Biopharmaceuticals [1 ed.] 9789350435298, 9788183186209

Citation preview

Biopharmaceuticals Prof. S. N. JOGDAND M. Sc.• M. Phil.

Department of Microbiology, Karrnaveer Bhaurao Patil College, Vashi, Nav; Mumbai.

( FIRST EDITION 2006 )

Hat GJlimalaya GpublishingGJIouse MUMBAI • DELHI • NAGPUR • BANGALORE • HYDERABAD

e

AUTHOR No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording and/or otherwise without the prior written permission of the author and the publisher.

ISBN: 97 8-81-83186-20-9 FIRST EDITION : 2006

Published by

Mrs. Meena Pandey

for HIMALAVA PUBLISHING HOUSE, NRamdoot", Dr. Bhalerao Marg, Girgaon, Mumbai - 400004. Phones: 23860170 & 23863863, Fax: 022-23877178

Email: [email protected] Website: www.himpub.com Branch Offices: Delhi

Nagpur

Bangalore

NPooja Apartments", 4-B, Murari Lal Street, Ansari Road, Darya Ganj, New Delhi -110 002. Phone: 23270392, Fax: 011-23256286 Kundanlal Chandak Industrial Estate, Ghat Road, Nagpur - 440018. Phone: 2721216, Telefax: 0712-2721215 No. 16/1 (Old 12/1), 1st Floor, Nextto Hotel Highlands, Madhava Nagar, Race Course Road, Bangalore - 560 001. Phones: 22281541 & 22385461, Fax: 080-22286611

Hyderabcld

No. 2-2-1 167/2H, 1st Floor, Near Railway Bridge, Tilak Nagar, Main Road, Hyderabad - 500 044. Phone: 55501745, Fax: 040-27560041

Printed by

Geetanjali Press Pvt. Ltd., Kundanlal Chandak Industrial Estate, Ghat Road, Nagpur - 440 018.

CONTENTS

1.

Introduction

2.

Market for Biopharmaceuticals

24-34

3.

Biopharmaceuticallndustry

35-41

4.

Biopharmaceutical Sector in India

42-52

5.

Drug Discovery and Drug Designing

53-66

6.

Pharmacokinetics

67-79

7.

Clinical Trials

80-89

8.

Regulatory

90-101

9.

Therapeutic Proteins

1 - 23

102-136

10.

Blood Products

137-153

11.

Monoclonal Antibodies

154-181

12.

Hormone Therapy

182-195

13.

Vaccine Production - New Developments

196-220

14.

Transgenic Production of Biopharmaceuticals

15.

Human Gene Therapy

244-265

16.

Antisense Technology (DNA Medicines)

266-277

221-243

"This page is Intentionally Left Blank"

INTRODUCTION 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.13 1.14 1.1$ 1.16

Introduction Advantages of Recombinant Biopharmaceutica1 Products Product Definition Protein Production before the Biotechnology Revolution Protein Glycosylation Cost of Production, Pricing, Pharmacoeconomics Biopharmaceutica1 Products Classification of Biologics Biotech Drugs in Development: Major Therapeutic Categories Classification of Protein Drugs Emerging Biopharmaceuticals FDA-approved Biopharmaceuticals and Vaccines Major Approved Protein Therapeutics Products Facing Patent Expiration Companies Producing Biogenerics Recent FDA Approvals

2

Biopharmaceuticals

1.1 INTRODUCTION More than 20 per cent of new medicines launched in 2003 were produced using biotechnology, and nearly 80 per cent of those under development either use biotechnology or are derived from it. The discovery of recombinant DNA and monoclonal antibody technologies in the 1970s markedthe birth of the biopharmaceutical industry. Biopharmaceuticals are complex macromolecules derived from recombinant DNA technology, cell fusion, or processes involving genetic manipulation. They include recombinant proteins, genetically engineered vaccines; therapeutic monoclonal antibodies; and nucleic acid based therapeutics (i.e. DNA based drugs), including gene therapy vectors. While small molecule drugs of traditional pharmaceuticals "re delivered orally, biopharmaceuticals are usually administered by subcutaneous, intravenous, or intramuscular injection. Proteins expressed by genes are the targets of most drugs on the market and in development. Companies look for a compound that binds to a protein, either activating it or block it from working. In some cases, a protein can also be used as a drug e.g. insulin or human growth hormone. The first drug produced via genetic engineering was human insulin, which appeared on the market in 1982. By mid-2000, 84 biopharmaceuticals had been approved for marketing with almost half launched during the past three years. Worldwide sales have grown more than seven-fold over the past decade to reach US $ 15 billion by 1998. The US represents 46% of the market; compared to 36% for conventional drugs, due to a combination of earlier regulatory approval, easier market acceptance, and greater pricing flexibility than other countries. Although biopharmaceuticals comprise only 5% of world prescription drug sales, they account for six of the top 50 selling drugs, 13% of new medicines approved by the FDA in the 1990s and about 18% of all drugs in development. At the end of 1999, there were 369 biotechnology drugs in US clinical development against 438 disease indications with 25% in Phase III. Despite decreases in regulatory approval times in the US and the EU, there has been a substantial increase in the time biopharmaceuticals spend in clinical development, from 39.4 months for products approved in the 1980's to 61.5 months in the late 1990's. A large number have also been discontinued in Phase III. Many of the earlier products were recombinant versions of natural hormones with relatively well-understood properties compared to newer biopharmaceuticals with more difficult therapeutic targets and undefmed disease mechanisms. Also, the hurdle for discontinuing development is higher for a biotechnology company because it only has a handful of products in clinical development and its lead product largely determines its stock price. The biopharmaceutical industry is a key part of the knowledge-based economy. Biopharmaceutical development, however, is inherendy a high-risk activity. BiopharmaceutiCalS are gaining significance worldwide as big pharma companies are realizing the need to fill their depleted pipelines and due to unmet medical needs. About 300 bio-ventures are in place in Korea, 200 in Australia, 170 in India, 140 in Japan, 100 each in China and Taiwan and 30 in Singapore alone. Asia is becoming a prominent region for the sector due to huge natural resources for biotech development and a huge pool of bio-scientists, positive demographic trends, market growth potential, favourable government policies and returning of scientists from the Western countries. Asian countries lack first class scientists and scientific business managers, lack strong research base, low R&D investments, shortage of private venture capital and financial skills to efficiendy allocate early-stage capital. In the US, venture capital community has a critical role in bioentrepreneurship. This critical part of value chain needs to be addressed for the Asian biotech industry to thrive. (Pharmabiz.com, 16th April 2005) In the next 15 years more than 20 per cent of chemical drugs would be replaced by biologicals/ biotech drugs.

Introduction

3

1.2 ADVANTAGES OF RECOMBINANT BIO-PHARMACEUTICAL PRODUCTS Conventionally proteins and other biological products, processed from human or animal serum or tissues, often are of low purity. On the other hand, rDNA promises around 99 per cent purity, higher efficacy (therefore lower dosage requirement) and lower side effects. The other advantages with recombinant technology are that the final protein can be changed by a slight modification of the amino acid sequence. In other words, gene expression can be modified easily with faster product innovations. Recombinant drugs can be manufactured without encountering shortfall of raw materials. Besides; companies do not have to depend on factors like shortage of donors or animals. Many products like plasma derived hepatitis vaccines, human growth hormone, erythroprotein are dependent on donors who have to pass through stringent tests to ensure good quality proteins. Recombinant technology is free from such issues. The unlimited supply of recombinant drugs means constant and steady flow of products, hence is likely to be a driving factor in the coming years. Since recombinant drugs can be produced en masse, they achieve economies of scale much faster than chemically developed/animal derived drugs. This in turn results in lesser costs of the final product. The lower price, combined with the benefits offered by them make them value for money products. For instance, recombinant erythropoietin is about 30 per cent less expensive than the currently available imported products in India. Similarly, many Asian companies (predominantly Indian & Chinese) manage to sell Hepatitis B vaccines at low prices. This has encouraged local governments to include Hepatitis B vaccme in its list for compulsory vaccines. Biotech attempts at understanding individual response to drugs. Traditional methods focused on having the same drug for the entire population. This meant savings for the manufacturer. Genetic testing could let pharmaceutical companies segment people into four groups, according to how they react to a given drug: high responders (those who respond especially well), low responders, non responders, and adverse reactants. Warner-Lambert's revenues from Rezulin would have grown much more quickly had it been possible to identify the patients susceptible to the liver problem, who could then have taken the drug provided their Jiver enzyme levels were monitored. Instead, the company lost about $200 million in potential sales while it investigated the problem. (Naik Nitin, 'Asian Biotech Industry' A checklist on challenges ahead, Pharmabiz.com, 18th April 2002). With approximately 350 products in clinical testing and more than 1,000 others in early development phases, biopharmaceuticals are growing at almost twice the rate of total pharmaceuticals.

1.3 PRODUCT DEFINITION Biopharmaceutica1 Products: Although small molecule drugs remain the standard trea.tment for disease, new strategies based on the engineering of proteins, genes and cells as therapeutic agents will revolutionize medicine in the coming decades. Areas may include gene therapeutics, tissue engineering, stem cell biology, cell-based therapeutics and gene correction technologies. There is a growing conflict between the development of small-molecule and large-molecule therapies-between the pharmaceutical industry's traditional synthetic organic chemicals and the complex proteins and antibodies that are emerging from biotechnology. Many new drug targets key on proteinprotein interactions, and traditional small molecule approach is getting abandoned. Industry sources estimate that about 30% of the drugs in the development pipeline are biopharmaceuticals-the majority at pre-clinical stages. Industry expects that the 40% failure rate for small-molecule drug candidates in late-stage clinical trials will probably translate into a bleak prospect for success in biotech drugs.

4

Biopharmaceuticals

30 biotech drugs missed critical milestones in phase IT or phase ill trials. Still, 20 biopharmaceuticals received final approval last year, and 15 received approvals for new indications. From their small base~urrently 8% of the $ 390 billion worldwide drug market-biopharmaceuticals are expected to reach 15% of a $ 550 billion market by 2006. Barriers to developing large-molecule therapies are: (i) The mainstream pharmaceutical industry is still geared to the production of small-molecule drugs and lacks the infrastructure to produce major biopharmaceutical products, (ii) Small-molecule drugs can be administered in oral dosage form, whereas large-molecule drugs are administered via injection-a less popular option with patients and (iii) The cost of producing biotech drugs. is much higher than that of manufacturing small-molecule drugs. Biologics: Biologics, include products, which may be regarded as conventional biologics and recombinant biologics. Conventional biologics consist of blood derived polyclonal antibodies and clotting factors, antibiotics, and classical vaccines based on live or killed viruses, and are frequently classified as biopharmaceuticals, but these products long predate the emergence of recombinant DNA and monoclonal antibodies. As per an estimate, there are over 1100 biologicals in pre-clinical stage, over 220 biologicals in phase I stage, over 340 in phase II stage, about 80 in phase III and about 380 are approved or waiting for approvals.

Biopharmaceuticals: Chemically synthesized small molecule drugs have been the mainstream of the traditional pharmaceutical industry. Biophannaceutica1s are complex macromolecules created through the genetic manipulation of living organisms using gene cloning, recombinant DNA (gene splicing), or cell fusion technologies. In terms of product type, these include: . • Recombinant proteins; • Recombinant antigen vaccines and vaccines crafted from genetic material such as DNA; • Therapeutic monoclonal antibodies; and • Oligonucleotides (short sequences of DNA or RNA) such as antisense molecules which interrupt the production of disease causing proteins by inhibiting gene function and gene therapy which can enhance the production of a missing protein through the addition of a synthetic gene. Conventional drugs have molecular weights of the order of several hundred daltons but biopharmaceuticals are 100 times or more larger e.g. interferons (20,000), interleukins (15,000), tPA (70,000), and Factor VIII and several monoclonal antibodies (close to a million). The first recombinant protein (human insulin) was launched in 1982, the first recombinant vaccine (against hepatitis B) in 1986, the first therapeutic monoclonal antibody (against kidney transplant rejection) also in 1986, and the first and only oligonucleotide in 1998 (against Cytomegalovirus retinitis in AIDS patients). No gene therapy product has yet been approved. Recombinant proteins dominate the biopharmaceutical market accounting for the bulk of sales to date. Generics (or, more appropriately, multisource pharmaceuticals) are typically defined as pharmaceutical preparations that: • are essentially similar to an original product; • involve an active substance with expired patent protection; • are approved through a simplified registration process; and • sell under a common name typically with very little (if any) promotional activities.

Introduction

5

Essentially, generic pharmaceutical companies offer consumers a cheaper version of a product for which patent protection has expired. Within this framework, a critical success factor is represented by the ability to develop the generic copy with relatively modest investments, in order to make the economics work. Generics are prescription medicines that have lost patent protection and are being marketed by one or more companies that did not originally develop the drug. They are essentially similar to the original product, are approved through a simplified regulatory process and are sold under a common name with very little promotion. By extension, the term 'BiogenericsJ is used for pharmaceutical preparations (involving a biologically active substance) stemming from modern biotech tools. As with pharmaceutical generics, these biogenerics are essentially similar to an original biopharmaceutical whose substance patent has expired, are approved through a simplified abbreviated registration process, and are sold under the generic substance name as opposed to a brand name (e.g., EPO as opposed to Epogen). Biogenerics refers to therapeutic products based on genetically engineered or recombinant technologies. Biogenerics are generic versions of pharmaceutical preparations involving a biologically active substance that has generally been created using modern biotech tools. These products are still in their infancy due to the relatively recent patent expirations of the first branded set of biotech products. Biotech-engineered drugs are products based on large molecule proteins. On the other hand, generics generally refer to non-biologi 1,000

$ 40-65

$ 32-55

1,000-5,000 30-100

< 40

(a) These costs are for bulk active purified proteins but do not include the fill and finishing costs associated with the production of biopharmaceuticals. (b) Many complex proteins cannot be expressed in active form by bacterial fermentation. Source: Nexia Biotechnologies Prospectus 2000.

Economics of Protein Production CHO Cells (lg/litre)

Transgenic (Sg/litre)

Capital Investment ($ MM) 100kg 500kg

$ 20 $ 75

$5 $ 10

COGS ($/g) 100kg 500kg

$ 500 $ 200

$ 100 $ 40

Source: SG Cowen, Genzyme Transgenics.

There is also the risk factor to consider. The drug discovery value chain can be broken down into five stages: from target discovery (5000-10,000) to lead discovery (500-1000) to clinical trials, one has about five from the original 10,000, making it to phase III and, possibly, one goes to the manufacturing stage, to the market. Biopharmaceuticals with exception of insulin are much more expensive than small molecule drugs. Monoclonal antibodies, for example, are in the range of US $ 15,000 to $ 25,000 per year, estimates for ex vivo gene therapy are over US $ 100,000, while certain orphan drugs e.g. genetically engineered blood clotting factor for heamophiliacs can cost as much as US $ 150,000 per year. The new generation of vaccines will not be affordable outside industrialized countries. Governments and third party payers are increasingly demanding evidence that pharmaceuticals generate benefits commensurate with their high costs .(e.g. reduced public health care expenditures, decreased mortality, functional improvement, increased quality of life, or shorter length of therapy). Ontario, BC, a number of countries (Australia, New Zealand, Finland, UK, France, Italy, Norway and the Netherlands), and several US managed care groups (e.g. Blue Cross, Blue Shield) have instituted mandatory cost-effectiveness submission guidelines. However, comparisons are difficult because of the lack of standard study methodologies, the use of placebos in clinical trials, problems in measuring quality of life benefits and improved workplace productivity, and in accounting for differences in the cost of health care inputs from country to country. Clinical trials have generally not been aimed at demonstrating long-term health outcomes and cost savings so the need to capture this additional information will increase trial costs and extend development times.

Biopharmaceuticals

10

1.7 BIOPHARMACEUTICAL PRODUCTS NfJ.

Examples

1.

Antianaemic Agents

Erythropoietin (EPO)

2.

Antihaemophilic Agents

Factor VII, Factor VIII, Factor IX, Tissue Factor, von Willebrand Factor

3.

Anti-inflarrunatory Agents

Angiogenic Factors, Inhibitors of Proteolysis, Colony Stimulating Factors, Transforming Growth Factors, HeparinBinding Growth Factors, Interferon-Gamma, Interleukins, Lipocortins, Lysozyme, Superoxide Dismutase, Tumor Necrosis Factor

4.

Antineoplastic Agents

Angiogenic Factors, Aproliferin, Inhibitors of Proteolysis, Colony Stimulating Factors, Interleukin-3, Granulocyte/ Macrophage-Colony Stimulating Factor, GranulocyteColony Stimulating Factor, Macrophage-Colony Stimulating Factor, Endothelial Cell Growth Factors, Epidermal Growth Factor, Erythroid Differentiation Factor, Interleukins, Interferons, Transforming Growth Factors, Tumour Necrosis Factors, Laminin, Monoclonal Antibodies

5.

Antithrombotic and Fibrinolytic Agents

Hirudin, Protein C, Protein S, Thrombomodulin, Pro-Urokinase and Urokinase, Streptokinase, Tissue Plasminogen Activator, Antithrombin III

6.

Antiviral Agents

Interferons, Tumour Necrosis Factors, Antisense Nucleic Acids, Recombinant Vaccines

7.

Cardiovas~lar

Alpha-I-antitrypsin, Angiogenic Factors, Apolipoproteins, Atrial Natriuretic Factor, Calcitonin Gene-Related Peptide, Lipid Exchange Proteins, Superoxide Dismutase

8.

Growth Factors

Nerve Growth Factor, Osteogenic Factors

9.

Hormone Replacement Therapies

Insulin, Proinsulin, Growth Hormone, Calcitonin

10.

Immunomodulators

Colony Stimulating Factors, Interferons, Interleukins, Tissue Necrosis Factors, Monoclonal Antibodies, Catalytic Antibodies

11.

Wound Healing Accelerators

Epidermal and Fibroblast Growth Factors (EGF and FGF), Platelet-Derived Growth Factor, Transforming Growth Factors, Somatomedinsflnsulin-Like Growth Factors

12.

Other Biopharmaceuticals

Analgesics, Gastrointestinal Agents, Anti-obesity Agents, Respiratory Agents, Elastase Inhibitors, Superoxide Dismutase, Human Pulmonary Surfactant Protein, Atrial Natriuretic Peptide, Sex Hormones and Related Products, Gonadotrophins and their Releasing Factors, Inhibins, Vasoactive Intestinal Polypeptide

System-Related Agents

Introduction

11

1.8 CLASSIFICATION OF BIOLOGICS No

Category

Class

(I) Blood and Blood Derivatives • Cellular Components. • Stem Cells. Platelets. Packed Red Blood Cells. Plasma Fractions - Human Serum Albumins. Plasma Fractions - Immune Gloulins. • Plasma Fractions - Other Plasma Products. Whole Blood. Blood Substitutes.

• • • • • • 1.

Conventional Biologics

(II) Conventional Protein Drugs. • Anticoagulants. Hormones. Antineoplastic Agents. Enzymes. • Other Conventional Proteins.

• • •

(III) Conventional Vaccines Pneumococcal Vaccine. DtaP Vaccine. MMR Vaccine. Influenza Vaccine. • Hepatitis A Vaccine. • Vercella (Chickenpox) Vaccine. Cancer Vaccine. AIDS Vaccine. Other Conventional Vaccines. Veterinary Vaccines.

• • • • • • •

•

2.

Recombinant Proteins

(IV) Diagnostic Reagents • Enzymes. Hormones. Antigens and Antibodies. Hematology Reagents.

• •

•

(I) Blood Related Agents. • Eryhropoietins. Colony Stimulating Factors. Antihemophilic Factor. • Thrombolytic Agents.

• •

3

Recombinant Vaccines

(II) Recombinant Hormones. • Human Insulin. Huffian Growth Hormone. Fertilty Hormones. All Other Hormones Interferons.

• • •

Biopharmaceuticals

12 No

Class

(III) Other Recombinant Proteins. • Enzymes. Monoclonal Antibodies

4

Recombinant Hepatitis B Vaccine Other Recombinant Vaccines. • In Vitro Diagnostic Monoclonal Antibodies. • In Vivo Diagnostic Monoclonal Antibodies. • Therapeutic Monoclonal Antibodies. • Anti-cancer Monoclonal Antibodies. • Anti-inflammatory monoclonal Antibodies. • Other Therapeutic Monoclonal Antibodies. - Anti-transplant Rejection Monoclonals Thrombolytic Agents - Monoclonal Antibodies.

1.9 BIOTECH DRUGS IN DEVELOPMENT: MAJOR THERAPEUTIC CATEGORIES Therapeutic Area

175 45 25 20 20 18 15 15

Oncology Infectious Diseases Autoimmune Diseases AIDSjHIV eNS Disorders Respiratory Disorders Hear Disease Skin Disorders

1.10 CLASSIFICATION OF PROTEIN DRUGS No

Category

1

Antibiotics

2 3

Antibodies Blood Proteins Enzyme inhibitors Enzymes Hormones Lymphokines Other Protein drugs Vaccines

4

5 6

7 8 9

Introduction

13

Proteins in living organisms are classified according to their biological roles. These include: • Enzymatic - proteins that trigger all the chemical reactions that occur in the cells of living organisms. • Transport - proteins that carry other substances throughout the body or molecules aC{"Qss cell membranes. For instance, the protein haemoglobin carries oxygen from the lungs to other parts of the body. • Structural- proteins that help in supporting functions in the body. For instance, Keratin is the protein that is important for supporting hair and other skin parts. • Storage - proteins that store amino acids. For instance, casein is the protein in milk that provides a source of amino acids for baby mammals. • Hormonal - proteins that coordinate bodily activities. For instance, insulin is the protein hormone secreted by the pancreas that regulates the level of sugar in the blood. • Receptor - proteins that are built into the membrane of a cell and detect chemical signals released by other cells. They contribute to the cell's response to the chemical stimuli. • Contractile - proteins that help in movement. For example, actin and myosin are responsible for the movement of muscles. • Defensive - protect the body against diseases. For example, antibodies are proteins that protect the body against viruses and harmful bacteria. • Immunoregulators - Proteins that act as molecular messengers in cell-cell interactions for immune response. For example interleukins. Protein-based drugs are: Cytokines, Hormones, Clotting Factors, Vaccines, Monoclonal Antibodies.

1.11 EMERGING BIOPHARMACEUTICALS Mimetics and peptidomimetics (small molecule compounds that mimic proteins and peptides) have advantages over proteins in terms of ease of delivery and cost, but there are considerable technical challenges in mimicking the large and intricate protein-protein interacting surfaces and overcoming the weak binding forces of small molecules, a major cause of side effects. Examples include mimetics of insulin and granulocyte colony stimulating factor (in early stage development), peptide based mimetics of erythropoietin (late stage), and Montreal based Neurochem's mimetics of glycosaminoglycans (GAGs - complex carbohydrates that promote amyloid fibril formation characteristic of Alzheimer's) which compete with naturally occurring GAGs to inhibit deposition of amyloids (early stage). Glycotherapeutics (complex sugars/carbohydrates) have significant potential both as drugs and drug targets because of their diverse biological roles (cell surface carbohydrates act as binding sites for other molecules, playing structural roles in cancer transformation, immune system regulation, tissue repair, and anti-infection responses, as well as genetic disorders such as Gaucher's and Fabry'S diseases) and their enormous structural diversity (more than 10 million polysaccharides can be formed from nine common monosaccharides compared to 16,000 peptides from 20 amino acids). However, their application as biopharmaceuticals has lagged far behind protein drugs because they are difficult molecules to analyse and synthesize. Fully Human Monoclonal Antibodies First generation mouse-based antibodies provoked a strong immune reaction in humans limiting their clinical utility. Strategies to generate more human compatible antibodies include mouse-human chimeras (60%-70% human protein) first introduced in 1995, followed by humanized antibodies (90%-95% human protein) in 1998 in which certain mouse antibody amino acid fragments are grafted onto a human antibody. The next generation now in clinical

14

Biophannaceuticals

development are fully human antibodies produced via methods such as transgenic mice containing human antibody genes, phage display, or collection of human B cells from infected individuals which are then fused with human tumor cells to create antibody producing hybridomas. Because of their high costs and large dose requirements, advances in manufacturing capability are needed to meet market demand. Vaccines Conventional vaccines based on inactivated (killed) viruses; live, attenuated (weakened) viruses; microbial toxoids; or antigenic fragments (subunit vaccines), have a number of disadvantages. 11 New vaccine' strategies include:

• recombinant antigen vaccines where the antigen is produced in bacteria or yeast rather than extracting it from chronic human or animal carriers (e.g. hepatitis B); • recombinant vector vaccines using weakened viruses such as poliovirus, vaCCInIa virus or salmonella bacterium as carriers of the disease causing organism to overcome the inability of inactivated whole antigens or recombinant antigen vaccines.to enter cells and express the desired gene product (hepatitis B, HIV, herpes simplex, in development); • DNA vaccines, produced from a core gene of a virus, are effective against those pathogens that change their exterior protein coat by attacking the interior proteins of a virus (HIV, malaria, and a number of anticancer applications are the closest to commercialization); and • RNA vaccines (particularly applicable to yellow fever and polio caused by RNA viruses). Genomics has the potential to improve the effectiveness of existing vaccines and ot:sign vaccines for diseases for which no protection currently exists: by sequencing invading bacterial and viral organisms, those critical genes that encode the proteins that cause disease can be identified, leading to the development of novel and more effective antigens. Complete DNA sequences of most human pathogenic viruses and more than 20 bacterial pathogens have already been determined. The first genomics derived vaccine (Group B meningitis) developed by Chiron is expected to enter clinical trials in 2001. Vaccines are also being developed as anti-cancer agents, involving attempts to activate immune responses against antigens in the tumour to which the immune system has already been exposed. Research is also being undertaken on edible vaccines e.g. transgenic bananas in order to offer an improved mode of delivery. Gene therapy Whereas recombinant proteins and antibodies are produced outside the body and administered to the patient,gene therapy has the potential for the body to produce the desired protein on its own if a synthetic gene with the correct DNA sequence for that protein can be inserted into targeted defective cells. A number of major challenges have to be solved: design of a safe and effective gene delivery system, the short duration of gene expression, control over where the transferred gene is expressed, and possible immune reaction caused by the vectors, genes or new proteins. Many efforts now concentrate on conditions that require only transient gene ~xpression, e.g. cancer and coronary artery disease, because sustained gene expression is not necessary once the targeted tumor cells die or new blood vessels grow to bypass blocked arteries.

15

Introduction

1.12 FDA-APPROVED BIOPHARMACEUTICALS AND VACCINES Product NR.me Actimmune

Generic NR.me gamma interferon

Activase

Company Genentech, Inc.

DR.te ofApprovR.l Dec. 1990 Nov. 1987

Genentech, Inc. March,1990

recombinant alteplase Enzon, Inc.

Adagen adenosine

OctOber, 1989.

deaminase AlferonN recombinant interferon beta 1-B

Interferon Sciences Inc. Berb:

August, 1993.

Betaseron Ceredase Engerix B Epogen

Alglucerase hepatitis B vaccine epoetin alfa Somatropin

Humatrope alpha interferon

Laboratories/ Chiron Corp. Genzyme, Corp. Cerezyme imiglucerase Genzyme, Corp. SmithKline Beecham

anti-haemophiliac factor

Sept., 1989. June, 1989.

Amgen, Ltd. Eli Lilly & Co.

Leukine

June, 1994.

October, 1982

Intron A KoGENate

April, 1991.

yeast derived GM-

CSF

Schering-Plough Corp.

Neupogen Oncaspar

pegaspargase

Orthocione

OKT-3

Miles, Inc. Immunex, Corp.

Procrit

epoetin alfa

Amgen,Ltd.

Proleukin

interleukin (IL-2)

Enzone/RhonePoulenc Rorer

Protropin

somatotrem

June, 1986 June, 1988 November, 1988 Februaty, 1991 July, 1992 Februaty, 1993 March,1991 Februaty,1991 June, 1994 Februaty,1994

Ortho Biotech Pulmozyme

Dnase

Recombinate rAHF

recombinant haemophiliac factor recombinant alpha interferon

Recombivax HB RoferonA

Ortho Biotech

June, 1986 December, 1990 April, 1993

Chiron Corp. Genentech, Inc. Approved for

May, 1992 May, 1985 December, 1993

Genentech, Inc.

December, 1992 July, 1986 June, 1986 November, 1988

Baxter Healthcare Merck & Co.

Applimtion

Management of chronic granulomatous disease Treatment of myocardial infarction Treatment of infants and children with severe immunodeficiency Use in genital warts Management of relapsing, remitting multiple sclerosis Treating Type 1 Gauchefs disease Treating Type 1 Gaucher's disease Treating anemia associated with chronic renal failure and anemia in Retrovir-treated, HlV-infected patients Treatment of diabetes Treatment of hairy tell leukemia, genital wa~ts, AIDS-related Kaposi's sarcoma, non-A, non-B hepatitis, and hepatitis :6 Use in the treatment lof hemophilia A Use in autologous botne marrow transplantation ' treating Use in chemotherapy-inducFd neutropenia and bone marrow transplantassociated neutropenia Use in treating acute lymphoblastic leukemia Use in reversal of acut\e kidney transplant rejection Approved for use in the treatment of anemia associated with chronic renal failure and anemia in Retrovir-treated, HIVinfected patients and chemotherapy-associated anemia Treatment of kidney (renal) carcinoma Treating human growth hormone deficiency in children Use. in the ,?anagement of cystiC fibrOSIS

16

Biopharmaceuticals

Product Name

Generic Name

Company

Date ofApproval

Hoffman-La Roche

Application Treatment of hemophilia A Hepatitis B prevention vaccine Treatment of hairy cell leukemia and AIDS-related Kaposi's sarcoma

1.13 MAJOR APPROVED PROTEIN THERAPEUTICS 1.13.1 Non-Antibody Proteins Product

No.

Company

Indication

I.

Actimmune

InterMune

Granulomatous Disease

2.

Activase

Chronic

Acute Myocardial Infarction

3.

Aranesp

Amgen

Anemia

4.

Avonex

Biogen

Relapsing Multiple Sclerosis

5.

BeneFIX

Genetics Institute

Hemophilia B

6.

Betaseron

Chiron/Shering AG

Relapsing, Remitting Multiple Sclerosis

7.

Cerzyme

Genzyme

Gaucher's Disease

8.

Epogen

Amgen

Anemia (Chronic Renal Failure)

9.

lntton A

Shering-Plough

Hairy Cell Leukemia

10.

Kineret

Amgen

Rheumatoid Arthritis

II.

Leukine

Immunex

Autologous Bone Marrow Transplant

'12.

Nattecor

Scios

Congestive Heart Failure

13.

Neumega

Genetics Institute

Thrombocytopenia

14.

Neupogen

Amgen

Neutropenia

15.

Novo Seven

Novo Nordisk

Hemophilia A Bleeding hGH Deficiency

16.

Nutropin

Genentech

Cutaneous T-Cell Lymphoma

17.

Ontak

Ligand Pharmaceuticals

Hepatitis C

18.

PEG Intton

Shering-Plough

Anemia (Oncology)

Introduction No.

Product

Company

Indication

19.

Procrit

Amgen/J&J

Renal Cell Carcinoma

20.

Proleukin

Chiron

Respiratory Infections (Cystic Fibrosis)

2l.

Pulmozyme

Genentech

Hemophilia A Bleeding

22.

ReFacto

Genetics Institute

Hairy Cell Leukemia

23.

Roferon-A

Roche

Sepsis

24.

Xigris

Lilly

17

1.13.2 Monoclonal Antibodies/Fusion Proteins No.

Product

Company

Indkation

1

Campath

ILEX Oncology/Berlex

Chronic Lymphocytic Leukemia

2

Enbrel

Immunex

Rheumatoid Arthritis

3

Herceptin

Genentech

Metastatic Breast Cancer

4

Mylorarg

Wyeth-Ayerst

Relapsed Acute Myeloid Leukemia

5

OKT-3

J&J Acute

Kidney Transplant Rejection

6

Rernicade

J&J Rheumatoid

Arthritis, Crohn's Disease

7

Reopro

Lilly

Anti-Blood Clotting Agent (PCTA)

8

Riruxan

Genentech/IDEC

Relapsed, Refractory Non-Hodgkin's Lymphoma

9

Synagis

MedImmune

Respiratory Syncytial Virus Infections

10

Zenapax

Protein Design Labs/Roche

Acute Kidney Transplant Rejection

11

Zevalin

IDEC

Non-Hodgkin's Lymphoma

18

Biopharmaceutica/s

1.14 PRODUCTS FACING PATENT EXPIRA'1;'ION Eleven products face patent expiration by 2006 Brand Name

Sources

Generic Name

2001

Global Sales $ Million

U.S Patent Expiration

Amgen, Johnson & Johnson, and Sankyo

$ 5,772

2004

Human insulin

Novo Nordisk

1,829

2005

Filgrastim Human insulin

Amgen and Roche Eli Lilly

1,533

2006

1,061

2001

Interferon beta-l a

Biogen 972

2003

Inrerferon alpha-2b

Schering-Plough 700'

2002

Alglucerase

Genzyme 570

2001

Epogen or Procrit

Epoetin

Novolin Neupogen

~fa

Humulin Avonex Intron A Cerezymeor Ceredase

Somatropin

Humatrope

Alteplase

Eli Lilly

Activase

311"

2003

Genentech, Boehringer Ingelheim, Mitsubishi, and Kyowa Hakko Kogyo

276

2005

Nutropin

Somatropin

Genentech

250

2003

Protropin

Somatrem

Genentech

250

2005

TOTAL (susceptible to generic competition) Filgrastin

GM - CSF

Amgen

$ 13,524

2007

a Estimate. Source: Datamonitor

Source: Rouhi A. Maureen Generics Next Wave: Biopharmaceuticals, September 23, 2002, Volume 80, Number 38, pp. 61-65

19

Introduction

1.15 COMPANIES WITH LATE-STAGE PRODUCTS NEED MANUFACTURING PARTNERS Company

Compound

Therapeutic

Clinical Trial Stage

Area Alexion Pharmaceuticals/Procter & Gamble Pharmaceuticals

Pexelizumab

Cardiovascular disease

Phase III

Alexion Pharmaceuticals

Eculizumab

Rheumatoid arthritis

Phase II

Immuno-Designed Molecules

IDM-2

Bladder cancer

Phase II

IDM-4

Leukemia

Phase II

Immunomedics

Labetuzumab

Various cancers .

Phase IflI

NeuTec Pharma

Mycograb

Yeast infection

Phase II

Aurograb

Methicillin-resistant Staphylococcus aureus infection

Phase II

Onyvax-l0S

Colorectal cancer

Phase II

Onyvax

Source: (1) Technology Catalysts (2) Rouhi A. Maureen, Generics Next Wave: Bwpharmaceuticals September 23, 2002, Volume 80, Number 38, CENEAR 80 3~, pp. 61-65.

1.16

NUMBER OF BIOPHARMACEUTICAL PRODUCTS DEVELOPMENT PHASE Stage

Phase I Phase II Phase III BLA Market

Number of Products

Mammalian

Microbial

60 68 21 3

22 22

42

Source: Compiled from - The future of Biogenerics

15 3 39

BY

20

Biopharmaceuticals

1.17 COMPANIES PRODUCING BIOGENERICS Prodmu

Campany Cangene (Canada)

All existing molecules; limited clinical trials also

GeneMedix (URC); ties with Chinese institutes

All existing molecules

Microbix (Canada)

All existing molecules

Rhein Biotech (Gernan); ties with India and Argentina

Hepatitis B

Teva (Israel); ties with Biotechnology General, USA

Market HGH

LG Chemicals (Korea); ties with Western companies

EPO; insulin and interferrous

Stada (German); ties with DSM Biologics, Canada

Bulk; active substances; erythropoietin, filgrastin, interferon alpha and interferon beta

E. Merck (German); ties with Technofarma, Argentina

HCG, interferon in early eighties

l.18 RECENT FDA APPROVALS: 2003,2004 Campany

Year

Recombinant Drug

2003

Immune Globulin Intravenous (Human) (Flebogamma)

Instituto Grifols (Probitas Pharma)

For treatment of primary immune deficiency.

2003

Hyaluronic acid/Medicis (Restylane)

Medicis Pharmaceuticals Corp.

For correction of moderate to severe facial wrinkles and folds, e.g., nasolabial folds (lines/folds near the nose and mouth). (bacterial fermentation-derived; considered a mainstream biophar-maceutical product)

2003

CDlla Mab, rDNA (Efalizumab - Raptiva; CDlla monoclonal antibody, recombinant)

Genentech, Inc and Xoma Ltd.

For the treatment of moderate-to-severe psoriasis in adults who are candidates for systemic or phototherapy.

2003

Botulism Immune Globulin Intravenous (Human) (BabyBIG)

California Department of Health Services

For treatmemt of infant botulism caused by type A or type B Clostridium botulinum.

Application

Introduction Tear 2003

2003

&combinant Drug

Company

21

Application

Somatropin, rDNAj Serono (Somatropin (rDNA origin) Serostim; hwnan growth hormone recombinant)

Serono Inc.

For the treatment of HIV patients with wasting or cachexia (upgraded from accelerated approval granted in 1996)

Factor VIII, rDNA, PFM (Antihemophilic Factor (Recombinant), PlasmafAlbwnin Free Method - Advate; Factor VIII, recombinant; rAHF -

Baxter Hyland Immuno

For treatment of hemophilia A

PFM 2003

TNF Receptor-lgG Fc, rDNA (Etanercept Enbrel; twnor necrosis factor receptor2-immune globlulin Gl FC fusion protein, recombinant)

Amgen Inc. for treatment of active ankylosing spondylitis

For treatment of active ankylosing spondylitis

2003

Antitrypsin, alpha-If Aventis (Alpha-lProteinase Inhibitor (Hwnan) - Zemaria)

Aventis Behring LLC

For chronic augmentation and maintenance therapy in individuals with alpha I-proteinase inhibitor defidency and evidence of emphysema

2003

CD20 Mab, rDNA-l 131 radioconj. (Iodine 131 tositwnomab Bexxar; CD20 monoclonal antibodyiodine I 131 radiommune conjugate)

Corixa Corp. (formely Coulter Pharmaceutical) with marketing by Glaxo SmithKline (GSK)

For the treatment of patients with CD20 positive, follicular, non-Hodgkin's lymphoma (NHL), with and without transformation, whose disease is refactory to RituXimba and has relapsed following chemotherapy

2003

Immunoglobulin E Mab, rDNA (Omalizwnab - Xolair, rhuMab-E25; immunoglobulin antibody, recombinant; IgE Mab, RDNAJ

Genentech, Inc. (with manufacture by Tanox, Inc, and co-marketing by Novaris Pharmaceutical Corp.)

For treatment of Moderate-to-severe allegric asthma

2003

Hirudin, desulfatorDNAjAventis (Iprivask; Desirudin; Revasc; desulfatohirudin; hirudin, desulfaterecombinant)

Aventis Pharma

For the prophylaxis of deep vein thrombosis, which may lead to pulmonary embolism, in patients undergoing elective hip replacement surgery

22

Biopharmaceuticals Year

Recombinant Drug

2003

Influenza Vaccine, Live Intranasal (F1uMist)

Medlmmune Vaccines, Inc. (subsidiary of Medlmmune, Inc.)

For Influenza prophylaxis in healthy persons frm 5-50 year of age

2003

Iduronidase, rDNA (Iaronidase; Aldurazyme; a1pha-LIduronidase)

Biormarin Pharmaceutical Inc. (and Genzyme Corp.)

For treatment of Mucopolysaccharidosis I (MPS I)

2003

Galactosidase, beta rDNA (Agalsidase beta - Fabrazme; alphagalactosidase AJ -

Genzyme Corp.

For treatment of Muccopolysacchariodosis I (MPS I)

2003

Somatropin antagonist, PEG-, rDNA (Pegvisomanti Somavert; somatropin antagonist, pegylated, recombinant)

Pharmacia Corp.

For treatment of acromegaly

2003

Enfuvirtide synthetic (T-20; Fuzeon; pentafuside; DP-178)

Hoffmann-La Roche Inc.

For treatment of HIV -infection (synthetic; not a biopharmaceutica)

2003

LFA-3IgGl, rDNA (Alefacept; Amevives; leukocyte functionassociated antigen-3/ immune globulin G (IgG) fusion protein; recombinant

Biogen Corp.

For treatment of moderate-to-severed chronic plaque psoriasis

2003

Antitrypsibn, alpha-II Baxter (a1pha-l Proteinase Inhibitor (Human); Aralast; a1pha-l antitrypsin; AAT; AIPl)

Alpha Therapeutic Corp. (for markeitng by Baxter)

For enzyme replacement therapy in patients with heredity emphysema (AAT deficiency)

2004

Enfuvirtide synthetic (T-20; Fuzeon; pentafuside; DP-178)

Hoffinann-La Roche Inc.

For the treatment of HIV-1 infection in combination with other antiretrovoiral agents in treatment-experienced patients with evidence of HIV -1 replication despite ongoing antiretrovoiral therapy.

2004

Immune Globulin (IGIV)/Octapharm (Octagam; Immune Globulin Intravenous (Human)

Octapharma AG

For treatment of primary immune deficiency

Company

Application

Introduction Tear

Recombinant Drug

Company 1STA Pharmaceuticals Inc.

23

Application For use as a spreading agent to facilitate the dispersion and absorpotion of drugs, particularly local anesthetics during ophthalmic surgery; for hypodermoclysis; and as an adjunct in in subcutaneous urography for improving resorption of radiopaque

2004

Hyaluronidase, ovine (Vitrase; hyaluronate 4-glycanohydrolase)

2004

Insulin gluisine rDNA Aventis Pharma (Apidra; (Lysb3, GluB29) insulin; insulin (human), 3B-I-Iysine, 19B-B-glutamic acid-, recombinant)

For use a a rapid-acting insulin for treatment of diabetes

2004

VEGF Mab, rDNA (Avastin; bevacizumab; vascular endothelia growth factor monoclonal antibody, recombinant)

Genentech, Inc.

For use 10 combination with 5fluorouracil for treatment of metastatic cancer of the colon or return.

2004

EGF receptor Mab, rDNA (CeruximabErbitux; IMC-C22S; epidemal growth factor receptor monoclonal antibody, recombinant)

ImClone Systems Inc. (for marketing by Bristol-Myers Squibb Co.) .

For use in combination with irinotecan in the treatment of patients with epidermal growth factor receptor (EGFR)-exprc:ssing, metastatic colorectaI cancer who are refractory to irinotecan based chemotherapy, and for monotherapy treatment of patients with EGFR-expressing metastatic colorectal cancer who are intolerant to irinotecanbased chemotherapy

2004

ZLB Bioplasma AG Rho(D) Immune Globulin/ZLB (Rho(D) Immune Globulin Intravenous (Human) Rhophylac)

2004

Hyaluronic acidfAnika (ORTHOVISC High molecular Weight Hyaluronan)

Anika Therapeutics, Inc. for U.S. marketing by Ortho Biotech Products, L.P. (Johnson & Johnson)

For antepartum and postpartum prevention of Rho(D) immunization in Rho(D)-negative women

For the treatment of pain associated with osteoarthritis of the knee. (Derived from rooster combs; considered a borderline biopharmaceutical product)

REFERENCES 1. http.lfwww.vrgtech.com/products.html)

2. Dey Bindu, "Biogenerics in Perspective," Pharmabiz.com, 4th March 2006.

3. Rathore) S. et at) Costing Issues in the Production ofBiopharmaceuticals) BioPharm International, Feb. 2004. http.lfwww.biopharmintemational.com/biopharm

MARKET FOR BIOPHARMACEUTICALS 2.1 Global Market 2.2 US Market 2.3 European Union Market 2.4 Asian Market 2.5 Indian Market 2.6 Challenges, Issues affecting Biopharmaceuticals 2.7 Demand Outlook for Biopharmaceuticals

2.1 GLOBAL MARKET Globally, it is estimated that about 100 new biopharmaceuticals ate currently undergoing final clinical trials in the world. The current global market for biopharmaceuticals is well in excess of $ 25 billion and is expected to approach $ 40 billion within the next couple of years. 200 million people world-wide use biopharmaceuticals and there are 2,500 biotech companies producing these products. It is forecasted that in 2005, 70 per cent of the drugs in the market will be from biopharmaceuticals and 20 per cent of the top 100 drugs will be developed using biopharma research. The largest biopharma market is the US that accounts for 46 per cent of total sales. Biogenerics drugs are set to replace 70 per cent of the conventional therapies by the year 2025. Drugs that go off patent during the 2004-2006 period include Erythropoietin a (Amgen, J&J and Sankyo), Sermorelin (Serono), Palivizumab (Abbott), Alteplase (Genetech, Boehringer Ingelheim,

Market for Biopharmaceuticals

25

Mitsubishi and Kyowa Hakko Kogyo), Somatrem (Genentech) and Filgrastim (Amgen and Roche). The global market for interferon product is estimated to be $ 1.8 billion. The global biopharmaceutical market is estimated at Rs. 33,000 crores and is said to be growing at the rate of 15 per cent annually, in a scene dominated by global majors. India is seen as a key source and market for biopharma and food. When several big drugs go off patent in the coming years, pharma companies from all over will be getting into this market quickly through the biotech mode. New analysis from Frost & Sullivan, Strategic Analysis of the World Plant Molecular Farming, reveals that the biopharmaceutical market generated revenues of USD 45.0 billion in 2004. It is likely to reach USD 98.2 billion by 2011. In 2004, overall generic and biogeneric revenues worldwide totaled $ 39 billion, with an ;lverage growth rate of 10%. The U.S. comprised the largest portion with $ 17.4 billion. This was followed by Europe with $ 12 billlion, Asia with $ 5.5 billion and the ROW region with $ 4.1 billion. By May 2005, some 160 biopharmaceuticals (recombinant therapeutic proteins, monoclonal antibody based products used for therapeutic or in vivo diagnostic purposes, and nucleic acid based products) have gained marketing approval in the US and EU. Approximately one-fourth of new drugs coming to market are biopharmaceuticals. These drugs have benefited some 325 million people worldwide. These biopharmaceuticals generate over $ 30 billion annually with sales projected to cross $ 50 billion by 2010. 12 biopharmaceuticals got approval in US and EU in the year 2004.

2.2 US MARKET According to one estimate, US Biologic products demand in 2001 was $ 32.3 billion. Recombinant proteins accounted for 44%, monoclonal antibodies - 15% and conventional biologics (vaccines, blood producs, diagnostics) accounted for 41%. US demand for biologics will advance almost 12 per cent annually to over $ 56 billion in 2006. New product introductions evolving from advances in recombinant DNA and monoclonal antibodies will spur growth. Applications in the treatment, prevention and diagnosis of complex viral, malignant and auto-immune disorders will see the strongest gains in demand due to the safety and performance shoncomings of available conventional products. There are around 45 competitors in the field of biologics, including Amgen, Biogen, Johnson & Johnson, Aventis, Eli Lilly, Genentech, Genencor, Wyeth, Baxter, GlaxoSmithKline, Schering-Plough, Merck, Biogen, Chiron, MedImmune, and Genzyme.

2.3 EUROPEAN UNION MARKET The research-based pharmaceutical industry in Europe accounts for about 3.5% of the total EU manufacturing value-added and for 15% of the whole EU business R&D expenditure and performs well on most standard measures, such as: • employment: about 588,000 jobs in Europe, including 100,500 in R&D units; • R&D investment:

E

20,200 million in 2002 (up from

E

7,900 in 1990);

• trade surplus: E 36,000 million in 2002 (up from E 7,100 in 1990). , According to latest research from Frost & Sullivan, Biopharmaceuticals Industry Analysis Quantification of Supply and Demand of Manufacturing Capacities, Europe generated an estimated US $ 8.30 billion in global biotech revenues with R&D spends totalling almost US $ 5 billion in 2002. However, since the early 1990s, the research-based pharmaceutical industry in Europe has been losing competitiveness with respect to the US. Data for 2002 and preliminary figures' for 2003 confirm the vulnerability of Europe's research-based pharmaceutical industry. Benchmarking and performance indicators show that the US has continued to improve its relative position as a locus of innovation.

26

Biopharmaceuticals

• In 1990, the global research-based pharmaceutical industry invested still roughly 50% more in Europe than in the US. Today, the same industry is investing 40% more in the US compared to investments in Europe. • Between 1990 and 2003, R&D investment in United States rose fourfold while in Europe it only grew 2.6 times. • In 1990, the European companies still invested 73% of their R&D spending in Europe, while in 2000 this figure declined to 59%. • In 1960-65, European companies invented 65% of new chemical entities (NCEs) placed on the world market, but by the end of '90s, this share had fallen to about 35%. • Although Europe's biopharmaceutical sector is growing steadily, it remains dominated by its American counterpart. While direct employment is estimated at 33,300 in Europe, about 142,900 employees work in the US. • In 1990 the European market was the world's largest market with a 37.8% share (against 31.1 % for the North American market); in 2003, North America accounted for 49.2% of the world pharmaceutical market against 27.8% for the European market. • Between 1992 and 2002, the US market grew by 11.8% per annum well ahead of Europe (weighted average growth of 7.0). Figures adjusted for inflation show a higher differential of market growth in relative terms, with the US market growing twice as fast as the European market. These market evolutions have particularly benefited to US-based companies which have significantly increased their share in sales of new medicines and development of new molecules. • According to IMS Health data, 70% of sales of new medicines marketed since 1998 are generated on the US market, compared with 18% on the European market. • The fragmentation of the EU pharmaceutical market results in a lucrative parallel trade which benefits neither social security nor patients but deprives the industry from additional resources to fund R&D. Parallel trade was estimated to amount to E 4,300 million (value at ex-factory prices) in 2002. It would be too simplistic to attribute the deterioration of the European pharmaceutical environment to a single factor. As a whole, Europe remains less attractive for R&D investments than the US. The economic and regulatory framework, the science base, the investment conditions, and societal attitudes towards new technologies all contribute.

2.4 ASIAN MARKET The Asian biotechnology market, which is dominated by the biopharmaceuticals that represents 68.4 per cent of the total size, was estimated at $ 9.63 billion in 2001. The other significant market is genomics (dominated by pharmacogenomics) and molecular diagnostics and the trend of the prominence of biopharmaceuticals is likely to continue in the future. The industry projections for 2002 stood at $ 10.72 billion in the Asian market. Biotech companies in the US typically spend about 50 per cent of their turnover on R&D. This is drastically different from Asian companies spending less than 20 per cent on the same. To have a faster turnaround time for producing new molecular entities, it is essential for biotech companies to have techniques like combinatorial chemistry and high throughput screening. However, Asian companies are not able to afford these. Hence, they focus on developing products that have already been developed by other companies worldwide. This has resulted in price wars in the biopharmaceutical markets.

Market for Biopharmaceuticals

27

Sales from imported products constiUlte a major chunk of the biopharmaceutical markets in Asia. Examples include recombinant streptokinase, recombinant insulin and recombinant interferon. However, the import duty for these products is phenomenally high (in countries like India the duty stands at 44 per cent). On the other hand, local companies have neither the technological infrastrucUlre nor the supply of raw materials to produce them indigenously. As a minimum volume criteria drives companies to import products future rationalization of the duty strucUlre is considered necessary.

2.5 INDIAN MARKET The areas that the biopharma industry could focus on are manufacruring and marketing of biogenerics, contract manufacUlring, contract research and development and over-the-counter products that are naUlral extracts. The biopharmaceutical industry in India is mainly restricted to two segments; recombinant drugs and monoclonal antibodies. The Indian Biopharmaceuticals is in the development stage with about 30 players currently in the business. With a host of activities happening, this market is likely to almost double in terms of its revenue turnover by 2004. The major advantage of entering this market is the short timelines needed to develop a product and make it available in the market. The total Biopharmaceuticals market is worth Rs. 2800.1 million in 2001. Revenues are expected to reach to Rs. 4028.3 million by 2004. The market for recombinant medicine such as vaccines in India is expected to rise by over 80 per cent to about Rs. 958 crores by the year 2005 from the current market of Rs. 535 crores in India. Biopharmaceuticals is restricted to recombinant drugs and monoclonal antibodies. The potential of the Biopharmaceuticals market in India is large. Many of the products in this market are imported, which is why they are expensive. However, a number oflndian companies have entered/are planning to enter this market. They are expected to introduce products at much lower prices. This is likely to result in larger volumes by the end of the forecast period. With over .350 biotech drug products in the pipeline across the world, this is also the chance for Indian pharma biotech companies. By 2005, the Indian market for recombinant medicine is also tipped to grow from Rs. 535 crore to Rs. 958 crores. Investments into biodrugs are also expected to grow to Rs. 500 crores. Currently, four companies are into making the hepatitis B and several pharma companies have active biotech plans.

2.6 CHALLENGES, ISSUES AFFECTING BIOPHARMACEUTICALS The challenges affecting the biopharmaceutical products market are: • Small biotech start ups find marketing of products difficult • Moderate success rates daunts research activities • Myths surround biopharmaceutical products. The driving forces of this market are: • Large scale production leads to lowering of production costs • Inadequacy of traditional drugs to fulfil a need and perception of the value and safety of the therapy • Unlimited production ensures constant supply The factors that restrain the Biopharmaceuticals mark~t in India are: • High investment results in companies focusing one or two products and hence enter price war zones

28

Biophannaceuticals

• High capital and recurring costs hinder investments • Absence of microorganisms patent laws deter companies from investing in basic research • A large number of regulatory bodies in the country slow down processes of approval • High duties on imported products deters distributors

2.7 DEMAND OUTLOOK FOR BIOPHARMACEUTICALS Most important factor in the growing demand for therapies is increasing elderly population in industrial nations over the next 25 years. The main causes of death are cardiovascular disease, cancer, and respiratory disorders, all of which are strongly age-related. Other age-related illnesses such as osteoporosis, arthritis, Alzheimer's, and Parkinson's are major factors affecting quality of life. Mortality from cancer, diabetes, liver and kidney diseases have hardly changed, offering significant areas for research breakthroughs. Development of drugs for treatment of cancer is a major opportunity area for biotech firms. The investment needed to develop a cancer drug is lower than other diseases: the field has high priority with regulatory authorities who are willing to give it fast track status on the basis of smaller (and therefore cheaper) clinical trials (a few extra months of survival could be enough to win FDA approval); the clinical community is highly concentrated; and the market size is often larger than the approved indication because of a high off-label use (for other cancers). Infectious diseases, the third most common cause of death in the US, highlight the pressing need for new vaccines and antibiotics with novel mechanisms of action to avoid growing drug resistance. Only one new class of antibiotics has been approved over the last 30 years. Global Biopharrnaceutical Sales are expected to increase by 5% p.a. over the next five years approaching US $ 20 billion by 2004. Growth will largely be driven by therapeutic monoclonal antibodies, new approved indications for drugs already on the market, and second-generation versions of existing blockbusters such as EPO and interferon alpha and beta. Many of the new biopharmaceuticals in development are aimed at niche markets and may never reach the blockbuster status of EPO, human insulin, or human growth hormone. . Products and Markets: By mid-2000, 84 biopharmaceuticals had been approved for marketing with almost half launched during the past three years. Worldwide sales in 1998 totalled US $ 15 billion. Although representing only 5% of world prescription drug sales of $ 300 billion, biopharmaceuticals comprise six of the top 50 selling drugs, 13% of new medicines approved by the FDA in the 1990s, and 11% of all drugs in development. The US is the largest and most rapidly growing market, accounting for 46% of sales, compared to 36% for conventional prescription drugs, reflecting a combination of earlier regulatory approval, easier market acceptance and greater pricing flexibility. Europe and Japan are the next most important markets, accounting respectively for 30% and 17%. Recombinant Proteins: Recombinant proteins will continue to dominate the biopharmaceutical market. Genome sequencing efforts are making it possible to identify new protein candidates; protein product life cycles are longer than synthetic molecules because they are harder to duplicate and manufacturing methods are unique; new delivery vehicles may make oral or inhalation delivery possible; and proteins such as growth factors will allow "firms to enter the market for regenerative medicine organ and tissue repair. The first drug candidates derived from genomics (an anti-obesity drug from Amgen and four drugs from Human Genome Sciences for wound healing, obstructed arteries, infections, and as an adjunct in cancer chemotherapy) are all protein drugs.

Market for Biopharmaceuticals

29

Protein Biogenerics: A number of US biopharmaceutical patents (e.g. interferon alpha, human insulin, human growth hormone, and hepatitis B vaccine) will expire within the next five years, potentially opening the market for biogenerics. However, competition may not be as intense as that currently experienced in the generics industry. With traditional chemical generics, extensive clinical trials are not required for marketing authorization as long as bioequivalence studies confirm that the rate and extent of drug adsorption and hence safety and efficacy are similar. Proteins on the other hand are much more complex molecules whose mode of action is not completely understood and whose properties are influenced by many factors during the production, purification, and formulation processes, such as host cells and culture conditions. Because biological products are produced in living organisms, they do not have the same manufacturing consistency as drugs based on synthetic chemistry. Regulatory standards for biogenerics are still being developed. To prove that impurities and inactive compounds, which might differ from the original process, do not affect the safety and efficacy of the generic, new clinical trials may be required to validate the manufacturing process. The costs required for these trials, as well as the fact that the manufacture of biopharmaceuticals is more difficult and expensive, could be a significant barrier to entry. In addition, patent holders are developing second-generation products which are longer lasting and do not have to be injected as often. Recombinant Vaccines: The first FDA-approved recombinant vaccine (against hepatitis B) came in 1986, followed in 1998 by a vaccine against Lyme disease. Chiron also developed a recombinant vaccine against pertussis (whooping cough) but this is only marketed in Italy. The scarcity of approvals is due to numerous technical challenges: devising the correct recombinant antigens; increasing the immune response (the engineered protein has a limited lifetime in the body which reduces its antigenicity compared to inactivated/innocuous viruses); and designing broad antigenic variants of rapidly mutating RNA viruses such as HIV and influenza. Monoclonal Antibodies (Mab): Since the first approval of a therapeutic Mab in 1986, it took a decade for the next product to reach the market (to treat blood-clot-related complications in patients undergoing angioplasty), and are now achieving notable successes e.g. respiratory syncytial virus. Mabs are more versatile than other drugs as they can be constructed to treat a wide range of diseases such as cancer, rheumatoid arthritis, and Crohn's disease; are one the fastest ways of translating genomicsderived targets into therapeutics by targeting novel cell surface receptors; and entail relatively fewer subjects for clinical trials as they are designed to target specific cells. As anti-cancer agents, antibodies can be used alone to disable cancer antigens; conjugated to cytotoxic drugs or radioisotopes to kill tumor cells; or used as a vaccine in which an antibody is designed to mimic a tumor antigen in order to stimulate the immune system to identify and eliminate cancer cells with that particular antigen on its surface. The first anticancer antibody (against non-Hodgkin's lymphoma) was launched in 1997 and the first antibody linked to a chemotherapy agent was approved in 2000 against relapsed myeloid leukemia. Gene Therapy: Gene therapy was originally proposed as a treatment for genetic disorders such as hemophilia, sickle cell anemia, and cystic fibrosis, but over 80% of clinical activity is currently focused on cancer, AIDS and cardiovascular disease, areas considered to be more commercially promising. Worldwide, approximately 4000 patients participated in nearly 400 trials since 1990 for a variety of diseases but no convincing evidence of a long term beneficial effect has been established. In the US, 25 products were in clinical development at the end of 1999 with over two-thirds in Phase I. Only in cancer gene therapy has progressed into Phase III with only two products (against head and neck cancer and melanoma). It is still to be established as to whether the entire solid tumor can be killed or only those cancerous cells around the site of the injection, and whether there will be any effect on malignant cells that may have migrated elsewhere in the body.

30

Biophannaceuticals

Antisense: Antisense involves the use of nucleic acids (oligonucleotides) to prevent the production of proteins expressed by specific genes through RNA targeting. It is still an experimental technology with considerable challenges such as finding the right gene to target, delivery mechanisms and costs. Only one product (Isis' Formivirsen for the treatment of CMV retinitis, a virus that causes blindness in AIDS patientS) has been approved. Most antisense drugs are a' long way from market and suffer from the same delivery problems as gene therapy. Market Segmentation for Pipeline and Capacity Analysis* Commercial Microbial: 2003 - 19 2007 - 21Clinical Microbial: 2003 - 32+ 2007 - 37+ Commercial Cell Culture: 2003 - 26 2007 - 31 Clinical Cell Culture: 2003 - 43+ 2007 - 53+ 2003: Estimated Demand (kg/year) for Mammalian C~ll Culture-Derived Products (currently marketed products) Rituxan

380

Enbrel

274

Remicade

222

Herceptin

108

Synagis

73

All other (34)

97

Market for Biopharmaceuticals

31

TABLE 2.1 SALES OF RECOMBINANT DRUGS

Product

Protein

Efficts/therRpeutic use

Marketed by

Worldwide sales 2004 in US$m

Epogen

Eythropoeitin

stimulation of the production of erythrocytes

Amgen

2601

Prority/Eprex

Erythropoeitin alfa*

stimulation of the production of erythrocytes

J&

3589

NeoRecommonl Epogin

Erythropoeitin beta *

stimulation of the production of erythrocytes

Genetechy Rochel Chugai

1842

Aranesp

Darbepoeitin alfa

stimulation of the production of erythrocytes

Amgen

2473

Peg.Intron (Intron-A)

PEGylated alphainterferon + ribavirin

anti-Hepatitis C

Schening-Plough

1851

Pegasys

PEGylated interferon alpha2a + copegus (ribavirin)

anti-Hepatitis C

Roche

1382

Avonex

Interferon beta-la

multiple sclerosis

Biogen Idec

1417

Rebif

Interferon beta-la

multiple sclerosis

Serono

1091

Betaseronj Betaferon

Interferon beta-l b

multiple sclerosis

Schering AG

1057

Neulasta

G-CSF

stimulation of the production of granulocytes

Amgen

1200

Neulasta

G-CSF PEG conjugate

stimulation of the production of granulocytes

Amgen

1700

Leukine

GM-CSF

stimulation of leukocytes

Schering AG

80

Proleukin

Interleukin

cancer

Chiron

129

Humulin

Insulin

diabetes

Eli Lilly

998

.

JIOrtho

Biotech

32 Product

Biopharmaceuticals

Protein

EJfoctsftherapeutic

Marketed by

Worldwide sales 2004 in US$m

use Humalog

Insulin

diabetes

Eli Lilly

1102

Rituxan (in EU-Mabthera)

rituximab, humanised Mab

leukemia and lymphomes

Genentech/Roche

2989

Herceptin

trastuzumab, humanised antiHER-2 Mab

breast cancer

Genentech/Roche

1270 .

Campath

alemtuzumab humanised Mab againSt CD52

B-cell chronic . hymphoma

Genzyme

77

Mylotarg

gemtuzumab

relapsed acute myeloid leukemia

Wyeth

20

rheumatoid arhritis

Amgen

2580

rheumatoid

J&J

2891

ozogamicin, humanised anti-CD33 Mab Enbrel

etanercept (fusion) protein of antibody Dc and p75-TNF receptor protein)

Remicade

Inftximab, chimaeric MAb

arthritis

Humira

Adalimumab

rheumatoid arthinis

Abbott

852

Humatrope

human growth hormone (HGH) Somatotropin

dwarfism

Eli Lilly

430

Protropin/ Nutropin

human growth hormone (HGH) Somatotropin

dwarfism

Genentech

396

Serostim

human growth hormone (HGH)

dwarfism

Serono

86.8

Saizen

huinan growth hormone (HGH)

dwarfism

Serono

182.1

Cerez;yme/ Ceredase

Glucocerebrosidase

Gauher's disease

Genzyme

839

Synagis

h!lffianised Mab

respiratory syncytia virus prevention

Abbott/ Medimmune

942

Market for Biopharmaceuticals Product

Protein

Effects/therapeutic

33

Marketed by

Worldwide sales 2004 in US $m

stimulation of ovulation coronary infact

Serono

572.7 n.a

use Gonal F

Folitropin alpha

Activase

tissue plasminogen activator

ReoPro

GBllb/lllaantibody

inhibition of thrombosis

Eli Lilly/Centocor

363

Kogenate

Factor VIII

hemophilia

Bayer

481

Engerix-B

envelope protein of the hepatitis B

vaccine

Smithkline Beecham

n.a.

Genentech

299

Genzyme

43

209

Genentech

virus Pulmozyme

human DNAse

mucopolysaccharidosis

Aldurazyme

rec. human alpha L-iduronidase

enzyme

rec. human alphaglatosidase A

enzyme

Genzyme

replacement theapy

Biogen Idec

Amevive

alefacept

severe plaque psoriasis

Genentech, Novartis

43

Xolair

omalizumab, humanise anti-lgE MAb

persistent allegic astharna

Genentech Novartis

188

Raptiva

efalizumab, humanised antiCDlla MAb

severe plaque psoriasis

Genentech Xoma

57

Erbitux

cetuximab

advanced coloretal cancer

ImClone, Merck KGaA

365 (partial year)

Mylotarg

gemtuzumab ozogarnicin

bone marrow cancer (CD33) positive acute myelid leukemia

Wyeth Ayerst

20

;

Fabrazyme

replacement theapy mucopolysac- . charidosis

Biopharmaceuticals

34

Prod,,"

Zevalin

Protein

lbrirurnomab Tiuextan

bevacizumab

Avastin

Worldwide lilies 2004 in

Effiets/thempeuti& use

Marketed by

relapsed 0 rafractory lowgrade, follicular, or transtomed B-cell nonHodgkin's lymphoma advanced cotorectalcancer

IDEC Pharmaceuticals

23

Genentech

555 (partial year)

US$m

REFERENCES 1. European Federation of Pharmaceutical Industries and Associations (EFPIA) Report 3 ....l!rW!tkJiudt·htm)

hrrp:/!www.ejJzia.01JJ/

2 Naik Nitin, Asian Biotech Industry A checklist on challenges ahead, Pharmabiz.com, 18th April 2002.

BIOPHARMACEUTICAL INDUSTRY 3.1 Biopharmaceutical Industry: Definition and Characteristics 3.2 Biopharmaceutical Industry 3.3 Biopharmaceutical Industry: Major Players

3.1 BIOPHARMACBUTICAL INDUSTRY: DBFlNmON AND CHARACTERISTICS The biopharmaceutical sector refers to firms that have incorporated biotechnology either in their production processes or in their R&D programs or are selling biotechnology based pharmaceutical products. In certain discussions, Biopharmaceutical is expressed as (Bio) pharma [name is purposely bracketed in parenthesis] used in a wider context, which includes traditional therapies using local plants and biological ingredients, bioprospecting, modern pharmaceutical industry based on organic chemistry synthesis and recombinant DNA (rDNA), and hybridoma technologies. Biopharmaceutical industry is a typical combination of biotechnology and pharmaceutical industry. Biopharmaceutical sector in developed countries is typically represented by hundreds of small start-up companies. Pharmaceutical companies are facing increasingly stringent regulatory requirements, intense competition, pressures to control costs, and demands for lower prices. For the rapidly growing biopharmaceutical segment of the industry, there are additional hurdles: (i) constantly changing technology, (ii) evolving regulations for biologics, and (iii) intense public and political scrutiny of biotechnologies such as gene therapy and stem-cell research.

36

Biopharmaceuticals

Bruce Babbitt, JanHasker Jonkman, and Paul McKim in their article on "Small Biopharmas Face Unique Challenges" "Outsourcing Early-Stage Drug Development Issues" describe important issues faced by small biopharmaceutical start-ups. Small start-up biopharmaceutical companies have to face additional hurdles than expressed above. These are limited funding, dependence on a single product, the need to find additional investors or partners to move their product forward, and pressure from investors to show results quickly. Small start-up biopharmaceutical companies have little or no experience in preparing regulatory submissions, interacting with regulatory agencies, designing and managing clinical trials, or producing investigational drug supplies for clinical trials under Good Manufacturing Practices (GMP). During the last 30 years, the biopharmaceutical industry has successfully launched nearly 1,400 new chemical entities as human therapeutics, and has achieved strong sales as a result. In 2000, the global biopharmaceutical industry was projected to have invested US $ 58 billion in R&D. In 2001, the output of the global biopharmaceutical industry in terms of new drugs was the lowest in ten years. Only 31 new drugs were recorded as having been launched by the industry as a whole during 2001. Many companies in the biopharmaceutical industry are beginning to focus on previously unidentified disease targets and on marketing innovative medicines designed to treat conditions for which there is no existing treatment, or where treatment is inadequate. This is going to involve entirely new R&D processes and also more complex and longer clinical development than in the past. The secret of a successful generic biopharmaceutical industry lies in preparing and tackling the manufacturing, regulatory and scientific hurdles that are coming its way. Outsourcing in the biopharmaceutical industry began in the 1980s and exploded in the 1990s in the three core areas of clinical trials, contract manufacturing and sales force solutions. Staffing for clinical trials is found to be burden even by large pharmaceutical company. So outsourcing of this laborious work became a practice. Large pharmaceutical companies in spite of the fact that they did have the production setup found very soon even contract manufacturing appealing. This happened at peak in 1990s. Tomorrow even R&D will get outsourced. "Biopharmaceutical Industry Contributions to State and US Economies" a study conducted by the Milken Institute focuses on the economic impact of America's biopharmaceutical companies including jobs, economic output, and taxes paid - both at a national level and on a state-by-state basis. According to this study, America's biopharmaceutical companies are creating over 2.7 million jobs across the United States. It found that over 400,000 Americans are directly employed in the biopharmaceutical industry and that, on average, 6 jobs are created economy-wide for each biopharmaceutical job. The biopharmaceutical industry cannot be identified using the Standard Industrial Classification, which distinguishes firms on the basis of their output rather than their technology or production process, and international data are difficult to compare. Many biotechnology firms, for example, are identified, as biopharmaceutical companies to differentiate them from the mainstream pharmaceutical industry, but their prime focus are small molecule drugs targeted against proteins thought to be important in the disease pathway (proteins can be used both as drugs i.e. biopharmaceuticals or drug targets). Biologics, an area that consists of blood derived polyclonal antibodies and clotting factors, antibiotics, and classical vaccines based on live or killed viruses, are frequently classified as biopharmaceuticals, but these long' predate the emergence of recombinant DNA and monoclonal antibodies. Insulin, for example, was originally obtained from porcine or bovine pancreas while human growth hormone was extracted from the pituitary glands of cadavers. The biopharmaceutical category also often includes drugs derived from plants, fungi or marine organisms, but these are more in the realm of traditional medicinal chemistry research based on the random screening of natural compounds.

Biopharmaceutical Industry

37

Industry Size: There are approximately 3,000 dedicated biotechnology companies worldwide employing 230,000 people with the US accounting for 40% of firms and 70% of employment. Publicly traded firms - including an estimated 100 that floated IPOs during 2000 - number approximately 460 of which 74% are based in the US and 18% in the EU. The segment involved in biopharmaceuticals numbers about 150 firms. Only a handful of biotech firms -:- primarily those involved in biopharmaceuticals- have a product on the market. The industry as a whole continues to experience major losses because of high R&D costs. The largest and most successful (e.g. Amgen, Chiron, Biogen and Genentech) are mainly US based. With the exceptions of Serono (Switzerland), Celltech (UK), and Bio-Technology General (Israel), few non-US firms have brought a biopharmaceutical to market, reflecting their later startup dates. The total market capitalization of all public European biotechnology companies, for example, is only a little larger than Amgen. Japan has few dedicated biotechnology companies, with the industry primarily consisting of large food and pharma firms with historic strengths in fermentation attempting to diversify into biopharmaceuticals.

It is important to understand the distinction between biotechnology as a new process technology and as a drug discupery research tool. The first uses genetic engineering to manufacture large molecular weight drugs that cannot be directly synthesized or extracted. The second involves understanding the molecular basis of disease and the search for new therapeutic targets using techniques such as cloned receptors as screens or transgenic organisms created through gene knock-out technologies to determine protein function; most of the focus is on small molecule drugs that interact against those targets. As the pharmaceutical industry is using biotechnology in drug discovery, it will likely maintain its dominant position in small molecules, but the development and manufacture of protein-based therapeutics requires a completely different set of core competencies. Industry Structure: The biotechnology industry consistS of three groups: dedicated biotechnology firms, usually university spinoffs, researching disease mechanisms at the molecular level; traditional pharmaceutical companies ("big pharma") marketing drugs developed by biotechnology firms; and a specialized tier of companies serving both the pharmaceutical and biotechnology industries with platform technologies that can speed up the drug discovery process or improve drug delivery. The boundary between these three groups has blurred. Big pharma is investing heavily in molecular biology and genomics, biopharmaceutical firms are moving into small molecule therapeutics because of production and delivery advantages, and platform companies are entering the drug development field because of the prospects for higher returns.

3.2 BIOPHARMACEUTICAL INDUSTRY A breakdown of clinical development candidates among the top pharmaceutical companies shows that major drug firms are paying increasing attention to large-molecule, primarily protein, therapeutics. Big drug companies are typically viewed as committed to small-molecule drugs-even if they employ biotechnology steps in discovery and early development. However, small purely biotech companies and labs owe the credit of advances in biopharmaceuticals. All of the major drug players had begun biopharma programs prior to their big acquisitions. Most biopharma products in the top drug companies' portfolios were acquired along with the biotech firms that developed them. Others were accessed through partnerships. Through acquisition, major companies have also taken over well-established biopharma R&D and manufacturing operations. In most cases, the big drugmakers avoided the risks taken by biopharma pioneers. Amgen, Wyeth, Roche, GlaxoSmithKline, Merck, are top in the ranking of biopharmaceuticals in terms of manufacturing of biologics (as compared to small molecules). Pfizer, Abbott are also strong biopharma players. Among the top 10 drug companies, Roche has the longest involvement in biopharmaceuticals. With 40% of its sales coming from biopharmaceuticals-top products include

Biopharmaceuticals

38

Rituxan (rituximab) for non-Hodgkins lymphoma and erythropoietin f~r treating anemia-Roche is the second largest biopharma firm in the world by sales. For global pharmaceutical and biotechnology industries, the primary value driver of the genomic era will be an explosion of targets. Today's 400 or so targets are estimated to increase to at least 4,000 during the next decade. The $ 300 billion pharmaceutical market could grow to $ 3 trillion by 2020. These targets will be used both as diagnostic and focal points for the development of new precision drugs. Companies Manufacturing Biologics

No.

ComJHIny

Product

I.

Abbott Laboratories

Humanized Monoclonal antibodies

2.

Abgenix Incorporated

Therapeutic Monoclonal antibodies

3.

Acambis

Vaccines

4.

Akzo Nobel (Organon) (Diosynh) (Intervet)

Human Monoclonal antibodies

5.

Amgen Incorporated

Erythropoeitin, G-CSF

6.

Avecia Group

Therapeutic Proteins, DNA medicines, Peptides

7.

Aventis

Hirudin, alpha-I-antitrypsin

8.

Baxter International

Factor VIII

9.

Bayer

Factor VIII

10.

Biogen Incorporated

BI-Intelferon, gamma interferon

II.

Biopure Corporation

Blood products

12.

Boehringer Ingelbeim

Activase

13.

Cambrex Corporation

14.

Chiron Corporation

Interleukin

15.

Covance Incorporated

Antibodies, Monoclonals

16.

Daiichi Pharmaceutical Company

f3

17.

Degussa (Proligo)

18.

Dow Chemical Company

19.

DSM

20.

EPIcte Pharmaceutical

interferon

Biophammceutical Industry

39

No.

Onnpany

Product

2l.

Genencor International

Protein therapeutics Enzymes, Immunotherapeutics, MAbs

22.

Genentech Incorporated

Human growth hormone (HGH), tissue plasminogen activator

23.

Genzyme Corporation

Humanized monoclonal antibodies, glucocerebrosidose

24.

GlaxoSmithKline

CD20, MAb, HBsAG

25.

IDEC Pharmaceuticals

Monolonal antibodies

26.

Invitrogen Corporation

Antibodies, molecular probes

27.

Johnson & Johnson (Centocor) (Ortho-Clinical)

Erythropoeitin, Chiemeric MAb, Dnase

28.

Kirin Brewery Company

Transgenic animals for immunobulin

29.

Laureate Pharma

30.

Lilly (Eli) and Company

3l.

Lonza Group

32.

MedImmune Incorporated

Humanized monoclonal antibodies

33.

Merck & Company

Hepatitis vaccine, Monolonal antibodies

34.

Nabi Pharmaceuticals

Antobodies to protect against hepatitis, rabies, tetanUS etc.

35.

Novartis International

Humanized anti-IGE MAb

36.

Novo Nordisk

r-factor VIII for Hemophilia A

37.

Novozymes

Enzymes

38.

Pftzer Incorporated

recombinant vaccines

39.

Pharmacia Incorporation

Somatotropin antagonist

40. 4l.

Protein Design Labs Roche Holding (Hoffman La Roche)

PEGylated alfa interferon

42.

&bering (Berlex Laboratories)

Therapeutics

43.

Schering-Plough

PEGylated Interferon alplaza, Interleuhins (IL-4)

44.

Serono

Human growth hormone (HGH), Folitopin alpha BI-Interferon

45.

Wyeh

Humanized antibodies

Human insulin, HGH, Somatotropin, anti-hemophiliac factor

40

Biopharmaceuticals

3.3 BIOPHARMACEUTICALS INDUSTRY: MAJOR PLAYERS 1. Abgenix: A biopharmaceutical company, Abgenix develops and intends to commercialize antibody therapeutic products for the treatment of a variety of disease conditions. 2. Amgen: Developing and delivering important, cost-effective therapeutics based on advances in cellular and molecular biology. 3. Biogen: The first independent biotechnology company in the world, Biogen is the creator of several important and successful medical therapies. 4. Centocor: A world leader in monoclonal antibody technology, Centocor's innovative products focus on the management of cardiovascular, autoimmune and cancer diseases. 5. Chiron: Participates in the global healthcare markets of biopharmaceuticals, blood testing and vaccines, and the development of innovative products for preventing and treating cancer, infectious diseases, and cardiovascular diseases. 6. Cubist Pharmaceuticals: Focused on becoming a global leader in the research, development and commercialization of novel antimicrobial drugs to combat serious and life-threatening bacterial and fungal infections. "7. Gene Logic: Leading provider of genomic information, enabling the discovery and development of pharmaceutical, biotechnology and life science products through the systematic and industrialized application of genomics. 8. Genentech: Biotechnology company using human genetic information to develop, manufacture and market pharmaceuticals that address significant unmet medical needs. 9. Genset Oligos: Fully integrated genomics company engaged in providing tailored genomics information to pharmaceutical" companies throughout the drug lifecycle. 10. Human Genome Sciences: Committed to the new gene-based medicines to patients around the world.

trea~ent

and cure of disease by bringing

11. Immunex: Based in Seattle, Washington, Immu~ex Corporation is a biopharmaceutical company dedicated to improving lives through immune system science innovations. 12. ImmunoGen: ~mmunoGen's tumor-activated prodrug technology uses a highly potent drug that is covalently attached to humanized monoclonal antibodies for targeting tumor cells. 13. Medarex: A biopharmaceutical company developing monoclonal antibody-based therapeutics to fight cancer and other" life-threatening and debilitating diseases, and has assembled a broad platform of patented technologies for antibody discovery and development. 14. MedImmune: Biotechnology company that develops and markets products for infectio\lS diseases and transplantation medicine. " 15. Millennium Pharmaceuticals: Biopharmaceutical company focusing on the discovery and development of small molecule, biotherapeutic and predictive medicine products. 16. Myriad Genetics: Biopharmaceutical company specializing in the use of proteomic and genomic technologies to create break-through medical, diagnostic and therapeutic products. 17. Novavax, Inc.: Biopharmaceutical drug delivery company engaged in the research and development of differentiated drug products primarily in the field of women's health, infectious diseases, and cancer.

Biopharmaceutical Industry

41

18. Peregrine Pharmaceuticals Inc.: Engaged in the research, development, and commercialization of novel cancer therapeutics. 19. Schering-Plough: A recognized leader in biotechnology, genomics and gene therapy whose core product groups are allergy/respiratory, anti-infective/anticancer, dennatologicals and cardiovascu1ars. 20. Titan Pharmaceuticals: Develops novel and proprietary products for the improved treatment of cancer and central nervous system disorders. 21. Transgene: Integrated gene therapy company, dedicated to the discovery and development of gene delivery technologies and gene therapy products for the treatment of acquired and inherited diseases.

REFERENCE 1. Mullin Rick, "Biopharmaceuticals", Chemical and Engineering News, May 10, 20CM, Volume 82, Number 19, pp. 19-25.

BIOPHARMACEUTICAL SECTOR IN INDIA 4.1 Scenario of Biopharmaceuticals Market 4.2 Scenario of Biopharmaceutica1 Products 4.3 Scenario of Biophannaceuticals' Manufa~ 4.4 Scenario of Biophannaceuticals' Research

4.1 SCENARIO OF BIOPHARMACEUTICALS MARKET Pharmaceutical industry of India is 4th largest in the world. The Indian Biopharmaceutical business presently is estimated at $ 200 million and is expected to grow to about $ 3.3 billion by 2007-08. It is dominated by recombinant proteins, vaccines and monoclonal antibodies. The potential of the biopharmaceuticals market in India is large. Many of the products in this market are imported, making them expensive. The biopharmaceutical sector is likely to witness doubling of its revenue turnover by '05 from the current levels of Rs. 33,000 crores. Wockhardt is aiming to launch a slew ofbiopharmaceuticals in the US and the European Union using India as a cost..effective base. Dr. Reddy's Laboratories is working through its arm Aurigene and through its biotech and diagnostics division, it is developing biopharmaceuticals, vaccines and diagnostics. While Ranbaxy Laboratories is investing about Rs. 120 crores in Hyderabad to cover biopharmaceuticals, Bangalore-based Biocon India is entering the sector through a joint venture company with Cuba-based Centre for Molecular Immunology (eMI) to manufacture and market a ~~lect range of products.

Biopharmaceutical Sector in India

43

Products like insulin and erythropoietin are doing well here, but interferons and monoclonal antibodies (MABs) are also considered interesting by some pharmaceutical companies. But in India, currently, only two MABs are available for therapeutic use, for the treatment of cancer. While one is used for the treatment of chronic myeloid leukaemia, the other is used for a subset of breast cancer patients. The global market for biopharmaceuticals, which is currently valued at $ 41 billion, has been growing at an impressive compound annual growth rate (CAGR) of 21% (over the previous five years). With over one-third of all pipeline products in active development being biopharmaceuticals, this segment is set to continue outperforming the total pharmaceutical market and could easily reach $ 100 billion by the end of the decade. Pharma is a very strong sector for India but it does not have the same excitement in the global context in terms of new products, whereas biotech offers you that scope, because it will be affordable to do research in biotech. With this potential, a number of Indian companies have entered or are planning to enter this market. They are expected to introduce products at much lower prices. This is likely to result in larger volumes by '05. For Indian pharma companies, the major advantage of entering this market is the short timelines needed to develop a product, availability of human resource and produce it at an affordable rate in the market. In India, Biopharmaceuticals is restricted to recombinant drugs and monoclonal antibodies. With a paucity of funds and infrastructure, biopharmaceutical research is restricted in these two fields. The large and diverse Indian population is a potential database for genetic analysis. It is estimated that there are as many as 28,000 endogamous groups in the country. A sizeable number of rare genetic disorders are found too. In spite of this genetic pool, pharmacogenomics, or the development of tailor made drugs suited to the gene type of a patient, has not yet made its mark in India. The essential aspect of pharmacogenomics is an understanding of all the genes that determine action and sensitivity to drugs. This would help in minimizing adverse drug reactions, finding the set of people who would respond best to a particular dosage and identify new drug targets. The challenges affecting the biopharmaceutical products market, the driving forces of this market and the factors that restrain the biopharmaceuticals market are already mentioned in Chapter 2 and are important while considering development of biopharmaceutical market in India. The Indian Biopharmaceuticals is in the development stage with about 30 players currently. The total Biopharmaceuticals market was worth Rs. 2800.1 million in 2001. Revenues are expected to reach to Rs. 4028.3 million by 2004. This represents a CAGR of 15 per cent between 2000 and 2004. Most of the present revenues stem from recombinant products. Bharat Biotech, producing the fist biotherapeutic molecule from India, and the first protein against staphylococcus infection has received a global patent for Lysostaphin, which works on staphylococcus infection. The market potential is $ 12 billion. Production facilities as manufacturing base and interdisciplinary base are important issues for India to develop in this sector.

4.2 SCENARIO OF BIOPHARMACEUTICAL PRODUCTS In biotech sector in India, biopharmaceuticals is the largest segment in market. Biopharma Bioservices Bio Industrial

75.24% 8.95% 6.74%

Biopharmaceuticals

44

Bio Agri

6.95%

Bio IT

2.09%

In biopharmaceuticals, share of different category of the product is: Vaccines

-

Sales of US $ 383.6 million (2004-2005)

Therapeutics

-

Marketing licenses granted for over 25 recombinant therapeutic proteins

Diagnostrics

Sales US $ 138.2 million (2004-200S)

Therapeutics

Accounts for 16.7% of total biopharma sales. Diagnostics is forcasted to grow by 35-40% in 2005.

Biotherapeutics The biotherapeutic business features generic biotherapeutics that is a pre-genome business and new biotherapeutics that is a post-genome. The question is how much is one lagging behind, even with pre-genomic biotherapeutics. Insulin was approved in 1982 and companies are still struggling to come out with insulin. There are multiple approaches to biotherapeutics; natural products can be biotherapeutic. So can stem cells, improved and modified vaccines, proteins, genes and monoclonal antibodies. Not just protein alone. One has to look at the bigger picture. Natural products as biotherapeutics include plant sources such as taxol and trypsin inhibitors, animal sources such as immunoglobin produced by horses and cow urea and through the mining of microbial diversity. This has traditionally been used for small molecule but has not been exploited from the biological and therapeutic points of view. Most of it is used for antibiotics. Two monoclonal antibodies currently available in India are Mabthera (rituximab) and Herceptin (Transtuzumab). Both are marketed by Nicholas Piramal/Roche. Mabthera is indicated for relapsed/ refractory low-grade B-cell Non Hodgin's Lymphoma (NHL). It was the first monoclonal antibody to receive approval for a cancer indication in 1997. The product was made available in India in January 2001. The parent company for developing this drug - IDEC, is also developing a murine version of Mabthera, Zevalin, which is linked to a radioisotope for refractory B-cell NHL. Coulter Pharmaceutical's Bexxar is a similar product that targets the same antigen on B-cells but is conjugated to a different radioisotope. Herceptin is used for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 (human epidermal growth factor receptor2) protein and who have received one or more chemotherapy regimens for their metastatic disease. Product patents are expected in India by 2005, and once the system is implemented, Indian companies would not be allowed to reverse engineer molecules that are under patent protection globally. However, Indian companies would still be able to compete in the domestic market for generics, that is, drugs on which the patent has expired. The option available to the Indian companies would be to either operate in the generics market or invest in R&D and invent new chemical entities and dosage forms so as to achieve growth in the long run. The global market for biopharmaceuticals is estimated to grow at nearly 30 per cent per year for the next two years. As against this, the Indian biopharmaceuticals market is expected grow at a CAGR of 15 per cent during the same period and that too driven mainly by volume growth rather than value. Experts expect the Indian biopharmaceuticals market to increase to Rs. 400 crores in 2004 from the current Rs. 280 crores in 2002. Recombinant products and monoclonal antibodies will be characteristic of the Indian market. Advances in genomics and rapid understanding and exploitation of genetic information to make tailormade drugs suited to the gene type of the patients will drive the industry in advanced markets.

Biopharmaceutical Sector in India

45

According to a Frost and Sullivan report on biopharmaceuticals, absence of microorganisms patent laws deter companies from investing in basic research and a large number of regulatory bodies in the country slow down the registration and approval process. High capital and recurring costs and herd mentality of focusing on me too products and the subsequent price wars are cited as the other factors that restrain the biopharmaceuticals market in India. The cost of biotech research per say may be cheap in India, but government policies still do not encourage investments in this high-risk area where success rate is moderate. There is a general opinion that the public sector will have a central role in the biotech field because biology is not ideal for a business centric or purely commercial exercise with short-term goals. Globally speaking, around 100 biopharmaceuticals are in late stage development. There are 700 biotech molecules in human clinical trials, 140 of which are cancer therapeutics and over 2,000 new products are in pre-clinical development. More precisely, there are six antibody therapies in phase III development. Currently, the focus for Indian companies is restricted to the development and manufacturing of biogenerics. The priority'is towards monoclonal antibodies, insulins, hepatitis B vaccine, interferon and erythropoetin as the demand is huge. Augustin Maria and Shyama V. Ramani in their paper on TRIPS and its possible impact on the Indian biopharmaceutical industry for the presentation in the conference (held in Toulouse) on "Markets for Pharmaceuticals and the Health of Developing Nations" have commented on Strategic positioning of Indian firms in biopharmaceutical sector. According to them, the Indian biopharmaceutical firms visualize six main areas of activity to improve their competitive position in India and abroad: 1. Entry into the biogenerics market and the market for off-patent diagnostics, and 2. Vaccines; 3. Contract research; 4. Creation of new pharmaceutical products; 5. Bioinformatics; 6. Clinical trials. The recombinant drugs that are approved for marketing in India include (1) Human insulin, (2) Streptokinase, (3) Erythropoietin, (4) Hepatitis B vaccine, (5) Human growth hormone, (6) Human interleukin 2, (7) Human Interleukin 11, (8) Granulocyte colony stimulating factor, (9) Granulocyte macrophage colony stimulating factor, (10) Alpha-Interferon-2a, (11) Alpha-Interferon2b, (12) Gamma-Interferon, (13) Blood factor VIII, (14) Follicle stimulating hormone, (15) Tissue plasminogen activator, (16) Injection-Forte - the bone building drug, (17) Xigris, (18) Epidermal Growth Factor (EGF), and (19) Platelet derived EGF. Market for Biopharmaceutical Products in India Market in India Product 1. Erythropoietin

Rs. 40 to 45 crores

2

Hepatitis B vaccine

Rs. 150 crores

3

Human Insulin

Rs. 200 crores.

4

Interferons

Rs. 70 crores.

46

Indigenous Manufacturers Name of Company

Products

Shantha Biotechniques

Hep B Vac, Alpha Interferon

Bharat Biotech

Hep B Vac

Panacea Biotech

Hep B Vac

Wockhardt

Hep B Vac, Erythropoietin

Serum Institute

Hep B Vac Human Growth Hormone, Follicle stimulating hormone of India

Dr Reddy's

Hep B Vac, GCSF

About 7 lakh doses of interferon is being imported in India but once indian company comes in with lower prices, that market could multiply.

4.3 SCENARIO OF BIOPHARMACEUTICALS' MANUFACTURING Pri~e pressure in the US is increasing. Healthcare market and people are looking for more and more generics. India has started assuming significance as a big generic supplier. India is well-poised to have an exciting time in biopharmaceuticals in the next five-ten years. Biopharma production is a very sensitive process and having reliable power supply, telecommunication system, lo~istics structure, etc, is very important. This is where China is ahead of India. However, the fact that En~;jsh language skills, higher compared to China, is great advantage, particularly in research collaborations. Indian companies already have a very good understanding of how to deal with WHO, US FDA and other international regulatory requirements.

The national biopharmaceutical industry posted a total revenue of over US dollar 500 million (Rs. 2,292 crores) 2003, accounting for 70 per cent of the whole biotechnology industry at 700 million (Rs. 3,213 crores) which is expected to test US dollar seven billion to US dollar 10 billion by 2010, according to an "optimistic" estimate by the Export-Import Bank of India which has set eyes on the sector. The biopharma sector also contributed 76 per cent of the total biotech export revenue at US dollar 300 million (Rs. 1,377 crores) during the period, with vaccine business contributing most of it. Although this industry is relatively small as compared to US or some countries in Europe, it can be considered as fairly developed amongst countries in the Asia Pacific region and is showing the highest growth rates than many countries in Europe. India with strengths in process engineering, trained manpower in plant management is perhaps best positioned to leverage on the bio-generics opportunity and can be expected to repeat its success in pharma-generics. More and more pharma companies in India are looking at biotech-based pharma products. In view of the forthcoming products patents regime, pharma companies have identified R&D as one of the key growth drivers. With a high premium on R&D and many new avenues such as clinical research, contract research, developing new drug delivery systems and new chemical entities opening up before the Indian pharma companies, the demand for qualified trained professionals in related fields is growing. Not just the Indian companies, but several MNCs are looking at India to set shop to gain the cost, time, and the trained manpower advantage as R&D costs in their home countries are very high.

Biopharmaceutical Sector in India

47

As far as human resources required, new demands will have to be faced. Traditional pharmacology is increasingly being considered as the downstream processing of biotech-based pharma. With this change in focus, the requirements of pharma companies at the entry level are also changing. Besides, regular pharma graduates and postgraduates, the industry is looking towards M.Sc. and Ph.D. candidates possessing specialized knowledge. The field requires people trained in new technologies with specialized skills, like immunoblotting, protein/genome analysis, western blotting etc. Today, candidates possessing these skills are rare. The responsibility and specialization of the individual will depend on the process, which he or she is handling-fermentation technology, downstream processing, proteornics, genomics, etc. Importers - Marketers of Biopharmaceuticals in India No.

Name of Company

Products

1.

Alkem Laboratories

Erythropoietin

2.

Aventis

Human Insulin

3.

Bharat Biotech

Human Insulin, Hepatitis B vaccine

4.

Biocon

Human Insulin

5.

Biogen

Erythropoeitin

6.

Boehringer Manhem

Erythropoietin

7.

Cadila Healthcare

Hepatitis B vaccine

8.

Cipla

Human Insulin

9.

Dr. Reddy'S Laboratories

Erythropoietin

10.

Dragon Biotech

Erythropoietin

11.

Eli Lilly

Human Insulin

12.

Emcure

Erythropoietin

13.

Glenmark Laboratories

Erythropoietin

14.

Hindustan Antibiotics

Erythropoietin

15.

INCON/Zydus

Erythropoietin

16.

Intas Pharmaceuticals

Hepatitis B vaccine

17.

Johnson & Johnson

Erythropoietin

18.

Kee Pharma

Erythropoietin

19.

Knoll

Human Insulin

20.

L G Chemicals

Erythropoietin

21.

Malladi Drug & Chemicals

Erythropoietin

22.

MJ Pharmaceuticals

Human Insulin

23.

Neon Lab

Hepatitis B vaccine

24.

Nicholas-Piramal

GCSF

25.

Novo Nordisk

Human Insulin

26.

Panacea

Hepatitis B vaccine

Biopharmaceuticals

48

No.

N",,,"

of Comptlny

Products

27.

Ranbaxy

Erythropoietin, Hepatitis B vaccine

28.

Sarabhai

Human Insulin

29.

Serum Institute

Hepatitis B vaccine

30.

Shantha Biotech

Hepatitis B vaccine

31.

Shreya Life -Sciences

Human Insulin, Hepatitis B vaccine

32.

Smithkline Beecham

Hepatitis B vaccine

33.

Torrent

Human Insulin

34.

Transgene Vaccine

Hepatitis B vaccine

35.

USV

Erythropoietin

36.

VB Bhagat

Hepatitis B vaccine

37.

Wockhardt

HUman Insulln,- Erythropoietin, Hepatitis B vaccine

Biogenerics Production in India Production of Biogenerics is considered as an opportunity area by India. India has advantage due to highly competitive downstream processing skills and 50% lower manufacturing cost. Indian Government has granted marketing as well as manufacturing license to about 25 recombinant therapeutics including recombinant insulin, human growth hormone, intererons, heatitis B vaccine, interleukins and Tumor Necrosis Factor. Different companies in India with biogenerics are -

,

(1) Shantha Biotechniques, Hyderabad

-

Hepatitis B Vaccine (Shanvac) Alpha interferon2b (Shanferon) GM-GSF, HGH, r-insulin, streptokinase are in the pipeline.

(2) Bharat Biotech International Limited

-

Recombinant Hepatitis B vaccine (Revac-B) Lysostaphine (Working towards typhoid vaccine streptokinase, EGF, recombinant human insulin.

(3) Wochardt

-

Recombinant Hepatitis B vaccine (Biovac-B), r-erythropoietin (EPOx)

(4) Dr. Reddy's Laboratory

-

GRASTIM (filgrastin), GM-CSF. There are 10 other products at various stages of development in cancer, cardiovascular, diabetes and immunology segments.

(5) Biocon

-

r-insulin (through joint venture with Shantha Biotechnics), Streptokinase, G-CSF, EPO

(6) CIPLA

-

interferon, erythropoietin, hepatitis vaccine

All biogenerics cleared so far in India have followed the approval procedure, which are mix of the ones required for New Biological Entity (NBE) and generics. The molecules and vaccines have been cleared through three-tier mechanism (IBSC, RCGM and GEAC) adopting special abbreviated procedures and constituting special committees.

Biopharmaceutical Sector in India

49

4.4 SCENARIO OF BIOPHARMACEUTICALS RESEARCH 4.4.1 Challenges of Drug Discovery and Development in India The Indian pharmaceutical industry has made rapid progress since 1970 largely due to the revised patent law which permitted manufacturing and marketing of patented novel chemical entities by alternative, non-infringing processes. The industry is slowly evolving into research-driven industry. WTO obligations have hastened the process. Some large companies· however initiated the process of in-house drug discovery programs. Acceptance of Product patent system from 2005 is going to put new urgency in the efforts of drug discovery by Indian pharmaceutical companies. India has good base of scientific manpower. According to an estimate by the Organisation of Pharmaceutical Producers ofIndia (OPPI) more than 15% of the scientists engaged in Pharmaceutical R&D in the U.S. are of Indian origin. Still, the declining interest in the pursuit of science and the consequent decline in quality observed might be attributed to lack of suitable career prospects in R&D on one hand and the emergence of attractive, alternative career options in other industries like the infotech industry on the other. Shortage of trained scientific manpower could become a serious bottleneck as more Indian companies make a foray into pharma R&D. Multi-national companies using scientific capabilities for cost advantages make the problem more serious. Government, Academia and the Industry should pay attention to curtail this decline by revamping the academic course content and aligning it with Industry requirements. Industry also faces the problem with its R&D managers in terms of judiciously applying/adapting management techniques to reduce risk and enhance R&D productivity and pay particular attention to the drug discovery ~d development processes. Indian companies also have inadequate in-house infrastructure required for complex, multidisciplinary drug discovery and development endeavour on this purpose. Common facilities and science parks should fulfll the needs. It is mandatory to establish the safety and efficacy of experimental drug candidates in animals prior to clinical trials in humans. Animal testing facilities are not satisfactory in our country and services of Contract Research Organisations for such purpose is a costly affair. To ensure prompt preclinical evaluation of drug candidates, it is important to facilitate easy import of the necessary animal models for in-house research. Creation of state-of-the-art animal testing facilities will provide a good alternative for research organisations that are unable to establish such facilities in-house. In fact, the first national facility for transgenic and gene knockout mice was recently inaugurated at the Center for Cellular and Molecular Biology, Hyderabad with funding from Council for Scientific & Industrial Research (CSIR) and the Department of Science & Technology (DST). In addition, in-house animal ethics committees must be empowered to approve the use of animals on the basis of adequate scientific rationale. In order to monitor compliance with applicable policies and guidelines, annual activity reports may be requested by the CPCSEA from research organisations. Additionally, periodic audits at the research sites may also be undertaken. The regulatory environment must provide a clear framework for operations; be it for the use of radiolabelled material for R&D, import of animal models/biological samples, clinical development etc. Currently, there are not many facilities in the country that can undertake custom radiolabelling of small organic molecules for R&D. A centralized resource might be crctated that will aid research organisations in sourcing radiolabelled chemicals for research. This would allow easy monitoring of the movement and use of such chemicals. The Union Ministry of Health and Family Welfare is learnt to have decided to consult the Law Ministry prior to moving a proposal to dilute import regulations for biological samples into India by research institutions and the Industry. The existing rules require compliance with stringent formalities

50

Biopharmaceuticals

making it difficult for importing biological samples for further analysis and research. The simplification of these rules is expected to save time as well as reduce the cost of research. Clear guidelines are required for the proper conduct of clinical trials. Currently, Schedule Y of the Drugs & Cosmetics Act deals with this subject. Recently, the Central Government has put together draft rules governing medical research involving human subjects in the country. It is also proposed to have a Biomedical Regulatory Authority that will be responsible for the implementation of the new rules. The revision of regulatory laws and guidelines must be done so as to harmonize them with the internationally accepted laws/guidelines as far as possible. The success of the regulatory laws/guidelines will ultimately depend upon the ease of implementation on one hand and the ease of compliance on the other. Strong Intellectual Property laws and the machinery to enforce them is a basic requirement for promoting high level of innovations and their commercialization. In the absence of a strong legal framework, there would be little incentive for innovators to undertake R&D, leading to a gradual decline in competitiveness of the economy. India is in the process of ushering in the product patent era by 2005 in compliance with the TRIPS provisions of the wrO. Even as the new laws are formulated, it is imperative that the supporting machinery be put in place as well. For instance, the patent offices in the country need to be strengthened both in terms of trained manpower and the resources required for managing the increased IP-related activities in future. The companies on the other hand do not have adequate professionally trained manpower to handle the various aspects of Intellectual Property Management. More training programs and academic programs must be initiated to develop a pool of trained IP personnel to cope with the rigors of the product patent era. Patent drafting and filing alone are not sufficient and it is imperative to build capabilities in patent strategy, patent audit, and handling legal issues relating to patent maintenance, interference and infringement. The advances in science and technology have made the drug discovery and development process highly sophisticated. If genomics and proteomics-based approaches have the potential to yield novel drug targets, combinatorial chemistry and high throughput screening have enhanced the capability to shorten the time taken to discover lead compounds. The cost of implementing these state-of-the-art technologies presently is beyond the reach of most of the Indian companies. Also it is difficult to find skilled professionals to develop and man these technologies in India. Indian pharma companies may therefore plan their drug discovery programs to leverage on the relatively less expensive approaches of drug discovery. For instance, the analog approach for discovering novel chemical entities will be guided by known validated targets and well recognized chemical scaffolds for which the clinical proof of principle is already established. However, as the discovery programs mature over time, companies must judiciously adopt new technologies for pursuing cutting-edge research. This is because; it is novel target based research that results in breakthrough therapies for the treatment of disease. Networking among research laboratories across various sectors (industrial, academic and the government) has long been recognized as a valuable strategy to optimize research productivity. Pharmaceutical companies in close proximity to academic centres of excellence are common in developed countries and should be initiated here. Drug discovery and development is characterized by high failure rates. It takes 10-12 years of R&D wherein 5,000-10,000 compounds are screened before a new drug reaches the market. The most recent estimate by the Tuft's Center for the Study of Drug Development (CSDD) pegs the cost of bringing a new drug to market at 802 million USD. Roughly, one-third of the R&D cost is spent on discovery phase and the remaining two-third for development phase. While drug discovery and

Biopharmaceutical Sector in India

51

development can be carried out in India at a fraction of the global cost, global clinical development will be just as expensive. The ability of Indian companies to reduce the failures in the initial phases will be critical for evolving into discovery-led entities. The government must actively support the transition of the Indian pharma industry into a discovery-driven industry. This is paramount considering the size of India's population. According to a recent elI-McKinsey Healthcare study, the government spending on healthcare sector is expected to increase to about 6% of GDP by 2010 from the current 0.9%. Of this 2% is estimated to be spent on public health alone. If the Indian pharma industry is a source of new, patented medicines, the burden of maintaining the healthcare of the Indian population can be mitigated.

4.4.2 Genomics Research and India A number of laboratories in India, some of them, with the active support of leading Indian pharmaceutical companies, have moved into genornics research. The Western model is to have specialized genornics companies, which act as service organisations involved in the identification of DNA sequences in genes, determining the functions of the gene and the specific protein it expresses and developing the molecular targets required by the pharmaceutical companies to develop new drugs. These companies also compare genomic data on normal and defective genes, which can lead to therapeutic products for genetic diseases. The skills required for a successful bio-informatics company and a drug development company is entirely different. The formation of a consortium of several companies particularly in USA, to meet the challenges of the new science is a pointer to the complexity and cost of the total exercise involved in meaningful target-oriented genornics research. In India, in most of the areas involved, there is no minimum critical mass of scientists, infrastructure, or resources to be cost-effective and very little collaboration between corporates and bio-informatics companies. If the proliferating genomic techniques and tools, are not properly tuned to specific and focused needs, an enormous amount of resources will be wasted and the costs for drug development in terms of time and money are unlikely to be less than those for classical drug discovery and development programmes. No one disputes the potential of genomics research as an effective tool, the question is how soon and at what cost can it deliver the much-awaited benefits hoped for by millions of patients, healthcare planners and investors. Strategic tie-ups between big pharma companies and small biotech start-ups are already happening for contract research and manufacturing. Drug makers in the United States and Europe are increasingly moving clinical trials and research work to India, which helped Indian firms earn $ 54 million in revenue in the last fiscal year. The practice of passing on jobs in drug development research, analysis of research data and clinical trials to India is a new but growing trend. Revenue from work outsourced by Western companies accounted for more than 13 per cent of India's biotechnology exports of $ 395 million in the year that ended March 31, 2004. Britain's GlaxoSmithKline, German drug maker Bayer, Aventis of France, and U.S.based Pfizer Inc. are some of the companies that have already begun outsourcing work to India. In five years, Indian firms are expected to be earning annual revenues of $ 5 billion from outsourcing of biotechnology-related, services. Outsourcing in this area growing exponentially over the next few years and giving us the same success that India had in software. The industry has been growing very well at the rate of 25 per cent for the last two or three years.

52

Biopharmaceuticals

REFERENCES 1. Economic Times, Online 26th Dec. 2003.

2. Pharmabiz,com 30 July, 2002.

3. Express PharmtJ, Pulse, 10 Jan 2002. 4. Biopharma awaits venture capital for rapid growth, Express Pharma Pulse 30th Sept. 2004. 5. Challenges of drug discovery and development in India, Dr. A. Venkateswarlu & Rajanikant Riw, Phamabiz.com.

DRUG DISCOVERY AND DRUG D.ESIGNING 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10

Introduction Faster Drug Research Based on Computers and Biotechnology Positional Cloning for Identification of Disease Gene Antibodies in Rational Drug Designing Classes of Therapeutic Targets in the Living Cell Drug Development in Past and Role of Biotechnology Today Role of Bioinformatics in Genome-based Therapy Future of Bioinformatics Antisense DNA Technology for Drug Designing Role of Genomics Research in Drug Discovery

5.1 INTRODUCTION The goal of phannacology is to design a chemical compound that would interact with characterized receptor on a cell in specific and predictable manner. Drugs which are more specific in action and with less side-effect are to be developed. In order to move towards rational drug design it is necessary to know as much as possible about the structure of drug and about the structure of the receptor. Study of structure of drug is less complex, than study of structure of receptors on membranes of cells. It is only recently that the information has become available about the structure of receptors and other small molecules. Most drug discovery programmes now combine rational drug-design, medicinal chemistry with sophisticated screening. Computer-aided compound design and chemical database searches help the medicinal chemist and pharmacologist to match structure with function to device novel organic therapeutics. X-ray crystallography can be applied to drugs for study of structure but it is difficult to apply it to the receptors. Recent developments in molecular biology have provided several important techniques, which can be used to study interactions of drugs with specific membrane receptors.

54

Biopharmaceuticals

Biotech has challenged drug companies to make fundamental changes to their routine. Molecular biology has helped in predicting the complete amino acid sequence of receptors from clones isolated by using DNA probes coding for small peptides (receptor fragments) which have been purified and sequenced. This has helped to determine primary structure of several receptors. Since most receptors belong to same gene families it has been possible to determine primary structure even of those receptors tor which no amino acid sequence was available. Receptor proteins thus share certain conserved amino acid sequences. It has been now possible to group receptors into families based on their structures. This further groups them into gene families. Diversity of receptors from today's data suggests existence of four such gene families. However, the diversity of receptors may be larger than anticipated. Molecular biology can playa role (i) to determine the structure of receptor, (#) to screen drugs for interactions with specific receptors. It is essential to isolate receptor of interest from other receptor subtypes and to reconstitute the receptor into functional environment. This can be done by cloning cDNA in proper mammalian cells and their expression. This gives large amount of receptor protein for various studies. Study of structure can be done then. Also sufficient amount of membranes can be obtained from such expressed mammalian cells to use them for filter binding assays with radioactive ligand. Same cells can be characterized electro-physiologically using sensitive patch-clamp technique and drugs can be rapidly applied over the whole cell surface. Molecular biology plays role in characterizing drug-binding sites. The availability of cloned DNA and the ability to express receptors at high levels provides an experimental framework in which the structure of receptor can be studied by making changes in the receptor through in vitro mutagenesis. Then the effect of changes can be studied at the level of binding and coupling studies. Molecular biology techniques will be applied in combination with traditional biochemical and biophysical techniques to provide large amounts of receptor proteins necessary for structural studies. TABLE 5.1 NEWER TOOLS AND TECHNIQUES IN DRUG DESIGNING (1) (2) ( 3) (4) (5 ) (6 ) (7)

Bioinformatics Molecular Biology Positional Cloning Antibodies Combinatorial Chemistry Vast database and genetic libraries Molecular Diversity Technique

5.2 FASTER DRUG RESEARCH BASED ON COMPUTERS AND BIOTECHNOLOGY Computers and biotechnology have united in a new field known as 'bioinformatics', which provides faster research in drug discovery. Instead of tedious process of trial and error in finding DNA sequence matches, a computer can reassemble the DNA, acclerating the whole process. The creation of vast genetic libraries is a major driving force for bioinformatics. There is now so much information available that it is impossible for scientists to conduct experiments without the use of a computer. It is the only way to provide the access to the huge amount of data floating around. With applications of bionformakics cost of drug discovery reduces by 33%, time of drug discovery reduces by 2 years and number of leads to be tested reduces. Based in Cambridge, Massachusetts, USA, Millennium Pharmaceuticals uses genetics, genomics and bioinformatics to identify the genes responsible for common, major diseases and to determine

Drug Discuvery and Drug Designing

55

their role in disease initiation and progression. The company hopes this knowledge will lead to the discovery of a new generation of diagnostic and therapeutic products that will be capable of addressing major diseases at their root causes, as opposed to simply identifying and treating symptoms. Millennium's strategy contrasts with traditional pharmaceutical approaches in that it begins by identifying and understanding the roles of genes that cause or increase the susceptibility to a disease. Drug candidates can tfien be selected and drugs designed based on their ability to intervene at this fundamental level. A new approach to the treatment of AIDS is being undertaken by Glaxo Wellcome and Affymetrix, the company which has developed the GeneChip to assay the gene sequences. Glaxo Wellcome will use the GeneChip to produce a database, which corelates the genetic sequences of the HIV virus with sensitivity to various drug regimes. There are three classes of drugs, which can be used in combination therapies: (i) Protease inhibitors (ii) Nucleoside analogue reverse transcriptase inhibitors (NARTI) (iii) Non-nucleoside analogue reverse tanscriptase inhibitors (NNARTI). The initiative of Glaxo Wellcome should enable clinicians to make sense of the vast number of different combinations of protease inhibitors, NARTIs and NNARTIs that are possible. A drug research company, which focuses entirely on the use of human tissue, has been established in the Royston, Herts, U.K. This company, Pharmagene Laboratories is first of its kind, and will work in collaboration with the pharmaceutical industry to discover new medicines through the expression and function of genes and gene products in human tissue. The company is founded by Dr. Smith Baxter (Ex Smith, Kline and Beecham) and Dr. Robert Coleman (Ex Glaxo Wellcome) and has a group of expert biochemists, molecular biologists and pharmacologists. The company has already started working since Dec. 1996, and is busy with collecting pre-clinical data for their drug discovery programmes. Progress in design of protein growth factor agonists and antagonists has been exceedingly slow due to rudimentary understanding of most proteins and their receptors. Abbott Laboratories is investing $ 42.5 million in a genomics company, Genset. They will have a joint venture in the new virgin field of 'pharmacogenomics', which aims to develop new drugs through study of genetic differences within different groups of populations.

5.3 POSmONAL CLONING FOR IDENTIFICATION OF DISEASE GENE Positional cloning is an important method for identifying disease genes. It requires the availbility of a large family with many aftlicted individuals. The cystic fibrosis defect was mapped utilizing linkage analysis in 1989. Here a comparison is made within a family for the coinheritance of markers of known chromosomal location with the mutant gene. The closer a marker to the given gene, the higher the chance that there will be no recombination between them during genetic reassortment and these genes will be co-inherited. Co-inheritance suggests that the marker and the mutant gene are juxtaposed on the chromosome. By linkage analysis CF gene has been mapped at its location on chromosome 7. The known gene then becomes a marker and provides a starting point to utilize recombinant DNA cloning techniques to walk and jump up to the gene of interest. The walks and jumps help to identify other markers that are closer and therefore more tightly linked to the defective gene. The availability of markers that saturate the genome from the CEPH collaboration will provide the higher resolution map needed in order to identify a desired gene with less random walking or jumping.

Biopharmaceuticals'

S6

The final identification of a gene relies on analyses of overlappiJ;lg sequence fragments from shotgun sequencing in the finely mapped zone. Absolute identification of the target gene, can only be made by the demonstration of mutational differences in the gene sequences between patients and control subjects. A number of"genomics" startup biotechnology companies are already exploiting human genome information in medical and industrial applications. The use of hybridization subtraction techniques between expression maps of cells allows "snapshots" of cellular processes in normal and diseased cells to be compared. Then by dissecting the expression of disease genes, the proteins they express can be determined. This will reveal the underlying mechanism in the cell of the disease genes on the disease.

5.4 ANTIBODIES IN RATIONAL DRUG DESIGNING Antibodies are one of the three 'ligand-selection' technologies to detect lead compounds for drug development. Antibodies provide a powerful tool to study protein and peptide ligands and receptors. The 3D structure of target serves as a starting point (template) for design of a drug to act on it. Understanding of the tertiary structure of most proteins and their receptors is rudimentary. Hence progress in the design of protein growth factor agonists antagonists (protein-mimetics) is also slow. Some companies are trying to find out the 3D information on receptors, enzymes and viruses with the use of antibodies. Through recombinant monoclonal technology, antibodies to key drug interaction sites in receptors, enzymes and viruses can be raised. These antibodies are mirror image of their targets in both spatial and electronic sense and are labelled as ideotypic antibodies. By using ideotypic antibody as an antigen, anti-ideotypic antibody can be produced which is a positive image of the target site. This will obviate the need of actual isolation and purification of the target molecule which is often difficult. More structural information on such positive image of the target site then can be obtained by X-ray crystallography and other studies. PCR will help to determine the sequence of CDRs. Also key residues in the target can be found out by site directed mutagenesis and its effect on antibody affinity. In principle, panels of monoclonal antibodies can be used to describe the essential binding features of receptors or their agonists. These panels of surrogate receptors could then be used as a general method to screen large batteries of organic molecules from fermentation broth or other sources. The essential features of complex, hard-to-manipulate receptor molecules can be encoded in the matrix of monoclonal antibodies. Ligands can be easily removed from the 'capturing' monoclonal antibody matrix for structure determination.

Methods are developed to generate so-called epitope libraries. Random peptides are encoded in non-essential region offllamentous phage. This library, containing a hundred million or more epitopes, is used to probe the antibody active site. The development of epitope-mapping techniques may permit the pharmaceutical chemist to use antibodies as a means to discover the essential peptide forms required to begin the synthesis of organic molecules.

5.5 CLASSES OF THERAPEUTIC TARGETS IN THE LIVING CELL (i) Extracellular proteins -

mediate cellular function in disease, mediate cellular function in disease, (iii) Membrane channels - allow the passage of molecules that signal cells to follow particular pathways that may underly the disease pathology, (iv) Transmembrane receptors - allow the passage of molecules that signal cells to follow particular pathways that may underly the disease pathology, (ii) Extracellular enzymes -

Drug Discuvery and Drug Designing

(v) Intracellular signalling proteins membrane to cell nucleus,

.57

"second messengers" that convey message from cell

(vi) DNA-binding proteins - regulate the expression of genes, (vii) mRNA which encodes information for production of proteins, (viii) DNA itself. Many of the diseases are better targeted by 'small molecule' drugs. Two revolutionary techniques in small-molecule drug discovery are: (a) Combinatorial chemistry combined with high-throughput screening, which enables testing of vast number of new and unique potential compounds, and (b) computer-assisted drug design which helps to decide physicochemical properties of candidate drug and its possible mode of interaction with target. Combinatorial chemistry and molecular diversity techniques are the experimental ones while computer-assisted techniques are computatioal ones and in combination they enable the drug designers to simulate, synthesize and screen large number of compounds in a rational manner. Structure-based drug design is rapidly becoming the core part of work done by all large pharmaceutical companies and many small biotechnological companies. Agouron, Vertx, Biocryst are the few names to cite as examples of companies using these techniques solely for drug design. Cloning, expression and isolation of the gene products and their analyses in order to deduce the needed structure-function information relation is required for novel drug discovery. Deciphering of genome sequence of Bacillus subtilis promises new developments in enzymes and drugs. Potential targets for developing new antibiotics can be determined.

5.6 DRUG DEVELOPMENT IN PAST AND ROLE OF BIOTECHNOLOGY TODAY 5.6.1Combinatorial Chemistry in Drug Development The biggest advantage of combinatorial chemistry over classical synthetic chemistry is that it can lead to compounds that otherwise might not be synthesized using traditional methods of medicinal chemistry. Traditionally drug development has kept same targets in mind as are considered today. Generally efforts used to be for the disease, for which large market was expected. Diseases were not studied at cellular level and at molecular level. Most drugs developed before biotechnology revolutions were developed through combination of luck and intuition regarding the basis of drug function. It incorporated lot of screening work by biologists and chemists. Large number of chemicals synthesized by microorganisms and plants and those synthesized by chemists were screened for use as drugs. Thus pharmaceutical companies have collected a large database of compounds with hope of their use for one or other target. Development of a drug from potential compounds includes lot many steps of deciding functional group, reducing side effects, improving pharmacological activity. Conventional drug discovery process has evolved in the last fifty years through

»

Random screening of synthetic molecules as well as extracts or single chemical entities from natural products, against a variety of in vitro and in vivo screening models.

» Rational design, primarily based on competitive inhibition of essential life processes, needed by an infecting organism or a vector.

Biopharmaceuticals

58 ~

Molecular manipulation for improvements of early leads and the development of new galenic forms and delivery systems for optimal activity.

~ 'Many drugs have been developed based on chapce observations of therapeutic utilities during the clinical phase or even after a drug has been marketed. ~ Fine-tuning of efficacy and additional safety of certain drugs were possible through marginal derivatizations of lead compounds and through modelling to refine appropriate threedimensional structures. ~

Complex and high molecular weight natural products with promising activity, not amenable to synthesis, were used for making semi-synthetic derivatives with improved efficacy, reduced toxicity and better pharmacokinetic properties.

~

Drugs, which are marketed as racemates, are being resolved into single enantiomers, or chiral drugs are being synthesized, which have the potential to lead to new drugs and new indications for existing drugs.

~

Very few synergistic combination drugs, which potentiate the activities of the individual components, have been developed since the dramatic breakthrough drug Cotrimoxazole.

Biotechnology companies have used the power of genetic engineering to develop understanding of a disease state at molecular level. Mapping the molecular pathology of a disease by genetic analysis allows the identification of the molecules iQ. a cellular cascade that result in diseased cell or tissue. Targets may include genes, gene regulatory factors, gene products (including enzymes, regulatory and signalling proteins), and both intracellular and extracellular receptors. The ability to clone, express and isolate virtually any desired targets has led to explosion in target strategies. Biotechnology companies are developing large number of molecules that moderate effects of target molecules. Biotechnology companies have developed rapid in vitro methods for generating, screening and amplifying drug candidate molecules using the same building blocks of life - nucleic acids, amino acids and small organic molecules. The goal of this technology is also to explore three dimensional "shape space" of the targets for requisite affinity and specificity. At molecular level this means determining steric and electronic complementarity between target and drug. Creating a large library of chemicals with novel structures is called Molecular Diversity (MD). Molecular diversity thus takes advantage of - (i) the ability to isolate target molecules in pure, crude extract or whole cell in vitro assay screens, and (ii) development of robotics and instrumentation to perform high-capacity screening on microtitre plates in a 'rapid and automated fashion. Today in fact phannaceutical companies have started using MD as an 'extension of their traditional work. Biotechnology companies use MD techniques to progress from molecular biology of large molecules to small molecules. The basic strategy of MD involves the synthesis of large compounds libraries from peptides, oligonucleotides, carbohydrates, to synthetic organic molecules. There are three strategies, which are used for generating molecular diversity. All methodologies assemble every possible combination of given set of molecular building blocks, simulatneously recodt those that have been used and then assay the resulting compounds simulatneously and select from the record, those, which are promising. (i) Those using mutatable molecules (peptides and oligonucleotides) and the process of directed molecular evolution to rapidly optimize promising molecules that can act as templates for next round of optimization. (ii) Those using small organic molecules as building blocks, which can not be mutated but exhibit large range of properties. (iii) To create and refine large numbers of peptides or oligonucleotides until a nanomolar-range molecule is found and then to convert that "lead" into small organic compound by using drug design methodologies.

Drug Discwery and Drug Designing

59

Two synthesis technologies allow rapid creation of combinatorial libraries. These are (i) "mix and hit" synthesis, and (ii) "parallel" synthesis. Identification of active molecule is done by use of monoclonal antibodies or with use of soluble receptor. Fluroscence tagged other antibody can be used to locate earlier attached monoclonal antibody, using microscopy.

5.6.2 Computer-assisted Drug Design The role of computational drug design is to aid in the discovery and optimization of new candidate drug molecules. The drug discovery cycle can be split into six phases (i) Discovery and lead generation (1-2 years)

(ii) Lead optimization (1-2 years)

(iii) In vitro and in vivo assays (1-2 years) (iv) Toxicology trials (1-2 years) (v) Human safety trials (1 year) (vi) Human efficacy trials (1-2 years)

Thus total 6-12 years are required for the development of a drug and costs are $ 100 to $ 200 million or more. Computational drug designing will mainly contribute to improve upon this cycle. Computer-based methods used for drug design may be for engineering of proteins, peptides, oligonucleotides or small organic molecules. Computational drug design includes new compound discovery by computer searching of chemical databases, compound optimization by systematic modification of functional groups to maximize potency and to minimize or eliminate side effects and toxicity, and de novo drug design (generaion of entirely new molecules that might fit a receptor site and act as antagonists or inhibitors). Most of the companies use computers in some part of their drug discovery and drug optimization process. The US company Net Genics has secured $ 6.5 million of investment to help it develop software that can be used to generate new drugs from genetic data. Net Genics will speed up the work on Synergy, its cross-platform bioinformatics software. NaviCyte Inc. has entered into an agreement with SmithKline Beecham pIc (SB) to collaborate on use of NaviCyte's proprietary computational biology tools to identify new candidates for drug development. One of the major problems in drug discovery and lead compounds selection is the high level of uncertainty in predicting the absorption and availability of drug in humans by using animal models. NaviCyte's computational tools are designed to increase the predictive power of animal models by improving the selection process of lead drugs chosen for animal testing in the first place. By using data obtained with proprietary in vitro High Throughput Pharmacokinetic technology in concert with computational tools, it provides valuable absorption information normally available after expensive animal testing. Also it comes at a very early stage in drug discovery process. The result is that once the lead compounds enter expensive animal testing they are more likely going to correlate favourably with subsequent human trials.

Computer-aided drug designing thus includes Quantitative modelling of chemical behaviour of compounds - search for generation of structures and their conversion to 2- and 3-dimensional structures, analysis of activities or properties; tools for analyzing experimental data (such as spectroscopic or diffraction studies); modelling and visualization systems for examining and predicting chemical properties and structures; and computer-assisted synthesis.

60

Biopharmaceuticals

5.7 ROLE OF BIOINFORMATICS IN GENOME-BASED THERAPY Bioinformatics provides informational framework that integrates data obtained from mapping and sequencing. This information then can be used as a starting point for development of new or improved molecules of therapeutic or clinical importance. Biomedical researcher is now equipped with a workstation, suitable software, databases and Internet. Bioinformatics has rapidly emerged as critical to gene based drug discovery. Bioinformatics encompasses (a) The use of software, databases and networks for gene and open-reading frame (ORF)

identification; (b) Database homology and pattern searching with both DNA and protein sequences;' (c) Comparative sequence analysis (which can pinpoint specific regions against which to target

drugs) and multiple sequence alignment; (d) Protein structure prediction and the mapping of functional sites; (e) Protein homology modelling and "inverse" folding as a means of probing protein structure

and function; and (j) The discovery or design of drugs against genes or their products. Comprehensive library of genes and protein sequences is required for above work. All databases are accessible and searchable via the Internet. Information Gene sequences Protein sequences. Sequence-tagged site database Expressed sequence tag database (cDNA sequences) • Polymorphic markers in human families • Patented DNA & protein sequences

• • • •

Database (Database in Europe, Japan) GenBank, GSDB (EMBL) (DDBJ) PIR (SWISS-PROT) dbSTS dbEST (CEPH) GENESEQ (published by Derwent, Inc./IntelliGenetic)

Japan archives DNA sequences obtained on the Asian subcontinent in DNA DataBank of Japan (DDBJ). GenBank, EMBL, DDBJ exchenge sequence data among each other regularly. Human Genome Project (HGP) is currendy generating 250 to 500 kbases per day and this is expected to increase to 10 Mbases per day within the next five years. Analysis of this data requires data centres both nationally and internationally to collect, manage and distribute this data. As genome project is generating information of the new type, novel electronic publishing systems (bulletin boards - the BIOSCITM newsgroups and hypertext links) are being developed. World Wide Web (WWW) on Internet and softwares such as MosaicTM and NetScapeTM are used for distributing information. Collaboration is established between database centres. In the United States the National Centre for Biotechnology Information (NCBI) has been established to see all aspects of Human Genome Initiative. Similarly in Europe, the EMBL has established European Bioinformatics Centre in Cambridge, England. A number of commercial concerns are also sequencing portions (cDNA fragment) of the human genome for commercial gains. Their proprietary database is not accessible to the public. There is a lot of debate on such maintaining of proprietary sequence information (and to allow patenting of genes or gene fragments for which function may not be known). The competitive value of genetic information is examplified by the acquisition of rights by Amgen Corporation for US $ 20 million

Drug DisclJVery and Drug Designing

61

to commercialization of therapeutic agents developed from the discovery of the leptin gene (conferring predisposition to obesity). The Institute for Genomic Research (TIGR) is a non-profit privately funded organization that is performing cDNA sequencing of fragments of human genome. They have sequenced about 100, 000 genetic fragments, which corresponds to half the genes of human genome. They aim to identify functions of the genes and to develop potential therapeutics. There are other commercial companies who have developed novel diagnostics and therapeutics from human genome technology. These are, (1) Genomyx and Hyseq (rapid sequencing technology), (2) Mercator Genetics (chromosome mapping), (work on cardiovascular disease, some cancers, endocrine disorders, psychiatric disorders) (3) Incyte Pharmaceuticals (cDNA and EST sequencing), (4) Seqana, Inc. and Myriad Genetics, Inc. (diagnostics from gene markers) (work on diagnostic kit for breast cancer, melanoma, prostrate and colon cancer, heart disease, hypotension), (5) Darwin Molecular Corp. (large scale sequencing and directed molecular evolution of targets to T-cell receptor genes), and (6) Millennium Sciences, Inc. (mapping and sequencing of multifactorial gene disorders such as diabetes). There are a number of service organizations which assist HGI. These are, Bios Labs, Inc. has developed kits for coupled DNA amplification, bulk DNNRNA isolations and characterizations, automated DNA sequencers and fast chips and dedicated boards for rapid gene comparison. ABI, Pharmacia, Genomyx, Hyseq have focused on the development of new automated or semiautomated DNA sequencers. Affymax/Affymatrics has done development of high-density arrays of oligonucleotide probes. Various genome databases store data in incompatible formats. Synergy uses CORBA to communicate with them. CORBA is a software architecture becoming widely accepted as a method for handling diverse database formats in standard ways. Synergy is designed to run on a computer behind a company Firewall (a gateway to the Internet that keeps out electronic intruders). Pharmaceutical companies will be able to access the software's functions via familiar Web browsers such as Netscape Navigator or Microsoft's Internet Explorer.

5.8 FUTURE OF BIOINFORMATICS Further developments in Bioinformatics might include • Linked entries from different databases; • New tools for the discovery of information from the data in current databases such as recognition of promoter sequences or gene start/stop sites; • Rapid and straightforward sharing of bioinformatics data; and • The ability to relate DNA information to complex control networks that regulate the immune system, intracellular signalling, and cell development. Higher-order databases of important value might include databases of enzyme activities, taxonomy databases, plant genetic databases, quantitative structure activity relationships, metabolic pathways, and drug interaction sites.

62

Biopharmaceuticals

5.9 ANTISENSE DNA TECHNOLOGY FOR DRUG DESIGNING Amongst the various therapeutic targets mentioned earlier, mRNA i.e. "sense stJ::aI1d" is the earliest target, which can be thought of to prevent the disease. Antisense technology is a novel drug discovery method. Antisense drugs work at the genetic level to interrupt the process by which diseasecausing proteins are produced. Proteins playa central role in virtually every aspect of human metabolism. Almost all human diseases are the result of inappropriate protein production (or disordered protein performance). This is true of both host diseases (such as cancer) and infectious diseases (such as AIDS). Antisense drugs can be designed to treat a wide range of diseases including infectious, inflammatory and cardiovascular diseases and cancer and have the potential to be more selective and, as a result, more effective and less toxic than traditional drugs. Specific genes contain information to produce specific proteins. The information required for the human body to produce all proteins is contained in the human genome and its collection of more than 30,000 genes. Genes are made up of DNA, which contains information about when and how much of which protein to produce, depending upon what function is to be performed. The DNA molecule is a "double helix" - a duplex of entwined strands. During transcription of information from DNA into mRNA, the two complementary strands of the DNA partly uncoil. The "sense" strand separates from the "antisense" strand. The "antisense" strand of DNA is used as a template for transcribing enzymes, which assemble mRNA - a process called "transcription." Then, using encoded information in mRNA amino acids are joined together to form specific proteins. This process is called "translation." Antisense drugs are complementary strands of small segments of mRNA. To create antisense drugs, nucleotides are linked together in short chains (called oligonucleotides). Each antisense drug is designed to bind to a specific sequence of nucleotides in its mRNA target to inhibit production of the protein encoded by the target mRNA. There are some basic differences between traditional drugs and antisense drugs in their designing and action. These become the advantages of antisense drugs over traditional drugs. The design of antisense drugs is rapid and less complex. Rational drug design usually begins by characterizing the three-dimensional structure of the protein target in order to design a prototype drug to interact with the target. Proteins, however, are complex molecules whose structure is difficult to predict. In contrast, antisense compounds are designed to bind to mRNA whose structures are more easily understood and predicted. Once the receptor sequence on the mRNA is identified, the threedimensional structure of the receptor site can be defined, and the prototype antisense drugs can be designed. Isis Pharmaceuticals, Inc. is the leader in the discovery and development of this exciting new class of therapeutic compounds based on antisense oligonucleotides. Isis has initiated programs to discover and develop antisense drugs active against a wide range of infectious, inflammatory and immune-mediated diseases and cancer. Isis' proprietary technology to discover and characterize novel oligonucleotide analogs has enabled Isis scientists to modify the properties of its oligonucleotide analog drug candidates for optimal use with particular targets and thus to produce a broad proprietary portfolio of compounds applicable to many disease targets. The effectiveness of the antisense mechanism continues to be proven in the laboratory, in animal studies, and in human trials. To date, Isis has five antisense compounds in clinical trials and a rich pipeline of preclinical compounds.

Drug Discovery and Drug Designing

63

5.10 ROLE OF GENOMICS RESEARCH IN DRUG DISCOVERY The development of diagnostic kits, prophylactics (vaccines), therapeutics and ultimately gene therapy, customized drugs and treatment will benefit from Genomics research. Genome sequences of disease causing organisms and vectors have aided pharmaceutical companies in the past and human genome sequencing will also be of help to them. The approach to new drug discovery in the future is going to be radically different from the ones practiced so far. In 1993, with the collaboration between the then SmithKline Beecham (SKB) and Human Genome Sciences (HGS), with the latter supplying SKB all its databases to develop drugs targeted at specific enzymes and receptors involved in disease processes, the new approach was set in motion. HGS has so far identified three proteins for cancer, wound healing and blood regeneration, which are under early clinical trials. These drugs are expected to reach market by 2005 (12 years after the start). Big pharma companies are bringing technologies like 'Genomics' in-house and are moving from traditional screening/chemistry capabilities to the front end of drug discovery research. They have a direct competition here with biotechnology companies. In terms of time frame the new approach is no different from the more conventional drug discovery process. In addition, whether the molecular targets developed are relevant to the therapeutic outcomes will be known only at the clinical trials stage. If indeed they are, the new drugs developed will be novel and specific for the disease conditions. The same applies to other drugs under the late pre-clinical development such as the osteoporosis drug of Amgen and the anti-cancer agent ofImmunex. All these products were developed years before the human genome was deciphered and were developed based on databases already available from Incyte, Millennium and others. Companies active in the field of genomics as a source of new drugs are - (1) American Home Products (AHP), (2) Bayet, (3) Roche, and (4) Glaxo Wellcome, The proportion of Genomi~ Research in the R&D portfolios of these companies, however, vary a great deal. For example, AHP with its ownership of Genetics Institute and collaboration with Millennium has half its projects based on genomics, while only 10% of Bayer's efforts are in this area. Glaxo SB still lays more emphasis on other technologies, including combinatorial chemistry libraries, supported by High Throughput Screening (HTS). Merck has shown minimal interest in shifting to development of genomics-based drugs. The company plans to develop custom-made drugs based on polymorphisms in people's genetic make-up. The resulting drugs, however, would belong to orphan drug category and therefore will have very restricted markets. Their emphasis in genomics research is therefore restricted to isolation of disease-related genes from affected populations. Amongst Japanese companies Takeda and Yamanouchi have been using databases in the public domain and perhaps will depend on Government funding for genomic research, which has been announced. Progress in Genomics Research as an aid to new drug discovery has been slower than anticipated and the prediction is that while several hundred new drug targets will be discovered, converting them to products will take time and money, not too different from the conventional drug discovery process. The recent trends to form consortiums of large pharma companies and genomic companies are meant to pool technical and financial resources to shorten developmental time and reduce costs.

5.10.1 Role of Genomic Companies A new breed of genomic companies has come up which collects and compiles the data of genomic sequences from genome databases available in the public domain and in some cases on subscription, and makes them useful for companies involved in hew drug discovery and development.

Biopharmaceutica/s

64

However, these companies need to have the capability to convert raw data into an appropriate information base, which then has to be annotated and correlated to relevant disease patterns. This process generally known as bio-informatics, involves using proprietary high-throughput sequencing technology, deriving genetic information and applying to the specific situation. Bio-informatics thus facilitates the selection of appropriate drug targets. Genomics-based drug discovery consists of identifYing the DNA sequences which make up the genes within the genome, detecting the precise function of the gene or the protein in the biological process related to the disease and fmally the information for designing new drugs through combinatorial chemistry and high-throughput screening. The potential of pharmaco-genomics leading to development of a large number of diagnostic kits is enormous, since a variety of tests can be generated using genome sequences. With the availability of human genome data, the number of potential targets has increased several folds since data on many genes related to the diseases or the infective agents will be now available. However, analyzing the massive data and making sense out of it requires sophisticated and high computational capacity. Overall, drug discovery has moved from a highly empirical expensive exercise to one that is highly data-oriented. Specialized companies provide not only integrated target validation systems, but also data on gene functions.

5.10.2 Drug Discovery Drug discovery is based on four core technologies: 1. Genomics (source of novel drug targets);

2. Combinatorial chemistry (molecules generated from small chemical building blocks to interact against those targets); 3. High throughput screening (methods to assay one against the other using cultured cells to mimic some aspect of a disease of interest); and 4. bwinformatics (computerized techniques for managing and analysing biological information stored in databases such as DNA sequences, and protein structure and function). While existing drugs are active against some 500 targets, genomics is expected to lead to 10,000 or more new targets for drug development. Whether these will be amenable to small molecule intervention or result in the discovery of new proteins with biological effects that can be useful as therapies in themselves remains to be seen. Big pharma is bringing these technologies in-house, moving from traditional screening/chemistry capabilities to the front end of drug discovery research, in direct competition with biotechnology companies. Despite the heavy investment in genomics, only a handful of genomics-based drugs (defined as those based on the identification of an unknown gene sequence followed by elucidation of its function and therapeutic potential) have reached the clinical stage. All are protein drugs. Also, no drugs have been discovered by the exclusive application of combinatorial chemistry/high throughput screening, nor are there is any evidence that the success rate has materially improved in the clinic. The multiplicity of targets has become a major bottleneck and many targets used in screening programs have not been properly validated (defining the role of the receptor, enzyme or target protein and confirming it is relevant to the disease process), as compounds are ultimately found to interact with irrelevant targets. There is a need for better target validation technologies and cell-based assays that more closely mimic a specific disease state, and for standardized bioinformatics software platforms so that biologists can sha re and compare data from multiple computer operating systems. The whole drug discovery field is being driven by the need for higher throughput resulting in advances in miniaturization; microfluidic

65

Drug Discovery and Drug Designing

chip based screening systems, automation and robotic chemistry devices, protein microarrays, novel signal detection and imaging systems, and advanced algorithms and pattern recognition software. DRUG DISCOVERY AND DEVELOPMENT PATHWAY

Developmental Stage

Discovery

Average Time To Complete

Safety Toxicity Testing in Animal models Total Pre-clinical

Purpose

Cost $ Million

% Probability

of Success

1-2 years

1-3

1

1 year 1-2 years 1-2 years

1 1-2 1-2

2 3 4

3-5 years

3-5

5

1-2

7

0.5-1

10

Pre-clinical

In vitro Testing In vivo Testing

Test Population

Laboratory and animal srudies

CMC (Chemistry, 1-2 years Manufacruring & Controls) 3-6 months IND (Investigational New Drug) (Preparing, Filing and FDA and FDA Approval) Clinicals Phase I 1.5 years

Phase II

2.0 years

Phase III

3.5 years

Total Clinical

6-9 years

CMC for NDA NDA (New Drug Application) Preparation and submission includes pre-NDA meeting with FDA Review Total NDA Process FDA Drug Approval Phase IV

1-2 years 6 months to 1 year 1·2 year

1.5·3 years 12·21.5

20 to 80 healthy volunteers 100 to 300 patient-volunteers 1000 to 3000 patient-volunteers

Assess safety and Biological Activity

Determine safety & dosage

15-25

Evaluate effectiveness, Look for side-effects Confirm effectiveness Monitor adverse reactions from long-term use

35-45

Can be parallel to Phase II & III

65

$ 13 $ 29 million 2-4 $ 1-2 million $ 1-2 million

Review processs Additional post·marketing testing required by FDA

$ 2·4 million $ 23.5 to 48 million 23.8·48

65

70

75

100

66

BiophRrma&euticRis

However, gene sequence data by itself provides little information about a gene's relationship to a particular disease. Efforts have now shifted to the determination of the biological function of each gene (functional genomics) using DNA micro arrays (also called gene chips or biochips) to detect mutations in specific genes as markers of the onset for a particular disease and to measure differences in gene expression (protein production) in healthy and diseased cells. Proteomics (identification of the protein profile of each cell type along with the protein's structure and function using 2-dimensional gel electrophoresis, mass spectrometry and X-ray crystallography) complements functional genomics. Because most diseases are due to protein imbalances or aberrations in protein-protein interactions, those proteins involved in critical interactions can be developed as targets for therapeutic intervention. Protein analysis has been slower to automate because proteins are much more complex than nucleic acids and because a gene may make several proteins, each of which may have a different role.

I Genome-wide Sequencing I I

Genome-wide SNPs

I

I Global mRNA expression profiling I Proteome-wide protein expression profiling

Protein

S~cture

Global Protein-Protein interactions

Industrial scale drug designing and testing

•

New Chemical Entities

RBFBRENCE 1. Nair M.D., Gmomits ReseM'Ch: Can it Speed up Drug Discovery? Pharmabiz.com

PHARMACOKINETICS 6.1 Pharmacokinetic Study 6.2 Explanation of Basics of Pharmacokinetics 6.3 Pharmacokinetic Studies on Biopharmaceuticals 6.4 Pharmacogenomics

6.1 PHARMACOKINETIC STUDY Pharmacokinetics is the study and characterization of the time course of drug absorption, distribution, metabolism and excretion, and the relationship of these processes to the intensity and time course of therapeutic and toxicologic effects of drugs. The physiologic concepts underlying the fundamental pharmacokinetic processes of absorption, distribution and elimination should be understood. The interrelationships among pharmacokinetic parameters and physiologic variables in healthy volunteers and in patients should be considered. In a typical PK study, blood samples are obtained from test animals following a single dose or a timed perfusion. Plasma samples are separated and analyzed. Typically, if the drug is going to be administered orally, both an intravenous PK and an oral PK should be run. From these two studies both the bio-availability and the pharmacokinetics of the drug can be calculated. The data is also used to generate concentration vs. time curves and allow the determination of fundamental PK parameters such as Cmax, Tmax, AVC, drug clearance, terminal elimination half-life, oral bioavailability and volume of distribution. Pharmacokinetic studies are carried out in mice, rats and in larger animals.

Animal studies of drug absorption, distribution, metabolism, and excretion are important during the early investigational new drug (IND) phase to aid in toxicity study interpretation, but need not all be completed prior to phase 1. Generally, for initial studies in humans, determining pharmacokinetic

68

Biopharmaceuticals

(PK) parameters, such as area under the curve (AUC), maximum concentration (Cmax ), and half-life (tl/2 ) in animals, is sufficient to provide a basis for predicting safe clinical exposure.

In vivo pharmacokinetic studies should be used to evaluate drug disposition and metabolism, degree of linearity and accumulation, dose proportionality, and, for oral dosage forms, food interactions. Some of these data can be gathered in a single study designed to evaluate a number of parameters. During formulation development, bioequivalence studies linking formulations may be recommended. The development of antibodies to biologicals is of concern because it may accelerate drug clearance or alter its distribution, resulting in changes in therapeutic benefit over time, or following repeated courses of treatment. Sponsors should build into their repeat-dose clinical protocols a coordinated evaluation of drug levels, receptor saturation, antidrug antibodies, and clinical responses. Optimally, these assessments would be conducted at the initiation of therapy and at several time points over the course of therapy. The presence of antidrug antibodies and their role in altering drug exposure, clinical activity endpoints, or adverse events would be evaluated. The goal of an integrated analysis of these parameters is to provide data to guide drug dosage or schedule changes to optimize therapeutic benefit. The best time for conducting these pharmacokinetic studies is prior to phase 3, before commitments have been made regarding dose and schedule. Many biotechnology-derived pharmaceuticals intended for human are immunogenic in animals. Therefore, measurement of antibodies associated with administration of these types of products should be performed when conducting repeated dose toxicity studies in order to aid in the interpretation of these studies. Antibody responses should be characterised (e.g., titre, number of responding animals, neutralising or non-neutralising), and their appearance should be correlated with any pharmacological and/or toxicological changes. Specifically, the effects of antibody formation on pharmacokinetic/ pharmacodynamic parameters, incidence and/or severity of adverse effects, complement activation, or the emergence of new toxic effects should be considered when interpreting the data. Attention should also be paid to the evaluation of possible pathological changes related to immune complex formation and deposition. It is difficult to establish uniform guidance for pharmacokinetic studies for biotechnology-derived pharmaceuticals. Single and multiple dose pharmacokinetics, toxicokinetics, and tissue distribution studies in relevant species are useful; however, routine studies that attempt to assess mass balance are not useful. Differences in pharmacokinetics among animal species may have a significant impact on the predictiveness of animal studies or on the assessment of dose response relationships in toxicity studies. Alterations in the pharmacokinetic profile due to immune mediated clearance mechanisms may affect the kinetic proftles and the interpretation of the toxicity data. For some products there may also be inherent, significant delays in the expression of pharmacodynamic effects relative to the pharmacokinetic profile (e.g., cytokines) or there may be prolonged expression of pharmacodynamic effects relative to plasma levels. Pharmacokinetic studies should, whenever possible, utilize preparations that are representative of that intended for toxicity testing and clinical use, and employ a route of administration that is relevant to the anticipated clinical studies. Patterns of absorption may be influenced by formulation, concentration, site, and/or volume. Whenever possible, systemic exposure should be monitored during the toxicity studies. When using radiolabeled proteins, it is important to show that the radiolabeled test material maintains activity and biological properties equivalent to that of the unlabeled material. Tissue concentrations of radioactivity and/or autoradiography data using radiolabeled proteins may be difficult to interpret due to rapid in vivo metabolism or unstable radiolabeled linkage. Care should be taken in the interpretation of studies using radioactive tracers incorporated into specific amino acids because of recycling of amino acids into non-drug related proteins/peptides.

Phannacokinetics

69

Some information on absorption, disposition and clearance in relevant animal models should be available rrior to clinical studies in order to predict margins of safety based upon exposure and dose. Now we can demonstrate equivalence through pharmacokinetic, pharmacodynamic, and clinical studies and overcome the fact that biopharmaceutical versions are not exactly the same. Obtaining Pharmacokinetic (PK) data is a key requirement in the evaluation of new chemical entities. PK data is also necessary for sound interpretation of pre-clinical efficacy studies and before performing of toxicology studies. PK, bioavailability, bio-distribution and bio-equivalence studies are done for new or experimental drugs. For traditional pharmaceutical products, generics are easily developed and gain market approval through an abridged procedure demonstrating physicochemical similarity and bioequivalence by pharmacokinetic and pharmacodynamic studies in normal volunteers. This concept of a generic drug cannot be applied to biopharmaceuticals due to the difficulty in developing equivalent agents for complex proteins. Because of their complexity, biopharmaceuticals cannot be adequately characterized using current analytical methods. Biological, physiological, and overall clinical qualities of biopharmaceuticals are highly dependent on specific processes of production, purification, and formulation. Biopharmaceutical innovators accumulate experience and data concerning the influence of the production process on the product that the producer of the second entry product simply does not have and cannot access. Thus, use of the same cell line and gene, and similar production, purification and formulation processes does not guarantee the product will be equivalent to the original compound.

6.2 EXPLANATION OF BASICS OF PHARMACOKINETICS Pharmacokinetics is a branch of pharmacology dedicated -to the determination of the fate of substances administered externally to a living organism. Pharmacokinetics studies the manner and extent of absorption of a substance, the distribution of this substance throughout the fluids and tissues of the body~ the successive metabolic transformations of the compound and its daughter metabolites and finally, the elimination of the parent substance and its metabolites or, in rare cases, their irreversible accumulation in a tissue. Bioavailability is a measurement of the rate and extent of therapeutically active drug that reaches the systemic circulation and is available at the site of action. Absolute bioavailability measures the availability of the active drug in systemic circulation after non-intravenous administration (i.e. after oral, rectal, transdermal, subcutaneous, etc administration). • The fraction of the administered dose that reaches the systemic circulation of the patient is affected by: - Dosage form Dissolution and absorption of the drug Route of administration Stability of the drug in the GI tract (if oral route) Extent of drug metabolism before reaching systemic circulation Presence of food/drugs in GI tract • Bioavailability Factor (F) Estimates the EXTENT of absorption Does not consider the RATE of absorption • Amount of drug reaching systemic circulation

= (F)

x (dose)

Biopharmaceuticals

70

In order to determine absolute bioavailability of a drug, apharmacokinetic study must be done to obtain a plasma drug cdhcentTation vs time plot for the drug after both intravenous and extravascular administration. The absolute bioavailability is the dose-corrected area under curve (AVC) extravascular divided by AVe intravenous. Note here that a drug given by the intravenous route will have an absolute bioavailability of 1 (F =1). Drugs given by other routes usually have an absolute bioavailability of less than one. Relative bioavailability measures the bioavailability of certain drug when compared with another formulation of the same drug, usually an established standard, or through administration via a different route. When the standard consists of intravenously administered drug, this is known as absolute bioavailability. Absolute bioavailability of a drug, when administered by an extravascular route, is usually less than one. This means that there are factors at work which reduce the availability of the drug prior to it entering the systemic circulation. Factors affecting bioavailability include • poor absorption from the gastrointestinal tract • hepatic first-pass effect • degradation of the drug prior to reaching system circulation

Half Life For a quantity subject to exponential decay, the half-life is the time required for the quantity to fall to half of its initial value. Quantities subject to exponential decay are commonly denoted by the symbol N. (This convention suggests a decaying number of discrete items. This interpretation is valid in many, but not all, cases of exponential decay.) If the quantity is denoted by the symbol N, the value of N at a time t is given by the formula: Where dN =-A.NorN =Ce-At dt A. is a positive constant (the decay constant) and

e is constant of integration

When t = 0) the exponential is equal to 1) and N (t) is equal N? As t approaches infinity) the It.,ocponential approaches zero. C is often written No (original quantity) Half life of drug is the time required to reduce the plasma concentration to half of its original value. t 1/ 2

=

In?)

where In (2) is naturallogrithm of 2 and A. is the decay constant

Toxicokinetics is the application of pharmacokinetics to determine the relationship between the systemic exposure of a compound in experimental animals and its toxicity. It is used primarily for establishing relationships between exposures in toxicology experiments in animals and the corresponding exposures in humans. Pharmacodynamics is the study of the biochemical and physiological effects of drugs and the mechanisms of drug action and the relationship between drug concentration and effect.

Pha'1'mRCOkinetics

71

Pharmacodynamics is the srudy of what a drug does to the body, as opposed to pharmacokinetics, which is the srudy of what a body does to a drug. Knowledge of pharmacokinetic data about a drug tells us: l.~ What dose to give? 2 .. How often to give it? 3. How to change the dose in cenain medical conditions? 4. How some drug interactions occur? The four processes involved in pharmacokinetics are • Drug absorption • Drug distribution • Drug metabolism • Drug excretion If a drug is going to have an effect in the body it needs to be present: • In the right place • At the right concentration • For the right amount of time

Factors affecting drug. distribution • Plasma protein binding - Competition for protein binding sites • Specific drug receptor sites in tissues - e.g. domperidone does not cause EP side effects because it doesn't reach dopamine receptors in the brain • Regional blood flow - Reduced blood flow e.g. diabetics - Enhanced blood flow e.g. liver • Lipid solubility - Blood/brain barrier - Membrane of GI tract e.g. vancomycin - Highly water soluble drugs e.g. gentamicin • Disease - liver disease can cause low plasma protein levels - renal disease causes high blood urea levels • Many drugs bound to circulating plasma proteins such as albumin • Only free drug can act at receptor site

Factors, which can increue the fraction of unbound drug: • Renal impairment due to rise in blood urea • Low plasma albumin levels «20-2Sg/L) - e.g., chronic liver disease, malnutrition • Late pregnancy - increased albumin production, but diluted by increased blood volume • Displacement from binding site by other drugs - e.g., aspirin, sodium valproate, sulphonamides, • Saturability of plasma protein binding within therapeutic range - e.g., phenytoin Factors affecting drug .metabolism (1) Main site of drug metabolism is liver. Drug metabolism can be affected by: - First pass effect - Hepatic blood flow - Liver disease - Drugs which alter liver enzymes (2) Genetic factors - e.g., acetylation status

72

Biopharmaceuticals

Other drugs - hepatic enzyme inducers - hepatic enzyme inhibitors (3) Age - Impaired hepatic enzyme activity - Elderly - Children < 6 months (especially premature babies) (4) Enzyme Inducing Drugs enhance the (production of) liver enzymes which break down drugs. This results in faster rate of drug breakdown. Larger dose of affected drug needed to get the same clinical effect. Examples are: Phenytoin, Phenobarbitone, Carbamazepine, Rifampicin, Griseofulvin (5) Chronic alcohol intake (6) Smoking (7) Enzyme Inhibiting Drugs - Inhibit the enzymes which break down drugs. This results in decreased rate of drug breakdown. Smaller dose of affected drug needed to produce the same clinical effect. (8) Hepatic Clearance is a measure of how much drug is cleared from the blood stream by the liver at each pass Hepatic Clearance

= Hepatic

blood flow

X

hepatic extraction ratio

If a drug has a high hepatic extraction ratio, drug clearance is highly affected by hepatic blood flow. If a drug has a low hepatic extraction ratio, drug clearance is independent of hepatic blood flow, but depends on the metabolic capacity of the liver. (9) Volume of Distribution (Vd): Drugs are distributed unevenly between various body fluids and tissues according to their physical and chemical properties - For example, gentamicin' Very good water solubility Very poor lipid solubility Gentamicin stays mainly in blood and body water. Reflection of the amount left in the blood stream after all the drug has been absorbed and distributed. We have to estimate because we can only measure the drug concentration in the blood stream. A low volume of distribution tells us that the drug is mainly confined to blood and body water. Very little has 'overflowed' into the tissues. A high volume of distribution tells us that the drug is widely distributed to the tissues A lot has 'overflowed' into the tissues Volume of Distribution will vary between different drugs according to: - Lipid and water solubility High lipid solubility lets the drug cross membranes. - Plasma or tissue protein binding properties. High protein binding leaves less drug circulating in the plasma. If a drug is highly distributed to the tissues the first few doses disappear immediately from the blood stream. Loading doses are required to fill up the tissues and the plasma. Important if the site of drug action is in the tissues ' Factors affecting drug excretion Main site of drug excretion is kidneys. Impaired renal function

= impaired drug excretion,

if drug is mainly renally excreted.

Pharmacokinetics

73

Glomerular filtration -

Normal GFR is about 120mlfmin

-

Rate of urine production is about 1-2ml/min.

-

Drugs can be filtered at the glomerulus

-

Extent of flltration proportional to GFR and fraction of unbound drug in plasma fu

Passive tubular reabsorption

-If total renal clearance < fu Active tubular secretion

X

-If total renal clearance > fu

X

GFR, drug is also being passively reabsorbed in renal tubules GFR, drug is also being actively secreted in renal tubules

Renal failure Renal drug elimination depends on: -Blood flow to kidney (normallSOOml/min) -Glomerular filtration rate (normal120mls/min) -Urine fl9W rate and pH which indirectly alter Passive reabsorption Active tubular secretion Patients with poor renal function will not eliminate renally excreted drugs very well • Drugs also excreted in bile, sweat, lungs, breast milk, tears, genital secretions, saliva. -Define half life and bioavailability and describe the relevance of these to drug action -Describe the effect on drug blood levels caused by impaired renal and hepatic function

6.3 PHARMACOKINETIC STUDIES ON BIOPHARMACEUTICALS 6.3.1 Cytokines Advances in molecular biology and recombinant DNA technology have led to the de.velopment of cytokines as therapeutic agents for a variety of disease states. The pharmacokinetic analysis of cytokines involves the understanding of analytical methods capable of detecting these agents in biological fluids and recognition of several factors, which may have an impact on the cytokine concentrationtime curves. The pharmacokinetic proflle of recombinant cyrokines is influenced by a number of variables: endogenous production, circulating soluble receptors and cell-associated receptors, immunocompetence and antibody production against the cytokine all may influence the disposition of the agent. The route of administration is an important variable since cytokines administered by subcutaneous injection may be partially metabolised by proteases present in the subcutaneous tissue. Other methods to simplify cytokine delivery are being actively investigated and include formulations for inhalation, topical and oral administration. A variety of cytokines (including interferon-alpha, interleukin-6 and tumour necrosis factor) are capable of inhibiting cytochrome P4S0 hepatic enzynr.!S and, therefore, possess the potential to cause ili"llg-cytokine interactions. Understanding of pharmacokinetics of cytokines is essential to the design of optimal dosage.

.

74

Biopharmaceuticals

6.3.2 Interferons Interferons (IFNs), have been widely used as antiviral and antitumor agents in humans. However, therapy with alpha IFN (IFN -cd has been complicated by the production of neutralizing antibodies to IFNs. Some reports suggest that antibodies appear to be of the immunoglobulin G class, and neutralizing antibodies have been found more frequently in patients treated with recombinant IFN-2a (rIFN-2a) than in those treated with rIFN-2b or with natural IFN preparations such as human lymphoblastoid IFNs or leukocyte IFNs. Generally, the response to the drug could be influenced by the sensitivities of living organisms to drugs and/or the pharmacokinetics of the drugs. Approaches are under investigation to improve their pharmacoefficiency. One approach to increasing the efficiency of pharmacotherapy is administration of drugs at a time of day at which they are most effective and/or best tolerated. Certainly, the use of a chronopharmacological strategy can improve the effects of drugs and reduce toxicity. IFN- is better tolerated by cancer patients when it is administered in the evening than when it is administered in the morning. There are significant dosing time-dependent differences in the antitumor and myelosuppressive activities ofIFN- in mice. Also, the rhythmic changes in IFN-induced fever and antiviral activity were examined in mice. Findings suggest that an appropriate dosing schedule and/or dosing time for IFNmay reduce the level of production of anti-IFN-neutralizing antibodies in experimental and clinical situations.

6.3.3 Interleukin-2 (IL-2) Proleukin (aldesleukin) recombinant human interleukin-2 (IL-2) has antitumor activity in patients, with significant responses occurring most frequently in renal cell carcinoma and malignant melanoma. The IL-2 receptor-positive T-Iymphocytes are thought to be primarily, but not exclusively, associated with efficacy and reside largely in the lymphoid organs. On the other hand, after IL-2 exposure, natural killer cells and neutrophils in plasma produce cytokines, reactive oxygen intermediates, and proteases, all of which have been shown to be necessary, but not sufficient, to produce the full spectrum of IL2 toxicities. Therefore, adverse in vivo activity ofIL-2 may be related to the plasma concentrations, but beneficial activity may be related to lymph concentrations. Absorption of macromolecules like IL-2 can be targeted to the lymphatic system by subcutaneous rather than intravenous administration, because the plasma concentrations of macromolecules are dependent on capillary and lymphatic absorption processes after subcutaneous dosing. Macromolecules like IL-2 diffuse through the interstitium and enter both blood and lymph capillaries. Proteins circulate within the lymph and are gradually returned to the blood. Because the primary and secondary lymphoid organs may be the sites of action of IL-2, the intensity of a pharmacological response may depend on both the systemic exposure (as measured by the plasma concentrations) and the route of administration. By simultaneously measuring the plasma and lymph concentrations, the rate of absorption directly from the injection site into the blood and lymph and the transfer rate from the lymph to the blood can be measured. As a small protein «50,000), IL-2 is rapidly cleared from the body by glomerular filtration, peritubular extraction, and, in humans, an inducible receptor-mediated mechanism. Therefore, frequent dosing is required for efficacy. To decrease IL-2 clearance, its molecular weight was increased by the addition of monomethoxy polyethylene glycol (PEG) molecules to form PEG-IL-2. PEG-IL-2 has an apparent molecular weight of 95,000 to 250,000, whereas IL-2 has a molecular weight of 15,000. There is a direct relationship between molecular weight up to 19,000 and the proportion of a dose transported lymphaticallyafter the subcutaneous administration of a neutral, water-soluble compound, and a diverse set of proteins between molecular weights 7,500 and 75,000. It has also been found that

Pharmacokinetics

75

positively charged proteins had decreased lymphatic absorption relative to negatively charged proteins with similar molecular weight. The addition of PEG to IL-2 increased the molecular weight and resulted in an overall decrease in positive charge of IL-2 after PEG attachment to lysines. It was expected that PEG-IL-2 would be preferentially absorbed via the lymphatics compared with IL-2. However, the most profound consequence of changing IL-2 to PEG-IL-2 was the increased water solubility. Therefore, the extent of absorption of PEG-IL-2 compared with IL-2 into the lymphatic system is not predictable from studies conducted so far. Modification of recombinant human interleukin-2 (IL-2) with polyethylene glycol (PEG-IL-2) decreases clearance and might favour absorption into the lymphatics, due to its increased molecular weight.

6.3.4 Recombinant Human Albumin A study carried out in Sweden for Comparison of Recombinant Human Albumin with Human Serum Albumin: Safety, Tolerability and Pharmacokinetics shows intravenous and intramuscular administration of recombinant human albumin to be well tolerated, with no treatment-related adverse events and no evidence of an immunologic response. Results of the study, which compared recombinant human albumin with human serum albumin (HSA), were presented at the 32nd Annual Meeting of the American College of Clinical Pharmacology (ACCP). According to the researchers, this is the first study reporting the safety, tolerability and pharmacokinetics of recombinant human albumin in healthy human volunteers.

6.3.5 rHuGM-CSF Information regarding the pharmacokinetics of rHuGM-CSF after intravenous or subcutaneous administration is available from studies in healthy adults, adults with malignancy or myelodysplastic syndrome, and children with recurrent or refractory solid tumors. The degree of glycosylation of synthetic rHuGM-CSFs influences pharmacokinetic parameters. Studies have determined that the pharmacokinetics of sargramostim are similar among healthy individuals and patients. The pharmacokinetics of sargramostim are dependent on the route of administration. Peak serum concentrations are higher after intravenous administration; however, bioavailability (as determined by the area under the concentration-versus-time curve) of sargramostim is similar between administration routes. The elimination of sargramostim occurs principally by nonrenal mechanisms. Serum concentrations are more prolonged after subcutaneous administration than after intravenous administration. The magnitude of the percentage of increase in absolute neutrophil count with a specific dose of sargramostim is greater after subcutaneous injection than after 2-hour intravenous infusion. The pharmacokinetics of molgramostim (0.3 to 30 fJ.g/kg) also were studied after subcutaneous and intravenous administration. Maximum serum concentrations and area under the concentrationversus-time curve increased with dose for both routes of. adminstration, but appeared larger after intravenous administration in comparison to the same dose administered subcutaneously. However, rHuGM-CSF concentrations greater than 1 ng/mL were maintained longer after subcutaneous administration. Immunoreactive molgramostim was detected in the urine of patients, ranging from 0.001 % to 0.2% of the injected dose, supporting nonrenal elimation. The half-life after intravenous adminstration ranged from 0.24 to 1.18 hours; the mean half-life was 3.16 hours after subcutaneous adminstration.

76

Biopharmaceuticals

6.3.6 Recombinant Human Erythropoietin There are substantial differences between intravenous (IV) and subcutaneous (SC) administration of rHuEpo. SC administration of rHuEpo induces lower peak plasma Epo concentrations, with an elimination half-life of about 19 to 22 hours versus the 4 to 5 hours of IV rHuEpo. SC administration of smaller doses of rHuEpo more closely resembles the physiology of Epo production and leads to greater efficacy than IV administration of larger doses. Therefore, rHuEpo should routinely be administered Sc. Recombinant human erythropoietin (rhEpo) is a support in the treatment of anaemia, primarily in renal failure. Because the half-life of circulating rhEpo is relatively short (4-8 h), the drug is usually administered 2-3 times weekly. Recendy, a novel erythropoiesis-stimulating protein (NESP) with a longer half-life (24-26 h) has been approved. NESP possesses two additional N-glycans compared to endogenous Epo or rhEpo. The pharmacokinetics of rhEpo and NESP in humans have been investigated in detail. The composition of the N-glycans is clearly important in determining the biological activity and the velocity of the degradation of Epo and its analogues. Investigators have implicated the liver, kidneys, and bone marrow as possible sites of the catabolism of Epo. However, while hepatocytes take up desialylated Epo, the liver does not appear to playa major role in the degradation of intact Epo. Likewise, renal Epo clearance is apparendy of secondary importance. Studies showing non-linear pharmacokinetics of Epo suggest that Epo is eliminated by saturable mechanisms. The hormone, as well as the recombinant drugs, can be incorporated by erythrocytic progenitors and other tissues expressing the Epo receptor. The affinity of the Epo receptor for rhEpo is 4.3-fold higher than for NESP. Taken together, it seems most likely that native Epo, rhEpo and NESP are degraded following Epo receptor-mediated uptake, mainly in bone marrow.

6.3.7 Recombinant Factor IX Recombinant factors possess essentially the same amino acid composition, but may still a different protein structure, e.g., due to differences in posttranslational processing between human cells and the produqion cell line. This may be the cause of differences in pharmacokinetic behaviour compared to the plasma products, as found with recombinant factor IX.

6.3.8 Pharmacokinetics and Biodistribution of Monoclonal Antibodies Radiolabeled mAbs are important clinical reagents for both tumor imaging and therapy and also provide an ideal evaluation of pharmacokinetics. Therapeutic administration requires a balance between prolonged retention at the target site and slow clearance-which can lead to liver accumulation and high radiation exposure of other tissues. The choice of radionuclide dictates both the application and the required pharmacokinetics: for example, 1241 for positron emission tomography (PET) imaging and 90y for radiotherapy. For solid tumors, there are the additional problems imposed by penetration through the vasculature and dispersion against an interstitial pressure. Recendy, algorithms have been formulated that identify appropriate design criteria for cell targeting by inclusion of rates for diffusion, binding, internalization and systemic clearance. Vascular penetration may be gready enhanced by targeting fragments to surface molecules on endothelial caveolae, because these specialized plasmalemmal invaginations can transcytose across the normally restrictive endothelial cell barriers to reach underlying tumor cells. This allows much greater penetration into, and accumulation throughout, solid tumors and should enable more effective delivery of imaging agents or therapeutic payloads. Systematic in vivo studies have provided striking confirmation that size is an important parameter in pharmacokinetics and biodistribution of mAb molecules. Large IgG molecules (150 kDa) specific

Pharmacokinetics

77

for tumor-surface molecules can penetrate solid tumors only slowly, are' nonuniform in their final distribution and have high serum levels and associated toxicities. Conversely, small scFv fragments (30 kDa) are cleared extremely rapidly and have poor tumor retention because of their monovalent binding properties. The ideal tumor-targeting reagents are intermediate-sized multivalent molecules (for example, bivalent diabodies, 55 kDa), which provide rapid tissue penetration, high target retention and rapid blood clearance. The most recent biodistribution studies have independently confirmed that diabodies, because of their small size, are rapidly eli'llinated through the kidneys, thereby limiting the exposure to the bone marrow, which is most often the dose-limiting organ with intact radiolabeled mAbs. Diabodies possess an excellent combination of rapid tumor uptake and clearance for in vivo imaging when labeled with 1231 or lllln and rapid xenograft visualization by PET when labeled with positron emitters such as 64Cu or 1241. These studies helped for effective diabody radiotherapy. Diabodies have also proven useful in imaging angiogenesis and atherosclerotic plaques. Larger bivalent molecules, such as minibodies (scFv-CH 3 dimers), and scFv2-Fc can accumulate to higher abundance in tumors. Minibodies may be ideal for tumor therapy because they achieve a higher total tumor uptake, substantially faster clearance and better tumor-to-blood ratios than either intact immunoglobulin (150 kDa) or Fab'2 (110 kDa;). Preclinical trials with Fv tetramers either as (scFv2h or as bispecific bis-tetrabodies have also been encouraging, although the additional linker residues can be exposed and thereby result in increased protease degradation. Tumor localization and uptake depends not only on size but also on other parameters such as interaction affmity. Avidity seems to be more important than affinity, with diabodies generated from lower-affinity single-chain VH- and VL -domain fragments (scFvs) consistently achieving higher tumor uptake. The pharmacokinetics of mAb fragments can also be modified using other strategies, such as linkage to polyethylene glycol (PEG). PEG linkage (PEGylation) has been very efficient for increasing half-life and scFv stability, conferring improved anti-tumor activity and apparently also reducing immunogenicity. Another strategy for increasing serum half-life of mAb fragments is through fusion or noncovalent interaction with long-lived serum proteins, such as albumin or serum immunoglobulin. Finally, if desired, systemic clearance can be accelerated by mannose glycosylation, which is provided by the use of yeasts, such as Pichia pastoris, as expression hosts.

6.4 PHARMACOGENOMICS 6.4.1 What is Pharmacogenomics? The way a person responds to a drug (positive and negative reactions) is a complex trait that is influenced by many different genes. Person's response to a particular drug could be predicted only if all of the genes involved in drug response are understood. Then genetic tests could be developed to predict the drug's response. Pharmacogenomics is a science that examines the inherited variations in genes that dictate drug response and explores the ways these variations can be used to predict whether a patient will have a good response to a drug, a bad response to a drug, or no response at all. High throughput genotyping for search of variants is the most critical technology in pharmacogenomics. Pharmacogenomics refers to the general study of all of the many different genes that determine drug behaviour. Pharmacogenetics refers to the study of inherited differences (variation) in drug metabolism and response. The two terms are used interchangeably. Pharmacogenomics and Pharmacogenetics will have major impact on the future of healthcare. Pharmcogenetics is the study of the genetic basis for individual's variable drug responses and is being

78

Biopharmaceuticals

applied with increasing frequency at clinical level. These efforts rely on pharmacogenomics, the genome-wide analysis of the genetic determinants of drug efficacy and toxicity. Currently the field of pharmacogenomics is immature. Wordwide market for pharmacogenomics was $ 1.24 billion in 2004 and is projected to rise to $ 3.7 billion by 2009. Genotyping technologies is expected to reach to $ 1. 7 billion while SNP identification technologies will reach $ 397 million in 2009. Pharmacogenomics comprises the study of variations in targets or target pathways, variation in metabolising enzymes or, in the case of infectious organisms, genetic variations in the pathogen. Pharmacogenomics does not include the use of genetic or genomic techniques for the purposes of biological product characterisation of quality contol (e.g. cell bank characterisation bioassays). Pharmacogenomics also does not refer to data resulting from proteomic or metabolomic techniques.

6.4.2 Gene Variation to Predict Drug Response Right now, there is a race to catalog as many of the genetic variations found within the human genome as possible. These variations or SNPs, as they are commonly called, can be used as a diagnostic tool to predict a person's drug response. For SNP to be used in this way, a person's DNA must be sequenced for the presence of specific SNPs. The problem is, however, that traditional gene sequencing technology is very slow and expensive and has therefore impeded the widespread use of SNPs as a diagnostic tool. DNA microarrays (or DNA chips) should make it possible for doctors to examine their patients for the presence of specific SNPs quickly and affordably. A single microarray can now be used to screen 100,000 SNPs found in a patient's genome in a matter of hours. As DNA microarray technology is developed further, SNP screening in the doctor's office to determine a patient's response to a drug, prior to drug prescription, will be possible. SNP screening will benefit drug development and testing because pharmaceutical companies could exclude from clinical trials those people whose pharmacogenomics screening would show that the drug being tested would be harmful or ineffective for them. Excluding these people will increase the chance that a drug will show itself useful to a particular population group and will thus increase the chance that the same drug will make it into the marketplace. Pre-screening clinically that subject should also allow the clinical trials to be smaller, faster and therefore less expensive. Resultantly the consumer could benefit from reduced drug costs. Finally, the ability to assess an individual's reaction to a drug before it is prescribed will increase a physician's confidence in prescribing the durg and the patient's confidence in taking the drug. This would encourage the development of new drugs tested in a like manner. Today patients are given medications that either don't work or have bad side effects. Treatment continues till the doctor can find a drug that is right for them. Pharmacogenomics offers a very appealing alternative. Now, the doctor will perform genetic test and decide which drug available in the market will be most appropriate for the patient. The tools for pharmacogenomics carry the promise of achieving improved drug safety, earlier attrition rates, decreased drug development costs, a reduced drug development cycle, and resuscitation of failed drugs. The practice of pharmacogenomics can potentially help clicnicians administer more tailored treatment. The Human Genome Project has aided scientists in identifying the function and location of only a small fraction of the genes that define the human race. Much more genetic data will thus have to be collected and deciphered before genetic medicine can reach its full potential. Moreover, testing patients to identify their genes is still a slow and expensive process that costs from $ 1,000 to $ 2,000 for each test, though a few simple tests for infectious diseases cost as little as $ 20. The cost of individualized drug therapy, too, is likely to be high.

Pharmacokinetics

79

Pharmacogenomics might reduce the length of trials, something that would be particularly beneficial for developing drugs to treat degenerative diseases whose early stages are relatively asymptomatic. If the three levers of cost reduction - a reduction of the number of compounds, the number of patients, and the length of trials - were applied across all phases of clinical development, pharmacogenomics could eliminate $ 60 million to $ 85 million in costs for each approved drug. The savings take into account the higher expense of pharmacogenomic testing-perhaps $ 5 million to $ 10 million for every approved drug. Inherited variations such as the genes that specify enzymes responsible for drug along metabolism or in the receptors that bind to the drugs contribute to variability in drug responses among population. The detection of a single nuclieotides polymorphisms (SNPs slight differences in DNA sequences among individuals leading to variations in protein expression) will enable companies to segment people according to how they react to a particular drug. The technologies employed (DNA microarrays and sequencing) are presendy too expensive for routine use and few doctors know how to interpret the results. Tests for drug responses are particularly relevant when the drug therapy is either expensive or risky, or a high probability of non-responders exists. For example, Monoclorial antibody "Herceptin", for breast cancer is effective only in 30% women who are overexpressing a particular growth factor. REFERENCES 1. Piscitelli SC, Reiss WG, Figg WO, Petros WP, Pharmacokinetic studies with recombinant cytokines. Scientific issues and practical considerations, Clin Pharmacokinet. 1997 May; 32(5):368-81. 2. Wang De-sheng, et aI. Effect of Dosing Schedule on Pharmacokinetics of Alpha Interferon and AntiAlpha Interferon Neutralizing Antibody in Mice, Antimicrobial Agents and Chemotherapy, January 2001, pp. 176-180, Vol. 45, No. 1. 3. Chen Sharon A. Plasma and Lymph Pharmacokinetics of Recombinant Human Interleukin-2 and Polyethylene Glycol-Modified Interleukin-2 in Pigs· The Journal of Pharmacowgy and Experimental Therapeutics, Vol. 293, Issue 1, pp. 248-259, April 2000. 4. Filion Mike, ACCP: Recombinant Human Albumin Gets High Marks For Safety, Tolerability and Pharmacokinetics htt.p:I}www.pslgroup.comltWc.guide.htm. 5. Armitage James O. Emerging Applications of Recombinant Human Granulocyte-Macrophage ColonyStimulating Factor, Blood, Vol. 92 No. 12 (December 15), 1998: pp. 4491-4508. 6. Jelkmann Wolfgang, The enigma of the metabolic fate of circulating erythropoietin (Epo) in view of the phamuuokinetics of the recombinant drugs rhEpo and NESP European Journal of Haematology Volume 69: Issue 5.

7. http:jwww.nebi.nim.guv/About/primer/intiex.htmt)

o CLINICAL TRIALS 7.1 Introduction 7.2 7.3 7.4 7.S

Types of Clinical Trials Phases of Clinical Trials Clinical Trials Opportunities lndia as Destination for Clinical Research

7.6 India's Strength 7.7 Players in Clinical Trials Field in India 7.8 Hurdles for Clinical Trials in India

7.1 INTRODUCTION 7.1.1 An Outline of the Drug Development Process Details of drug discovery and development pathway are already given in Chapter 5. Drug development involves a complex, FDA-regulated process of clinical trials. The discovery phase or the preclinical phase can cost $ 3-5 million. During this phase, the in vitro and in vivo (animal) testing is performed to determine the efficacy, safety and any toxic side-effects of the drug. If promising results are obtained, the scale-up conditions for the Chemistry, Manufacturing and Controls (CMC) are set-up to manufacture the drug according to Good Manufacturing Practice (GMP) regulations.

Clinical Trials

81

Following this, the Investigational New Drug (IND) application is filed for FDA approval before progressing to Phase I clinical trials. At this stage, the success of a drug can be rated at 10%. After the approval of IND, the drug's safety and efficacy are tested, in different sub-set of population in different doses, formulations, parameters, etc. during 3 distinct phases of clinical trials; Phase, I, Phase II and Phase III. This period of clinical trials can range from 6-9 years and cost 25 million dollars to 80 million dollars. The probability of success of a drug with good results at this stage is 65%. The results obtained, during the Phase I, II and III trials, are compiled for a New Drug Application (NDA) at this stage for FDA approval. This is a very critical step and the total process involves pre-NDA meetings with FDA, preparation, submission of NDA, followed by the review process by the FDA. There is a 75% possibility of success at this stage. FDA approval can be extended from 1-3 years. The FDA approval allows the drug to be marketed, after the revisions, restrictions and other suggestions by the FDA are implemented. On the average it takes $ 800 million and 12 to 15 years to develop a new drug and bring it to the market. However, the alternatives based biotedmology cur down the costs and time by more than half due to limited side effects. Industry studies show that some 371 biotech-based drugs are now under development by 144 companies worldwide against 200 diseases. So far, the US government has approved 95 biotechnology drugs.

7.1.2 Clinical Trial A clinical trial (also clinical research) is a research study in human volunteers to answer specific health questions. A clinical trial is a research study to answer specific questions about new drugs or treatments for disease. Clinical trials are used to determine whether new treatments are both safe and effective in humans. These trials generally occur after extensive work in the laboratory and in animal studies. Clinical trials are regulated by the Food and Drug Administration (FDA) and other regulatory agencies around the world. Carefully conducted clinical trials are an established way to find treatments that are safe and effective. Interventional trials determine whether experimental treatment or new ways of using known therapies are safe and effective under controlled environments. Observational trials address health issues in large groups of people or populations in natural settings.

7.1.3 Protocol Protocol is a study plan on which all clinical trials are based. The plan is carefully designed to safeguard the health of the participants as well as answer specific research questions. A protocol describes what types of people may participate in the trial; the schedule of tests, procedures, medications and dosages; and the length of the study. While in a clinical tiral, participants following a protocol are seen regularly by the research staff to monitor their health and determine the safety and effectiveness of their treatment.

7.1.4 Expanded Access Protocols Most human use of investigational new drugs takes place in controlled clinical trials conducted to assess safety and efficacy of the new drugs. Data from the trials can serve as the basis for the drug marketing application'. Sometimes, patients do not qualify for these carefully-controlled trials because of other health problems, age, or other factors. For patients who may benefit from the drug use but don't qualify for the trials, FDA regulations enable manufacturers of investigational new drugs to provide for "expanded access" use of the drug. For example, a treatment IND (Investigational New

82

Biopharmaceuticals

Drug application) or treatment protocol is a relatively unrestricted study. The primary intent of a treatment IND/protocol is to provide for access to the new drug alternative treatment. A secondary purpose for a treatment IND/protocol is to generate additional information about the drug, especially its safety. Expanded access protocols can be undertaken only if clinical investigators are actively studying the experiment treatment in well-controlled studies, or all studies have been completed. There must be evidence that the drug may be an effective treatment in patients like those to be treated under the protocol. The drug cannot expose patients to unreasonable risks given the severity of the disease to be treated. Expanded access protocol are generally managed by the manufacturer, with the investigational treatment administered by researchers or doctors in office-based practice.

7.2 TYPES OF CLINICAL TRIALS Treatment Trials test experimental treatments, new combinations of drugs, or new approaches to surgery or radiation therapy. Prevention Trials look for better ways to prevent disease in people who have never had the disease or to prevent a disease from returning. These approaches may include medicine, vitamins, vaccine, minerals, or lifestyle changes. Diagnostic Trials are conducted to fInd better tests or procedures for diagnosing a particular disease or condition. Screening Trials are the trials which test the best way to detect certain diseases or health conditions. Quality of Life Trials (or Supportive Care Trials) explore ways to improve comfort and the quality of life for individuals with a chronic illness.

7.3 PHASES OF CLINICAL TRIALS After a new drug or treatment is ready, government regulations in most countries require it to go through four phases of clinical trials prior to approval for sale to the people without too many restrictions. Phase I trials are the toughest where the new drug or treatment has to be tested on a small group of 20 to 80 healthy people. This has to be done to evaluate the drug's safety, determine the dosage requirement and identify any side effects. It also aims i:o fInd out how quickly drug is absorbed, metaboilised, and excreted from the body. In Phase II trials, the drug or treatment is administered to a select group of patients ranging from 100-300 to test its effectiveness against signs and symptoms of disease and safety further. In Phase III trials, the drug has to be given to a large group of patients (usually 1000-3000) to consolidate data on effectiveness, safety, best dose and rare side effects. This data is then compared with existing drugs or treatments used for the disease to facilitate permission for large scale. If all goes well, the drug manufacturer applies to the Food and Drug Administration for an NDA, a new drug application. If it is granted, the generic name of the drug is replaced by a brand name chosen by the manufacturer. For example, one of the fIrst drugs used against AIDS was azidodideoxythymidine (AZT). When placed on the market, this name was replaced by the brand name zidovudine. In the post-marketing era, Phase IV trials are done to collect additional information on the drug's additional risks and benefIts and study its optimal use. Even after a drug is available for prescription, its use is carefully monitored and unexpected side effects are reported.

Clinical Trials

83

7.3.1 Phase Zero Development costs for new drugs are rising dramatically. A large factor in the increased costs

is that many drugs are failing late in development (Phase III trials). The approval rate for innovative new drugs is declining. Some of these failure can be attributed to poor or poorly understood pharmacokinetic (PK) parameters and the fact that regardless of how well characterised a compound's behaviour is in vitro or in animal models, these systems are imperfect representatives of human physiology. Some 30%-40% of new drugs fail due to poor performance at the tranistion from animal to human trials. To mitigate the risks and costs associated with large-scale failures, companies have recently looked to a new method of testing compounds earlier in humans. Phase Zero. These microdosing studies involve the administration of sub-pharmacologic or sub-therapeutic doses (on the order of micrograms) of a drug candidate to humans, who are monitored to generate a preliminary ADME or PK proflle. It is hoped that giving companies earlier, safer data on how the drug is processed in the body will dramatically accelerate the more expensive clinical testing phase. Although the Phase approach is not appropriate for all compounds, when thoughtfully applied, Phase 0 techniques help developers select only the most promising drug candidates for further development by mitigating the risk of failure due to PK and bioavailability characteristics in humans. For early-stage pharma and biotech flrms, Phase Zero testing is a cost-effective way to increase value by providing flrst-in-human data earlier in the development/investment cycle. European Agency for the Evaluation of Medicinal Products (EMEA) put out a position paper in early 2003 which supported the use of microdosing as nonclinical safety studies in support of further clinical studies, and it deflned a microdose as 1/100th dose required to present a pharmacological effect, and no more than 100 grams. FDA went a step beyond the EMEA paper by issuing a draft guidance document relating to exploratory Investigational New Drug (IND) applications and which included reference to the use of microdosing as part of this process. The EMEA position paper deals only with microdose studies that allow only single, nonpharmacologic doses and provide information only on pharmacokinetics. The FDA guidance also discusses the option of performing repeat-dose clinical studies using doses designed to induce pharmacological effects. These latter types of studies provide much more information regarding potential efficacy. The new guidance should save companies millions of dollars in development costs in short order. In the traditional IND, preclinical toxicology and safety requirements cost more than $ 650,000 and can take as long as six months to perform. However, a human microdosing experiment can be initiated with less than $ 150,000 in preclinical toxicology and safety testing which can be completed within one month.

7.4 CLINICAL TRIALS OPPORTUNITIES Every year over 80,000 clinical trials of various drugs and the treatment are conducted in the world. Estimated to cost around $ 13 billion (Rs. 60,000 crores), these trials happen mostly in the developed countries. Out of this, approximately $ 4 billion (Rs. 19,000 crores) is spent on doctors and the remaining $ 9 billion (Rs. 41,000 crores) goes to organisations conducting the trials. It is estimated that about 20 per cent of all clinical trials conducted gloablly will be from India by 2010. Over two million people will be participating in clinical studies in India by that time. In 2004, over nine million patients participated in clinical trials globally. Clinical trial industry has grown from Rs. 100 crores to Rs. 250 crores and is expected to touch Rs. 5,000 crores by 2010. Clinical research and trials are expected to grow exponentially over the next 5 years.

84

About 10 per cent of global clinical trials take place in Latin America, Asia and Central Europe, and the figure would rise to 25 per cent by 2008.

7.S INDIA AS DESTINATION FOR CLINICAL RESEARCH Several top pharmaceutical companies in India are venturing into contract research on a big way as they find it was a potential area for growth in the next few years. The current Indian CRO market is estimated at 70 to 100 million, which is growing at a rate of 60 per cent annually. Some of the major pharma companies which have set up their own CROs recently include (1) Wockhardt, (2) Zydus Cadilia, (3) Cipla, (4) Sun Pharma, (5) Torrent, (6) Unichem, (7) Alkem, (8) Intas, (9) Ranbaxy, and (10) Nicholas Piramal are already having their CROs namely SRL Ranbaxy and Well Quest. Along with CROs promoted by pharma companies (pharma CROs), there are independent CROs like (1) Siro Clinpharma, (2) Clinivent Research, (3) Lambda, (4) Synchron, (5) Quintiles, and (6) Coance doing business in the country. Most of these CROs have restricted their activities to bioequivalence (BE) and bioavaliability (BA) studies. However, a few like Zydus Cadila's CRO, SRL Ranbaxy and Well Quest have extended their work in the area of clinical trial as well. BE/BA studies in India can be conducted at about one-third costs compared to US or Europe. So, foreign companies could outsource the BE/BA studies to India, considering cost benefits, even if the product is not meant to be marketed in India. According to DiMasi et al, the cost of NCE development for US companies has reached $ 1.1 billion per molecule. The clinical costs have gone up by 8 per cent compared to few years back. US health care expenses are expected to go up by 14.7 per cent in 2012 compared to just 9.5 per cent in 2002. In the area of clinical trials, US is expected to have a shortfall of 56,000 investigators. India is seen as a key emerging destination for conducting clinical trials. A large number of these trials could be conducted at much lower costs in developing countries like India. However, due to a variety of factors including regulatory issues, inadequate facilities and issue related to safety of sensitive data, this has not been happening in a big way. According to a Rabo-India Finance report, China seems to have attracted more interest in clinical trials despite India's English-speaking doctors and technicians. The recent global guidelines making it mandatory for testing new drugs across a variety of gene pools may have increased the interest in India. India definitely offers a large patient diversity and the numbers. It is easier to do multi-centre, large scale trials in India for many diseases. For example, India has over 25 million patients suffering from type II diabetes, accounting for 20 per cent of the global diabetic population. Many biotech companies are looking in India for clinical trials. Eli Lilly is a major user of Indian expertise. In 2002 it spent Rs. 10 crores on clinical trials in India. Participation in global clinical trials is a win-win situation for the medical profession, patients, medical institutions, laboratories and data management specialists. Patients will have access to 'state of the art' medical care at no expense and medical professionals will get an opportunity to work with 'cutting' edge technologies and exposure to modem research methods. In addition, participating medical centres will get the latest equipment to facilitate these trials; the standard of laboratories will increase to match global levels. The IT industry will benefit from data manager service orders. And the nation will earn on average $ 3,000 per patient taking part in the trials. Pharma companies, spend $ 300-500 million for developing any new drug, of which expenses account for 75 per cent on clinical research and evaluation. This is an area we should really exploit. The country's large disease population provides a great degree of statistically superior clinical data and India is in a unique position to convert its disease disadvantage into a research advantage. However, clinical trial research could provoke criticism of the country's population being made guinea pigs.

Clinical Trials

85

Specifications of schedule Y of Drugs and Cosemetic Act in India guide the clinical trials legislative requirements. Recently the Ministry of Health, along with DCGI and Indian Council for Medical Research (ICMR) has come out with draft guidelines for research in human subjects. These are essentially based on Declaration of Helsinki, WHO guidelines and ICH requirements for GCP, (Pharmabiz.com 18th July 2002). The government has to strengthen the implementation and monitoring of adherence to Indian Council for Medical Research (ICMR) guidelines on good clinical pratices (GCP), trial professionals in GCP and good laboratory practices (GLP) in tune with global standards and ensure accreditation to laboratories. Facilities should be of international standards, and ensure accreditation to laboratories. Facilities should be of international standards established by World Health Organisation (WHO) or Federal Drug Authority of the US (FDA). Clinical trials for around 20 r-DNA products are going on in India. 70% of New Chemical Entities (NCEs) under clinical testing are all products of r-DNA or gene based Biotechnology, most of these originating from small and medium sized biotech companies.

7.6 INDIA'S STRENGTHS 1. Prequalified physicians 2. There are many hospitals in India - small and big, municipal and Government, which conduct clinical trials. Large private hospitals and clinics too perform this work. Hospitals capable of conducting clinical trials for oncology, central nervous system, endocrinology, infectious diseases, internal medicine and cardiovascular drugs are available in India. 3. It costs less to conduct clinical trials in India than in developed countries. 4. India has a largest patient pool. Availability of patients of genetically distinct population groups.

5. Availability of investigators. 6. Networks of academic medical centers. 7. Poor population may benefit by gaining access to latest treatments. 8. Speed. 9. English language speaking field workers, doctors, nurses, technicians etc. All the source documents, hospital papers, laboratory reports, clinical notes are generally written, printed in English avoiding need of translation of documents that arises in countries like China or Japan for auditors from the West. The monitors and other project management staff are also generally fluent in both English and the local language enabling building of easy Qlld quick rapport. 10. Quality, responsiveness are some of the other advantages India has, to make clinical trials as field of opportunity. 11. It does not take more than six months for the setting up of multi-centric trials by taking permissions from DCGI, import licensing and clearances and local ethics committees. This makes India a comfortable place for investigators for such work. 12. Highly motivated and committed clinicians to comply with the protocols and willing and eager to work to international standards is a further plus point.

86

Biopharnuu:euticals

7.7 PLAYERS IN CLINICAL TRIALS FIELD IN INDIA Broadly Contract Research Organisations (CROs) are classified into three categories:Large companies having a global presence and serving as sponsors for phase III trials for a three-year phases, national presence and the university born CROs which operates. Clinigene International a subsidiary of Bangalore-based Biocon Group has positioned itself well to offer clinical trials to foreign companies. They have initiated clinical studies in diabetes in collaboration with a California-based biotech company. These studies are expected to extend to other disease segments - asthama, lipidemia and arthritis - in the coming years. Clinigene also has the capability to conduct drug-related bio studies, pharmacogenomics based clinical studies as well as other specialised longitudinal studies on select patient populations. Clinigene's competencies, include conducting bioequivalence, bioavailability studies, Phase I to Phase IV clinical trials and other special clinical studies in line with internationally accepted norms and practices. Irish company ICON Clinical Research has started operations in India in April 2002 to conduct clinical trials for foreign companies. ICON offers a range of services including biometrics and statistics, clinical laboratory services, clinical research consultancy, clinical research management, data management, interactive voice response system, medical writing, pharmacovigilance and regulatory and consultancy services. Quintiles Transnational Crop. (QTRN, Research Triangle Park, N.C.) has conducted more than 35 trials, primarily Phase III trials to support regulatory submissions in the US and Europe. The Hyderabad-based Vimta Labs is setting up a 2,00,000 sq. ft. laboratory complex at SP Biotech Park in Hyderabad which is expected to be commissioned by September 2005. The new facility is expected to cost Rs. 45 crores and Rs. 12 crores will be through internal accruals and rest of Rs. 33 crores funding will be by way of term loans. The facility will comprise molecular biology laboratories, microbiology laboratories, captive laboratories for drug testing, pre-clinical toxicology laboratories, secure data centre and network, and knowledge centre. The company will also set up a training centre where it will train 50 people per batch on various parameters of contract research. The facility is to meet good laboratory practices, good manufacturing practices and good automation lab practices. Vimta is also planning to take up capacity expansion at its Chennai facilities. It is to add bed capacity in its voluntary health centre (VHS) for facilitating clinical trials. At present it offers clinical pathology and clinical trial services at Chennai. The Company also carries out clinical pathology services in Visakhapatnam and Vijayawada facilities. The company's services for overseas markets accounted for 40 per cent of the total services in the financial year 2004-05 and are expecting it to grow to 50 per cent during the current financial year. The company finds good opportunities in the US, Europe, Gulf countries and Asia Pacific. Vimta is expecting to recruit 100 more scienctists during the current fiscal, and it has a strength of 500 employees at present. Mumbai-based Haffkine Institute will begin clinical research activities after its drug testing labs are upgraded. Maharashtra Government has recently sanctrioned a funding of Rs. 3 crore in this regard. Currently the tests conducted by the institute include toxicology, bacteriology and virology, and polymerase chain reaction-based testing. The Government of India has introduced guidelines for conducting clinical trials in the country. The Biotech Commission of Maharashtra, in consultation with the pharmaceutical industry, has worked on the same and finalised the guidelines for the state. The pharmaceutical companies are

Clinical Trials

87

discussing with the health department in this regard. This is an innovative area the state is looking at. Though companies are looking at the private hospitals for clinical trials but their client base is very small. Public hospitals can provide them with a list of patients and a huge database to carry out the trials. As per the guidelines finalised by the Biotechnology Commission, the public hospitals would maintain privacy and follow ethical guidelines while carrying out the trials. We are also looking at providing facilities to carry out clinical trials on animals so that the pharma companies need not look at other sources for their new molecules. Companies .like Workhardt, Nicholas Piramal, Glaxo and Lupin are investing in biotechnology. Glaxo SmithKline Pic would be setting up a clinical research facility in India soon. The facility will be used to undertake trials of drugs which are in the company's R&D pipeline and intended for introduction in the Indian market. The company's decision to have a full-fledged CR set up for global trials here has come now as the country is approaching the new patent regime. Many other companies such as Quitntiles, SIRO Clinpharm, Wellquest, Lambda, Lotus Labs, Synchron are active in this area. MNCs like Novartis and Pfizer also have established data analysis centres in India. Quintiles in Ahmedabad, Clinfarm Consultants and Specially Ranbaxy in Mumbai and Kumar in New Delhi are active in clinical trials area. Quintiles and Specially Ranbaxy have foreign tie-ups giving them the technological advantage over the purely local ones. India will have to implement product patents from 2005. This will lead to widening of the market for indigenous (say, Ayurvedic products) as they will have the authentiity of intenationally recognised product patents. Manipal AcuNova, a collaborative venture between Maipal Education and Medical Group (MEMG) International India cind AcuNova Life Sciences Pvt. Ltd. is a new entrant in the field of clinical trials. Bangalore's Manipal Hospital and at Kasturba Medical College (KMC) Manipal with 'patient-physician' availability, access to the steam cell research and genetic centre at Bangalore, University of MARE providing all the resources including the animal house and Pharmacogenomics core facility situated at the KMC Life Science (set up under the research and development scheme from the Department of Science and Technology (DST) and Technology Information Forecasting and Assessment Council (TIFAC), Government oflndia) are the support facilities for this company. Primarily KMC will focus on bio-availability and bio-equivalence (BA/BE) studies, phase I to III trials performed for genetic drugs, new drug delivery systems and new chemical entities. They will do special studies like research into the drug performance with reference to the genetic background of patients to assess efficacy parameters. The Institute of Clinical Research (ICR), the largest professional clinical research body in Europe, will soon start operations in India to train professionals and to raise the standards of clinical research activities in India. ICR can help in develQping professionals, sharing knowledge and raising standards.

7.8 HURDLES FOR CLINICAL TRIALS IN INDIA 1. Government regulations do not permit conducting of phase I trials (on small group of healthy volunteers) except when the drug or treatment originates in India. Apparently these restrictions have been retained to prevent potential risks to Indian volunteers from untested drugs. 2. Lack of full-scale protection to intellectual property rights (IPRs). Protection to research data (data exclusively) is an area of concern to foreign companies. Foreign companies can not

88 TABLE 7.1 LIST OF COMPANIES ENGAGED IN CLINICAL TRIALS IN INDIA No.

Name of Company

1

BioMix Networks Ltd.

Software for clinical trials

2

Clinigene International Pvt. Ltd (Biocon Group)

Offers laboratory services to clinical research organisations (CROs) for a range of clinical trials.

3

Cytogenomics (P) Ltd.

Clinical trials for diagnostic and therapeutic products.

4

Lambda Therapeutic Research

Monitoring Services for clinical trials (Phase Ifll/ I1IfIV), Bioavailability studies.

5

Pfizer India Limited

Proposals from domestic biotech companies for clinical research.

6

Quintiles Spectral (India) Ltd. (QSL) (subsidiary of Quintiles transnational)

Providing management services for logistics supporr for clinical trials of drugs.

7

Siro Clinpharm Pvt. Ltd. http://www.siroindia.com/intro.htm

End-to-end clinical trials operation group, clinical trials supplies management.

8

SRL Ranbaxy Limited

World Cass Central Clinical Reference Laboratories

9

Wellquest Clinical Research (clinical research division of Nicholas Piramal)

Clinical trials (Phase I - III) for evaluaing safety and efficacy ofNCEs and other products, Clinical Pharmacokinetic Studies.

do anything if other Indian players manage to get the trial data and use it to form alternative products.

3. Long-time period (four to nine months) to get regulatory approvals, unpredictability of regulatory guidelines is also an hurdle. On an average such approvals are available in countries such as Australia, Germany, Belgium, Norway, UK and South Africa in 6 to 12 weeks. 4. Need to strengthen the implementation and monitoring by Government of good clinical practices (GCP), train professionals in GCP and good laboratory practices (GLP) in tune with global standards, and ensure accreditation to laboratories. 5. Need to develop facilities of international standards established by World Health Organisation (WHO) of Federal Drug Authority of the US (FDA). REFERENCES 1. Clinical Trials.gov.

2. Feb., 2002, 'India walks gingerly towards biotech dream', Yahoo, India, Technology)

Clinical Trials

89

3. N. Suresh, Clinical trials: the new mantra for growth, Pharmabiz.com 10th April 2003. 4. Pharmabiz.com 26th April 2005. 5. Pharmabiz.com 9th April 2005. 6. http:/www.biospectrumindia.com. 7. Dr. Kiran Mazumdar-Shaw, Chairman of the Biocon India Group in Business Line, 21st March 200l. 8. http:/www.indiaonestop.com/clinicaltrials.htm 9. Wills Randal, "What is Phase Zero", The Scientist, Volume 19, Issue 20, page 38, Oct. 24, 2005.

REGULATORY 8.1 Difficulties in Approval of Biologics/Biogenerics 8.2 Approval System for Biotech Products in India 8.3 Regulatory Requirements for Recombinant Product Application Processing in India 8.4 Regulations and Approval Times

8.1 DIFFICULTIES IN APPROVAL OF BIOLOGICS/BIOGENERICS Under current law, generic small-molecule drugs are approved on the basis of essential similarity of the active ingredient and the bioequivalence of the drug to the brand-name product. For chemical products, proving these characteristics is straightforward. A small molecule can be precisely characterized. Bioequivalence can be established with lab and limited clinical studies. The situation for biopharmaceuticals, which are proteins is different. Proteins are more difficult to characterize exactly than small molecules, because their activity depends not only on composition but also on conformation. In addition, a clear understanding of how process changes affect structure and biological activity does not exist. A minor change in process might trigger changes in biological activity. Hence, using the innovator product's data to support a marketing application-as is done with small-molecule drugsis invalid with biopharmaceuticals. Equivalence can be demonstrated through pharmacokinetic, pharmacodynamic, and clinical studies and overcome the fact that biopharmaceutical versions are not exactly the same.

Regulatory

91

Generic drug companies consider generic biopharmaceuticals-or biogenerics-to be the next big wave in their industry, but it's a wave they can't ride without legislative action. In many parts of the world, especially in Eastern Europe, biogenerics are already in the market. But in the US and European Union, the absence of specific regulations for biogenerics is shutting off access to low-cost versions of biopharmaceuticals. On both sides of the Atlantic Ocean, the generics industry is clamouring for clear guidelines for approval of biogenerics. But Biogenerics are not likely to gain an entry into the market without stiff resistance from current patent holders. Meanwhile, generic companies are preparing for the coming of biogenerics in the US and Western Europe.

In the US generic versions of small-molecule drugs are approved through an Abbreviated New Drug Application, or ANDA. With an ANDA, generic manufacturers use the safety and efficacy data of the already-approved brand-name product and only have to conduct limited studies to prove that their product is biologically equivalent. An abbreviated process for generic biopharmaceuticals does not exist. "Anyone who has to make a biogeneric seems to have to go through the same process as the innovator. And it almost seems that what a generic company must prove has to be negotiated with FDA on a case-by-case basis. Costs are higher then for generic company since they have to deal every time as innovator. More and more of the new chemical entities being approved by FDA are biopharmaceuticals. Some of these products are coming off patent, but they aren't facing any generic competition, even though it's not unusual for them to cost $ 100 per dose. There's no monopoly like a regulatory monopoly. It can't be broken. The generics industry believes that FDA has the authority to make regulations for biogenerics, but FDA believes otherwise. So the generics industry is looking to American Congress to force FDA to establish clear guidelines for generic competition in biopharmaceuticals. The biotech companies will throw a lot of money to make sure they don't get competition.

In the United States, FDA would shift oversight of biotechnology drugs from its biologics division, which regulates products made from living organisms, to its main drug division, which oversees more traditional drugs made using chemistry. The move appears to be a response to criticism from the biotechnology industry that the biologics division takes longer to approve drugs than the drug division. It also appears to be a response to criticism of the biologics division by a Congressional committee that has been investigating insider trading and other aspects of the controversy surrounding ImClone Systems, whose cancer drug was not even accepted for review by the biologics division. The biologics division, known formally as the Center for Biologics Evaluation and Research, will retain jurisdiction over blood products, vaccines, tissue transplants and gene therapy. Biological drugs, like the protein EPO for anaemia, have until now fallen into this category because they are made in genetically modified animal cells. According to FDA, rules governing biological drugs would remain largely the same. Biotechnology drugs will still not be subject to generic competition in the same way chemical drugs are. The Generic Pharmaceutical Association (GPhA) has urged the US Food and Drug Administration (FDA) to move forward to accelerate the approval of affordable generic versions of biopharmaceutical drugs. The GPhAs comments come in the wake of a public meeting to discuss the formulation of a regulatory route to market for so-called biogeneric or biosimilar products. Some of the biggest-selling biological drugs developed during the first phase of the biotechnology revolution in the 1980s - including human growth hormone (hGH) and insulin - are set to lose patent

92

Biopharmaceuticals

protection in the US in 2005 and beyond. This opens up a market currendy worth $ 30 billion and growing at 10 per cent a year - but only if a regulatory route to market can be teased out. API and generic companies are hoping that biologicals can provide additional impetus to the strong growth being seen in the market for chemical-based generics worldwide. Biogenerics could quickly address a $ 10 billion market in which many treatments cost upwards of $ 10,000 per patient per year. The generic pharmaceutical industry can provide consumers with safe, effective and affordable biopharmaceuticals. But it is accused that Pharmaceutical Research and Manufacturers of America and the Biotechnology Industry Organisation - which represent the interests of the research-based pharmaceutical and biotech sectors - are dragging their feet into the world of competition by undertaking efforts to block consumers' timely access to affordable biopharmaceuticals. These delay tactics are harming the millions of Americans who need access to affordable health care. In response, the companies that research biological drugs point out that the complexity of their production means that it is hard to guarantee equivalency - the benchmark for approval of generic drugs - for both efficacy and safety. This is backed up, for example, by the serious side effects that emerged with one of the erythropoietin drugs, which eventually was attributed to changes in its production schedule. This has placed regulators in a difficulty, amply evidenced by the FDA's inability to reach a decision on the marketing application by Sandoz of a generic human growth hormone product, Omnitrope. The FDA said at the time that it did not identify any deficiencies in the application, but 'uncertainty regarding scientific and legal issues' meant it had been unable to reach a final decision. Given escalating pharmaceutical expenditures and the broader healthcare crisis, the time is now for the FDA and Congress to commence competition for expensive biopharmaceutical medicines so that consumers can have timely access to these lifesaving products. But to date approval of biogenerics has been patchy to say the least. Last year, SICOR (now part" of Teva) gained approval in Lithuania for a generic version of granulocyte colony stimulating factor (G-CSF), the active principle in Genentech's Neupogen (filgrastim) drug for treating neutropenia in patients undergoing cytotoxic cancer chemotherapy. Meanwhile, other companies, for example Wockhardt (with erythropoietin) and GeneMedix (with granulocyte macrophage colony stimulating factor or GM-CSF), have concentrated on Asian markets until such time as a route to market for these drugs in the US and Europe opens up. Biotech drugs are among the most expensive medications, yet there are no generic versions on the horizon. That's mainly because the Hatch-Waxman Act of 1984, which made it cheaper and easier for conventional generic drugs to win FDA approval, did not include similar provisions for all biologics. Biotechnology was just to begin then. The FDA isn't pushing for regulatory changes that would open the door to generic biologics. ''There are significant unresolved scientific issues about how to show 'sameness' between complex biological macromolecules so that FDA can be assured that any generic biologic is safe, pure, and potent as well as 'equivalent' to an innovator product. USP committee is exploring the possibility of developing quality standards for biologics that may help both innovator and generic manufacturers. The drug, BeneFIX (Recombinant Coagulation Factor IX), is synthesized in a laboratory, using nonhuman cells, so it is "inherendy free" from the risk of major human blood-borne pathogens,

Regulatory

93

according to its manufacturer, the Genetics Institute of Cambridge, Mass. BeneFIX is a biotech drug (a synthetic version of a natural biologic substance) to replace the clotting factor missing from his blood.

Bioequivalence Studies The Vision Group in biotechnology, Government of Karnataka State in India, has called for a revamp in the present Schedule Y of the Drugs and Cosmetics Act as it imposes lot of restrictions in product development in the area of biopharmaceuticals. The revised Schedule Y of the Drugs & Cosmetics Act, is likely to cover the bioequivalence and bioavailability studies of existing drugs on human volunteers as well. A new set of rules and regulations to be followed for conducting these studies is to be incorporated in the schedule. Currently Schedule Y of the Drugs & Cosmetics Act deals only with the studies of new drugs on human i.e. clinical trials. A fresh thought among the CDSCO (Central Drugs Standard Control Organization) officials including Drug Controller General of India towards bringing the BE and BA labs under the regulatory scan has led to the preparation of new mandatory guidelines for such studies. A draft guideline in this regard would be prepared and submitted to DCGI. By including BE studies under Schedule Y, the institutions and hospitals conducting bioequivalence and bioavailability studies of drugs are to come under the direct monitoring of the DCGI. With this move the licensing of the BE study centres and labs would become mandatory and the studies should get due approval from the DCGI. Currently there is no such requirement of approval from either DCGI or any other authority as per the Drugs and Cosmetics Act, 1940 to conduct such studies. Since the studies are conducted on the existing drugs and they were not conducting clinical trials on new drugs, the government so far ignored this area. Number of controversial cases reported in recent years regarding unethical drug studies on human v:olunteers prompted the government to make the change. In India at least 24 major institutes and another 60 small laboratories and hospitals conduct BE/BA studies on human volunteers for pharmaceutical companies for the purpose of new formulations. Recognition and approval of such centes would be required now. Clinical trials: Government regulations in most countries require new drug or treatment to go through four phases of clinical trials prior to approval for sale to the people without too many restrictions. Phase I trials: Phase I trials is the toughest where the new drug or treatment has to be tested on a small group of 20 to 80 healthy people. This has to be done to evaluate the drug's safety, determine the dosage requirement and identity any side effects. Phase II trials: In phase II trials, the drug or treatment is administered to a select group of patients ranging from 100-300 to test its effectiveness and safety further. Phase III trials: In phase ill trials, the drug has to be given to a large group of patients (usually 1000-3000) to consolidate data on effectiveness and side effects. This data is then compared with existing drugs or treatments to facilitate permission for large scale. Phase IV trials: In the post-marketing era, phase IV trials are done to collect additional information on the drug's additional risks and benefits and study its optimal use. On an average it takes $ 800 million and 12 to 15 years to develop a new drug and bring it to the market. However, the alternatives based on biotechnology cut down the costs and time by more than half due to limited side effects. Industry studies show that some 371 biotech-based drugs are now under development by 144 companies worldwide against 200 diseases. So far, the US government has approved 95 biotechnology drugs.

94

Biopharmaceuticals

8.2 APPROVAL SYSTEM FOR BIOTECH PRODUCTS IN INDIA Regulatory approvals for rDNA products in India have become more complicated and timeconsuming. On the contrary, countries like US and Japan have centralized systems and one umbrella committee looks into biopharmaceuticals and evaluates initial data generated by the company and gives approval for clinical trials. The clinical data is thus evaluated for marketing approvals in the final stage. There is an urgent need for India to be self-reliant in vaccines and other life saving drugs. Currently these drugs are mainly being imported and are not available at affordable cost. If congenial atmosphere is created, India can achieve excellence in developing these drugs. The companies working on recombinant products fmd the approval process complicated with the long chains of approvals right from the Institutional Bio-Safety Committee (IBSC) to Genetic Engineering Approval Committee (GEAC) functioning under multiple ministeries. IBse and DBT nominee approves project (category I and II) and recommends to RCGM Then comes the RCGM approval for Category III followed by the approval for toxicity study by RCGM subject to animal ethics committee clearance. Animal ethics committee approval is obtained followed by the toxicology studies. IBSC reviews toxicity studies and clinical trial protocols and recommends to Review Committee on Genetic Material (RCGM). RCGM approves toxicity reports and recommends to Drugs Controller General of India (DCGI) and GEAC for human trials. DCGI permission for human trials is in addition to GEAC permission. IBSC reviews human clinical trial data and recommends to RCGM. RCGM approves clinical trial data and recommends to DCGI and GEAC. Next comes the review of human clinical data by DCGI constituted expert committee. DCGI approval for manufacturing and marketing is subject to product testing, facility inspection and GEAC clearance. GEAC approval is then obtained. Final manufacturing license is obtained from local drug authority after facility inspection and clearance from product testing from the National Laboratory. Then fmally comes the stage, of commercial launch for manufacturing and marketing of product. In India, company thus takes 4 years to commercialize the rDNA product. It was proposed by the Association of Biotechnology Led Enterprises (ABLE) (a representative body of the Indian Biotech Industry) that rDNA Expert Committee constituted by DCGI consisting of the nominees from Ministry of Environment, DBT, ICMR and practicing medical experts should be given the sole authority to examine clinical trial protocol and to give the permission for clinical trials. The Union Ministry of Environment and Forests has set up a task force to provide recommendations on streamlining the regulatory process for recombinant products for the biotech-pharma industry. The task force is headed by Dr. R. A. Mashelkar, director general, CSIR and Secretary, Government of India.

Regulatory

95

8.2.1 GEAC to Weigh IBSC Report for Bio-safety Compliance in r-drug Manufacture The Genetic Engineering Approval Committee (GEAC) under the Ministry of Environment and Forests has decided to consider the report of the Institutional Biosafety Committees (IBSC) on biosafety aspects involved in the manufacture (If recombinant drugs for according manufacturing clearances. The IBSC report will not be routed through the Review Committee on Genetic Material (RCGM) for opinion but will be directly consid('red by GEAC thereby cutting down the time taken for clearances. Regarding the suggestion of GEAC to seek the report from RCGM on the adequacy of containment facilities and other biosafety aspects involved in the manufacture of r-pharma products, GEAC decided in the recent meeting that it could take its view on the basis of IBSC report without routing it through the RCGM. Many members of GEAC felt that RCGM is responsible for bringing out bio-safety guidelines as well as the evaluation of safety requirements in the manufacturing units even though implementation of Protocols and Guidelines are under the supervision ofIBSC. Ordinarily, the report of IBSC, which has a nominee of DBT and has advantage of on site supervision, should be adequate. However, the need for a review by an impartial technical body after the recommendation of IBSC, which is primarily company outfit was also emphasized. After the deliberations, the view taken was that the report of IBSC could be considered by RCGM on case-to-case basis and after due consideration by RCGM may make the recommendation to GEAC for fmal clearance. The committee considered the need for a report from RCGM regarding the biosafety aspects including the containment facilities for subsequent products manufactured within the same premises. On this matter the Committee's view was that after taking into consideration the type of product to be manufactured and type of containment facilities installed by the company the RCGM may make its recommendation to GEAC in every case. GEAC also decided that since the pre-clinical trials data are evaluated and approved by RCGM prior to DBT recommending the proposal for phase III clinical trials, there is no need to refer the case to DBT at this stage. The issue of delay in decision-making due to delay in obtaining the opinion of experts (who were not members of GEAC) was also discussed. The Member Secretary apprised the Committee that as per prevailing practice expert opinion was sought at the initial stage of receipt of the proposal before conducting phase III clinical trials. Noting that phase III clinical trials are conducted to establish safety and efficacy of the product, the proposals are to be referred to outside experts along with clinical trials data at the manufacturing stage. However, this would be applicable to approved products. In the case of new products, experts' opinion will be obtained prior to phase III clinical trials. Inspite of the ministry's decision to hold the GEAC meeting every month, the minimum time given to experts for the review of proposals was decided at 30 days.

8.2.2GEAC Approval before DCGI Renewal of Import of Recombinant Drugs and Vaccines The Genetic Engineering Approval Committee (GEAC) under the Ministry of Environment and Forests has requested the office of Drugs Controller General of India (DCGI) to check for prior GEAC approval while renewing the import license of recombinant drugs and vaccines. DCGI should give necessary directions for this. Necessary amendments could be considered for the purpose.

96

Biopharmaceuticals

8.2.3 GEAC Nod for Phase III Clinical Trials only after DCGI Approval The Genetic Engineering Approval Committee (GEAC) under the Ministry of Environment and Forests has decided to give clearance for Phase III clinical trials of the recombinant drugs only after making sure that the clinical trial applications have got approvals first from the office of Drugs Controller General of India. The clearance for Phase III clinical trials under 1989 Rules would be issued only after the proposals have been recommended by RDAC. It has also requested DCGI to forward the minutes of the RDAC meeting to the member secretary in all such cases in future. GEAC asked Virchow Biotech Pvt Ltd, Hyderabad to conduct clinical trials on r-h-Interferon Alpha 2b (Inferon) subject to clearance from DCG!. GEAC accorded approval for manufacturing finished dosages from imported crystals of rhuman Insulin to Shreya Life Sciences Pvt. Ltd., Mumbai. GEAC gave permission for the import of 1500 vials of Monoclonal antibody hR3 (thera CIM manufactured by r-DNA technology) to Biocon Biopharmaceuticals Pvt. Ltd., Bangalore from Centre of Molecular Immunology, Havana city, Cuba. GEAC asked Indian Immunologicals Limited, Hyderabad not to initiate clinical trials for its indigenously developed combined Rabies DNA vaccine (Veterinary use) unless it provides detailed information on the association of DNA with germline of recipients to DBT and get their positive comments. The company will have to submit clarification from DCGI on the status of approval for conduct of Phase-ill clinical trials for the combined rabies DNA vaccine for veterinary use. The company will also have to submit information as per GEAC proforma.

8.3 REGULATORY REQUIREMENTS FOR RECOMBINANT PRODUCT APPLICATION PROCESSING IN INDIA Most of the recoI11binant products used as drugs are within the category of "new drugs" under Schedule Y of the Drugs and Cosmetics Act and Rules. For the production of recombinant products, there are certain regulatory mechanisms involved under the Environment (Protection) Act, 1986, and the Rules thereunder (1989) as they involve production of a GMO capable of manufacturing a particular product. Any company involved in the manufacture of recombinant product has to follow the EPA and the Rules thereunder before the production of a recombinant product as a drug. The regulatory procedure under the EPA notified by the Govt ofIndia on December 5, 1989, defines competent authorities and composition of such authorities for handling all aspects of GMOs and the products thereof. Since the involvement of GMOs apprehends various effects on the environment, as they may have impact on the human and animal health, flora and fauna, if released into the environment without proper assessment, the Rules and Procedures under EPA have to be followed strictly. The Rules and Procedures, which govern the handling of GMOs, have been notified vide GSR 1037(E) dated 5.12.1989 from the Union Ministry of Environment & Forests. The Rules cover all kinds of GMOs and products thereof, which are controlled commodities for handling and use in the country under the EPA. Once the product of GMO as a drug after following EPA is available then the Drugs and Cosmetics Act and Rules step in for their commercial use. The application of 1989 Rules are for the manufacture, import and storage of micro-organisms and gene technology products; genetically engineered organisms and cells and correspondingly to any substance and products and food stuff etc of which such cells, organisms or tissues form part; new gene technologies in addition to cell hybridisation and genetic engineering methods. As stated earlier, there are six competent authorities involved in the regulation of GMOs.

Regulatory

97

The Recombinant DNA Advisory Committee (RDAC): This committee constituted by the Department of Biotechnology in the Ministry of Science & Technology is to monitor the developments in biotechnology at national and international levels. The RDAC submits its recommendations from time to time that are suitable for the implementation for upholding the safety regulations in research and application of GMOs and products thereof. This committee prepared the first Indian rDNA Biosafety Guidelines in 1990, which were adopted by the government for conducting research and handling of GMOs within the country. The Institutional Biosafety Committee (IBSC): This committee is constituted by the organisations/industries involved in research with GMOs or where rDNA work is being undertaken. The IBSC has a nominee ofDBTwho oversees the activities to ensure that safety aspects in accordance with the Biosafety Guidelines are fully adhered to by the organisation/industry. Every R&D project where rDNA technology/GMO is to be used is reported to the IBSC by the investigator apart from the status of the results of the experiments as well as the proposed experiments to be carried out. All experiments belonging to the contained conditions can be permitted by IBSC, however, the synopsis of all such experiments has to be prepared and submitted to the Review Committee on Genetic Manipulation (RCGM). IBSC also ensures experimentation at designated locations and adherence to approved protocol. Review Committee The Review Committee on Genetic Manipulation (RCGM) is constituted in the DBT as per the Rules 1989 of the EPA. The main functions of RCGM are to review all ongoing projects involving high-risk category and controlled field experiments. RCGM is also to lay down procedures for restriction or prohibition, production, sales, import and use of GMOs both for research and applications. RCGM authorises import of transgenics, to carry out experiments with category III risks aIld above with appropriate containment, generation of relevant data on rDNA products in appropriate system and field experiments, issues clearance for the import/export of etiological agents, vectors, germplasms etc related to experimental work for rDNA products, research, training etc. All experiments using GMOs, which belong to the Category III risks and above as elaborated in the Biosafety Guidelines, require a permit to be issued by the Department of Biotechnology authorising such experiments, based on the recommendations of the RCGM. Genetic Engineering Approval Committee (GEAC): The GEAC constituted in the Ministry of Environment & Forests (MOEF) and is responsible for approval of activities involving large-scale use of GMOs in research, industrial production and applications. The clearance of GEAC is only from environment angle under the EPA. GEAC permits the use of GMOs and products thereof for commercial applications. The drugs made through GMOs would require separate approval for manufacture and use under the Indian Drugs & Cosmetics Act; the production of GMOs is also authorised under Indian Industries (Development and Regulation) Act and, therefore, these clearances are also mandatory. Large-scale experiments beyond the purview of RCGM are also authorised by GEAC. GEAC can impose prohibition of the import, export, transport, manufacture, processing, use or sale of any GMOs or their product. State Biotechnology Coordination Committee (SBCC): The Chief Secretary of state government in each state where research and applications of GMOs are proposed heads this committee. This committee has the powers to inspect, investigate and take punitive action against violations of Statutory Provisions under Biosafety Guidelines. This committee also nominates state government representatives in the activities requiring field/premises inspection etc.

98

Biopharmaceuticals

,

,

District Level Committee (DLC): This committee is constituted at the district level td monitot the safety regulations in installations engaged in the use of GMOs and its applications in the environment. The District Collector heads the committee who can induct representatives from state agencies to enable the smooth functioning and inspection of installations with a view to ensure the implementation of Biosafety Guidelines while handling GMOs. This committee is authorised tCl formulate the information chart, find out hazards and risks associated with the installations and to coordinate activities for meeting any emergency. IBSC notes the intention of the work at the institutional/industrial level and based on the risk category recommends to RCGM for information/approval to conduct research. The RCGM directs the applicant to generate biosafety data on the GMO and products thereof on case-by-case basis. After RCGM satisfies itself about the safety of the GMO/rDNA product, it recommends to the GEAC for granting approval for environmental clearance of a recombinant product. . The GEAC may satisfy with the recommendations of the RCGM or may direct the applicant to generate more data on the safety aspects depending upon the type of the product. Based on the data available, the GEAC grants approval for environmental clearance of a product and the applicant has to follow other statutory requirements applicable to the product for commercialisation. Within the country, the recombinant drug products are either imported and marketed or manufactured and marketed after satisfying all environmental biosafety clearances and after the approval of the DCGI taking into account the data on phase I to phase III clinical trials. The existing biosafety institutional framework for the use of GMOs and recombinant products thereof, in case of rDNA drugs, pharmaceuticals and therapeutics is thus as follows: Proposal Institutional Biosafety Committee with DBT nominee RCGM's approvals based on the pre-clinical data RCGM conveys its recommendations to the applicant and copy to the DCGI and to GEAC RDAC approves the protocol and recommends for conducting human clinical trials IBSC examines the human clinical trial data and sends it for RCGM and DCGI for recommendation to GEAC for environmental release GEAC approval for Environmental Release The applicant is to follow the provisions of the Drugs Act for commercial release of the product. This shall include inspection of the production facilities, according temporary license to produce trials batches, sending products from five trial batches to CRI, Kasauli or CDL, Kolkata, receiving the test report by DCGI and finally granting approval to manufacture and marketing the product. Approval procedure for recombinant drug products developed/produced' indigenously: IBSC meetings are convened at the institute/organisation with the agenda. Minutes of all IBSC meetings are sent to RCGM. IBSC can approve experiments of the Category I and Category II risk experiments. For experiments of Category III and above risks, IBSC sends its recommendations to RCGM for approval. Applicants submit applications in prescribed formats to IBSCjRCGM. RCGM grants approval for conduct of experiments of Category ill and above risks, and asks for progress reports periodically from the applicants/project-investigators through IBSC. '.xperiments concerning drugs produced from GMOs require generation of animal toxicity data. Before such data are generated, results of at least five trial batches (including each bulk and formulation batches) are evaluated first by the IBSC and then by the RCGM to ascertain if a reasonably stable proces has been developed.

Regulatory

99

Product acceptability criteria for both bulk and formulated materials are then fixed. Protocols for carrying out animal toxicity studies are to be submitted for RCGM's approval. Protocol must address risks emanating from host-related contaminants as well as residual toxic chemicals used in processing. RCGM approves the conduct of animal toxicity studies; thereafter, Animal Ethics Committee of the institution/organisation has to approve the' proposed protocol by the organisation/investigator. Animal toxicity has to be carried out at least on two relevant species of animals (often, one rodent and one non-rodent species are selected). The species of animal is to be indicated in the protocol. Animal toxicity studies are carried out and IBSC meeting is convened by institution/organisation to discuss the report. If satisfactory results are obtained, protocols for Phase I, Phase II and/or Phase III studies are prepared by the institution/ organisation, and placed before IBSC members for approval. Phase I is needed for a new product/ molecule. Phase II is needed when the dose is not known or when the dose finding has to be carried out. Phase III studies can be carried out straightaway if the drug is well-known (like biogenerics) and the host is assessed as safe. Above documents, including protocols for human studies are submitted by the applicants to RCGM and DCGI for approval. RCGM approves and sends its communication to the applicants and asks applicants to apply to GEAC for clearance under EPA. DCGI examines the protocol and recommends carrying out of clinical trial only after GEAC has cleared such trials first from environmental angle. Human clinical trials/studies are completed and data relating to human safety including risks to human health are generated and data is submitted to IBSe. IBSC meeting is convened by the institution/organisation for approval of the human clinical trial report. Clinical trial report is submitted to RCGM, GEAC and DCGI. The applicant submits information in a format seeking clearance under EPA. For clearance under Drugs Act, a separate format is used. GEAC sends the application to four or five experts including DBT, and, on the basis of the recommendations of the experts, GEAC members evaluate and approve the r-DNA drug under EPA for open environmental release, i.e.) for marketing. The joint inspection of the production site may be required, and can be done by the representative of GEAC and DCGI. Thereafter, DCGI allows production of trial batches, and five such batches are sent to Central Drugs Laboratory (CDL), Central Research Institute (CRI), Kasauli for testing under the Drugs Act. Mter the receipt of Testing Reports from CRI, Kasauli, the DCGI grants the fmal approval to manufacture and market the product. The DCGI has set up an expert committee for r-DNA drugs and the applicant is required to make a presentation before that committee. DCGI takes a decision on the basis of the recommendations of the expert committee. Both DCGI and GEAC can impose conditions of surveillance on the product during marketing. Marketing under EPA can be for a period of two to four years initially and this can be renewed on the basis of an application. Post-market surveillance data may be required to be generated and submitted to DCGI and GEAC by the applicants. Approval procedure for rDNA products imported and marketed in India: Such drugs cannot be considered for import ,if they are not approved in the country of origin. The applicant/institution/organisation makes an application simultaneously to the Genetic Engineering Approval Committee (GEAC) in a format (in about six copies), and to the Drugs Controller General of India (DCGI) in another format in five sets. GEAC sends the application to four to five experts including the Department of Biotechnology (DBT) seeking their evaluation/comments on th~ report. DCGI also examines the report in-house as

100

Biopharmaceuticals

well as consults experts. After receipt of comments of all the experts, the GEAC examines the application along with the comments in its meetings. At this stage, GEAC may ask for 21 sets of application from the applicants. GEAC may decide to direct the applicant to conduct further clinical trials if the committee has any doubt about the product in terms of evaluation of safety, lack of information on certain aspects of safety, the" inadequacy of the sample size used in the evaluation etc. Phase III clinical trials may be ordered incorporating in the protocol questions that address certain issues of human risks/adversary actions. If the data are found to be satisfactory by the GEAC, the latter sends its recommendations to the DCGI incorporating conditions that require the generation of post-market surveillance information (Phase IV data). If not approved, the GEAC directs the applicant to apply afresh after generating the necessary data under Phase III clinical trial. After receiving the authorisation from the GEAC, if the DCGI is also satisfied with the data, it grants its initial approval in accordance with 122A. Applicants then apply for form 10 along with 122A approval. Authorisation for imports is issued by the DCGI incorporating its conditions as well as those of the GEAC, and the applicant imports and markets the drug.

8.4 REGULATIONS AND APPROVAL TIMES Biopharmaceutical companies are more sensitive to approval times than big pharma because of their cash constraints and pressures from investors for positive news. During 1997/98, the mean approval time for biopharmaceuticals was 12.2 months at the US FDA and 10.6 months at the EMEA. However, the European advantage disappears because companies must then negotiate prices with each country before the products can be marketed, adding one to three years to the process. To improve approval times, the EMEA plans to establish a new ISO-day approval for a fast track products and initiate meetings with industry to review clinical trial design. The lack of a competitive regulatory climate in Canada could lead to a decline in domestic clinical trial activity and the later introduction of breakthrough therapies. Clinical trials are important in keeping a country's medical system at the forefront of modem treatment. The International Conference on Harmonization (ICH) has been working since 1990 towards standardizing test protocols and drug registration procedures. The ultimate goal is the mutual recognition of approvals to reduce certification costs and to facilitate the simultaneous launch of new drugs in multiple markets. As a first step, a Mutual Recognition Agreement on GMP inspections has been negotiated, which will eliminate the need for pharmaceutical companies to seek certification of " manufacturing facilities in various markets (the agreement would not affect the approval of individual drugs). There is a three-year transitional period with the expectation that as of December 1, 2002 the parties will mutually accept each others' GMP certification. The ICH recently reached agreement on a Common Technical Document which outlines a common format for a registration dossier yet accommodates country-specific administrative requirements. The FDA permits drug sponsors to include clinical data conducted in other countries but there must be at least one US trial in order to validate the foreign data. This is due to concerns about differences in trial designs and adherence to clinical protocols by foreign investigators; difficulty in reviewing foreign clinical records; and differences in population characteristics, diagnostic procedures, and therapeutic practices. Japan, long reluctant to accept foreign clinical data because of perceived differences in diet and race, has relaxed requirement that the trials be repeated as long as they are supported by a abridging study demonstrating its relevance to Japaneo;e patients. On the other hand, Japanese clinical trial practice will have to be raised to international standards because of their looser efficacy requirements.

Regulatory

101

REFERENCES 1. Rouhi A. Maureen, "Generics Next Wave: Biopharmaceuticals," September 23, 2002. Volume 80, Number 38, CENEAR 80 38 pp. 61-65. 2. Andrew Pollack, FDA Shifts Biotech Drugs Into Main Oversight Unit, The New York Times, Sept. 2002. 3. Tripathi K. K., Express Phanna Pulse, 13th Nov. 2003. 4. Tripathi K. K. Express Phanna Pulse, 20th Nov. 2003. 5. Biopharma Overview Report, http://stmtegis.ic.gc.cR/pics/bo/bo01879e.pdf.

THERAPEUTIC PROTEINS 9.1 Introduction 9.2 What are Cytokines? 9.3 Some Features ofCytokines 9.4 General Physiological Roles of Cytokines 9.5 Points to be Considered in Application of Cytokines for Therapy 9.6 Lymphokines and Therapy 9.7 Therapeutic Uses of Cytokines 9.8 Preparation of Lymphokines from Natural Sources 9.9 Preparation of Lymphokines by r-DNA Technology 9.10 Interferons 9.11 The Interleukins 9.12 Colony Stimulating Factors (CSFs) 9.13 Tumor Necrosis Factor (TNF) 9.14 Future Developments in Cytokine Therapy 9.15 Breakthrough in Manufacture of Therapeutic Proteins 9.16 Market for Therapeutic Proteins 9.17 Product Development Strategies and Challenges 9.18 Comparison of Bacteria-Based Route Vs Use of Animal Cells

Therapeutic Proteins

103

9.1 INTRODUCTION Proteins in living organisms are classified according to their biological roles. These include: (i) Enzymatic proteins (ii) Transport proteins (iii) Structural proteins (iv) Storage proteins (v) Hormonal proteins (vi) Receptor proteins (vii) Contractile proteins (viii) Defensive proteins. Protein-based drugs are: Cytokines, Hormones, Clotting Factors, Vaccines, Monoclonal Antibodies.

Sources: 1. Human and animal corpses - For instance, the human growth hormone was taken from human corpses and insulin required to treat diabetes was collected from slaughtered pigs. These drugs were available in limited supply and they were expensive, given their sources. Organs and secretions like urine have also been used as source of some useful proteins. 2. Recombinant DNA technology 3. Hybridoma cell technology - Hybridomas are fusion cells of lymphocytes and cancer cells. Unlimited production of monoclonal antibodies became possible with this. 4. Transgenic animals - The DNA gene for the desired protein is coupled with a DNA signal directing its production in the mammary glands. The new gene functions in the mammary glands so that the protein drug is made only in the milk. 5. Transgenic plants As a class "Protein Therapeutics" include (1) naturally occurring human proteins; primarily plasma proteins (2) recombinant copies of naturally occurring proteins (3) mutated or modified versions of naturally occurring proteins having higher efficiency, lower toxicity or higher functionality and (4) monoclonal antibodies. Recent developments in Genomics are facilitating the discovery of proteins at unimaginable pace. Important Groups of therapeutic proteins manufactured today are (1) Interferons (2) Interleukins (3) Colony stimulating Factors (4) Blood products such as Factor VIII and Factor IX (5) Structural proteins such as serum albumin, collagen, fibrinogen (6) Insulins (7) Human Growth Hormones (8) Erthropoietins (9) Enzymes like Alfa Glucosidase, Cerazyme/ Ceredase (10) Monoclonal Antibodies (11) Recombinant Protein Vaccines. Since 1982, more than 95 therapeutic proteins, or peptides ("biologics"), have been licensed for production using bacterial, fungal, and mammalian cells grown in sterile cultures, and hundreds of additional therapeutic proteins are currently being developed and tested. They are difficult to produce, expensive and involve time-consuming production processes. Today, there are more than 371 new biotechnology medicines in the pipeline. Cell cultures are grown in large stainless-steel fermentation vats under strictly maintained and regulated conditions. In some cases the cells secrete the proteins; in other cases the cells must be broken open so the protein can be extracted and purified. Therapeutic proteins can be used in a variety of applications including surgery, trauma, cancer therapy, urinary and fecal incontinence, cosmetic reconstruction, and chronic diseases. However, current production methods are limited by the inability to produce high value complex proteins, high cost of production, and long lead times before production facilities are operational. Producing therapeutic proteins in the milk of cloned transgenic animals can increase efficiencies in the industry. Transgenic cows have the potential to produce enormous quantities of therapeutic proteins in their milk. For example, transgenic cows have produced human Collagen at a concentration of 8 gil and human Fibrinogen at 2.4 gil. Both molecules have been purified and shown to be bioactive. Many drugs, which could earlier be obtained only by sacrificing animal life, can now be obtained through biotechnological route. Example of one such biotech drug is a-interferon. The cost of this

104

Biopharmaceuticals

drug was earlier US $ 16 million/50 mg approximately costing a patient US $ ISO/day. So it was out of the range of purchasing power of the common man. In this context, biotechnology played a significant role in reducing cost to US $ l/day. This drug at present can be manufactured by recombinant microorganisms in large quantities. The corresponding gene from human has been cloned through plasmid vector in bacteria. But the present cost is also very high as compared to drugs obtained via the chemical synthesis route (though this drug itself cannot be obtained via chemical synthesis route). The demand of this drug is increasing in the world market. To reduce the cost of these types of drugs, large-scale production is necessary. Cytokines play fundamental role in control of many physiological functions in human body and are implicated in a large variety of disease states. Cytokines are involved in many different cancers, autoimmune disorders, virus infections (including AIDS), and inflamatory diseases. There is an obvious interest in the structure, function and mechanism of regulation of their activities. The advances in recombinant DNA technology, has enabled molecular biologists to isolate and sequence the genes of cytokines. It was further possible to clone the isolated genes and produce these cytokines in suitable expression systems. Production of monoclonal antibodies specific for different cytokines proved to be a powerful technique for purification of these regulatory proteins. Availability of purified cytokines in large quantities and the knowledge of their role in health and medicine has prompted us to study their practical applications as therapeutic agents. Today the informatiop. on cytokines is in a very primitive stage. Cytokines can not be used like hormones for therapy because the relevent cytokine is rarely completely absent. Also dose for therapeutic use needs to be studied. Recombinant human cytokine produced in other expression systems like bacterial, yeast or mammalian cells may lack in posttranslational modifications (e.g.) glycosylation). Although many such problems exist today cytokines are an important and potentially powerful class of agents which will prove to be valuable medicines of tomorrow. This chapter sets out to explain in brief, various aspects of cytokines including their possible use in clinical medicine.

9.2 WHAT ARE CYTOKINES? Cytokines are a large family of proteins, which function in modulation of cell-mediated immunity. Cytokines are produced by their producer cells and then they influence the behaviour of target tells. Classical polypeptide hormones although similar in this regard, are not classed as cytokines, because hormones are produced by specific endocrine glands whereas cytokines are produced by a variety of producer cells. Cytokines are produced by lymphocytes, monocytes and macrophages. Cytokines produced by lymphocytes are termed as lymphokines while those produced by monocytes are called monokines. Cytokines are synthesized and secreted in minute quantities. Cytokines are extremely potent in action. Cytokines after release in secretion by producer cells bind to specific receptors on surface of target cells. The resulting effect is brought about by signal transduction to the interior of cell. Three types of cytokine-cell interaction is illustrated in the figure (Fig. 9.1). The term cytokine, or immunocytokines, was used initially to separate a group of immunomodulatory proteins, also called immunotransmitters, from other growth factors that control the growth and activities of non-immune cells. These proteins also mediate interactions between cells directly and regulate processes taking place outside the cell. Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death. Today the term cytokine is used as a generic name for a diverse group of proteins and peptides that act as regulators. Examples of different cytokines are - Interferon, Interleukins, and Colony stimulating factor and Tumor necrosis factor and some growth factors. The cytokines exhibit different effects including: • Pleiotrophy - the same cytokine can act on several different cell types and can cause different responses, depending on the cell type upon which the cytokine acts.

Therapeutic Proteins

105

• Redundancy - several different cytokines acting on a cell can individually cause the same response. • Synergy - two (or more) cytokines can individually cause a certain level of response; however, if the two or more cytokines are simultaneously present, the sum of the response is greater than the sum of the individual responses (the cytokines act synergistically). • Antagonism - the presence of one cytokine inhibits the action of a different cytokine; one cytokine blocks the effect of the other.

9.3 SOME FEATURES OF CYTOKINES (i) They are small proteins of 15-30 kd. (ii) Some are modified after secretion (addition of carbohydrate side chain). (iii) Some of them are first synthesized as precursors and are later cleaved to biologically active

molecule. (iv) They are rarely produced at co~tant rate. (v) Their life-time in the blood stream or other extracellular fluid is usually short and this ensures that they act only for a limited period. ' (vi) Synthesis, clearance and destruction of cytokines is a tightly regulated process.

1

CyIotia8

1

TaptceD

11\ I

~

eIfec:II

I

(a) Producer cell

---+

same cell has receptor ---+ biological effects

(b) Producer cell

---+

nearby different ---+ biological effects cell type with receptor

(c) Producer cell

---+

blood stream or other body fluid

---+ target cell with biological receptor elsewhere effects

in body Fig. 9.1 Cytokine-cell interaction

9.4 GENERAL PHYSIOLOGICAL ROLES OF CYTOKINES .(i) Control of cell proliferation

(ii) Control of cell differentiation

Biopharmaceuticals

106

(iii) Regulation of immune response (iv) Regulation of hematopoiesis (v) Control of viral and parasitic infections

(vi) Regulation of inflammatory response

(vii) Control of cytotoxic and phagocytic cells (viii) Wound healing (ix) Influences on cellular metabolism

Interferon was the first cytokine to get FDA approval for clinical use in 1986. Recombinant DNA technology has enabled production of cytokines in large quantities. Different cytokines are effective in treatment of wide variety of human diseases including AIDS, rheumatoid arthritis, asthama, multiple sclerosis, allergies and cancer.

9.5 POINTS TO BE CONSIDERED IN APPLICATION OF CYTOKINES FOR THERAPY Although there is great research going on in the field of cytokines on therapeutic applications, certain points need attention on the way. (i) The role of cytokines in health and disease is quite complex and increase or decrease of levels may not directly correlate with effectivity. (ii) Cytokines are produced locally, released into microenvironment and act on adjacent cells. Systemically administered cytokines may not reach target environment. (iii) Target cell specificity of cytokines is very limited. (iv) Since lymphokines work together, both synergistically and in cascades, it is likely that 'cocktails' of them may produce greater benefit than a single factor. (v) The half-life of lymphokines is only minutes in the circulation. Because cytokines have short half-life in vivo, repeated therapy may be necessary. Sufficient high level of lymphokines should be maintained without any severe toxicity. Or, short intense therapy be carried out. (vi) Toxicity of cytokines when given in larger doses (to reach desired level at targeted site) is of serious concern and needs solution if to be used for therapy. (vii) Many side effects have been noticed with cytokine therapy. These are: (a) For IL-2 - fever, fatigue, anaemia, eosinophilia, rashes, nausea, diarrhoea, weight gain, dyspnoea, hypotension, congestive heart failure etc. (b) For IFN-a - fever, chills, influenza-like symptoms (c) For GM-CSF - bone pain, fever, malaise, myalgia, arthralgia, anorexia. eSFs --+ Bone marrow --+ Haematopoiesis stimulated ILl 1'NFs --+ Anterior --+ Fever IFNs hypotballamus IL6 ILl--+ TNFs

Induction of Liver

---+ acute phase

proteins

Fig. 9.2 Role of cytokines in combating the cause of inflammation and aid in re&trPe1'J.

Thempeutic Proteins

107

9.6 LYMPHOKINES AND THERAPY The interest in lymphokines has two sides. They can be used directly in therapy and there may be need to inhibit their production or action. Lymphokines are involved in chronic inflammation and hence there is an interest in search for natural and synthetic inhibitors of lymphokines. However, since they also mediate normal immune response, it will be necessary to control abnormal lymphokine production and action without damage to the normal immunoregulatory processes. Direct therapy with lymphokines (1) Lymphokines may be used to reconstitute failed immune system as in AIDS, or to overcome temporary immunodeficiency (due to cytotoxic treatment) caused after treatment of tumors or bone transplantation. (2) Lymphokines may be used to stimulate immune response to tumors or overwhelming infections. (3) Lymphokines have an effect on haematopoiesis and can be significant for regeneration of respective cells in many situations (bone marrow transplantation, plastic anaemia, agranulocytosis, congenital or acquired neutrophil dysfunction and infections) which adversely affect haematopoiesis. (4) Stimulation of immune response with lymphokines has beneficial application to treatment of infectious diseases (e.g., lepromatous leprosy) and tumors. Specific uses of different lymphokines have been discussed under respective heading. Control of production and action of lymphokines -

9.7 THERAPEUTIC USES OF CYTOKINES • Wound healing • Stimulation of haematopoiesis • Treatment of immunodeficiency syndrome • Activation of killer cells • Control of autoimmune diseases • Control of infectious diseases

• In cancer therapy • Control of inflammatory diseases

9.8 PREPARATION OF LYMPHOKINES FROM NATURAL SOURCES Large-scale culture of cells is carried out generally in serum-free medium and supernatant is collected. Peripheral blood mononuclear cells may be used. Hybridomas or cell lines from both lymphoid or non-lymphoid sources may be used. Cells are induced with mitogens or phorbol esters or mixtures of these to get more yields. Lymphokines are secreted in medium by 24 hours. Lymphokines are purified from supernatant by one or more protein purification methods in sequence. For purification of the product; Dye affinity chromatography, irnrnunoaffinity chromatography, Reverse-phase HPLC, , Gel filtration, Preparative isoelectric focusing are the various methods used, either alone or in combination in proper sequence. Presence of lymphokines is determined by bioassay or immunoassays (RIA or ELISA) using monoclonal antibodies or polyclonal antibodies. Further characterization oflymphokine is done for molecular weight, homogeniety, pI, amino acid sequence etc.

Biopharmaceuticals

108

h

[~--,r--II Cell culture of lymphoid or non-lymphoid or hybridoma cells

Concentrate

Supernatant

~

I

oo SM!

........"00

1 + Cbaractrisation SJ;>S-PAGE Isoelectric focussing Amino-acid analysis Sequencing

1

Purification Dye affinity chromatography Immunity chromatography Reverse phase HPLC Gel filtration Preparative isoelectric focussing

Fig. 9.3 Purification and charaterisation of lymphokines from natural sources

9.9 PREPARATION OF LYMPHOKINES BY RECOMBINANT DNA TECHNOLOGY (i) Isolation of lymphokine gene (ii) Cloning of lymphokine gene (iii) Growing cloned cells

(iv) Purification of the product (i) Isolation of lymphokine gene - Two' approaches may be used. In the first, oligonucleotides are prepared to match the portion of the amino acid sequence of the protein and these are used to probe cDNA libraries. Pure protein (lymphokine) will be the primary requirement of this procedure. In second approach, mRNA is collected from appropriate cell which produces lymphokine. This is then translated in vitro, or in Xenopus oocytes, and translation product is detected by bioassay and by binding with appropriate antibody. The fraction of mRNA which synthesizes the protein is used to construct the cDNA library. The library is probed with mRNA to identify a clone making a full-length cDNA insert. (Ii) Cloning of lymphokine gene - A double-stranded cDNA is inserted via plasmid into E.coli or yeast. Cloned lymphokine genes can be sequenced and investigaed in various ways. Lymphokine genes may be alternately engineered into yeast or mammalian cells to produce more natural product. Site directed mutagenesis may be used to alter single amino acid so that they no longer glycosylate or form disulfide bridges. (iii) Growing Ctm"ed cells (iv) Purification of Therapeutic Recombinant Proteins

Expanded Bed Adsorption Chromatography Expanded bed adsorption chromatography is a convenient and effective technique for capture of. proteins directly from unclarified crude sample. This technique can be successfully used in the purification

Therapeutic Proteins

109

of a therapeutic protein in compliance with current Good Manufacturing Practices (cGMP). SO processing cycles could be run on the same STREAMLINE adsorbent with consistent high recovery. The recovery is high and reproducibility is excellent. Compared with other methods, STREAMLINE expanded bed adsorption eliminates one clarification step and increases overall yield. After fermentation, the broth is centrifuged and the supernatant discarded. The cells are then suspended in chromatographic buffer and subjected to high pressure (up to 1000 bar) to disrupt them. The physico-chemical properties of the p,'otein suggest that cation exchange chromatography would be suitable as the first step, which was to capture the target protein from the starting material. Target protein is captured on the adsorbent, whilst cells, cell debris, particulates and most contaminants passed unhindered through the expanded bed. The flow direction is then reversed to allow the expanded bed to sediment. STREAMLINE SP is then used in conventional packed bed mode according to the instructions supplied. Increasing the ionic strength of the buffer elutes the target protein. The remaining contaminants are removed with 1 M NaC!. Cleaning-in-place (CIP) is applied after each cycle. STREAMLINE SP adsorbent is exposed to a CIP solution containing 0.5 M NaOH for one hour without flow. This solution is then replaced with 0.1 M NaOH or 20% ethanol for storage until the next run.

9.10 INTERFERONS Alick Isaacs and Jean Lindenmann, in 1957 in London, discovered this protein material elaborated by virally infected cells and having the property of inducing resistance to other invading viruses in other cells. All vertebrates produce a variety of interferons. These proteins are species specific and exhibit a wide range of biological activities. Interferon can be considered as body's first line of defense against viral infections. Interferons were initially thought to be only antiviral proteins, but later on two more important biological effects of interferon were noticed - (1) inhibition of cell proliferation (therefore useful as potential anticancer drugs) (2) modulation of immune system. For most mammals there are three types of interferons - interferon alpha (IFN-a), interferon beta (IPN-f3) and interferon gamma (IFN-y) according to type of cells that produc~ them in abundance. Also this grouping is based on their distinct antigenic properties. Alpha and beta interferons are derived from fibroblasts of connective tissue and leucocytes. Gamma interferons are derived from cells of immune system. While lPN-a and IPN-f3 are synthesized in cells exposed to viruses or viral RNA, IPN-y is synthesized in response to cell growth stimulating agents. Both CD4+ and CDS+ lymphocytes can produce IFN-y although the former are considered to be the major producers in response to antigenes. lPN-a is a single polypeptide chain with 165-166 amino acids. At least 16 different types of human lPN-a showing different specificities are known to exist. Molecular weight of lPN-a ranges from 16000 to 26000 Da. IPN-a.2 is 7 times more effective than IPN-al when human cells are treated with virus. lPN-a, IPN-f3 bind to same cell surface receptor, are acid-stable and these interferons are collectively termed as type I interferons. IPN -y binds to a distinct cell surface receptor which is widely distributed on many cells, is acid-labile and is termed as type II interferon. Comparison of 3 types of interferons is given in Table 9.1. Interferons are the cytokines that can "interfere" with viral growth. They also have the ability to inhibit proliferation and modulate immune responses. Four types of interferons have been identified: lPN-a, IPN-f3, IPN-ro and IPN-y. The first three are Type I IPNs which have relatively high antiviral potency. IPN-y is the Type IT lPN, also called immune IPN.

Biopharmaceutica/s

110

TABLE 9.1

COMPARISON OF IFN-a,

IFN-~

and IFN-y

IFN-a

IFN-~

(1) subtypes

16

nil

nil

(2) number of amino acids (3) monomer (4) Molecular weight

166-172 monomer 20 kDa

166 dimer 26 kDa

143-146

(5) synthesis by

lymphocytes, monocytes, macrophages

fibroblasts, some epithelial cells

activated T lymphocyte

(6 ) Location of gene

Chromosome 9

Chromosome 9

Chromosome 9

(7)' stability

acid-stable

acid-stable

acid-labile

(8) Type

Type-I

Type-I

Type-II

(9) produced in

viral and other infections, presence of foreign cell types or antigens

viral and other infections, presence of foreign cell types or antigens

cell growth stimulating agents

(10) Acid stability

Yes

Yes

(11) receptor

same for IFN-a and IFN-j3

response to

IFN-y

17 kDa

Yes

(12) Glycosylation in increasing order from IFN-a to IFN-y

Type I IFNs are produced by macrophages, neutrophils and other somatic cells in response to, infection by viruses or bacteria. After they are released, they may bind to their receptors that are expressed on most cell types, resulting in the production of over 30 different proteins in the target cell. Among them, two enzymes play a critical role in the inhibition of viral replication: RNA-dependent protein kinase (PKR) and 2'-S' oligoadenylate synthetase (2-5A synthetase). Doublestranded RNA can activate both enzymes, which may be present in some viruses. The activated PKR can phosphorylate a protein (eIF2) to inhibit protein synthesis. The 2-SA synthetase produces oligoadenylate that can bind and activate a cellular endonuclease to degrade mRNA. Type n IFNs IFN-y is produced in activated THI and NK cells, particularly in response to IL2 and IL-12. Its production is suppressed by IL-4, IL-I0, and TGF-fi. Binding of IPN-y to its receptor increases the expression of class I MHC on all somatic cells. It also enhances the expression of class II MHC on antigen-presenting cells. lPN-a may also activate macrophages, neutrophils and NK cells. Interferon is a copy of a protein found naturally in low levels in the human body. It was approved by (US) FDA (Feb. 25, 1991), to treat hepatitis C. The product, alpha interferon, is the first effective treatment against this form of hepatitis, which affects an estimated 150,000 Americans each year. According to the manufactUrer's literature for using Interferon in the treatment of Hepatitis C: 3 million units per dose 3 times a week. Interferon has an effective cure rate of about 25%.

111

Therapeutic Proteins

Besides hairy cell leukemia and hepatitis C, alpha interferon is licensed for treatment of AIDSrelated Kaposi's sarcoma and genital warts. Schering-Plough Corporation of Kenilworth, N.J., which markets a version of the product under the trade name Intron-A, received approval for the product's use for hepatitis.

9.10.1 Useful Activities of Interferons (1) IFN-a. is effective in Hepatitis B viral infection and in treatment of genital wart caused by Papiloma virus. (2) IFN can modulate immune response. IFN induces expression of MHC gene. IFN also increases number of cell receptors for cytokines. IFN stimulates cytotoxic activity of macrophages, immune cells and NK cells. (3) Promising results are seen in preliminary studies for treatment of rabies, hepatitis, shingles, various herpes infections and Cytomegalovirus infection (fever, pneumonia and even death in newborn children and weakness in adults is produced). (4) Patients of cancers of skin, bone, breast and blood are found to be benefiting from use of interferons as suggested in tests. Interferons may have supplementary role in cancer therapy. (5) Prophylactic use of interferons is considered more important. (6) Interferons may prove useful in treatment of arthritis, asthma, multiple sclerosis. (7) IFN-Y has potent immunoregulatory effects on many cells, since many cells have been shown to have receptors (protein in nature) for it. These include activation of macrophages, induction of class I and class II major histocompatibility complex (MHC) gene products on both macrophages and other cells of nonhaemopoietic in nature. IFN-Y is useful as adjuvant in the treatment of acute and chronic infections.

9.10.2 Mechanism of interferon action in control of viral infections Two mechanisms are suggested for the control of viruses by interferons - (1) The interferons affect the cellular membranes, so that viral particles are not disseminated to other cells; (2) interferons control the multiplication of viruses by inhibiting their protein synthesis. Interferons set off a number of enzymes in the cell which can stop the virus using the cell's protein synthesizing machinery for its own protein synthesis. Ribosomes from interferon containing mammalian cells do not form polysomes with viral RNA, but when they are exposed to host (mammalian) RNA they readily form polysomes and initiate the protein synthesis. Thus ribosomes in presence of interferon recognize specific mRNA and regulate the overall protein synthesis. (3) Hybrid interferons induce test cells to synthesize (2'-5') oligoisoadenylate synthetase. This enzyme generates (2'-5')-linked oligonucleotides, which in turn, activate a latent cellular endoribonuclease that cleaves viral mRNA.

+ • Virus

--. Infected Cell Fig. 9.4 Aetion of Interforrm

Altered Cell

Interferon

Biopharmaceuticals

112

Interferons - - Ccll ,uda