vasodilation [3 - 5]. Central projections of trigeminal and cervical nociceptive afferents terminate on the trigeminal nucleus caudalis (TNC), which extends from lower medulla oblongata to upper cervical spinal cord, forming the

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trigeminocervical complex (TCC) [6]. A monosynaptic reflex connection between the TNC and superior salivatory nucleus causes release of vasodilatory acetylcholine and nitric oxide (NO) from the post-ganglionic parasympathetic fibers innervating the dural vessels by way of sphenopalatine ganglion [7, 8]. This sequence of events leads to a sterile (neurogenic) inflammation in meninges, which adds to the sustained activation and sensitization of dural nociceptors [9, 10]. The sensitization of nociceptors generates an increased mechanosensitivity to head motion and vascular pulsations and, is thought to be responsible for the throbbing nature of the migraine headache. Central projections of the TNC terminate in contralateral ventral posteromedial thalamic nucleus, from where connections to somatosensory cortex and other cortical areas like insula, limbic structures and hypothalamus originate [7, 8, 11]. Sensitization of the TNC neurons and the thalamic neurons causes head/neck and body allodynia, respectively [7]. Cortical spreading depression (CSD), a neuronal and glial depolarization wave spreading at a velocity of 2 to 6 mm per minute has been established as the putative cause of migraine aura [13, 14]. In animal models, CSD has been shown to activate the TVS, suggesting a mechanistic link between migraine aura and headache [15 - 17]. Recent experimental data have shown that CSD stimulates TVS by initiating a parenchymal response triggered by neuronal pannexin-1 (Panx-1) channel opening (which may also involve purinergic P2X7 channel opening but this remains to be demonstrated) inflammasome activation and resultant HMGB1 and IL-1β release [18]. These pro-inflammatory mediators activate NF-κB in astrocytes, possibly inducing sustained release of prostanoids, cytokines and NO from glia limitans to subarachnoid space, which stimulate the trigeminal nociceptors [18]. The parenchymal signals are then amplified by release of peptides from meningeal nociceptive terminals and development of neurogenic inflammation. Individuals with migraine cluster within families and, hence, it is accepted that migraine has a genetic basis [19]. The genetic loci underlying common migraine are still being explored, however, a number of mutations in genes coding for ion channels and transporter proteins have been identified in familial hemiplegic migraine. Interestingly, these mutations increase susceptibility to CSD [20, 21], supporting the idea that CSD plays an important role in migraine with aura.

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ACUTE ATTACK TREATMENT Most patients learn by themselves that mild attacks can be subsided with the overthe-counter pain killers like acetaminophen or NSAIDs, whereas severe attacks may require administration of more expensive migraine-specific agents or may eventually lead to emergency department care. Primary measure of efficacy for acute abortive treatment is the percentage of patients being pain-free by the end of second hour [22]. Sustained response at 2 days with no relapse requiring use of rescue medication is regarded as the final goal in acute migraine treatment [22]. In this section, individual classes of drugs that are currently available and those being developed will be reviewed. Currently Available Drugs Nonsteroidal Anti-inflammatory Drugs (NSAIDs) NSAIDs are frequently used for acute management of migraine pain. Because of their favorable side-effect profile and availability as over-the-counter agents, they are usually the drugs of first choice by many patients. Their action is mediated by nonselective inhibition of cyclooxygenase (COX)-1 and -2 enzymes, which convert arachidonic acid to prostanoids including prostaglandins, prostacyclin and thromboxane. Application of an inflammatory mixture containing PGE2 over the dura causes activation and sensitization of meningeal nociceptors in rodents, showing that prostaglandins have the capability to stimulate the trigeminovascular system [23]. PGE2 can also induce CGRP release from both peripheral and central trigeminal nerve terminals [24, 25]. An induction of COX-2 enzyme expression within cortex as well as in astrocytes forming the glia limitans is observed approximately 30 min after CSD in mice, possibly inducing sustained release of prostaglandins into subarachnoid space to reach to the nociceptive fibers around the pial and dural vessels [18, 26, 27]. Indeed, naproxen inhibits the sustained middle meningeal artery vasodilation, a manifestation of trigeminal activation and neurogenic inflammation triggered by CSD [18]. Nonselective COX inhibitors like acetylsalicyclic acid, naproxen and ibuprofen have been shown to inhibit peripheral sensitization of meningeal nociceptors and suppress meningeal vasodilation as well as neurogenic inflammation after

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electrical stimulation of dura [28, 29]. The main target affected on systemic administration can be the peripheral trigeminocervical terminals, however, since most NSAIDs can cross the blood-brain barrier (BBB) [30], they can also exert central actions. Indeed, intravenous injection of naproxen, aspirin, ketorolac or indomethacin has been shown to inhibit central trigeminal sensitization [31 - 33]. Interestingly, NSAIDs can abort the sensitization hours after it has been established, while triptans cannot [23, 32]. It is likely that COX-2 inhibition has also a role in the analgesic effects of these drug as dural application of a selective COX-2 inhibitor NS-398 can abolish meningeal nociceptor sensitization with an efficacy comparable to topically applied naproxen [29]. In line with these experimental data, intravenous infusion of PGE2 and PGI2 in migraine patients triggers typical headache attacks [34, 35]. Similarly, the PGE2 level increases in jugular venous blood and saliva early during an attack while interictal PGE2 levels are not different in migraineurs than in controls [36 - 38]. Experimental efficacy and observational clinical benefit of NSAIDs have been validated in several placebo-controlled trials. Aspirin (900-1000 mg) [39], ibuprofen (400 mg) [40], metamizole (1000 mg) [41] and diclofenac (50-100 mg) [42] have been found to be more effective than placebo in aborting mild to moderate migraine attacks within 2 hours and providing pain-relief for at least 24 hours. Intravenous ketorolac (30 mg) was found to be as effective as sumatriptan [43]. This formulation was shown to terminate headache even after allodynia has emerged and also in triptan unresponsive patients, a finding that agrees with animal studies [44]. Intravenous ketorolac injection was therefore suggested as rescue treatment in emergency departments [43, 45]. An intranasal form of ketorolac has also been developed with good tolerability but in its phase II trial, the primary endpoint of significant pain-free response at 2 hours, was not met [46]. A trial of intravenous ibuprofen, another formulation as a potential rescue treatment is currently recruiting patients (NCT01230411). On the other hand, selective COX-2 inhibitors celecoxib (400 mg), rofecoxib (25-50 mg) and valdecoxib (20-40 mg) were found to be equivalent to therapeutic doses of ibuprofen or naproxen sodium for acute migraine treatment [47 - 49]. However, despite their lower risk of gastrointestinal side effects by sparing COX-1 inhibition in gastric mucosa, chronic use of selective COX-2 inhibitors is

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associated with increased cardiovascular risk, which hampers their routine use as analgesics [50]. Acetaminophen, despite not being an NSAID with low anti-inflammatory activity but with COX inhibitory effects, is one of the first-choice drugs in acute migraine being a widely available and cheap over-the-counter agent with high tolerability. A recent Cochrane review documented its superiority (1000 mg single dose) against placebo but with relatively high number-needed-to-treat value when used alone, compared to other commonly used NSAIDs [51]. Therefore it is usually preferable for treatment of mild to moderate attacks. Combination preparations of acetaminophen, on the other hand, has much better efficacy comparable to triptans (see below) [51]. In summary, it is generally agreed that NSAIDs are effective for migraine, even though Class-I evidence for the efficacy of most drugs is limited. Interestingly, a patient may respond more favorably to one NSAID than others. Unfortunately, there are no trials comparing effectiveness of different NSAIDs against each other. NSAID choice and dosing generally depend on best clinical judgment [40]. Ergot Alkaloids and Triptans Drugs targeting the 5-HT1B/1D serotonergic receptors constitute the first specific drug class for migraine. 5-HT1B receptors are predominantly located on smooth muscle cells of intra and extracranial arteries [52], whereas 5-HT1D receptors are expressed by trigeminal nociceptive afferent terminals [53]. Both peripheral and central terminals of nociceptors as well as second-order neurons in TNC express 5HT1B/1D receptors [54, 55]. Ergot alkaloids were first tested in migraineurs to reverse the abnormal dilatation of cerebral and dural arteries that were thought to be the cause of headache at that time [56]. Ergot alkaloids constrict cerebral arteries via 5-HT1B agonism [57]. However, by their action on neuronal 5-HT1B/1D receptors they also suppress peptide release from trigeminal afferents and, hence, dural vasodilation and plasma protein extravasation [58 - 61]. Ergot alkaloids, however are rather “dirty” drugs with additional binding to other 5-HT receptor subtypes, dopaminergic D2 and adrenergic (alpha-1, alpha-2) receptors [62]. The vasoconstrictive effect of

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ergot alkaloids by way of vascular 5-HT1B receptors is responsible for their cardiovascular side effects [63]. They are also contraindicated in pregnant women due to their contractile effect on uterine muscle. 5-HT2B agonism on the other hand is held responsible for fibrotic complications in heart valves [64]. Ergot derivatives may also worsen nausea and vomiting due to their agonistic effects on dopamine receptors [65 - 67]. Rectal ergotamine administration has better clinical outcome with more favorable pharmacokinetics compared to oral form [68]. Nevertheless, considering lack of well-designed randomized clinical trials and, its highly variable clinical efficacy and significant side effects, today, ergotamine is seldomly used for migraine treatment. DHE, on the other hand has better tolerability than ergotamine and higher efficacy in aborting migraines [70]. However, its gastrointestinal absorption is negligible and it must be administered parenterally [69]. Both intravenous and intramuscular DHE has 100% bioavailability while time to peak plasma level is 1-2 min and 24 min, respectively [71]. In an open-label trial, 1 mg im DHE provided almost complete pain relief at 24 hours in 90% of migraineurs experiencing a moderate or severe attack [72]. DHE has less side effects then ergotamine [70]. DHE is also less likely to cause medication overuse headache (see below) [70]. An intranasal form of DHE is also available and, compared to parenteral form, it has lower but acceptable bioavailability and has been found superior to placebo in clinical trials [73 - 75]. A novel orally inhaled formulation of DHE is suggested to have similar efficacy with parenteral forms and less side effects due to lower Cmax and less 5HT2B receptor binding, decreasing fibrotic complications [76]. However, general contraindications of parenteral and oral ergots are still valid for the novel pharmaceutical forms. Triptans are selective 5-HT1B/1D agonists. Their lack of significant binding to, dopaminergic, adrenergic or other subtypes of serotonergic receptors provide a more favorable side effect profile. They are also superior to ergots in terms of efficacy. Triptans show similar efficacy with ergots in neurogenic inflammation models by way of 5-HT1B/1D receptor agonism. They inhibit CGRP release from nociceptive terminals and the resultant neurogenic inflammation in experimental animals [77, 78]. Elevated jugular CGRP levels during migraine attacks can be normalized by triptan application in patients as well as in cats [79]. Triptan action

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can be abolished by application of 5-HT1B/1D antagonists and, it is now generally agreed that anti-migraine effects of triptans depend on 5-HT1B/1D receptor agonism [80]. Their action is however far more complicated, because, a group of molecules that are mainly triptan derivatives with selective agonistic properties on 5-HT1B/1D receptors but with less potent vasoconstrictive effects (plasma protein extravasation inhibitors), inhibits neurogenic inflammation preclinically [81] but clinically ineffective for migraine treatment [82]. There is controversy about the main site of triptan action since sumatriptan, the prototypical drug of this class, was reported not to cross the blood brain barrier [83], possibly restricting its pharmacodynamic effects to the peripheral nociceptive compartment. Indeed, supporting this view, sumatriptan can prevent but does not reverse central sensitization once it is established [23, 44, 84, 85]. One would expect more lipophilic triptans like naratriptan and zolmitriptan that are readily accessible to the CNS [86, 87] to have higher clinical efficacy. While this may be the case for naratriptan [88], zolmitriptan is almost equivalent to sumatriptan [89]. However, naratriptan’s superiority might depend on its 2-3 fold higher receptor affinity (hence, potency) compared to sumatriptan [90]. Indeed, triptans with less lipophilicity are unable to reverse headache and allodynia once central sensitization occurred [85]. Triptans are therefore highly effective for symptomatic treatment of moderate to severe migraine attacks [91] if they are taken early during the attack before the onset of central sensitization, that is, emergence of cutaneous allodynia [85, 92, 93]. Sumatriptan was first introduced for clinical use in 1992. Today, several approved triptan molecules are available. Although they all have the same agonistic effect on 5-HT receptors, their pharmacokinetic and pharmacodynamic characteristics differ, which offer choices for different conditions. A patient may respond to one triptan formulation while not benefiting from another. For instance, almotriptan and eletriptan were found to provide headache relief within 2 hrs for a subset of sumatriptan-nonresponders in two Class-I studies [94, 95]. Most triptans have short onset of action within 2 hours, a desired feature. They mostly have an elimination half-life of 2-6 h [96], predisposing to a high rate of relapse [97]. On the other hand, frovatriptan can be promising for certain patients experiencing relapses and can also be used as an intermittant prophylactic (see prophylactic

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treatment section) due to its extended half-life of about 26 hours [98]. Although triptans are advantageous over ergot alkaloids by acting selectively on 5-HT1 receptors, they still bear potential for cardiovascular side effects because of 5-HT1B receptor agonism and should not be concomitantly used with ergot alkaloids and MAO-A inhibitors to avoid the risk of serotonin syndrome, severe coronary vasospasm or cerebral vasoconstriction syndrome [96]. One should also be cautious when combining them with SSRIs or SNRIs [99]. However, triptans also act on 5-HT1F receptors in addition to 5-HT1B/1D receptors [100]. These receptors show a similar distribution to 5-HT1B/1D but they do not induce vasoconstriction [101], which offers a major advantage by eliminating the risk of cardiovascular side effects. This led to development of specific 5-HT1F agonists that are structurally distinct from triptan molecules. Such a molecule, lasmiditan was recently tested in two phase-II trials, enrolling 520 subjects and, was found both intravenously and orally effective with somehow comparable response rates to triptans. However, it had moderate dose-dependent adverse effects like somnolence, dysequilibrium or paresthesias [102] and, its efficacy should be validated in larger phase-III trials. One major problem in migraineurs during attacks is limited usability of oral drugs due to presence of nausea and vomiting and also to the reduced gastric motility [103]. Subcutaneous forms are always more effective than oral forms of the same agent possibly because of more rapidly rising serum levels [104]. For example, subcutaneous route increases therapeutic gain of sumatriptan at 2h compared to oral administration. A nasal spray of sumatriptan was developed but it exhibited low bioavailability with unpredictable responses and increased adverse events [105]. Rapidly dissolving oral tablets of sumatriptan, zolmitriptan and rizatriptan may be another alternative for patients bothered by nausea while swallowing a tablet. Systematic studies comparing those formulations are limited but rapidly dissolving form of sumatriptan appears to be acting faster and better than the classical tablet [106] and fast-dissolving rizatriptan is reportedly effective [107]. Oral dissolving tablets of zolmitriptan and rizatriptan have been approved by the Food and Drug Administration (FDA) for migraine treatment. Other novel triptan

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formulations include intranasal application of sumatriptan powder (OptiNose), iontophoretic transdermal sumatriptan, and intranasal zolmitriptan. Combination Regimens As clinical efficacy of individual symptomatic treatment options for migraine episodes discussed above is limited, the combination of multiple drugs, targeting the molecular cascades at more than one point has been tried. There are several reports showing that two drugs from different classes can act synergistically or additively. For example, efficacy of sumatriptan and naproxen combination was found significantly higher than either monotherapy [108] and this combination has been approved by the FDA. Sumatriptan and naproxen can suppress neurogenic inflammation by way of 5-HT receptor agonism and COX inhibition, respectively. Caffeine is another drug frequently used in anti-migraine drug combinations. While acetaminophen alone was not highly effective with a high NNT value, acetaminophen (1000 mg) and caffeine (130 mg) combination was found to be almost equivalent to oral sumatriptan use (50 mg) and, highly tolerable [109]. A fixed combination of aspirin, acetaminophen and caffeine (250, 250 and 65 mg respectively or combination of their double doses) has been shown to be superior to placebo [110], individual components [111], ibuprofen [112] and subcutaneous sumatriptan [113]. This combination is the first non-prescription medicine approved by the FDA for migraine treatment. Addition of caffeine to analgesics appears to produce a significant but modest effect with a NNT of about 15[114]. Dopamine antagonists metoclopramide and prochlorperazine are also part of various combination preparations to suppress nausea. Oral combinations of metoclopramide with acetaminophen or aspirin also add to their analgesic effects [39, 51]. Metoclopramide increases gastric motility and its efficacy in migraine is attributed to its effect in increasing plasma concentrations of analgesic agents [115, 116]. However, risk of extrapyramidal side effects should always be considered when administering dopamine antagonists like metoclopramide. Narcotic Analgesics Narcotics, despite being the most powerful analgesics, are not recommended for routine management of episodic migraine attacks. Besides not being more

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effective than most NSAIDs, DHE and antiemetic medications, they have been shown to cause higher recurrence rates and addiction [117 - 119]. They also cause a condition similar to medication overuse headache, named as, “opioid-induced hyperalgesia” [120]. Hyperbaric Oxygen Therapy Administration of 100% oxygen in an above-atmospheric pressure environment has been evaluated in several trials for termination of acute attacks and there is evidence that it can provide relief within 40 minutes in more that 70% of patients with no significant adverse effects [121]. Therefore it may be considered as an alternative in refractory status migrainosus, If the effect is validated in further high-quality multicenter trials. Its high cost and availability issues, however, may restrict its use to a highly selected patient group. Medication Overuse Headache Excessive use of acute migraine medications like NSAIDs, ergot alkaloids and triptans for extended periods of time may lead to a chronic form of headache named as “medication overuse headache” [122]. International Classification of Headache Disorders 3rd beta edition [123], defines this form as headache occurring at least 15 days monthly, associated with an overuse of at least one drug used for acute treatment of headache for more than 3 months. For medication overuse headache treatment, prompt cessation of the offending medication together with administration of a suitable prophylactic drug are recommended [122]. The pathophysiology of medication overuse headache and rationale treatment approaches remain to be identified. Novel Targets for Attack Treatment CGRP The first antagonist developed against CGRP receptors and tested in preclinical models was a peptide, CGRP8-37 [124] not suitable for oral use. The first smallmolecule, high-affinity CGRP antagonist, BIBN-4096 (olcegepant) was highly successful in terminating migraine headaches [125] with no significant adverse

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events and hypotensive effects [126, 127]. However, it had poor oral bioavailability and had to be administered intravenously [125], therefore, it was not tested beyond Phase-II trials. High-throughput screenings led to the development of another small-molecule CGRP antagonist, telcegepant. The major advantage of this molecule was its oral availability. A phase-III trial demonstrated its efficacy in aborting migraine headache as well as associated symptoms comparable to zolmitriptan [128]. Its efficacy against placebo was confirmed in another phase-III trial [129]. Unfortunately, due to elevated liver enzymes in some patients after long-term prophylactic use of telcagepant [130], its further clinical testing was halted. Development of another structurally related and clinically effective oral CGRP antagonist MK-3207 [131]was stopped for the same reason. Those safety issues halted further research on development of CGRP antagonists and currently there is no molecule expected to be introduced to the market in the upcoming years. However there are still some efforts on identification of new CGRP-targeting drugs. For instance, BI 44370 TA is an orally available antagonist, which was found dose-dependently effective against placebo in a Phase-II trial [132]. It has good tolerability and has also been found to be more effective in preventing relapses than eletriptan, having better rates of 24-hr pain relief [132]. BMS-927711 was also found effective compared to placebo in phase II trials [133]. The clinical efficacy and safety of those two promising drugs remains to be established in multicenter phase-III trials. There is still ongoing preclinical research to identify new candidate molecules for CGRP antagonism: MK-8825 is another potent CGRP antagonist showing good oral bioavailability in rats [134]. Thiazolidinones as highly CNS-penetrant CGRP antagonists [134a] and BMS-742413, an intranasally applicable CGRP antagonist have been tested in rabbits [135]. In addition to the small molecules, antibodies against CGRP itself or its receptor are an alternative approach. A monoclonal antibody against a C-terminal epitope of CGRP appears to block meningeal artery dilatation following dural electrical stimulation in animal models [136]. The antibody’s effect extends up to 1 week, however, it has a slow onset of action. Therefore, it could have a potential for prophylaxis rather than as an acute therapeutic [136]. It is highly likely that these antibodies primarily affect CGRP release from peripheral nociceptors as they cannot cross the blood brain barrier

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[136]. Clinical studies on CGRP antibodies are further discussed in the “preventive treatment” section. CGRP receptor is a heteromeric complex of two proteins: RAMP1, a transmembrane-spanning protein and CLR, a G-protein-coupled receptor [137]. Both components are essential for a functioning CGRP receptor. Overexpression of RAMP1 in mice generates a phenotype with enhanced tendency for photophobia and allodynia [138]. An aminoacid modification in RAMP1, for instance, reduces the binding affinity of olcegepant for CGRP receptor complex [139]. Similar findings have been reported for telcagepant as well [140]. Development of new CGRP antagonists with better efficacy and less toxicity can be facilitated by better understanding of the interactions among RAMP1, CLR, CGRP and antagonist molecules. NOS Inhibition NO is another molecule that has a potential role in migraine. NO donors are employed in several migraine models. For instance, glyceryltrinitrate (GTN) administration induces dural inflammatory changes, degranulation of mast cells [141], increased sensitivity in meningeal nociceptors [142] and TNC [143], painrelated behaviour [144], hind paw allodynia [145] and light-aversion [146]. GTN infusion also induces a migraine-like attack in human volunteers [147, 148]. iNOS inhibition suppresses plasma protein leakage from rat dural vessels after GTN infusion [141]. Nonselective inhibitors and selective nNOS inhibitors can also reverse neurogenic and CGRP-induced dural vasodilatation, whereas selective iNOS inhibitors cannot [149]. Activation of nNOS in TCC by NO donors may also point to a specific role for NO in central trigeminal activation [150]. A nonselective NOS inhibitor, L-NMMA was tested and found effective for aborting headache and photophobia in a small population of migraine patients [151], however the hypertensive effect of the drug due to eNOS inhibition prevented its clinical use [152]. Two specific iNOS inhibitors, GW274150 and GW273629 were found clinically ineffective in two Phase-II trials [153, 154]. NXN-188 is combination drug comprising a triptan plus nNOS inhibitor [155]. A single Phase-I study of NXN-188 demonstrated linear pharmacokinetics and no

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serious side effects with the drug [156]. A randomized multicenter Phase-II study testing the effectiveness of the drug against placebo and sumatriptan as the active comparator was completed in 2010 (NCT00920686) [157] but has not been published to date. TRP Channels TRP channels are emerging targets for migraine treatment based on animal experiments. These excitatory cation channels are activated by environmental stimuli like cold temperature, volatile irritants and cigarette smoke [158, 159], therefore, they are thought to underlie the increased sensitivity of migraineurs to irritants. Out of the six subfamilies of TRP receptors, TRPA1 and TRPV1 channels have been more extensively studied in preclinical migraine models. CGRP release, dilatation of meningeal blood vessels and headache-like behavior can be inhibited by selective TRPA1 antagonists (e.g. HC-030031) [160]. For TRPV1, on the other hand, there is inconsistent preclinical data: An antagonist, SB-705498 suppressed TCC activity following dural stimulation [161], whereas A993610 did not [162]. A clinical trial with the TRPV1 antagonist SB-705498 failed to show any benefit for migraine [163]. Glutamate Receptors Glutamatergic receptors, mainly AMPA, are expressed in central nociceptive neurons [164] and modulate TNC activity [165]. In accordance with a central role, AMPA antagonism by LY466195 suppressed c-fos immunoreactivity in TNC following stimulation of the trigeminal ganglion, whereas it did not inhibit dural vasodilation induced by capsaicin or peri-arterial electrical stimulation or exogenous-CGRP [166, 167]. LY293558 was found effective for acute migraine in a proof-of-concept study enrolling a small number of patients [168] but it had to be administered intravenously. A multicenter Phase II trial of BGG492, another AMPA antagonist, failed to show any superiority against placebo for 2-hr painfree response [169]. PACAP-38 PACAP-38 is a vasodilatory molecule which, when infused, can trigger migraine-

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like attacks [170]. PACAP-38 is increased in the plasma of migraineurs during ictal period compared to attack-free period [171]. It can contribute to neurogenic inflammation by causing dural vasodilation and mast cell degranulation [172], and also facilitates central nociceptive transmission by inducing CGRP release in TNC [173]. Three different receptors for PACAP have been identified: PAC1, VPAC1 and VPAC2 [174]. PACAP receptors are expressed by dural mast cells, meningeal arteries (both on smooth muscle wall and perivascular nerve fibers) and TNC neurons [174]. PACAP6-38 is a peptide antagonist of the PAC1 and VPAC2 receptors. It has been shown to inhibit PACAP-mediated vasodilation in experimental models [175, 176]. However, none of the PACAP-targeting peptides are suitable for clinical use because of lack of oral availability. Cannabinoids Anandamide, an endogenous ligand of CB1 and CB2 cannabinoid receptors, can abolish GTN-induced c-fos expression in TNC and hyperalgesic behavioral responses [177]. TCC responses evoked by dural stimulation as well as neurogenic dural vasodilatation can also be attenuated with intravenous administration of anandamide in rats [178, 179]. However, anandamide or other cannabinoid agents have not been tested in clinical trials for migraine probably due to concerns about substance abuse. The Parenchymal Inflammatory Cascade Recent evidence on the parenchymal inflammatory process triggered by CSD, leading to trigeminovascular activation identifies novel targets for migraine [18]. As noted on page 6, CSD can promote neuronal inflammasome formation and release of proinflammatory mediators HMGB1 and IL-1β [18]. Indeed, HMGB1 and IL-1β have both been shown to mediate various types of pain [180, 181]. Glycyrrhizin [182], and metformin [183] can either suppress HMGB1 release and both drugs show beneficial effects in neuropathic pain models [184, 185]. Strongly supporting a role for IL-1β in migraine headache, it has been found to be elevated during migraine attack in the internal jugular vein [186], the vein draining mainly the blood of intracranial origin in humans. Migraine attacks are frequently observed in cyropyrin-associated periodic syndrom (CAPS) [187], in

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which IL-1β is overproduced due to inflammasome mutations. Anakinra, an IL-1β receptor antagonist [188] diminishes neuronal sensitivity and pain-related behaviour in a neuropathic pain model [189]. In line with experimental data, antiIL1β treatment provides near-complete resolution of headaches and neurological symptoms in CAPS [187, 188]. Ability of anakinra to cross the BBB [190] makes it an attractive but expensive candidate for headache treatment. PROPHYLACTIC TREATMENT If migraine headache frequency exceeds one per week or if individual attacks are severe to interfere with daily activities and resistant to symptomatic therapy, prophylactic treatment should be considered [191]. It is also indicated for patients with chronic migraine or medication overuse headache. The aim of migraine prevention is to reduce the frequency as well as the severity and duration of headache episodes. A successful preventive treatment should provide at least 50% reduction in attack frequency [191]. An ideal prophylactic drug should be orally taken, have no significant side effects, drug interactions, potential for abuse or addiction and, should cause no rebound headache when treatment is ceased. The effect of preventive treatment is usually not apparent until 8 weeks and treatment should be continued for 3-12 months. Before discussing various drug classes available for migraine prophylaxis, it is appropriate to combine medical therapy with non-drug approaches for an effective prophylaxis. The patient should be aware about the nature of disease and avoidance from physical and psychological stressors as well as lifestyle modification should be assured. Keeping a headache diary may also be helpful in identification of individual triggering factors. Intake of food containing triggers like monosodium glutamate, tyramine, alcohol and excess caffeine should be reduced [192, 193]. Because of the risk of increased stroke and cardiovascular events, combined oral contraceptive use is not recommended in migraine with aura [194]. Regular sleep and meals are crucial as well as avoidance from noisy environments with bright lights. Regular physical exercise may also be beneficial [195]. There is also evidence that effect of complementary treatments like relaxation training, biofeedback, cognitive-behavioral therapy and acupuncture may be comparable to that of medical therapy [196] and this effect is even more

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amplified when both treatment methods are combined together [197, 198]. Currently Available Drugs Antiepileptics Antiepileptic drugs topiramate and valproic acid have received FDA approval and are widely used for prophylactic purpose [96]. Actions of antiepileptics in migraine pathophysiology are likely to be complex but suppression of CSDs is probably one of the main mechanisms [199]. Topiramate and valproic acid, when administered chronically for a few weeks but not when given acutely, decreases CSD induction threshold and propagation velocity in rats [199, 200]. This effect is dose-dependent and increases with longer duration of treatment [199]. These experimental data are in line with the common clinical observation that preventive effects of those drugs appear after at least 3 to 4 weeks of use [201, 202]. The delayed action might depend on the drug-induced changes in the expression of various genes and/or reorganization of synapses, which await clarification. A major clinical issue with valproic acid is its teratogenicity [203] which limits its use considerably in child-bearing age. Chronic administration of other antiepileptics has also been tested in CSD models. Lamotrigine, similar to valproic acid and topiramate exerts a suppressive effect on CSDs [204]. Gabapentin also increases CSD threshold when administered intravenously to rats, without affecting CSD duration, amplitude or propagation speed [205]. However the effect of gabapentin was shown to be acute, appearing after a single dose that is 2-4 times higher than usual human dose equivalent. Carbamazepine and oxcarbazepine had no CSD-suppressing action [206, 207] . A recent review has found that, out of a pool of antiepileptics, only topiramate and valproic acid had sufficient evidence for reducing the mean monthly headache frequency compared to placebo [208]. Both drugs decrease headache frequency by more than 50%. In line with preclinical studies, oxcarbazepine is clinically ineffective for migraine prophylaxis [209] and no clinical study has been performed with carbamazepine. Likewise, gabapentin, although suggested to be protective in one clinical trial at a 1200 mg/d dose [210], its effect was not confirmed in two later trials utilizing even higher doses [211, 212]. It has not been

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included in the recommendations list of a recent Cochrane review [208]. Antidepressants The tricyclic antidepressant amitriptyline has inhibitory effects on CSD [199]. However, its extremely complex pharmacological features make it difficult to elucidate how it acts in migraine. Like those of topiramate and valproate, CSDsuppressing as well as migraine-preventing effects of amitriptyline are not immediate [199]. Anti-migraine effect of amitriptyline is probably not directly related to its antidepressant action although both effects appear within weeks of drug use because the anti-migraine dose (25-150 mg/d) is lower than its effective antidepressant dose (100-300mg/d). However, FDA has not approved amitriptyline based on its low-class evidence for its clinical efficacy [213]. Selective serotonin-reuptake inhibitors (SSRIs, e.g. fluoxetine) or serotoninnoradrenaline reuptake inhibitors (SNRIs, e.g. duloxetine or venlafaxine) are effective antidepressant drugs. There is no convincing clinical evidence for most SSRIs in migraine prophylaxis [214, 215], although fluoxetine may have a modest effect [216]. Meanwhile, fluoxetine and citalopram were shown to slow down propagation velocity and reduce amplitude of CSDs in rats [217, 218], this effect was observed even after a single topical application of drugs and their effect on CSD threshold was not determined [217]. The effectiveness of SNRIs, has also been tested for migraine prevention. In a Class-I study, venlafaxine 150 mg/d was reported to be more effective than placebo to reduce the frequency, severity and duration of attacks at the end of 2 months [219]. The effect of 150 mg/d venlafaxine was comparable to 75 mg/d amitriptyline in a 12-week randomized crossover study [220]. However, based on this Class-I evidence, it is regarded as “probably effective” and has not yet received FDA approval. No placebo-controlled trial is available for duloxetine, an SNRI widely used to treat neuropathic pain. Milnacipran, another SNRI, has recently been tested in a small open-label study, suggesting a prophylactic effect, however, there was no placebo arm in this trial [221]. Importantly, when using SNRIs for migraine prophylaxis, the risk of serotonin syndrome with addition of triptans for acute migraine management should be considered [222].

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Beta Blockers Propranolol, a beta adrenergic blocker that can readily cross the blood-brain barrier [223] suppresses CSD on chronic administration [199]. Two-week propranolol use also relieves headache attacks induced by an NO-donor in migraine patients [224]. Effect of propranolol does not depend on its direct vascular actions, as it does not inhibit neurogenic dural vasodilatation [225]. Metoprolol is a selective beta-1 blocker again with high central nervous system penetration and effective for migraine prophylaxis [213]. Interestingly, other selective beta-1 antagonists that are BBB-impermeable, like timolol, nadolol and atenolol, still show prophylactic efficacy [213]. This attributes a role to betablockers outside the central nervous system, which remains to be identified. Propranolol at a dose of 80-240 mg/d is an established migraine preventive drug and has been approved by the FDA along with timolol [213]. Propranolol was found to be equivalent to valproic acid for prevention [226]. Beta blockers with intrinsic sympathomimetic activity, like acebutolol and pindolol are ineffective for migraine prevention [96]. Calcium Channel Blockers Flunarizine is a nonselective calcium channel blocker widely used in many countries where available. It has not been tested in currently valid CSD models, whereas supportive results had been gained from rather old preclinical studies [227]. Flunarizine may show inhibitory effects on neurogenic inflammation [228] but its exact mechanism in migraine prevention remains to be elucidated. In randomized controlled trials it was found superior to placebo [229, 230] and equivalent to propranolol [231] for migraine prophylaxis. Verapamil, a widely used L-type calcium channel blocker, is an excellent prophylatic drug for paroxysmal hemicrania and cluster headache [232]. It was also suggested to abort neurological symptoms on familial and sporadic hemiplegic migraine in case reports [233 - 235], in which voltage-gated calcium channels of the P/Q type are dysfunctional. However, data on its prophylactic benefit in non-familial migraine is conflicting and of low-quality [213]. Other nonselective calcium channel antagonists like nimodipine and diltiazem have no

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proven effect for migraine prevention [236, 237]. Glutamate Receptor Antagonists Considering that antagonism of NMDA but not AMPA receptors effectively blocks CSD initiation and propagation [206, 238], NMDA receptor antagonists could have a role in migraine prevention. Moreover, NMDA receptor antagonists, like dizocilpine maleate (MK-801) can also act on glutamatergic targets in TCC to block its activation in response to dural stimulation [239, 240]. Memantine, a weak NMDA antagonist, is already used in Alzheimer’s disease with a good safety profile and long-term clinical experience. In addition to a number of single case reports, memantine has been tested in an open-label pilot study. In 28 cases with migraine refractory to at least one approved prophylactic medication, memantine (10-20 mg/d) significantly reduced headache frequency and severity [241]. This study was, however, not placebo-controlled and patients were using other medications for migraine prophylaxis or other indications. Triptans Triptans, although being principally used for symptomatic treatment of attacks, can also be considered for migraine prophylaxis in specific circumstances. A fraction of female migraine patients suffer from attacks exclusively around the menstrual period, attributed to estradiol withdrawal. Keeping in mind that longterm use of triptans has a risk for medication overuse headache; frovatriptan could have potential for intermittant prophylaxis in menstrual-related migraine owing to its long half-life of 26h [242]. The patients can estimate timing of migraine attacks in relation to the menstrual cycle. Therefore, frovatriptan can be started one week before the beginning of menses and used daily for a week to cover this vulnerable period [213]. Once or twice daily regimens of 2.5 mg frovatriptan achieve steady-state therapeutic blood concentrations starting the second day [243]. Frovatriptan, although not approved by FDA for this indication, was found effective in reducing menstrual migraine incidence and severity in two Phase-III trials [244, 245] and is recommended by American Neurological Association (ANA) guidelines [213]. In a recent meta-analysis comparing various triptan drugs for menstrual migraine, zolmitriptan 2.5 mg (three times daily) and

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frovatriptan 2.5 mg (twice daily) regimens were superior to other triptans [246]. Novel Targets for Prophylactic Treatment CGRP Targeting CGRP may potentially be instrumental for attack prevention. In addition to its inhibitory effects on neurogenic inflammation, TCC activation and sensitization, CGRP antagonism may also reduce CSD propagation and intensity as shown in rat neocortical slices with the antagonists MK-8825 and olcegepant [206]. A Phase-II trial of CGRP receptor antagonist, telcagepant, had to be terminated due to elevated liver enzymes despite the data suggesting effectiveness against placebo beginning within 4 weeks [130]. In contrast, a recent trial of subcutaneous anti-CGRP monoclonal antibody (LY2951742) administered every 2 weeks has documented its tolerability as well as efficacy against placebo [247]. Taking the advantage of its long half-life, allowing infrequent repeat injections [136], it could be an acceptable option if further studies prove its efficacy and safety in larger patient populations. Also its use in chronic or refractory migraine should be assessed to define if it could be an option for these difficult conditions. A trial is currently recruiting participants to test the safety, tolerability and pharmacokinetics of subcutaneous form of the antibody (NCT01337596). Another humanized anti-CGRP antibody, ALD403, has recently completed its first exploratory phase-II trial, presenting no safety issues and suggesting efficacy for migraine prevention [248]. Prophylactic efficacy of one other CGRP antibody, LBR101 [249] and one anti-CGRP receptor antibody (AMG334) are currently being tested in clinical trials for chronic migraine. NOS Inhibition NOS inhibition has not yet been shown to have any benefit for migraine prevention. A selective iNOS inhibitor GW274150 was not superior to placebo at doses predicted to provide at least 80% iNOS inhibition for reducing headache frequency at the end of 12 weeks of use [250]. No data is available in the literature for the role of eNOS or nNOS inhibition in migraine prophylaxis.

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Gap Junction Blockers Tonabersat, a modulator of neuronal and glial gap junctions was shown to inhibit CSD initiation and propagation [251, 252] as well as CSD-induced NO release [253]. In a study that was prematurely terminated due to drug-induced hypotension, tonabersat exhibited no preventive effect on GTN-induced migraine attacks in a small number of patients suffering from migraine without aura studied [254]. It was also found ineffective in two multicenter Phase-II studies enrolling migraine patients with or without aura [255], one of which was not published (TEMPUS trial, NCT00534560) and in the other one, the placebo responses were unexpectedly high, complicating interpretation of the study results [255]. However, in a more recent study conducted on spontaneous migraine patients with and without aura attacks, the drug was found to significantly decrease the frequency of aura as well as headache attacks following the aura but had no effect in reducing frequency of migraine without aura [256]. Renin-Angiotensin System Incidental findings in patients with concomitant migraine and hypertension that are using drugs targeting the renin-angiotensin system (RAS) have identified these drugs as possible migraine preventive agents [257]. Several observations indirectly suggest a possible involvement of RAS in migraine pathogenesis: Migraine patients are reported to have higher interictal activity of plasma angiotensin converting enzyme [258]. Angiotensin-II can activate the inflammatory NF-κB pathway [259] and induce mast cell degranulation [260]. In turn, mast cells may release renin and increase local production of angiotensin-II in the meninges [261], creating an environment of sustained inflammation. Further supporting its potential role, angiotensin-II is co-expressed with substance-P in trigeminal ganglia neurons [262]. In randomized double-blind trials, lisinopril [257] and enalapril [263] reduced the [257] migraine frequency and headache severity by 20-50% compared to placebo and no serious side effects including hypotension apart from cough were noted. Angiotensin receptor blockers were also tested in several trials. Candesartan and telmisartan were found superior to placebo [264, 265] and candesartan was found

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equivalent to propranolol [266] for prophylaxis. These drugs seem to have effective antimigraine action at their antihypertensive doses and the results of Phase III studies with higher number patients are being awaited. Acid Sensing Ion Channels One of the recently emerging migraine targets is acid sensing ion channels (ASICs). ASICs, which sense the changes in extracellular pH are expressed in trigeminovascular system neurons, mainly in dural afferents [267]. Experimentally induced pH changes in dura in acidic direction leads to activation of ASIC3 channels and dural afferents and cutaneous allodynia in rats [268, 269]. This effect can be abolished by an ASIC blocker, amiloride, which is currently used in clinic as a potassium-sparing diuretic [268]. Moreover, amiloride has been shown to inhibit CSD with an ASIC-dependent mechanism identified in ASIC1 knockout mice as well as neurogenic vasodilatation of MMA [270]. An open-label study enrolling a small number of migraine-with-aura patients showed a possible prophylactic effect of daily intravenous amiloride, reducing frequency of both headache and aura [270]. Botulinum Neurotoxin Chronic migraine, as defined in ICHD-3 beta diagnostic criteria [123], is a rare (prevalance is 1-3% [271]) but highly debilitating condition [272] with unsatisfactory response to most therapeutics. So far, FDA has approved only one treatment for chronic migraine: botulinum toxin-A (BoNT-A, onabotulinumtoxinA) injections. BoNT-A targets the synaptosomal associated protein SNAP-25 and inhibits neurotransmitter release from cholinergic terminals [273]. Principally it is used for decreasing involuntary muscle contractions in spasticity or dystonias. BoNT-A failed to show any efficacy for episodic migraine prevention in multiple randomized controlled trials [274 - 276] and subsequent meta analyses [277, 278]. On the other hand, regarding chronic migraine, the drug passed two Phase-III trials (PREEMPT 1 and 2) enrolling a total of 1384 patients and was found superior to placebo with regard to change from the baseline number of headache days [279, 280]. It should be noted that, according to pooled analysis of PREEMPT data, the difference between reduction in monthly headache days

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between BoNT-A and placebo arms is only 1.8 days (8.4 vs 6.6 days) [281, 282]. This difference, although statistically significant, is modest [281].

ACUTE TREATMENT

Preclinical CGRP8-37 MK-8825 BMS-742313 NPITCa HC-030031 Parthenolide LY466195 Anandamide

Preclinical

PROPHYLAXIS

MK-801 Ezogabine

Phase I / Open label efficacy NXN-188 GW273629

Phase I / Open label efficacy Milnacipran Memantine Amiloride LBR-101 AMG-334 Verapamil

Single positive RCT OptiNose Olcegepant MK-3207 b BI44370TA BMS-927711 L-NMMAh LY293558 PNU-142633 Transdermal sumatriptan GW274150 SB-705498 BGG492

Single positive RCT Telcagepant b ALD403 LY2951742 (sc*) Enalapril/Lisinopril Candesartan/Telmisartan Nadolol/Atenolol Zolmitriptan MM SSRIs f Carbamazepine Oxcarbazepine GW274150 Tonabersat Pindolol/Acebutolol Lamotrigine LBR-101 * AMG334 *

Multiple positive RCTs Telcagepant b Ketorolac (iv/im) Lasmiditan Metoclopramide (iv) Aspirin Diclofenac Naproxen c Acetaminophen c

Multiple positive RCTs Venlafaxine Amitriptyline Metoprolol Flunarizine Frovatriptan MM BoNT EM Gabapentin

Approved (FDA) Ibuprofen Excedrind Ergotamine/DHE Triptanse

Approved (FDA) Valproic acid Topiramate Propranolol Timolol Methysergide g BoNT CM

* Ongoing trial Selective 5HT1D agonists PNU-142633 Selective 5-HT1F agonists Lasmiditan CGRP antagonists CGRP8-37 MK-8825 BMS-742413 MK-3207 BI 44370 TA BMS-927711 Olcagepant Telcagepant CGRP antibodies LBR-101 AMG334 ALD403 LY2951742

NOS inhibitors NPITC NXN-188 L-NMMA (+) GW274150 GW273629 TRP antagonists/partial agonists HC-030031 (+) Parthenolide SB-705498 AMPA Antagonists LY466195 LY293558 BGG492 NMDA antagonists MK-801 Memantine ASIC Blockers Amiloride

a

N-(1-(piperidin-4-yl)indolin-5yl)thiophene-2-carboximidamide b

Halted due to toxicity

c

High NNT

d Aspirin+Acetaminophen+Caffeine

combination e

Naratriptan, almotriptan, frovatriptan, sumatriptan, rizatriptan, zolmitriptan (including nasal sumatriptan and zolmitriptan; orally disintegrating rizatriptan and zolmitriptan) f

Sertraline, fluoxetine, fluvoxamine, paroxetine g

Abandoned

MM

Menstrual migraine

CM

Chronic migraine

EM

Episodic migraine

Fig. (2). An overview of migraine therapeutics. Green color indicates the success of the molecule at that stage, while red color marks failure. RCT: Randomized controlled trial, FDA: the Food and Drug Administration.

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CONCLUDING REMARKS There has been significant progress in understanding the migraine pathophysiology within the last 3 decades. Several classes of pharmacological agents designed based on the recent discoveries on migraine are in various stages of development (Fig. 2). Progress in animal models is expected to facilitate this progress by identification and targeting of novel pathways related to cortical spreading depression and trigeminovascular nociception. Therefore, we are optimistic and expect a rapid progress in the understanding and treatment of this debilitating disease in a near future. CONFLICT OF INTEREST The authors confirm that they have no conflict of interest to declare for this publication. ACKNOWLEDGEMENTS Turgay Dalkara's work is supported by Turkish Academy of Sciences. REFERENCES [1]

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CHAPTER 6

The Now and Tomorrow of Ischemic Stroke Treatment Ethem Murat Arsava1, Turgay Dalkara1,2,* 1

Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

2

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey Abstract: The last decade has witnessed a number of major developments in the field of stroke. A major breakthrough in the treatment of ischemic stroke was attained by the demonstration of improved functional outcomes with intravenous tissue plasminogen activator therapy in the hyperacute period. Recent efforts of pre-hospital thrombolysis facilitated by the use of specialized stroke ambulance systems and newer generation thrombolytics provide promising results in terms of optimizing the benefit obtained from intravenous thrombolysis. These advances, together with successful results observed by endovascular recanalization, especially by the use of thrombectomy devices, unambiguously demonstrate that the penumbra concept (presence of a salvageable ischemic brain tissue) is valid. Despite these encouraging developments, other approaches like neuro-glial protection or restoration have not been so successful in patients with acute ischemic stroke. Nonetheless, there is still hope for these therapies, especially if clinical and radiological algorithms are developed for appropriate patient selection and the recanalization is supplemented by measures aiming neuroprotection and restoration of blood flow not only at the arterial but also microcirculatory level. As for secondary prophylaxis of ischemic stroke, the availability of new anti-platelet and anti-coagulant agents combined with the progress attained in risk factor control has led to an impressively significant reduction in stroke related mortality and morbidity over the last decades.

Keywords: Anticoagulants, Antiplatelet agents, Drug treatment, Endovascular recanalization, Ischemic stroke, Microcirculation, Neuroprotection, Penumbra, Corresponding author Turgay Dalkara: Department of Neurology, Faculty of Medicine and Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey; Tel: +90 312 3052130; Fax: +90 312 3117908; Email: [email protected]

*

Atta-ur-Rahman (Ed.) All rights reserved-© 2016 Bentham Science Publishers

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Thrombolytics.

ADVANCES IN TREATMENT OF ACUTE ISCHEMIC STROKE Over the years numerous therapeutic approaches have been developed and tested in experimental models of cerebral ischemia and human stroke, with the ultimate goals of establishing recanalization/reperfusion, neuro-glial protection and prevention of secondary complications. Recanalization/Reperfusion A major breakthrough in acute ischemic stroke treatment was achieved in 1995 by the publication of the results of the National Institute of Neurological Disorders and Stroke (NINDS) recombinant tissue plasminogen activator (t-PA) study [1]. The randomized trial assessed the efficacy of intravenous t-PA against placebo treatment administered within 3 hours of symptom onset in ischemic stroke patients. Patients receiving intravenous t-PA at a dose of 0.9 mg/kg were more likely to harbor a favorable neurological outcome at 3-month follow-up in comparison to patients receiving placebo; specifically, the proportion of patients achieving a modified Rankin scale (mRS) of 0 to 1 was 42% in the t-PA arm and 27% in the placebo arm (absolute benefit ~15%, number needed to treat ~7, p=0.019) (Table 1). This benefit of intravenous t-PA treatment was observed despite an approximately tenfold increase in the rate of intracerebral hemorrhage. The findings of the study were replicated when the data was pooled together with those of other t-PA trials [2]. The analysis showed a statistically significant absolute benefit of 12% for mRS of 0-1, and 8% for mRS of 0-2 at 3 months and, 8% for mRS of 0-2 in favor of t-PA (Table 1). These publications consolidated the position of intravenous t-PA treatment as the first worldwide accepted and approved treatment strategy for acute ischemic stroke. As an additional important observation, the pooled analysis highlighted the importance of the ‘time is brain’ concept, by showing that the benefit obtained by intravenous thrombolysis being highest when the treatment was administered within 90 minutes of symptom onset. The efficacy persisted, but gradually declined and crossed the line of insignificance at 4.5 hours after symptom onset. Due to the hints of a potential efficacy between 3 and 4.5 hours after symptom onset, t-PA treatment was

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compared against placebo specifically within this time window in the ECASS-III trial [3]. Of the patients receiving intravenous thrombolysis, 52% achieved a mRS of 0-1, whereas the corresponding figure was 45% in the placebo arm (absolute benefit 7%; number needed to treat ~14) (Table 1). Based on these findings and replication of the trial data in the real-world experience [4] as well as in metaanalyses [5], intravenous t-PA treatment is currently considered as the standard of care in ischemic stroke patients who can be treated within 3 hours of symptom onset, unless there is a contraindication for the thrombolytic agent [6]. On the other hand, the use of t-PA for acute ischemic stroke within the 3-4.5 hour time window, although being recommended by major guidelines and approved by the European Medicinal Agency, is still not approved by the FDA. Despite the proven efficacy of intravenous t-PA in acute ischemic stroke care, only 4-7% of ischemic stroke patients actually receive the treatment, primarily due to late admissions to the hospital after the therapeutic window is closed [7, 8]. More importantly, the treatment can be initiated within 90 minutes of symptom onset– the golden period with the maximum benefit obtained by treatment – in only a minority of patients [9]. Therefore, a number of strategies have been developed in order to increase the rates of t-PA administration to eligible patients. Telemedicine is one of them by which rural hospitals or centers without stroke neurologists available on site can consult patients for their eligibility for intravenous thrombolysis. They can even apply the so-called ‘drip and ship’ procedure in which t-PA treatment is initiated and the patient is transferred to a stroke center thereafter. The use of telemedicine and teleradiology systems has been shown to increase t-PA administration rates and decrease the onset-totreatment time [6, 10]. In addition, by establishing strategies that promote population awareness, rapid transport and in-hospital infrastructure as well as organization, the delay between emergency department admission and treatment initiation could be cut down below 20 minutes [11]. However, recent studies have highlighted the fact that there is still room for improvement in optimizing treatment times by novel pre-hospital treatment strategies. These strategies, which probably will become an inherent part of future stroke care, make use of ambulance systems which include all the essentials for initiating on-site thrombolytic treatment: primarily a computed tomography mounted into the

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vehicle for ruling out intracerebal hemorrhage and point of care devices for a basic laboratory work-up. The initial results observed by implementation of prehospital thrombolysis have demonstrated a significant decrease in symptom onset to treatment times and this decrease hopefully will reflect positively on patient outcomes [12, 13]. Table 1. Summary of 90-day outcomes (mRS scores in % of all patients) in positive intravenous thrombolysis and endovascular treatment trials.  

 

NINDS  (1) 

iv‐tPA    Control 

NINDS,  ECASS,  ATLANTIS  pooled  (2) 

0  No  Symptoms 

2 Slight  Disability 

3 Moderate  Disability 

18    11 

1 No clinically  Significant  Disability  24   16 

8   12 

iv‐tPA    Control 

19    14 

23   16 

ECASS III  (3) 

iv‐tPA    Control 

28    22 

PROACT II  (20) 

ia‐prourokinase    Control 

MRCLEAN  (29) 

5 Severe  Disability 

6 Dead   

13   14 

4  Moderately  Severe  Disability  14    20 

6   7 

17   21 

7   11 

14   15 

12    20 

7   8 

18   18 

25   23 

14   16 

9   11 

9    14 

8   5 

7   8 

13    12 

13   5 

14   8 

12   22 

14    19 

9   7 

25   27 

Endovascular  treatment  Best medical  treatment 

3    0 

9   6 

21   13 

18   16 

22    30 

6   12 

21   22 

ESCAPE  (31) 

Endovascular  treatment  Best medical  treatment 

15    7 

21   10 

18   12 

16   15 

13    24 

7   12 

10   19 

EXTEND‐IA  (30) 

Endovascular  treatment  Best medical  treatment 

26    17 

26   11 

20   11 

17   11 

3    17 

0   11 

9   20 

SWIFT  PRIME  (32) 

Endovascular  treatment  Best medical  treatment 

17    9 

26   11 

17   16 

12   17 

15    22 

3   14 

9   12 

REVASCAT  (33) 

Endovascular  treatment  Best medical  treatment 

7    6 

18   7 

19   16 

18   19 

8    17 

12   20 

18   16 

Issues with use of t-PA in acute ischemic stroke management is not only limited to its applicability in a timely manner. Despite intravenous thrombolysis, approximately 50% of acute ischemic stroke patients die or left with moderate to

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severe disability by the end of three months after the stroke. Among many other factors, interaction of t-PA with matrix metalloproteinases is considered to contribute to its potential hemorrhagic complications [14]. Newer generation and more fibrin specific thrombolytic agents may bypass this disadvantage; The first hint in this regard have emerged by the publication of the results of a phase II study comparing tenecteplase and t-PA [15]. Tenecteplase, administered as a single intravenous bolus, rather than a bolus followed by an 1-hour infusion of tPA, did better in terms of imaging and clinical end-points in this trial [15]. Desmoteplase, is another thrombolytic that is being tested in acute ischemic stroke for some time, but has not been shown to be superior than placebo treatment when administered between 3 to 9 hours after symptom onset [16, 17]. Nonetheless, we will hear more about new generation thrombolytics in the upcoming years hoping that any one of them would appear as a better alternative to t-PA with better recanalization rates, longer time-window opportunity and lower hemorrhagic complications. The recanalization rate with intravenous thrombolysis dramatically decreases in patients with proximal cerebral artery occlusions because the clot is generally resistant to intravenously administered thrombolytic agent [18, 19]. This observation has led to the use of local (i.e. intra-arterial) means of thrombolysis. Initial efforts, aiming direct local delivery of thrombolytic agents to the site of occlusion have shown promising results; 40% of patients with middle cerebral artery occlusion receiving intraarterial pro-urokinase within 6 hours of symptom onset achieved an mRS of ≤2, while the corresponding figure was only 25% in the control arm [20] (Table 1). Despite these positive and significant findings, another phase III study was not performed to replicate these observations and therefore administration of intra-arterial thrombolytics for acute ischemic stroke has never been approved by the regulatory agencies. Nonetheless, the treatment was offered to patients giving an informed consent as an experimental treatment in many centers, and the field continued to grow by addition of intra-arterially applied mechanical devices for establishing recanalization. Mechanical approaches were not only advantageous as they led to higher recanalization rates in proximal arterial occlusions especially when compared to figures from intravenous thrombolyis trials, but also achieved this goal without interfering with the

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coagulation cascade of the system and thereby were amenable for use even in patients with increased risk of bleeding (e.g. coagulopathy, recent surgery, etc.). Some of these devices – including MERCI retriever, Penumbra system, Solitaire revascularization device, Trevo stent retriever –, based on their success and swiftness in recanalization of proximal cerebral occlusions (upto >80% recanalization rate), obtained Investigational Device Exemption approval from regulatory agencies for use in patients with acute ischemic stroke. Among these, newer generation mechanical thrombectomy devices, the so-called stent retrievers, seemed to be superior in terms of recanalization rates in comparison to older versions of thrombectomy devices [21, 22]. However, as the data primarily relied on observational registries [23 - 25] and did not come from controlled randomized trials, the issue of whether mechanical thrombectomy is efficacious in improving clinical outcome when compared to the best medical treatment (including intravenous thrombolysis) was unresolved. In this regard, MR-RESCUE study (comparing mechanical embolectomy against standard care), SYNTHESIS trial (comparing endovascular treatment against intravenous thrombolysis) and the IMS-3 trial (comparing endovascular therapy on top of intravenous thrombolysis against intravenous thrombolysis only), were unable to provide evidence in favor of endovascular therapies in patients with acute ischemic stroke [26 - 28]. The trials although being criticized heavily, among many other factors by the use of older generation thrombectomy devices which were considered as outdated by the time the trials were terminated, was a major setback for the endovascular therapy. However, the hopes bloomed by the recent publication of the results of the MR CLEAN, EXTEND-IA, ESCAPE, SWIFT PRIME and REVASCAT trials, which demonstrated a significant increase in the proportion of patients with favorable clinical outcome undergoing endovascular recanalization in comparison to the group receiving best medical treatment (Table 1) [29 - 33]. Appropriate patient selection by imaging criteria, use of modern thrombectomy devices and rapid recanalization times were considered as the major contributors to the positive findings in these trials. As majority of the patients in the endovascular groups, have also received intravenous thrombolysis as part of these trials, the approach of bridging intravenous and intraarterial treatments – which was shown to be unsuccessful per the IMS-3 trial– is still not out of the picture. Therefore, it seems that we will hear about endovascular therapies more commonly in the near future

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and this approach will remain as an important treatment modality in patients with proximal arterial occlusions and sufficient salvageable ischemic tissue. The options for establishing recanalization are not only limited to thrombolytic agents or endovascular therapies. In analogy with treatment of acute myocardial infarction, other pathways involved in fibrin formation have been targeted in ischemic stroke for the purpose of restoring flow to the ischemic cerebral tissue. These included defibrinogenating drugs like ancrod [34] and glycoprotein IIb/IIIa inhibitors like abciximab [35], tirofiban [36] and eptifibatide [37], which were administered either alone or in combination with intravenous/intraarterial thrombolysis approaches. Despite hints for some efficacy, none of these drugs provided a meaningful improvement in terms of clinical outcome and are therefore not part of acute ischemic stroke treatment algorithms for the time being. Another interesting means of recanalization that has been evaluated in ischemic stroke has been sonothrombolyis, which is performed by application of ultrasound waves to the occluded artery via transcranial ultrasound delivery systems. A phase II study has shown an increased rate of recanalization by application of ultrasound together with intravenous t-PA treatment [38]. A phase III study is underway, which will test the efficacy of a fully automated, operator independent ultrasound system for targeting the arteries in patients with ischemic stroke. As an additional concept indirectly related to recanalization but directly involved with improved flow hemodynamics, collateral augmentation strategies have been evaluated for improving cerebral perfusion in patients with ischemic stroke. Various pharmacological agents have also been used for the purpose of hemodilution, volume expansion and induced hypertension, but were either unsuccessful or still await confirmation by large clinical trials [39 - 41]. A randomized trial of mechanical perfusion augmentation by partially occluding the aorta was also unsuccessful [42]. However, efforts for augmenting cerebral collaterals are not over yet, and there are upcoming trials, which aim to improve the collateral flow by manipulating the parasympathetic nervous supply of the relevant vasculature [43]. Although recanalization has been the hallmark strategy in acute ischemic stroke management, evidence is accumulating over the last decade that recanalization may not necessarily translate to successful reperfusion at the microcirculatory

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level, a phenomenon called incomplete microcirculatory reperfusion (also known as no-reflow phenomenon) [44]. In concordance with the experimental data, observations from humans have shown that recanalization of proximal arteries may not be always accompanied by restoration of perfusion metrics within the tissue, thereby hindering favorable tissue and clinical outcome [45]. Therefore, future stroke treatment algorithms should combine strategies that will target both recanalization of arteries and reperfusion of microcirculation. Over the last decade it has also become clear that a ‘one fits all’ approach is not feasible for recanalization therapies; the ischemic vulnerability of the cerebral tissue is highly variable among humans and appropriate tools are needed to identify patients that would get the optimum benefit with minimum harm by establishing reperfusion. For now, the time criterion constitutes the backbone in patient selection for acute stroke therapies. However, the time after symptom onset, by itself only, can not pinpoint accurately the group of patients with highest benefit to harm ratio in terms of successful reperfusion; we know from experimental and human studies that reperfusion is accompanied by tissue viability and improved functional outcome if the magnitude of the irreversibly damaged tissue (i.e. core) is small and the volume of salvageable but ischemic tissue (i.e. penumbra) is high [46]. In this regard, observational studies have come up with certain tissue profiles based on diffusion and perfusion magnetic resonance imaging or computed tomography perfusion parameters that could differentiate patients with favorable and unfavorable response to thrombolytic treatments [47 - 49]. Nonetheless there are discrepant observations from randomized controlled trials regarding the benefit of such patient selection algorithms [17, 28, 30, 31]. We probably need more sophisticated algorithms considering multiple variables for implementing these systems into the clinic. These will probably include, but will not be limited to, metrics that would determine the hemorrhagic risk of the patient (e.g. by the use of permeability maps in order to assess the integrity of blood brain barrier), hemodynamic reserve of the patient (e.g. by assessing the collateral flow), magnitude of the actual clot burden (e.g. the use of clot burden scores on computed tomography or more sophisticated imaging tools like fibrin imaging by magnetic resonance imaging) and individual patient characteristics (e.g. age, admission glucose level, leukoaraisosis burden, etc.) [50 - 56].

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Neuro-Glial Protection Maintenance of the viability of neural and glial tissue during the period of hemodynamic compromise has been one of the most studied targets in ischemic stroke management. The standard supportive care for ischemic stroke including the avoidance of hypoxia, hyperthermia, hypotension, malignant arrhythmias and hyperglycemia promotes neuro-glial protection by maintaining physiologic conditions around the ischemic core [6]. However, as for specific pharmacologic agents that could fulfill this promise, no success has been attained in human stroke despite positive results observed in experimental models with numerous agents. These molecules were administered for various purposes, consisting of (but not being limited to) prevention of the excitotoxicity, free radical damage, inflammation, and interference with necrotic and apoptotic pathways. The major reasons for the negative results in human trials include execution of large scale clinical trials without sufficient preclinical and phase I/II evidence, administration of neuroprotectants at inappropriate and late time windows, and especially, their use without restoration of cerebral blood flow – which is highly critical for tissue survival [57]. Therefore, in order to prevent further disappointments, it is suggested that the development of novel neuroprotectants should strictly follow the criteria developed by Stroke Therapy Academic Industry Roundtable (STAIR) [58, 59]. Still, a number of promising developments are occurring in the field of neuro-glial protection. The FAST-MAG trial, although being a negative trial, introduced an important paradigm change for testing of pharmacological agents in the acute ischemic stroke; the initiation of therapeutic agents in the pre-hospital setting, which will probably become a standard approach in future trials [60]. Another progress in neuro-glial protection comes from the field of hypothermia, where data is rapidly accumulating. Hypothermia, the safety and feasibility of which was established in small sample sized trials, is now being tested for efficacy in phase III trials (NCT01123161, NCT01833312). The mounting evidence regarding untoward effects of inflammation on tissue prognosis following cerebral ischemia and success obtained in limiting infarct expansion in experimental studies by antiinflammatory agents, underscore the potential of these treatments for translation to humans in the near future [61].

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Prevention of Secondary Complications Secondary complications contribute significantly to morbidity and mortality following ischemic stroke and therefore should be handled with utmost care. Infections, the most common being pneumonia, is one of the leading complications. Although traditionally pneumonia is considered secondary to aspiration and the urinary tract infections, secondary to indwelling catheters, recent research has highlighted the role of neurogenic immunodepression triggered by the stroke itself as a contributing factor to infections [62]. As activation of the sympathetic system and hypothalamo-pituitary-adrenal axis has been implicated in this pathologic response, various pharmacological approaches have been attempted in order to manipulate the relevant pathways. The prophylactic use of beta-blockers, immunomodulators and antibiotics have been tested in preclinical and preliminary clinical studies, and need to be evaluated in larger cohorts to prevent these devastating complications [63, 64]. Ischemic brain edema is another frequent complication, and has a highly complex and dynamic pathophysiology. The initial cytotoxic edema, which is primarily driven by the movement of osmotically active molecules from extracelluar to intracellular space and is observed as restricted diffusion on magnetic resonance imaging studies, contributes little to elevation of intracranial pressure, as there is no net water movement into the brain parenchyma. On the other hand the ensuing ionic (secondary to sodium and accompanying water transport across blood brain barrier due to endothelial dysfunction) and vasogenic (secondary to transfer of sodium along with water and other macromolecules following breakdown of blood brain barrier) edema, is associated with elevated water content in the cerebral tissue, is reflected as decreased T1 and increased T2 signal on magnetic resonance imaging and might become a life-threatening condition in patients with large cerebral infarcts [65]. Over the years, many agents including hyperosmolar solutions have been used in order to fight with this clinical condition, yet the evidence for their benefit is rather limited [6]. On the other hand, decompressive craniotomy has been found as a life saving treatment in randomized controlled trials when applied within the first 48 hours [6]. A recent development in management of cerebral edema arose from identification of the role of sulfonylurea receptors and transient receptor potential cation channels in the

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pathophysiology of ionic edema. Preclinical studies and a case controlled human study have shown a trend for reduction in edema by the blockage of these receptors and channels by intravenous glyburide [66]. An ongoing phase II study (NCT01794182) is currently evaluating whether this observation could be replicated in a larger cohort. In summary, the future of ischemic stroke therapy in the acute period is open to a multitude of exciting advances. However, it should be emphasized that the acute stroke care needs to be performed in specialized units capable of performing all treatment approaches ranging from reperfusion to rehabilitation. The establishment of stroke centers has been shown to improve patient outcomes [6] and thereby will definitely play the central role in implementation of future therapies. ADVANCES IN SECONDARY PROPHYLAXIS OF ISCHEMIC STROKE The management algorithms designed for prevention of future strokes in patients with a history of cerebral ischemic event include three main strategies. The first focuses on optimization of cardiovascular risk factors present and includes a multitude of therapeutic plans for control of blood pressure, dyslipidemia and blood glucose, smoking cessation, reduction of heavy alcohol use, fighting with obesity, malnutrition, physical inactivity, and other issues related to cardiovascular health like obstructive sleep apnea [67]. This highly challenging task, both from the perspective of patients and treating physicians, necessitates a multi-disciplinary approach and plays a key role in minimizing recurrent vascular events including heart attacks in the post-stroke period. The wide variety of pharmaceutical agents used as part of this treatment strategy, including antihypertensives, anti-diabetics and anti-hyperlipidemics among many other drugs, will not be discussed further within the perspective of this review. The second strategy in secondary stroke prevention focuses on interventions for elimination of specific stroke etiologies. The most studied and applied intervention in this regard is carotid revascularization (either surgical or endovascular) in patients with severe and symptomatic carotid artery stenosis [67, 68]. Other interventions like left atrial appendage occlusion (in patients with atrial fibrillation who are ineligible to anticoagulation) or closure of patent foramen ovale are reserved for highly selected patient populations. The final and most commonly applied strategy in this context is the use of anti-thrombotic therapies with the aim of

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preventing recurrent thromboembolism. The first group of anti-thrombotic therapies includes drugs that interfere with aggregation and activation of platelets. Unless there is a contraindication or there is an underlying stroke etiology that necessitates anticoagulation (please see below), anti-platelet administration is a standard of care for all ischemic stroke patients [67]. The classical anti-platelet agent that has been extensively studied in the field of cardiovascular diseases is aspirin, an irreversible inhibitor of the cyclooxygenase enzyme. The recommended daily dose ranges between 50 to 325 mg, and higher doses are not recommended due to gastrointestinal side effects and no additional benefit for cardiovascular protection [67, 69]. The cyclooxygenase pathway constitutes only one of the several mechanisms that play a role in platelet activation. In this regard, there has been intensive research for agents that target these other mechanisms with the hope of more efficient platelet inhibition and better safety profile. ADP receptor antagonists, ticlopidine and clopidogrel (both belonging to the thienopyridine family) were specifically tested for their efficacy in the prevention of recurrent stroke. Based on head to head comparisons with aspirin, these agents are practically considered equivalent to aspirin in terms of efficacy [70, 71]. Ticlopidine, however, is relatively infrequently used in clinical practice due to its risk for agranulocystosis. Data regarding the use of newer generation ADP receptor blockers, like ticagrelor and prasugrel for secondary stroke prevention is currently accumulating (NCT01994720) and they seem as promising agents due to their lower degree of interaction at the level of cytochrome p450 enzymes in the liver and thereby more predictable pharmacokinetics especially when compared to clopidogrel. Another antithrombotic regimen that has been tested in the ischemic stroke is the combination of extended release dipyridamole and aspirin, which leads to inhibition of the phosphodiesterase system in platelets in addition to the effects produced by aspirin. This approach leads to significant reduction in the number of recurrent strokes both in comparison to placebo and aspirin alone [72 - 74]. However, such superiority is not evident in comparison to clopidogrel [75]. Importantly, the utility of this combination in clinical practice is diminished by its side effects including gastrointestinal intolerability and headache. Based on the evidence from aspirin trials, it is recommended to initiate anti-platelet therapy within 48 hours of

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symptom onset [76, 77]. In addition, accumulating data suggests that patients with minor ischemic stroke and transient ischemic stroke might benefit from combined anti-platelet therapy (e.g. aspirin and clopidogrel combination) in the first few months of stroke, during which the recurrence risk is highest [78, 79]. However, this advantage of combination therapy is lost in the long run due to potential hemorrhagic complications, hence, combined anti-platelet treatment regimens should not be administered for long periods of time unless definitely indicated [80, 81]. For now, aspirin, clopidogrel or aspirin/extended release dipyridamole are the most commonly used anti-platelet regimens in patients with ischemic stroke. However, new agents are expected to enter the clinic in near future. Triflusal, having both cyclooxygenase and phosphodiesterase blocking actions, has been shown to a have similar efficacy with respect to aspirin in terms of secondary stroke prevention [82]. A similar observation was attained with sarpogrelate, a 5hydroxytryptamin receptor inhibitor [83]. More importantly, studies performed in Asian populations have suggested a hint for superiority in comparison to aspirin, with the phosphodiesterase-3 inhibitor cilostazole [84]. On the other hand, the thromboxane receptor antagonist terutroban failed in its head to head comparison with aspirin in patients with ischemic stroke [85]. The utility of other newly developing anti-platelets, like protease-activated receptor antagonists, in the setting of stroke is also an active field of investigation [86]. On the other hand, for ischemic strokes secondary to fibrin-rich embolism, antithrombotic treatment with anti-platelets do not provide sufficient secondary prevention and administration of another class of anti-thrombotics, the anticoagulants is needed. The conditions that necessitate prophylaxis with anticoagulants include cardiac pathologies like atrial fibrillation, acute myocardial infarction and left ventricular thrombus, rheumatic valve disease and prosthetic heart valves, and other pathologies like hypercoagulable states and antiphospholipid antibody syndrome [67]. In contrast to anti-platelets, the issue regarding the timing for initiation of anti-coagulants following the ischemic stroke is not clear, as their use in the acute period might be complicated by intracerebral hemorrhage. Therefore, routine use of parenteral anticoagulants like heparin, lowmolecular weight heparins or heparinoids in the acute stage for secondary

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prophylaxis is not recommended except for preventing deep venous thrombosis [6]. Once the acute stage is over, however, oral anticoagulant therapy becomes the standard approach for the above listed conditions. Warfarin inhibits the synthesis of the active forms of coagulation factors II, VII, IX and X by its vitamin K epoxide reductase blocking action. Numerous trials have consistently demonstrated that warfarin is superior to placebo, anti-platelet monotherapies and anti-platelet combination therapies in prevention of cardio-embolic strokes [87 90]. Despite its well-proven efficacy, warfarin has a narrow therapeutic index, is open to numerous drug-drug and drug-food interactions, and necessitates monitoring of the prothrombin time in order to optimize the level of anticoagulation and prevent hemorrhagic complications. In the real-world scenario, approximately 50% of the patients could be kept in the therapeutic range and the treatment is associated approximately with a 1% annual risk of major bleeding. Therefore, there have been efforts to develop new oral anti-thrombotics, which do not harbor the shortcomings of warfarin therapy and more importantly have a more predictable dose-response relationship. The first agent in this class that was tested in large-scale clinical trials was ximelagatran, which specifically inhibited thrombin in the coagulation cascade. Although ximelagatran was found to be non-inferior to warfarin for prevention of strokes in the setting of nonvalvular atrial fibrillation, the marketing of the drug was halted due to its hepatotoxicity [91, 92]. This was followed by randomized trials of dabigatran (also a direct thrombin inhibitor) versus warfarin in patients with non-valvular atrial fibrillation, once again showing the non-inferiority of the newer agent, however this time without the presence of any hepatotoxicity [93]. The success obtained by development of direct thrombin inhibitors was then extended to another class of new oral anticoagulants, the factor Xa inhibitors. Three agents from this class, rivaroxaban, apixaban and edoxaban were shown to offer risk reduction for stroke and systemic embolism comparable to that of warfarin in randomized trials [94 - 96]. The efficacy of new generation oral anticoagulants in terms of preventing ischemic complications and decreasing mortality varied slightly according to the type and dosage of oral anticoagulant; however, all of these agents led to a consistent and significant reduction in the prevalence of intracerebral hemorrhage, when compared to warfarin [93 - 96]. These findings has led to the approval of dabigatran (150 mg bid or 110 mg bid [75 mg bid in

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US]), rivaroxaban (20 mg qd or 15 mg qd), apixaban (5 mg bid or 2.5 mg bid) and edoxaban (60 mg qd or 30 mg qd) by central regulatory agencies, as alternative treatments to warfarin for prevention of embolic complications in patients with non-valvular atrial fibrillation. The major advantage offered by these newer generation oral anticoagulants is the presence of a fixed dose regimen that does not necessitate any monitoring by coagulation assays. The dose needs to be lowered in patients with renal impairment, older age, lower body weight or those using pharmaceuticals that may interfere with the metabolism of these drugs. On the other hand, the lack of a specific antidote for these agents remains as a challenge during management of their bleeding complications. Therefore there is currently an intensive effort to develop fast and reliable methods for detecting the level of coagulapathy induced by new generation oral anticoagulants and control the bleeding with specific antidotes [97]. Initial large scale clinical studies have shown that it is possible to rapidly nullify the effects of the newer generation anticoagulants by antidotes like idarucizumab, andexanet alfa and aripazine [98, 99]. Other direct thrombin inhibitors and factor Xa inhibitors, together with other modulators of coagulation cascade will probably be tested for their efficacy in stroke prevention in the near future. Whether the success attained in the field of non-valvular atrial fibrillation will be extended to other conditions necessitating oral anticogulant therapy is also an active area of research. Initial efforts, however, testing the comparability of dabigatran with warfarin in patients with prosthetic heart valves have failed, and warfarin is still the treatment of choice in these patients [100]. Nonetheless, there is still a potential for new generation oral anticoagulant use in other clinical scenarios like arterial dissections and venous sinus thrombosis. In fact, there are ongoing trials testing the efficacy of rivaroxaban and dabigatran in patients with cryptogenic stroke (NCT02313909, NCT02239120). CONCLUDING REMARKS The last two decades have witnessed tremendous advances in acute stroke management and secondary stroke prevention. These developments contributed significantly to the decrease observed in mortality and morbidity related to ischemic stroke. Nonetheless, there is still need for improvement; in the acute period, there is a requirement for therapeutic approaches that would provide

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effective recanalization and reperfusion with minimal side-effects, and could be applicable to the majority of ischemic stroke patients. Similarly, the future of secondary stroke prevention would involve novel approaches that would diminish the thrombotic risk without elevating hemorrhagic complications. On the other hand, the holy grail of stroke treatment would be the establishment of therapeutic strategies that would target neuro-glial restoration and recovery. Unfortunately, no pharmacological agent except for fluoxetine was able to provide promising results in that aspect until now [101]. The demonstration of neural stem cells in cerebral tissue has raised the hopes for the use of stem cells for neuro-restorative purposes in various neurological disorders, including ischemic stroke. Nonetheless, there is still a long way ahead before such approaches could be safely implemented in clinical practice, and a combined approach involving pharmacological agents, neuro-glial stimulation, robotics and brain-computer interfaces would probably play critical roles in fulfilling these long awaited goals in the future. CONFLICT OF INTEREST The authors confirm that they have no conflict of interest to declare for this publication. ACKNOWLEDGEMENTS Turgay Dalkara's work is supported by Turkish Academy of Sciences. REFERENCES [1]

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SUBJECT INDEX

A Actovegin 228, 277, 295 AD biomarkers 115, 132, 143 AD imaging 115 Aldose-reductase inhibitors 228 Alemtuzumab 4, 45, 46, 102, 103 Alpha-lipoic acid 252, 253, 266, 278, 281, 287 Alzheimer's disease 115, 116, 162, 224 Amyloid 115, 123, 128, 130, 148, 149, 155, 166, 196, 204, 207, 212-225 Angiotensin converting enzyme inhibitors 228 Anti-inflammatory drugs 145, 201, 217, 228, 269, 292, 301, 324 Anti-Lingo-1 antibody 4 Anticoagulants 346, 370 Antiepileptics 297, 314, 339 Antioxidants 227, 228, 243, 252, 254, 258, 277, 281, 289 Antiplatelet agents 346 APOE 115, 117, 133, 134, 125, 131, 138, 142, 150, 152, 153, 160, 162, 167, 196, 197, 201, 206, 215, 217, 295 APP 115, 130, 131, 141, 146, 152, 153, 164, 166, 167, 187, 188, 198, 199, 206, 208, 210, 213, 214, 222

C C-peptide 228, 231, 247, 248, 270, 271, 286, 292, 293 CGRP antagonists 297, 309, 310 Chronic complications 228, 233, 239, 240, 251, 281

D

Daclizumab 4, 54-56, 60, 95, 106 Diabetes compensation 228, 229 Diabetes mellitus ii, 232, 233, 235, 242, 250, 266, 290, 294 Diabetic neuropathy i, ii, 240, 242, 255, 256, 259, 289-296 Disease-modifying drugs 4, 29 Drug treatment 51, 297, 330, 346

E Electrical nerve stimulation 228, 279 Endovascular recanalization 346, 351 Epigenetic modifications 228, 280, 281 Erythropoietin 152, 186, 228, 276, 277, 294, 295 Ethiopathology 4 Experimental studies 228, 354

G Growth factors 42, 228, 246, 268

H Headache ii, 41, 264, 266, 322, 323, 357 Helminthes 4 Histopathology 4, 20 Hyperexcitability i, ii, 192, 193, 205, 207, 221

I Immune system ii, 3, 4, 14, 18, 30, 38, 40, 42, 45, 46, 53, 60, 62, 65, 66, 68, 70, 73, 78, 87, 91, 111, 159, 168, 193, 197, 199, 230, 249, 282 Immunotherapy 114, 115, 162, 172, 216 Inflammation 5, 18, 19, 25, 26, 35, 61, 65, 67, 68, 75, 92, 93, 101, 106, 108, 122,

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134, 136, 161, 174, 178, 193, 197, 198, 202, 204, 207, 212, 230, 249, 250, 256, 257, 275, 276, 280, 282, 286, 297, 300, 301, 304, 305, 307, 312, 316, 318, 319, 328, 334, 335, 337, 354, 367 Intervention ii, 130, 170, 190, 192, 193, 200, 202, 208, 219, 230, 251, 262, 356 Ischemic stroke 89, 369

M Magnetic field therapy 228, 279, 296 Management ii, 75, 91, 96, 258, 259, 269, 273, 281, 293, 301, 307, 315, 349, 352, 360, 362, 363 Masinitib mesylate 4 Microcirculation 252, 268, 269, 275, 346, 353 Migraine i, ii, 280, 297, 298, 300-345 Monoclonal antibodies 4, 41, 42, 47, 52, 57, 60, 78, 102-104 MOR103 4, 58, 60 Multiple Sclerosis i, ii, 3, 4, 52, 106-112

N Neurogenic inflammation 297, 300, 301, 304, 305, 307, 312, 316, 318, 328, 335 Neuroprotection 189, 346, 366 Neurotrophic factors 228, 238, 249, 270, 273, 281 New therapies 115, 193 NSAIDs 201, 217, 270, 297, 301-303, 308, 325

O Ocrelizumab 4, 104 Ofatumumab 4, 49, 51, 52, 60, 104

P Pain relief 228, 265, 280, 296, 304, 309

Penumbra 346, 351, 353, 365 Prodromal 140, 163, 193, 201, 203, 205, 208, 217 PSEN 115, 130, 131

R Remyelination strategies 4, 74, 78 Rituximab 4, 17, 47-49, 52, 53, 60, 103-105 Ruboxistaurin 228, 271, 272, 286

S Secukinumab 4, 59, 60, 108 Spinal cord stimulation 228, 280, 296 Spreading depression 297, 300, 338-343 Stem cells 4, 52, 65, 68, 91, 110, 153, 186, 361 Synaptic 95, 121, 122, 126, 127, 139, 141, 153, 169, 173, 186, 189, 193-196, 204207, 209-215, 217, 224-226

T Tabalumab 4, 57, 60, 106, 107 Thrombolytics 346, 347, 350, 366 Tolerogenic vaccines 4, 61, 62, 78, 108 Trigeminovascular system 301, 320, 323, 328 Triptans 297, 308, 315, 317, 318, 329, 330, 340, 342

V Vaccine 72, 78, 109, 115, 154, 187, 199 Vascular endothelial growth factor 228, 247, 272, 293 Vitamin D 4, 5, 7, 8, 26, 73, 74, 79, 82, 84, 111, 112, 262, 263, 290 Vitamins 251, 252, 257, 259, 262, 281

Ischemic Stroke Treatment

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