Translational Animal Models in Drug Discovery and Development [1 ed.] 9781608054695, 9781608055913

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Translational Animal Models in Drug Discovery and Development Editor

Xinkang Wang Director Translational Science Agennix Incorporated 101 College Road East Princeton, NJ 08540 USA

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CONTENTS Foreword

i

Preface

iv

List of Contributors

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CHAPTERS 1.

Animal Models of Atherosclerosis and Spontaneous Plaque Rupture

3

Andrew R. Bond and Christopher L. Jackson 2.

Experimental Models of Heart Investigation of Cardiac Drug Safety

Failure:

Translational 24

Xinkang Wang 3.

Animal Models of Ischemic Stroke: Issues in Translational Congruency

42

Frank C. Barone, Daniel M. Rosenbaum, Jie Li, Jin Zhou and Xinkang Wang 4.

Mouse Models of Coagulation Factor Deficiencies for Translational Research

67

Meghann P. McManus and David Gailani 5.

Xenograft Models of the Normal and Malignant Human Breast

122

Michael T. Lewis 6.

BALB-neuT Female Mice as a Dynamic Model of Mammary Cancer 139 Manuela Iezzi, Raffaele A. Calogero, Michela Spadaro, Piero Musiani, Guido Forni and Fedrica Cavallo

7.

Modeling Human Asthma in Animals Jason H.T. Bates, Mercedes Rincon and Charles G. Irvin

167

8.

Cigarette Smoke-Induced Translational Animal Models of Chronic Obstructive Lung Disease 196 Andrew Churg and Joanne L. Wright

9.

Mouse Models of Inflammatory Bowel Disease: Mechanistic Insight into Current and Future Therapeutics 217 Karen L. Edelblum

10. In Vivo Veritas: Preclinical Models of Human Arthritic Disease in Non-Human Primates 247 Michel Vierboom and Elia Breedveld Index

274

i

FOREWORD The Pharmaceutical Industry has been a long time leading industry in delivering effective and safe vaccines, drugs and diagnostics. In fact, new medical entities continue to be launched to the great benefit of patients and society at large underwriting life span extension and improvement of quality of life worldwide. However, grave concerns have emerged regarding the ability of the industry to maintain the pace of innovation and productivity due to exponential R&D cost escalation and diminished revenues. These pressures emanate from patent expiration, regulatory requirements and health management cost-containment that no doubt contribute to the emerging stagnation in effective deliveries of new medical entities (NME). A direct consequence of this unfolding reality is manifested by accelerated mergers and acquisitions, driven by dwindling new products from internal R&D pipelines that force acquisitions of product from liquidated companies with the ultimate contraction in R&D and overall diminished work force of skilled professionals in the Pharmaceutical R&D sector at large. While ‘external’ factors contribute significantly to the dire situation that the pharmaceutical industry is facing, the ‘forces’ that govern this new reality are more complex and beyond the ‘external’ ones. Analysts of the sector as well as officials of experience and knowledge in the Pharma business have pointed to major deficiencies in the ‘internal’ milieu of the Pharma R&D that directly contribute to high attrition rate of products and ever growing challenges in translational medicine. These ‘internal’ R&D issues have been subject of many reviews in recent years, all pointing at deficiencies in translational principles from discovery through pre-clinical development and early clinical proof of concept studies. Such examples include insufficient target validation, lack of in depth pharmacokinetic and pharmacodynamic workout, limited use of biomarker to guide mechanism of compound action and most importantly, patients’ selection for pilot proof of concept clinical studies. This eBook on “Translational animal models in drug discovery and development” is an important addition to the subject matter – why Pharma fails to deliver innovative, effective new medicine in spite of rapidly escalating costs?

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The editor and his co-authors have placed the pendulum on a cardinal aspect in successful translation- the animal models used in pre-clinical pharmacology and safety assessment, which are a critical element in drug discovery. The editor and co-authors have focused on this important segment in drug discovery and development for the following reasons: First, while animals share genetic, biochemical, physiological and anatomical elements of some similarities to human biology, large variations in regards to humans persist in most fundamental ways. It is, therefore, imperative that prudent choices are made in regards to the extent of congruency that the chosen species carries in regards to the respective targets and systems in humans. In this regards, the excessive use of rodents, while clearly cost and time effective, as compared to larger species (dogs, pigs, non-human primates) also carries risks of more remote and diminished relevance to humans. Thus ‘positive data’ in rodents often turn to be ‘over-promising’ or misleading as also the lack of safety issues in rodents that turn to miss harmful pharmacology in humans. Second, disease processes in humans that are induced/simulated in rodents (often driven simply by costs and conveniences) could present phenotypic similarities (such as gross systemic or behavioral variables) yet often differ from the humans on mechanistic grounds, leading to grave disappointments in clinical development at painful costs and wasted time. Third, procedures that are implemented in animal models to represent the human disease often do not follow the human condition(s) in regards to: A. The correct cause for onset of the disease (even when well known); B. The time frame of the human disease evolution from initiation to recovery (or death); C. Therapeutic interventions in the animal model that are not adhering to the “targeted product profile” intended for patients; D. Safety parameters that are not studied for the duration and exposure that secure proper “therapeutic index”;

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E. Use of biomarkers for selection of “responders” vs. “non responders” that are rarely considered in animals due to the homogenous (mostly rodents) nature of the laboratory animals (especially rodents) vs. the expected large variability of humans/patients. This eBook addresses all these issues via specific case studies. Each particular case study presents to the readers the importance of global, comprehensive congruency of animal models to humans biology, disease processes as well as “targeted product profile” most suited to each disease. The editor has chosen prominent diseases where failure in translation has plagues progress in advancing medicines in these difficult diseases. Stagnation in realizing needed medicines to treat chronic heart failure, stroke, malignant neoplasms, asthma and COPD is escalating the burden of management of these diseases on national and international scale. The eBook thus adds an important reading, education and implementation guidance that is most timely and essential if Pharmaceutical productivity and innovation are to make the turn for better success in this decade.

Giora Feuerstein MD, MSc, FAHA Chief Medical and Technical Officer Department of Defense, DTRA Ft Belvoir, Virginia USA

iv

PREFACE Animal models of diseases play a pivotal role in drug discovery and development, ranging from proof of concept studies such as target validation, efficacy, pharmacokinetic and pharmacodynamic correlation to drug safety and tolerability assessment. Since considerable variables and differences exist between animal models and humans (such as genetics, physiology, anatomy, gene expression, or heterogeneity of disease conditions), none of the animal models fully and authentically represents the human biology and disease pathology. Therefore, partly due to the same reasons, enormous therapeutic agents demonstrated to be effective in preclinical models have failed in clinical studies in regards to efficacy, safety and tolerability. This eBook has undertaken the mission to address translational gap that emanates from the lack of congruency between preclinical models and humans, via systematic analyses and ‘case studies’ exemplifying animal models of disease from translational medicine perspectives. It is hoped that the readers will benefit from the specific case studies illustrated in this eBook, and eventually incorporate such experiences in their own schemes of drug discovery and development. In spite of the significant advances in modern diagnostics, therapies and improvement in patient management, a substantial number of acute and chronic diseases manifested remain significant unmet medical needs. Such diseases include chronic heart failure, malignant neoplasms, cerebrovascular diseases, and chronic lower respiratory diseases, which ranked the highest according to the latest US National Vital Statistics Reports (www.cdc.gov/nchs). While it is not possible to encompass all the diseases, we felt that focusing on three major therapeutic fields (cardiovascular diseases, cancers, and inflammatory disorders) is able to provide for sufficient representative examples that contain generic issues across other therapeutic disciplines. In all these case studies, the generic nature of “translational model” principles and their impacts on successful drug development are clearly demonstrated. The editor has been particularly fortunate to obtain high quality contribution from prominent authors with long standing experience and accomplishment in animal modeling and implementation of translational medicine

v

principles. In summation, all chapters were crafted to ‘fit for purpose’ with regard to the objective of this eBook. The first therapeutic area deals with the “number one” killer of cardiovascular diseases. Chapter 1 describes animal models of atherosclerosis, the underlying cause of most types of cardiovascular diseases. Numerous and diverse animal models of atherosclerosis have been offered over the past decades yet a key pathophysiological process, the rupture of the atherosclerotic plaque that leads to thrombus formation and vessel occlusion remains a major translational medicine issue in respect to monitoring drug effect on this enigmatic process. Heart failure is the leading cause of adult morbidity in developed countries. Animal models of heart failure are instrumental in assessing compound efficacy but much less so with regards to safety. Chapter 2 describes the strategy and provides examples for the use of heart failure models to investigate cardiac safety. Animal models of stroke are another example of most challenging disparity of congruency between preclinical models and humans. Over the last several decades, a large number of investigational compounds have demonstrated success in preclinical models but failed in late clinical studies. Chapter 3 discusses issues and opportunities for the use of animal models of ischemic stroke from translational perspectives. In contrast to stroke, coagulation factors and their roles in thrombosis and hemostasis are probably the best case to show the congruency between animal models and humans. As depicted in Chapter 4, coagulation factor deficiencies in both humans and mice are associated with remarkably similar phenotypes in regards to hemostasis and thrombosis, which could benefit significantly for the anticoagulant drug development in preclinical models. Cancer is another premier therapeutic area most suitable for translational medicine scrutiny with respect to translational medicine and personalized medicine. Tumor xenografts are the most commonly used preclinical models for efficacy in part due to the availability of many human cancer cell lines and tissues. Chapter 5 provides specific examples of xenografts using human breast cancer cell lines. While xenograft models are of value in establishing compound efficacy, limitations are apparent such as the need to use immunocompromised animals that restrict their broader translational value. Complimentary to xenografts, tumor models such as genetically engineered transgenic mice of mammary carcinoma are presented in

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Chapter 6. As shown in this example, over-expression of the rat neu oncogene drives the development of mammary carcinoma in BALB/c female mice. Because of the immunocompetent state of this model, various therapeutic agents and vaccines, including those that rely on the effective host immune system, can be evaluated successfully in the model. Inflammation is associated with many diseases, including not only those with non-immune etiological origins such as atherosclerosis, ischemic heart disease, stroke, as well as cancers as described in early chapters, but also those with immune system disorders such as asthma, inflammatory bowel disease (IBD), rheumatoid arthritis, and likewise chronic obstructive pulmonary disease (COPD). Chapter 7 presents animal models of asthma. Reproducing features of asthma in animals is a significant challenge, since the nature of this disease in humans is not fully understood: the human disease is defined mostly by phenotype and functional respiratory tests (FEV1) rather than by molecular mechanisms. Similarly, modeling COPD in animals is difficult, in part because COPD in humans often encompasses four different pathological processes (i.e., emphysema, small airway remodeling, pulmonary hypertension and chronic bronchitis) and the variable clinical syndrome of acute exacerbation. The currently available animal models only produce some, but not all, of this disease pathophysiology. Cigarette smoke-induced COPD models are described in Chapter 8 in respect to the traditional insult that underwrites the human disease. IBD involves chronic inflammation of all or part of the digestive tract. In spite of a large number of IBD models that have been developed, none of them sufficiently mimics the human disease because of the complex role of the diverse gastrointestinal flora, the genetic predisposing factors and the host immune system. Chapter 9 describes various models of IBD, their translational challenges, and their values in current and future therapeutics. Rheumatoid arthritis is one of the most frequent immune-mediated inflammatory disorders. While most rodent models of rheumatoid arthritis provide acceptable congruency to the human disease, the evolutionary distance between rodents and humans clearly impacts their translational value. Chapter 10 presents a lucid review of unique models of arthritic diseases in non-human primates. These non-human primate models are of great value in assessing both efficacy and safety pharmacology of investigational

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drugs because of their genetic, physiological, immunological and microbiological proximity to humans. In summary, this eBook is the first of its kind compilation of translational medicine discipline applied to pre-clinical modeling in drug discovery and development. We hope that the strategy and examples presented here will inspire scientists to consider practicing translational medicine discipline in all disease models used in preclinical development phases that no doubt will enhance decision points in committing investments towards clinical development.

Xinkang Wang Director Translational Science Agennix Incorporated 101 College Road East Princeton, NJ 08540 USA

viii

List of Contributors Frank C. Barone, SUNY Downstate Medical Center, Brooklyn, NY, USA Jason H.T. Bates, University of Vermont College of Medicine, Burlington, VT, USA Andrew R. Bond, University of Bristol, Bristol, UK Elia Breedveld, Biomedical Primate Research Centre, Rijswijk, The Netherlands Raffaele A. Calogero, University of Turin, Turin, Italy Fedrica Cavallo, University of Turin, Turin, Italy Andrew Churg, University of British Columbia, Vancouver, BC, Canada Karen L. Edelblum, University of Chicago, Chicago, IL, USA Guido Forni, University of Turin, Turin, Italy David Gailani, Vanderbilt University, Nashville, TN, USA Manuela Iezzi, University of Turin, Turin, Italy Charles G. Irvin, University of Vermont College of Medicine, Burlington, VT, USA Michael T. Lewis, Baylor College of Medicine, Houston, TX, USA Jie Li, SUNY Downstate Medical Center, Brooklyn, NY, USA Christopher L. Jackson, University of Bristol, Bristol, UK Meghann P. McManus, Vanderbilt University, Nashville, TN, USA Piero Musiani, University of Turin, Turin, Italy Mercedes Rincon, University of Vermont College of Medicine, Burlington, VT, USA Daniel M. Rosenbaum, SUNY Downstate Medical Center, Brooklyn, NY, USA

ix

Michela Spadaro, University of Turin, Turin, Italy Michel Vierboom, Biomedical Primate Research Centre, Rijswijk, The Netherlands Xinkang Wang, Agennix Incorporated, Princeton, NJ, USA Joanne L. Wright, University of British Columbia, Vancouver, BC, Canada Jin Zhou, SUNY Downstate Medical Center, Brooklyn, NY, USA

Send Orders of Reprints at [email protected] Translational Animal Models in Drug Discovery and Development, 2012, 3-23 3

CHAPTER 1 Animal Models of Atherosclerosis and Spontaneous Plaque Rupture Andrew R. Bond* and Christopher L. Jackson Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK Abstract: Atherosclerosis is a potentially fatal disease of the arteries affecting everyone, yet there are relatively few animal models that enable research into the events leading up to the rupture of an atherosclerotic plaque (the underlying cause of the majority of fatal thrombotic events). The apolipoprotein E knockout (ApoE-/-) mouse has been used for over a decade now, because when fed a high-fat diet it develops lesions in the brachiocephalic artery that spontaneously rupture at a known time point. Critics argue that the ApoE-/mouse does not exactly replicate human atherosclerotic plaque rupture, yet this model gives us valuable insight into the mechanisms and processes leading up to this clinically significant event. In this article, atherosclerosis shall be discussed, followed by some examples of animal models of atherosclerosis and plaque rupture used before the development of the ApoE-/- mouse model. Differences between mice and humans, and also the reasons that the ApoE-/- mouse model is of great benefit to the field of atherosclerotic plaque rupture are discussed, followed by recent translational applications of the model.

Keywords: Animal model, apolipoprotein E, atherosclerosis, cardiovascular disease, drug discovery, drug development, mouse, plaque rupture, statin, thrombosis, translational model. CARDIOVASCULAR DISEASE AND ATHEROSCLEROSIS Cardiovascular disease is the biggest global cause of morbidity and mortality. In 2009 there were 180,000 cardiovascular deaths in the UK – a third of all deaths in men and women [1]. The underlying cause of most types of cardiovascular disease is atherosclerosis. Numerous risk factors for the development of atherosclerosis have been identified. They can be divided into two groups: modifiable factors, such as obesity, tobacco *Address correspondence to Andrew R. Bond: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK; Tel: 0117 342 4139; Fax: 0117 342 2534; E-mail: [email protected] Xinkang Wang (Ed) All rights reserved-© 2012 Bentham Science Publishers

4 Translational Animal Models in Drug Discovery and Development

Bond and Jackson

smoking, physical inactivity, hypertension, hypercholesterolaemia, diabetes mellitus, and blood viscosity [2]; and non-modifiable factors, such as age, gender, family history and the menopause [3]. What Is Atherosclerosis? From a young age, even without the influence of external risk factors, low density lipoproteins (LDL) migrate from the blood into the subendothelial space in the arterial wall promoting the release of monocyte chemoattractants. Monocytes migrate through the wall and differentiate into macrophages and, along with smooth muscle cells, take up oxidised LDL becoming foam cells and forming what are known as fatty streak lesions. Fatty streaks have been detected during foetal development and maternal hypercholesterolaemia has been shown to increase the size of streak [4]. In one study all patients of 3 years of age onwards had at least minimal sudanophilic intimal deposits indicative of fatty streak development [5]. At these early ages, rather than being thought of as a disease, this process is seen more as a protective response to potential damage to the endothelium and underlying smooth muscle cells [6]. However, the location of these fatty streaks is the same as that of more advanced lesions [5], suggesting that this initial protective response eventually causes damage to the endothelium, provoking the development of intermediate lesions characterised by further accumulation of macrophages and smooth muscle cells [6]. Why Is Atherosclerosis a Problem? If atherosclerotic lesion growth occurs at a rate faster than the vessel can outwardly remodel, to accommodate the increased burden, the lumen area can decrease, leading to reduced blood flow. If the blood flow falls below the critical level needed to adequately oxygenate and perfuse an organ, the clinical consequences can be severe – myocardial infarction, stroke, or critical limb ischaemia. One way in which this clinical horizon can be reached very rapidly is if the plaque develops to the point where it ruptures, causing a thrombus to form over it. The thrombus can either restrict the lumen further, or occlude it completely, or shed emboli that occlude the artery further downstream. Thrombosis as a result of plaque rupture is thought to account for approximately 75% of acute coronary events [7]. Fortunately, some ruptures are clinically silent,

Animal Models of Atherosclerosis

Translational Animal Models in Drug Discovery and Development 5

as they do not lead to lumen-occluding thrombosis, and can heal themselves by the accumulation of smooth muscle cells at the site of rupture. This leads to secretion of fibrous extracellular matrix [8, 9] giving the plaque a striated appearance, so-called buried fibrous caps/layers (humans [10, 11], mice [12]). Plaque Vulnerability Atherosclerotic plaques can be divided into two types depending on their likelihood to rupture; vulnerable or unstable (high chance of rupture), and stable (unlikely to rupture). Based on retrospective analysis of human autopsy tissue looking at intact and disrupted plaques, those that were vulnerable had a large lipid-rich core occupying 40-50% of the plaque’s total volume, increased macrophage content and few smooth muscle cells, covered by a thin fibrous cap (1500mg/dl after 2 weeks, and ‘clinically apparent’ atherosclerotic lesions developed 5 months after initiation of fat feeding [43]. Spontaneous plaque rupture and subsequent thrombosis have been observed after fat feeding in LDL-R knockout mice, however the incidence was too infrequent for statistically accurate estimates [44]. Double knockout mice combining ApoE with LDL-R deficiency have been developed. These mice, when fed an atherogenic diet also become hypercholesterolaemic, and when exposed to mental stress or hypoxia undergo ischaemia and myocardial infarction [45]. They exhibited complex coronary lesions, but neither acute plaque rupture nor plaque-associated thrombosis.

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Non-Spontaneous Plaque Rupture Models Spontaneous plaque rupture has only been studied in animals since about 2000. Early studies into plaque rupture had to rely on mechanical or pharmacological methods to artificially induce the plaque to rupture. Examples include injecting Russell viper venom and histamine into cholesterol-fed rabbits leading to acute plaque disruption in ~30% of animals [46], implanting a balloon catheter into the thoracic aorta of a cholesterol-fed rabbit and, once an atherosclerotic lesion had developed around it at least one month later, inflating the balloon to disrupt the plaque [47]. Other models used the apolipoprotein E knockout mouse. These involved ligation of the left carotid artery followed by polyethylene cuff placement to cause ruptures with intraplaque haemorrhage and associated thrombus [48], blunt forceps to compress the lesion [49], or a needle inserted into the luminal surface of the lesion [50] to rupture the plaque. Even adenovirus expressing p53, the tumour suppressor protein, has been used to cause thinning of the fibrous cap leading to subsequent rupture when phenylephrine is given [51]. Whilst these models are all useful in determining the processes that occur after the rupture event (post rupture thrombosis, vessel remodeling), and enable us to develop potential therapies for the associated problems, they do not give us information about the events leading up to spontaneous physiological rupture. Spontaneous Plaque Rupture Models The brachiocephalic artery is a short vessel ~1.5mm long that emanates from the top of the aortic arch and branches into the right subclavian and right carotid arteries (Fig. 1). In mice it has a diameter of ~0.5-0.6mm (diastolic and systolic dimensions respectively) and in apoE knockout animals develops complex atherosclerotic lesions on the right lateral wall. Studies of ApoE-/- mice aged 4254 weeks, fed a normal chow diet, show loss of fibrous cap tissue, intraplaque haemorrhage and fibrotic conversion of necrotic zones in lesions in the brachiocephalic artery [52]. However, despite fibrous cap thinning, the plaques did not rupture. The findings of an acellular necrotic core through to the lumen may suggest a resemblance to plaque erosion, defined as loss of endothelium, leading to thrombus formation, without any demonstrable fissures or rupture [53].

Animal Models of Atherosclerosis

Translational Animal Models in Drug Discovery and Development 9

Figure 1: Resin cast of murine aortic arch and associated branches. BCA, Brachiocephalic (innominate) artery; RSC, Right subclavian artery; RCC, Right common carotid artery; LCC, Left common carotid artery; LSC, Left subclavian artery; x, Aortic Root.

Models of spontaneous plaque rupture eluded scientists until 2001 when ApoE-/mice were fed a modification of the Paigen diet that contained 21% pork lard and 0.15% cholesterol for up to 14 months [54]. These mice developed complex lesions in the brachiocephalic artery and showed clear evidence of plaque rupture (Fig. 2). Ruptures of ≤60μm in length (as shown by serial sections) were shown to occur consistently within the proximal 150μm of the vessel, enabling straightforward comparison of data across studies [55]. Brachiocephalic artery ruptures in these mice had several common features. They were relatively small, lipid-rich and globular, and were overlying large advanced lesions. They had intraplaque haemorrhages as evidenced by the presence of erythrocytes, and were associated with luminal thrombus formation [54].

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Figure 2: Stable (A) and unstable (B) murine atherosclerotic plaques in the brachiocephalic arteries of male apolipoprotein E knockout mice fed a high-fat diet for up to 9 months. Rupture site indicated with an asterix.

APOE KNOCKOUT MOUSE MODEL ADVANTAGES/DISADVANTAGES

AND

TRANSLATIONAL

In an ideal world we would be able to use ex vivo imaging techniques to track the progression of atherosclerosis from early atherogenesis up to the development of more advanced lesions, and observe the processes that lead to plaque destabilisation and rupture. According to Rudd et al. [56] the perfect imaging technique would be non-invasive, identify all plaques regardless of their impact on vessel lumen, and would characterise plaque structure as well as cellular composition. In the real world, we have to rely on human post mortem tissue and herein lies the quandary. Patients that die of cardiovascular disease have a high probability of a previous plaque rupture and the data will be skewed towards the vulnerable plaque phenotype. This issue of interpretation has been summarised as follows [57]: 1.

Patients are self-selecting – they have suffered a fatal cardiovascular event – and thus may not be representative of the general population or even of patients with non-fatal unstable plaques. This may bias any analysis towards particular underlying mechanisms.

Animal Models of Atherosclerosis Translational Animal Models in Drug Discovery and Development 11

2.

The elapse of time between symptom onset and specimen retrieval will be accompanied by changes in tissue composition.

3.

Within-subject temporal histopathological analysis is impossible, and cohort-based between-subject analysis is extremely difficult.

4.

Post hoc interpretation involves speculation that is not backed up by experimental verification.

The ApoE-/- mouse model is useful for overcoming some of these issues, as follows: 1.

It is possible to carry out longitudinal studies up to the point of rupture and observe the processes up to (and beyond) this clinically significant event.

2.

Mice can be culled at set time points and all tissue can be handled promptly and in a similar manner to minimise changes in tissue composition.

3.

It is possible to carry out temporal histopathological analysis and cohort-based between-subject analysis.

4.

It is possible to experimentally verify findings as one can go back and manipulate, or modify, one or more factors that may be involved.

Differences and Similarities between Humans and Mice An important feature of human atherosclerosis is that it occurs in the large and middle-sized arteries but extremely rarely in veins. Interestingly, if a vein is transplanted into the arterial circulation, for example during saphenous vein grafting, the vessel takes on the structure of an artery and usually develops atherosclerosis. This suggests that local haemodynamic factors, wall properties and pressure play a pivotal role in lesion formation. Further evidence for this is that atherosclerotic lesions do not occur randomly throughout the vasculature, but are found at regions of altered wall shear stress such as bends, bifurcations and Tjunctions [34]. Lesions found in mice are located at similar regions (aortic root,

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brachiocephalic artery, lesser curvature of the aortic arch, branch points of left common carotid and left subclavian arteries, iliac bifurcation, and to a lesser extent in the descending thoracic aorta) suggesting that the arterial wall is affected in the same manner, despite allometric predictions suggesting a 20-fold difference in actual wall shear stress between man and mouse [58, 59]. Humans are ~3000 times larger than mice which leads to predictable differences in cardiac mass, ventricular stroke volume and heart rate. However, cardiac mass and therefore stroke volume scale isometrically (in direct proportion to body mass) [60, 61]. Heart rate, on the other hand, scales allometrically with body mass (heart beat frequency  body mass-0.25) [62]. Human coronary arteries are typically 2.4 mm in radius with a wall thickness of 0.76 mm [63], whereas the mouse brachiocephalic artery is ~0.36 mm in radius with a wall thickness of 0.04 mm [12]. This therefore leads to very different tensile forces on the cap. Vessel diameter (or radius) is seen as one of the most important factors to determine resistance to blood flow through the vessel, as shown by Poiseuille’s equations. Poiseuille showed that the resistance to flow is inversely proportional to the tube radius raised to the fourth power, r4 [64] showing that small decreases in diameter caused by e.g., an atherosclerotic plaque, will greatly increase the resistance, and thus frictional forces of the blood flowing over the plaque. For a given radial pressure within a cylinder, there is a compensatory tension. This circumferential wall stress of a vessel is a result of the radial wall stress, the radius of the vessel, and the thickness of the artery wall. It follows that the tensile forces in the mouse lesion are a lot smaller than those in the human [65], and suggests that higher circumferential stresses can develop in a thin fibrous cap, possibly causing the mechanical failure of the plaque [66]. For two vessels with fibrous caps of the same tensile strength, caps covering mildly or moderately stenotic plaques will be exposed to greater circumferential strain, and be more prone to rupture, than those covering severely stenotic plaques [67]. Somewhat surprisingly there are important similarities between mice and humans. Murine systolic and diastolic blood pressures (125 and 90 mmHg respectively) are similar to those found in the human coronary arteries [68]. Doppler ultrasound studies show that the average peak aortic root blood velocities are strikingly similar at 1.04 m/s in mice [69] and 1.03 m/s in humans [70]. An interesting

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finding, and strong evidence in support of using ApoE-/- mice as a rupture model, is the similarity between biomechanical stress patterns in the brachiocephalic artery of mice and human vulnerable plaques, and aortic lesions (which do not show evidence of rupture in mice) when compared to stable lesions in humans, when determined using finite element models [68]. Vessel Remodeling A remarkable feature of blood vessels is that they adapt to their environment throughout life, even in the absence of lesions, maintaining a constant flow of blood through the lumen as part of a homeostatically regulated mechanism. In the presence of lesions it was first observed that in coronary [71] and femoral [72] arteries of non-human primates, and subsequently in coronary arteries of humans [73], localised radial enlargement occurs (outward, or expansive, remodeling) to compensate for the increased bulk of the lesion. Glagov noted that the vessel will only expansively remodel up to a certain point, after which the lesion mass will continue increasing and start to encroach upon the lumen. However, it has also been observed that in some cases the lumen can decrease with no apparent changes to the lesion size (inward remodeling), suggesting that the arterial wall itself actually shrinks, decreasing the cross-sectional lumen area [74, 75]. Expansive remodeling is also seen in the ApoE-/- mouse model as would be expected. Studies looking at the brachiocephalic arteries of ApoE-/- mice fed highfat diet for up to a year showed that an increase in plaque area was accompanied by an almost identical increase in vessel area [55]. Strain-matched wild-type mice fed the same diet did not develop any lesions and their brachiocephalic arteries did not expansively remodel, suggesting that this phenomenon is not simply a consequence of aging and growth. Further analysis showed that brachiocephalic arteries containing stable plaques did not increase in size (no change to external elastic lamina area), allowing the lesion to increase in size and encroach upon the lumen, causing stenosis. Unstable plaques increased in size at the same rate as stable plaques but interestingly the vessels expansively remodelled. This suggests that the growth of the plaque itself is not driving the expansive remodeling, supporting the finding that there is vessel expansion even in the absence of plaques [55]. These findings suggest that mechanical forces are perhaps

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responsible for physically tearing the fibrous cap open leading to ruptures. As the vessels remodel they put large amounts of strain on the thin fibrous cap and when the strain overcomes the cap strength a rupture ensues. These findings have been echoed in an in vivo human study using intravascular ultrasound whereby expansive remodeling (and larger plaque area) was associated with patients with unstable clinical presentation [76, 77], and negative, inward remodeling was associated with stable clinical presentation [77]. This led the researchers to the conclusion that there was ‘a greater tendency of plaques with positive remodeling to cause unstable coronary syndromes’. TRANSLATIONAL APPLICATIONS OF THE ApoE KNOCKOUT MOUSE MODEL Statins HMG-CoA reductase inhibitors (e.g., pravastatin, simvastatin, atorvastatin, fluvastatin, lovastatin) are a group of drugs that are used to lower plasma cholesterol levels, and have been shown to cause regression of atheroma [78]. HMG-CoA reductase plays a central role in the hepatic production of cholesterol. Aside from their lipid-lowering effects, statins are also thought to work by modifying endothelial function, reducing the size and stabilizing the lipid core and fibrous cap of lesions, inhibiting platelet thrombus formation and deposition, and reducing the thrombogenic response [79]. A recent clinical study showed that patients that received statins before the onset of ST-elevation myocardial infarction had a lower incidence of plaque rupture, implying stabilising effects [76]. These effects on humans have led to statins being investigated in ApoE-/mice. Earlier studies using mice on chow diet investigated the administration of simvastatin for up to 24 weeks, from 30 weeks of age, which resulted in reduced frequency of both intraplaque haemorrhage and calcification in the brachiocephalic artery, suggesting plaque stabilising effects [80]. A study using fat-fed ApoE-/- mouse showed that if pravastatin treatment was started at the same time as fat feeding (6 weeks of age), atherogenesis was impaired. However, if treatment was delayed until 16 weeks of fat feeding had elapsed, when lesions were fully established and unstable, it had no effect on plaque size but caused a decrease in lipid content, increased the thickness of fibrous cap nearly 5-fold, and decreased the incidence of plaque rupture [81].

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An alternative cholesterol-lowering agent is ezetimibe, a potent inhibitor of cholesterol absorption which acts by blocking the uptake of biliary and dietary cholesterol [82]. Clinical trials of a combination of ezetimibe with simvastatin suggest that this drug also leads to reduced atherosclerosis and adverse cardiovascular events [82]. When given to ApoE-/- mice on a high-fat diet there was a significant decrease in lesion surface area in the aortic arch, thoracic aorta, abdominal aorta, and proximal right carotid artery [83]. Unfortunately, plaque rupture was not investigated. Calcium Channel Blockers Calcium channel blockers, also called calcium antagonists, are a class of drugs that inhibit the movement of Ca2+ through L-type calcium channels, and are used clinically to treat angina pectoris, hypertension, and arrhythmia [84]. Treatment with the calcium channel blocker amlodipine produced a significant slowing in the development of carotid atherosclerosis, which was not associated with changes in blood pressure [85]. A number of key processes in atherosclerosis are also influenced by calcium channel blockers, such as lipoprotein oxidation [86], monocyte adherence and migration into the intima [87], formation of foam cells, proliferation and migration of vascular smooth muscle cells [88], and synthesis of matrix components such as collagen. It is thought that calcium-dependent factors, including cholesterol-induced changes in membrane calcium permeability and calcium deposition into lesions, may contribute to plaque formation and stability [85, 89]. These anti-atherosclerotic effects have also been seen in ApoE-/- mice treated with felodipine [84], and nifedipine [90]. However, effects on plaque vulnerability were not investigated. Bisphosphonates Bisphosphonates are used to prevent loss of bone mass, an effect mediated through their affinity for hydroxyapatite, and are used in the treatment of osteoporosis and similar diseases. Recent work is now focusing on the link between bone remodeling and atherosclerotic plaque remodeling. There is inconclusive evidence concerning bisphosphonate effects on atherosclerotic plaque vulnerability [91]. Koshiyma et al. [92] showed that patients with Type II diabetes and osteopenia treated with etidronate for a year had a significant

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decrease in carotid intima-media thickness. A recent study found that in women over 65, arterial calcification decreased after treatment, but there was an increase below the age of 65 [93]. In ApoE deficient mice fed a normal chow diet and dosed with risedronate or alendronate for 8 weeks, plaque ruptures were precipitated in the aortic root [94]. Vitamin D Vitamin D not only plays a role in bone and calcium regulation, but also in the immune system. Skin exposure to the UVB spectrum of sunlight leads to increased levels of vitamin D, and epidemiological data suggest that factors that have an influence on exposure to sunlight (i.e., geographical location, altitude, and season) are associated with altered cardiovascular disease mortality [95]. Clinical trials looking at levels of vitamin D (by measuring plasma levels of 25dihydroxyvitamin D, the principle circulating storage form of vitamin D) provide evidence that vitamin D deficiency is associated with increased incidence of cardiovascular disease, and therefore may be indicative of increased plaque rupture [96]. Further evidence for increased risk of atherosclerosis when vitamin D levels are decreased is provided by studies looking at hypovitaminosis D in type 2 diabetic patients, who are at high risk from cardiovascular disease, and measuring levels of foam cell formation, macrophage cholesterol uptake [97], and carotid intima-media thickness [98]. These studies suggest that low vitamin D increases foam cell formation and macrophage cholesterol uptake, and causes an increase in carotid intima-media thickness. The results from human studies imply that increasing the levels of vitamin D could have a beneficial effect on the incidence of cardiovascular disease and perhaps could stabilise plaques. This has been studied in ApoE-/- mice by administering calcitriol, an active form of vitamin D3, for 12 weeks. After this time, atherosclerotic lesion size, macrophage accumulation, and T-cell infiltration were all decreased, suggesting the possibility of a vitamin D-based therapeutic approach against atherosclerosis [99]. Thiazolidinediones These drugs are agonists of PPARγ. Rosiglitazone, a thiazolidinedione anti-diabetic insulin sensitizer and PPARγ agonist, has been associated with an increased risk of heart attack, though this is disputed [100]. Controversy aside, it has been shown that

Animal Models of Atherosclerosis Translational Animal Models in Drug Discovery and Development 17

in human blood, the expression of anti-inflammatory factors implicated in plaque rupture is decreased [101], suggesting the potential for increased plaque vulnerability. Experiments in fat-fed ApoE-/- mice have shown that in the aortic root rosiglitazone can reduce the vulnerability index of the plaque as well as the average number of buried layers [102]. Caution should be used when interpreting these results as the aortic root is a site not known for developing plaque ruptures. Anti-Platelet Therapy In humans, antiplatelet drugs such as clopidogrel have been shown to reduce the incidence of acute myocardial infarction, stroke and vascular death. It is unclear whether this is due to a stabilising effect on the plaque, or due to inhibition of thrombosis and subsequent embolism after the rupture event. There is little evidence that antiplatelet drugs prevent plaque rupture. A study in cholesterol fed ApoE-/- mice using clopidogrel alongside aspirin showed that in the aortic root there was no effect of either drug in the primary prevention of atherosclerosis: however, there was reduced platelet adhesion and subsequent thrombosis was decreased by 50% [103]. However, a study by Afek et al. showed that clopidogrel significantly reduces atheroma burden and stabilises plaques in the aortic root in ApoE knockout mice [104]. CONCLUSIONS Animal models are vital to the understanding of atherosclerosis, and in particular atherosclerotic plaque rupture. The fat-fed apoE knockout mouse proximal brachiocephalic artery is an excellent model to study the mechanisms and events involved when plaques begin to grow, then become more complex, and eventually rupture. There are currently relatively few well documented translational applications using this model, but wider recognition and acceptance of the model will perhaps enable more studies in the future. ACKNOWLEDGEMENT Declare none. CONFLICT OF INTEREST The author(s) confirm that this chapter content has no conflict of interest.

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Shimshi M, Abe E, Fisher EA, Zaidi M, Fallon JT. Bisphosphonates induce inflammation and rupture of atherosclerotic plaques in apolipoprotein-E null mice. Biochem Biophys Res Commun 2005; 328: 790-3. Zittermann A, Schleithoff SS, Koerfer R. Putting cardiovascular disease and vitamin D insufficiency into perspective. Br J Nutr 2005; 94: 483-92. Wang TJ, Pencina MJ, Booth SL, et al. Vitamin D deficiency and risk of cardiovascular disease. Circ 2008; 117: 503-11. Oh J, Weng S, Felton SK, et al. 1,25(OH)2 vitamin d inhibits foam cell formation and suppresses macrophage cholesterol uptake in patients with type 2 diabetes mellitus. Circ 2009; 120: 687-98. Targher G, Bertolini L, Padovani R, et al. Serum 25-hydroxyvitamin D3 concentrations and carotid artery intima-media thickness among type 2 diabetic patients. Clin Endocrinol 2006; 65: 593-7. Takeda M, Yamashita T, Sasaki N, et al. Oral administration of an active form of vitamin D3 (calcitriol) decreases atherosclerosis in mice by inducing regulatory T cells and immature dendritic cells with tolerogenic functions. Arterioscler Thromb Vasc Biol 2010; 30: 2495-503. Ajjan RA, Grant PJ. The cardiovascular safety of rosiglitazone. Expert Opin Drug Saf 2008; 7: 367-76. Mohanty P, Aljada A, Ghanim H, et al. Evidence for a potent antiinflammatory effect of rosiglitazone. J Clin Endocrinol Metab 2004; 89: 2728-35. Zhou M, Xu H, Pan L, Wen J, Liao W, Chen K. Rosiglitazone promotes atherosclerotic plaque stability in fat-fed ApoE-knockout mice. Eur J Pharmacol 2008; 590: 297-302. Schulz C, Konrad I, Sauer S, et al. Effect of chronic treatment with acetylsalicylic acid and clopidogrel on atheroprogression and atherothrombosis in ApoE-deficient mice in vivo. Thromb Haemost 2008; 99: 190-5. Afek A, Kogan E, Maysel-Auslender S, et al. Clopidogrel attenuates atheroma formation and induces a stable plaque phenotype in apolipoprotein E knockout mice. Microvasc Res 2009; 77: 364-9.

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CHAPTER 2 Experimental Models of Heart Investigation of Cardiac Drug Safety

Failure:

Translational

Xinkang Wang* Translational Science, Agennix Inc., Princeton, NJ 08540, USA Abstract: Heart failure (HF) is a major cause of morbidity and mortality. While the current therapies for inhibition of the rennin-angiotensin-aldosterone and sympathetic adrenergic systems have provided clear benefits to HF patients, the disease still progresses in most patients and development of novel therapeutics is still needed. Moreover, concerns about cardiac safety in drug development have taken center stage following recent reports of adverse cardiovascular effects with the use of PPARmodulators for the treatment of type-2 diabetes. Issues related to drug efficacy and safety call for appropriate preclinical models of HF that mirror the complex pathophysiological conditions in patients. In particular, cardiovascular drug safety is conventionally assessed pre-clinically in young healthy animals, whereas patients subjected to treatment with drugs such as PPAR-modulators often have multiple risk factors for cardiovascular disease. While a number of animal models have been developed to explore the pathophysiology of HF and to develop novel therapies, only a few reports address use of these models to assess cardiac drug safety. This chapter focuses on the use of experimental models of HF to assess drug-induced cardiovascular liability by means of biochemical, pharmacogenomic, physiological and imaging biomarkers. Rosiglitazone, a PPARactivator that has cardiovascular liability in humans, is used as an example for translational investigation in HF models.

Keywords: Animal model, biomarker, cardiac safety, drug safety, heart failure, myocardial infarction, PPAR, rosiglitazone, translational medicine, type-2 diabetes. INTRODUCTION Heart failure (HF) is a major cause of morbidity and mortality worldwide. The most recent statistics released by the American Heart Association (AHA) indicate that death rates from cardiovascular diseases have declined, in part because of currently available therapies and improved patient management, but that the *Address correspondence to Xinkang Wang: Agennix Incorporated, 100 College Road East, Princeton, NJ 08540, USA; Tel: 609-524-1017; Fax: 1-609-524-1089; E-mail: [email protected]

Xinkang Wang (Ed) All rights reserved-© 2012 Bentham Science Publishers

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Translational Animal Models in Drug Discovery and Development 25

burden of disease remains high [1]. Therefore, the development of novel therapeutics is still needed. Robust translational models of disease that mimic the complex pathophysiology of HF are required to address questions during development of novel therapeutics related to target validation, pharmacokinetic and pharmacodynamic investigation, and biomarker discovery. A number of experimental models of HF have been developed in a variety of species including rodents, rabbits, dogs, pigs and monkeys that have demonstrated value during drug discovery and development for treatment of HF [2-6]. Recent clinical observations have raised serious concerns about our capacity to assess cardiotoxicity during drug development. This is illustrated by recent findings regarding peroxisome proliferator-activated receptor-gamma (PPAR) modulators, a class of drugs used in the treatment of type-2 diabetes (T2D). PPARs are a group of nuclear receptors that function as transcriptional regulators of metabolic pathways in carbohydrate, lipid and protein metabolism [7]. The PPAR family is composed of three proteins: PPAR-, PPAR-and PPAR-. PPAR- is the molecular target of thiazolidinediones (TZD), which upon activation produce metabolic benefits in T2D [8, 9]. While diabetes itself is a risk factor for heart disease and stroke, meta-analysis of controlled clinical trials identified an increased risk of myocardial infarction and mortality associated with cardiovascular events in patients treated with the prototype TZD rosiglitazone (RGZ) [10]. Large scale clinical studies (29 trials involving 20,254 patients) showed that TZD therapy is consistently associated with a significantly higher risk of HF in patients with T2D or at high risk for T2D, [11]. However, the benefit/risk profile of TZDs remains controversial because, in addition to the benefits of improved insulin sensitivity, there appeared to be additional cardiovascular benefit with use of another TZD (pioglitazone) in several studies [1214]. To address increasing concern regarding the safety of anti-diabetic therapies containing RGZ, the United States Food and Drug Administration (FDA) restricted access to the drug by requiring the sponsor to submit a Risk Evaluation and Mitigation Strategy (REMS) [15]. The European Medicines Agency recommended in September 2010 that RGZ be suspended from use in the European market. From drug discovery and safety perspectives, it is extremely important to establish a translational model for cardiac safety assessment. However, there is a significant gap between efficacy and safety assessments for therapeutic agents like the TZDs.

26 Translational Animal Models in Drug Discovery and Development

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Efficacy is routinely conducted in animal models of diabetes that have features of human disease. Safety analysis, on the other hand, is usually performed young and healthy animals with or without diabetic background. This chapter focuses on the use of experimental models of HF to investigate cardiovascular drug safety. The effects of RGZ, a drug with known clinical cardiovascular side effects in humans, in HF models of will be discussed. TZDs are also known to be involved in fluid retention [16] and/or cardiac hypertrophy, both conditions having been observed in cardiomyocyte-specific PPARdeficient mice and in mice treated with agonists of PPAR [17]. The use of biochemical, pharmacogenomic, physiological and imaging biomarkers will be presented as they are applied to the study of drug assessment related to cardiovascular toxicity and safety. CLINICAL DEFINITION OF HF HF, also termed congestive heart failure (CHF), is a chronic condition that develops over a period of years in humans. In HF the heart cannot pump blood adequately to meet metabolic requirements. The clinical signs and symptoms of HF vary, but most patients develop fluid overload and pulmonary congestion, leading to dyspnea and orthopnea. Patients with right ventricular failure usually have jugular venous distention, peripheral edema, hepatosplenomegaly, and in more severe cases ascites. Others have signs and symptoms of low cardiac output, including fatigue, effort intolerance, cachexia, and renal insufficiency related to hypoperfusion (http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/cardiology /heart-failure/). HF may be caused by myocardial injury (e.g., myocardial infarction or dilated cardiomyopathy), chronically increased vascular resistance (e.g., hypertension, or stenosis of the aortic valve), or a high output state (e.g., severe anemia) [18]. When chronic, it is associated with multiple pathophysiological alterations and adaptations, such as marked anatomic and biochemical changes of the myocardium, left ventricular dysfunction and dilatation, increased systemic vascular resistance, and activation of neurohumoral and cytokine systems, which may aggravate myocardial injury or necrosis [18, 19]. The American College of Cardiology (ACC) and AHA have developed a classification system for HF based on clinical symptoms and diagnostic data [20]. Stage A includes patients who are at risk for developing HF but who have no

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Translational Animal Models in Drug Discovery and Development 27

structural heart disease. Stage B patients have structural heart abnormalities but do not have symptoms. Stage C patients have structural heart disease with current or prior symptoms of HF. Finally, Stage D patients have severe refractory HF. In order to determine the best course of therapy, physicians often use the New York Heart Association (NYHA) functional classification system [21], where Class I HF patients have no limitation on physical activity; Class II patients have slight limitation of physical activity, but are comfortable at rest; Class III patients have marked limitation of physical activity, even with relatively mild exertion; and Class IV patients are symptomatic at rest and may be bedbound. In addition to life style management, several types of medications are available for the treatment of HF to improve the symptoms and prolong survival, including angiotensin converting enzyme (ACE) inhibitors (e.g., captopril and enalapril), beta-blockers (e.g., carvedilol and metoprolol), digoxin and diuretics [20]. EXPERIMENTAL MODELS OF HF A number of animal models of HF have been developed in a variety of species (rodents, rabbits, dogs, pigs and monkeys) that mimic parts of the complex pathophysiology of the human condition [2-6]. Table 1 summarizes the most commonly used models of HF classified according to experimental approach: (1) induction of pressure overload (e.g., the creation of aortic or pulmonary artery stenosis by banding), (2) induction of volume overload (e.g., arteriovenous shunt or mitral regurgitation), (3) induction of myocardial ischemia (e.g., coronary artery ligation or coronary embolism), or (4) pacing-induced HF. Additional models, such as spontaneously hypertensive HF and/or Dahl salt sensitive HF models in rats (based on chronic pressure overload with no surgical operation) [22, 23], and genetically modified mice to address the genesis of cardiac arrhythmias and HF [5, 24], have been developed to study disease progress and response to therapy. Each model has advantages and limitations. While none perfectly reproduces the conditions responsible for HF in humans, they have provided new insights into many aspects of the complex pathophysiology of HF, and have been invaluable tools for investigating the efficacy of many novel therapeutic strategies.

28 Translational Animal Models in Drug Discovery and Development

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Table 1: Commonly used animal models of heart failure. Model Pressure Overload

Species Procedure Advantages rat, mouse, Aortic or pulmonary artery Simple; mechanism cat, dog, pig stenosis (e.g., banding) associated with hypertrophy and failure

Volume Overload

rat, dog, rabbit

Arteriovenous shunt, mitral regurgitation or aortic regurgitation

Myocardial rat, mouse, Ligation or embolism of Ischemia cat, dog, pig the coronary artery PacingInduced

dog, pig, rabbit

Induced by a pacer; for both ventricular and supraventricular

Weakness Lack of clinical relevance

Compensated and High cardiac output (unlike decompensated heart human heart failure) failure; rapid onset of heart failure Low cardiac output; Low survival rate; slow leading cause of heart onset of heart failure failure in patients No major surgical trauma; Mainly in large animals; reversible clinical syndrome of biventricular failure; rapid onset and reversible

In spite of the limited amount of work with these models to study the cardiac toxicity of therapeutic agents, the pathophysiological conditions of the model may suggest their value to study cardiovascular drug safety since most patients subjected to the therapeutic treatment (such as TZDs) often carry multiple risk factors of HF. The following sections discuss the use of HF models to assess cardiovascular toxicity for the agent RGZ. Myocardial Infarction-Induced HF in Rats MI is the most common precursor of HF in humans. An MI-induced HF model has been developed in male Lewis rats, involving surgical ligation of the left coronary artery [25]. Rats (~9 weeks of age) are anesthetized with pentobarbital while body temperature is maintained at 37 oC. An incision is made in the left side of the chest exposing the fourth intercostal space. A 6-0 surgical suture is placed under the left coronary artery and tied to completely occlude the vessel. Animals undergoing sham surgery without coronary artery ligation are used as a control. In animals with coronary artery ligation, approximately 50% survive. Half of the survivors are able to deliver a good quality ultrasound imaging and be used for the study. Cardiac structure and function are evaluated by echocardiography prior to surgery, and at 4 and 8 weeks post-surgery [26, 27]. A high-frequency real-time microvisualization scan-head can be used to obtain B-mode-guided parasternal

Experimental Models of Heart Failure

Translational Animal Models in Drug Discovery and Development 29

short axis M-mode images for cardiac function and structure, including ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter, systolic and diastolic (LVID), left ventricular volume (LVV; systolic and diastolic), stroke volume, cardiac output, and heart rate (HR). Echocardiographic parameters from one study are shown in Table 2 along with physiologic parameters such as heart rat, arterial blood pressure, body weight, heart weight, and lung weight in rats 4 weeks after MI-induced HF. All measured parameters were significantly different in animals with ligated coronary arteries compared to sham-treated controls with the exception of body weight and heart rate. The marked changes in cardiac structure following MI-induced HF were confirmed by histological assessment (Fig. 1). Table 2: Echocardiographic and physiological parameters in HF rats (n=6). LVIDs: left ventricular internal diameter, systolic; LVIDd: left ventricular internal diameter, diastolic; LVVs: left ventricular volume, systolic; LVVd: left ventricular volume, diastolic; HR: heart rate; BP: arterial blood pressure.

Ejection Fraction (%) Fraction Shortening (%) LVIDs (mm) LVIDd (mm) LVVs ( l) LVVd ( l) Stroke Volume ( l) Cardiac Output (ml/min)

Sham 79.6 ± 2.6 50.4 ± 2.6 4.1 ± 0.3 8.1 ± 0.1 74 ± 11 359 ± 10 285 ± 8 109 ± 4

HF 36.5 ± 0.8** 18.6 ± 0.4** 8.0 ± 0.4** 9.9 ± 0.5** 360 ± 39** 566 ± 59** 206 ± 21** 79.6 ± 9.9**

Body Weight (g) Heart Weight (mg) Heart/Body weight (mg/g) Lung Weight (g) Lung/Body weight (mg/g) HR (bmp) BP (mmHg)

399 ± 7 986 ± 19 2.3 ± 0.1 1169 ± 20 2.8 ± 0.1 379 ± 20 121 ± 4

391 ± 6 1071 ± 31* 2.5 ± 0.1* 1911 ± 393* 4.6 ± 0.9* 374 ± 8 88 ± 4**

*, p