Plavix: Platelets, Patents and the Pay-For-Delay Paradigm by Kalina King

Plavix: Platelets, Patents and the Pay-For-Delay Paradigm

by Kalina Shaw King

April 24, 2008

A Senior Thesis presented to the Faculty of the Department of Molecular Biology in partial fulfillment of the requirements for the degree of Bachelor of Arts, and to the Faculty of the Woodrow Wilson School of Public and International Affairs in partial fulfillment of the requirements for a Certificate in the Woodrow Wilson School.

For the scientists I was just guessing at numbers and figures, Pulling the puzzles apart Questions of science, science and progress, Do not speak as loud as my heart… Tell me your secrets, Ask me your questions, Oh let’s go back to the start –

– Coldplay

Acknowledgements

This Thesis Exists. Wow. Its existence has been contingent on the assistance of many people, a few of whom I would like to acknowledge individually below. I am deeply grateful for the chance to have worked under the tutelage of Dr. Leon Rosenberg for the past year and a half. He sparked the initial idea to study Plavix, and I have been astonished and pleased at how versatile a topic it has been, allowing me to dabble and grapple with not only molecular biology and public policy concerns, but also explore their relationships with economics, the pharmaceutical business, and legislative review. He warned me to choose a topic carefully, as it needed to be something close to my heart, something that I would be excited to study and write on for sixteen consecutive months. I thank him for the gentle guidance, careful editing, sharp wit and warm encouragement through this process. I probably tested his patience as much as he challenged my analytical abilities; a deep thanks both for his noodging and for our shared enjoyment of words. Patricia Fox deserves great thanks for all of her assistance – and cheery smile – in conducting the logistics of our meetings and directing the ‘air traffic control’ of our document transfers. I am indebted to the generosity of the Round Table Senior Thesis Fund’s support of my research by enabling both the collection of research materials and personal interviews. I would like to extend my gratitude to Aaron Barkoff, David Balto and Raoul Kennedy, who lent their time and expertise in legal matters. Kenneth Weg and Leon Rosenberg provided a nuanced and personal perspective on the initial partnership between Sanofi and BristolMyers Squibb to develop Plavix, and Wilbur McDowell assisted with the BMS archives. To the nameless people in my “carrel” in 028 Robertson Hall, thank you for your silent presence reminding me to work. To Jessica Lucas and Molly Kantor, my fellow majoring-inMolecular Biology-certificating-in-Woodrow-Wilson classmates and friends – We did it, and I’m so glad we did it together. Thanks to my friends, for listening to me refine my ideas, for the Wa runs and pool runs, and for all the laughter. The thesis has been the bane of my Princeton experience, but also its crowning achievement.

Abstract Plavix, a drug manufactured by Sanofi-Aventis and Bristol-Myers Squibb (BMS), is an antiplatelet agent used to treat cardiovascular disease, primarily for controlling acute coronary symptoms in ischemic heart disease and reducing the risk of future cardiovascular events. Plavix is a thienopyridine known in its generic form as clopidogrel bisulfate and targets a different pathway than its blood-thinning counterpart, aspirin (acetylsalicylic acid); clopidogrel selectively and irreversibly modifies an adenosine diphosphate receptor on platelets, thereby inhibiting platelet aggregation that is normally induced by an interaction with adenosine diphosphate. As a result of their complementary mechanisms, Aspirin and Plavix are prescribed in conjunction as a standard long-term non-invasive antithrombotic therapy. An evaluation of clinical trials gave a more thorough perspective on Plavix’s polytherapeutic approaches. However, problems with Plavix – documented platelet resistance to the drug and incomplete platelet abrogation, risks of bleeding, and high prices – have left room for other potential oral antiplatelet agents to enter the market. A review of the literature revealed that the adenosine diphosphate (ADP)-receptor mediated pathway of platelet aggregation has continued to elicit scientific investigation and several promising ADP receptor antagonists are in late-stage clinical development and could compete with Plavix before its patent expires in 2011. Eli Lilly is currently in Phase III trials with a novel thienopyridine blood thinner that acts along the same ADP-receptor-mediated pathway and has performed better than clopidogrel and placebo controls. AstraZeneca is developing an ATP-derivative that antagonizes the same receptor to inhibit platelet function but does not need to be metabolized in vivo. Plavix recently faced competition on legal and business fronts, so the scientific dialogue is extended to consider the business and legal impact of Plavix in light of the recent court case and generic drug competition. Plavix was the world’s second-best selling drug with annual sales of almost $6 billion before a generic manufacturer, Apotex, challenged the validity of the Plavix patents and launched generic clopidogrel in August 2006 at-risk of legal retribution. The drug slipped to fourth-place in worldwide sales after the brief generic entry. Besides the weakened sales, Sanofi and BMS were embroiled in litigation to protect the drug’s patents; the District Court upheld the validity of the patents, setting discerning precedents on the issue of racemate versus enantiomers in intellectual property rights. Plavix intersects many potential policy concerns, chief among them the issue of “pay-for-delay” settlements in the pharmaceutical industry and the causal role of the Hatch-Waxman legislation in promulgating such conduct. The policy analysis is directed at the Federal Trade Commission, and recommends that (1) in the arena of the Courts, the Commission alter its goal away from attempting to obtain a per se illegality ruling on such settlements, and instead, push for a more moderated framework of analysis that incorporates a considered opinion of patent validity into the antitrust considerations; (2) in its interactions with Congress, the Commission shift focus away from amending the legality of “pay-fordelay” settlements to reevaluating the (anti)competitiveness of the 180-generic-exclusivity clause within the Hatch-Waxman pharmaceutical legislation.

Table of Contents:

CHAPTER 1 - INTRODUCTION: PLAVIX AND CARDIOVASCULAR DISEASE ............................. 1 CHAPTER 2 – ATHEROTHROMBOSIS AND THE ROLE OF ANTIPLATELET THERAPY ....... 9 . ATHEROTHROMBOSIS: PLAQUE RUPTURE TRIGGERS CLOTTING.................................................................. 9 . ASPIRIN AND CLOPIDOGREL: COMPLEMENTARY ANTICLOTTING AGENTS ................................................ 10 . Aspirin Targets TXA2-Mediated Platelet Activation ............................................................................. 11 . Clopidogrel Inhibits the ADP Receptor-Mediated Pathway to Platelet Aggregation ........................ 13 CHAPTER 3 –ADP RECEPTORS AND PLATELET ACTIVATION ................................................... 16 . CHARACTERIZATION OF THE P2Y1 RECEPTOR ............................................................................................. 18 . CHARACTERIZATION OF THE P2Y12 RECEPTOR ........................................................................................... 26 . INTRINSIC REGULATION OF THE ADP RECEPTOR ....................................................................................... 36 CHAPTER 4 - PRE-CLINICAL DEVELOPMENT OF PLAVIX: THIENOPYRIDINE DERIVATIVES, ENANTIOMER SEPARATION AND THE SANOFI PATENTS.................................. 38 . Thienopyridine Chemical Synthesis ....................................................................................................... 39 . PCR 1033 and PCR 3349 ....................................................................................................................... 42 . The Plavix Precursor: PCR 4099 and the ‘596 Patent......................................................................... 45 . Toxic Convulsions & the Decision to Resolve the Enantiomers of PCR 4099.................................... 47 . Resolving the Racemate: Pharmacologic Implications ........................................................................ 49 . Bisulfate, an Unexpectedly-Acceptable Pharmaceutical Salt .............................................................. 51 . One Active Enantiomer: Absolute Stereoselectivity of the ADP Receptor .......................................... 53 . Selective Synthesis of (+)-Clopidogrel .................................................................................................. 55 CHAPTER 5 - CLINICAL AND METABOLIC STUDIES ON PLAVIX AND NOVEL ANTIPLATELET AGENTS............................................................................................................................ 57 . CLINICAL INDICATIONS OF PLAVIX .............................................................................................................. 57

. Prevalence of Prescribing Plavix........................................................................................................... 57 . PLAVIX MORE FAVORABLE THAN TICLOPIDINE FOR SECONDARY PREVENTION ....................................... 58 . CAPRIE: Clopidogrel Comparable to Aspirin and Safer than Ticlopidine ........................................ 58 . CURE Trial for Dual Therapy Treatment of UA/NSTEMI ................................................................... 59 . COMMIT and CLARITY-TIMI for Short-Term Treatment of STEMI .................................................. 60 . CLINICAL BENEFITS OF PLAVIX TREATMENT DURING AND A FTER CORONARY STENTING PROCEDURES 61 . CLASSICS: Clopidogrel Has a Favorable Tolerability Profile ........................................................... 61 . PCI-CURE: A Role for Clopidogrel During Stenting........................................................................... 61 . PRONTO: A Loading Dose Before Coronary Stenting ........................................................................ 62 . PLAVIX NOT INDICATED FOR PRIMARY PREVENTION ................................................................................. 63 . MATCH: Increases Bleeding Without Conferring Benefits in High-Risk Ischemia ........................... 63 . CHARISMA: No Role in Primary CVD Prevention .............................................................................. 64 . CLOPIDOGREL METABOLISM AND RESISTANCE .......................................................................................... 64 . In Vivo Metabolism of Clopidogrel ........................................................................................................ 64 . Hepatic Function and Clopidogrel Metabolism .................................................................................... 66 . Genetic Polymorphisms and Variable Clopidogrel Resistance ........................................................... 67 . Cytochrome-Dependent Drug Interactions ........................................................................................... 68 . Dual Resistance to Aspirin and Clopidogrel ......................................................................................... 69 . BEYOND CLOPIDOGREL: THE SEARCH FOR THERAPEUTIC A NTAGONISTS AT AND DOWNSTREAM OF THE P2Y12 RECEPTOR ............................................................................................................................................. 70 . Prasugrel, the Third-Generation Thienopyridine ................................................................................. 70 . Cangelor, A Promising ATP Derivative ................................................................................................ 73 . Specifying Targets Downstream of P2Y12 .............................................................................................. 77 . Conclusions on the Pharmaceutical Horizon for Plavix & Potential Competitors ............................ 80 CHAPTER 6 - FROM A PROFITABLE PHARMACEUTICAL PARTNERSHIP TO THE PLAVIX PATENT DISPUTE .......................................................................................................................................... 83 . FORGING THE SANOFI AND BMS PHARMACEUTICAL PARTNERSHIP .......................................................... 83 . The Generic Threat to Plavix ................................................................................................................. 88

. A Sweetheart Deal, Turned Sour ............................................................................................................ 91 . The BMS Decision to Settle with Apotex: Dumb or Desperate? .......................................................... 92 . Apotex on the Attack: The Launch of Generic Plavix At-Risk of Legal Action ................................... 96 . Injunction, In Limbo ................................................................................................................................ 99 . TAKING PLAVIX TO TRIAL .......................................................................................................................... 103 . ANTICIPATION ............................................................................................................................................. 105 . A Genus Cannot Anticipate a Species without a “Pattern of Preferences” ...................................... 106 . A Disclosure Describing the Racemate Does Not Render the D- and L-Enantiomers Obvious – Especially When Results of Separation Are Not Predicted .................................................................. 108 . OBVIOUSNESS-TYPE DOUBLE PATENTING: A NARROWER INTERPRETATION ......................................... 109 . Plavix as a Legal Precedent ................................................................................................................. 110 . THE PERMANENT INJUNCTION: A HARD PILL FOR A POTEX TO SWALLOW .............................................. 111 CHAPTER 7 - IMPLICATIONS FOR “PAY-FOR-DELAY” POLICY ............................................... 115 . THE PAY-FOR-DELAY PROBLEM ................................................................................................................ 115 . Problem Statement: ............................................................................................................................... 117 . THE COMMISSION V. THE UNITED STATES COURT SYSTEM ..................................................................... 117 . Pay-For-Delay in Schering-Plough ..................................................................................................... 119 . Pay-For-Delay in Tamoxifen ................................................................................................................ 121 . PLAVIX AND PAY-FOR-DELAY ................................................................................................................... 123 . Congress and Courts: Calibrating the Reach of Antitrust Enforcement ........................................... 125 . ARGUMENTS JUSTIFYING PAY-FOR-DELAY .............................................................................................. 127 . 1.

A Judicial Intuition to Promote Settlement................................................................................. 128

. 2.

Distortion of the Incentives to Innovate and Challenge Patents............................................... 129

.3.

The risk of legal extrapolation from pharmaceutical cases ....................................................... 130

. 4.

Reverse Payments are a “Natural By-Product” of Hatch-Waxman Regulation ...................... 130

. THE COMMISSION AND CONGRESS ............................................................................................................. 131 . History of Pharmaceutical Regulation in the United States............................................................... 131 . Legislation to Amend Hatch-Waxman ................................................................................................. 140

. Current Proposed Legislation and Future Directions ........................................................................ 143 . Recommendations to the Commission and Congress: ........................................................................ 145 APPENDIX A– THERAPEUTIC TARGETS IN THE MOLECULAR REGULATION OF PLATELETS AND THROMBOSIS ............................................................................................................ 146 . COAGULATION CASCADE............................................................................................................................ 146 . HEMOSTASIS – THE COAGULATION CASCADE AND PLATELET PLUG FORMATION ................................. 147 . Platelets.................................................................................................................................................. 147 . .PLATELET ACTIVATION: ............................................................................................................................ 155 .Therapeutic Utility of Antithrombotics ................................................................................................. 158 . ADP Receptors: ..................................................................................................................................... 158 . HEMOSTASIS: FIBRIN CLOTTING ................................................................................................................ 164 . HEMOSTASIS: CLOT DISSOLUTION ............................................................................................................. 167 REFERENCES ................................................................................................................................................ 168

Plavix and Cardiovascular Disease

Chapter 1 - Intr oduction: Plavix And Cardiovascular Disease

Few things conjure up more fear in people than the idea of themselves or a loved one contracting a heart attack or being paralyzed by a stroke. This fear, well-justified given the mortality statistics for cardiovascular disease, was exploited in an ad campaign that aired last year for the antiplatelet drug, Plavix. The dramatic television campaign featured middleaged professionals in high-powered positions – succumbing to heart attacks; the ad spots drove in the life- (and life-style) saving power of Plavix (Armstrong, 2008). What the ad didn’t mention was that the Plavix brand had itself been recently resuscitated with a legal victory, after a long battle over the validity of the patents covering its active ingredient, clopidogrel bisulfate. The introduction will serve to ground the relevance of Plavix in the context of cardiovascular disease and public health. The chapters to follow will address the drug’s molecular basis of action, and the pre-clinical and clinical development of clopidogrel and of future potential drugs in its class. Additional chapters will review the history of Plavix and the results of recent patent litigation, as well as analyze a proposed side deal to delay the entry of a generic Plavix and consider the policy implications of pharmaceutical “pay-fordelay” settlements. Cardiovascular disease (CVD) is a major and growing public health concern in both developed and developing countries. About one in three American adults have some form of CVD (79.4 million) (American Heart Association, 2006.). Over 20 million people survive a heart attack or stroke every year; as the risk of recurrence or death is high, most of these afflicted individuals go on to require expensive and long-term clinical care (WHO

Plavix and Cardiovascular Disease Cardiovascular disease: prevention and control). In 2004, CVD as the primary diagnosis was responsible for 72.648 million physician office visits, 4.164 million visits to emergency departments, and 6.369 million outpatient department hospital visits in the United States (Hing, 2006; McCaig, 2006; Middleton, 2006). In that same year, CVD was the underlying cause of death for 36.3% (871,517) of U.S. deaths (Rosamond, 2007). This staggering statistic can only be tempered by the realization that the physician and hospital visit numbers are overwhelmingly high in comparison to the mortality rate, giving confidence to the healthcare industryâ&#x20AC;&#x2122;s ability to successfully combat CVD. In 2003 alone, cardiovascular disease killed 910,600 people in the United States, and was the underlying or contributing cause to 58% of deaths. One-third, or 16.6 million, of global deaths in 2001 were attributed to various forms of CVD, and per year CVD causes more deaths than the combined number of deaths due to cancer, chronic lower respiratory diseases, accidents and diabetes mellitus. The World Health Organization estimates that CVD will be the leading cause of death in developing countries by 2010 and the leading cause of death and disability world-wide by 2020 (Leeder, 2004). The economic cost of cardiovascular disease in the United States is extremely high and has traditionally been measured in terms of both direct health care costs as well as indirect costs stemming out of productivity lost from morbidity and mortality. CVD had an economic cost of $403.1 billion in 2006 and an estimated cost of $431.8 billion in 2007 (American Heart Association, 2006; Rosamond, 2007). Coronary artery disease alone was projected to cost the United States $151.6 billion in 2007 (Rosamond, 2007). In contrast, the estimated economic cost of cancer to the US was $190 billion in 2004 (Lozano, 2001). Of the $431.8 billion figure for 2007, hospital stays accounted for $133 billion, or 30.8%, while 2

Plavix and Cardiovascular Disease drugs and other medical durables totaled $47.2 billion or 10.9 percent. Nursing home costs and physician and medical professional expenses rang in with the next highest price tags, at $45.3 billion and $43.3 billion respectively. The loss of productivity from morbidity was estimated to be $36.3 billion and the equivalent loss from mortality, discounted at 3%, weighed in at $112.3 billion (Rosamond, 2007). The segmented lump costs present challenges to the visualization of these costs on a per-patient basis; one study found that for Medicare patients in 2001, the average cost per discharge for short-term hospital stays due to CVD was $8,354 (Hing, 2006). The growing epidemic of heart disease is especially notable in the context of historical epidemiological patterns. Cardiovascular disease shifted from accounting for less than 10% of deaths world-wide at the beginning of the 20th century to its current position as the leading cause of death and disability in the developed world in addition to being responsible for 25% of deaths in developing countries (Murray, 1996; World Health Organization, 1999). A paper in the journal Preventive Cardiology identified the three major factors behind this paradigm shift in epidemiology (Levenson, 2002). The first of the three major drivers behind the growing threat of CVD is the explosion in the world’s population, predicted to be 60% in the thirty-year period from 1990 to 2020 (Murray, 1996). Secondly, reduced mortality due to infectious disease has made cardiovascular disease relatively more important and relatively more lethal. And finally, the prevalence of risk factors for CVD has expanded greatly due to changes in lifestyle patterns. Perhaps the more important contribution of studies such as the one above, however, has been to place cardiovascular disease in the larger context of “epidemiological transition” – changes in life expectancies and disease burdens as they evolve along with societies (Levenson, 2002; Pearson, 1999).

Plavix and Cardiovascular Disease Much of the developed world finds itself in the fourth and final phase of epidemiologic transition, the age of delayed degenerative disease, wherein medical and public health advancements have prevented or successfully treated cardiac events, while CVD and cancer become the top two causes of morbidity and mortality. Non-acute heart conditions such as angina pectoris, congestive heart failure and cardiac arrhythmias persist in the population, though aggressive treatments and primary and secondary prevention stem the damage from acute manifestations of heart disease (Levenson, 2002). Chronic heart disease has typically been a burden for industrial nations â&#x20AC;&#x201C; it has been the leading cause of death in the United States for the past 80 years -- due to lifestyle changes such as overeating, smoking and sedentary routines (United Nations Population Division, 2003). Developing countries, in contrast, were considered immune to the ravages of chronic illnesses such as CVD, at least in comparison to the impact of infectious and nutritional diseases. This immunity has in large part been shattered by two factors: first, the public health initiatives, which stemmed infectious disease and suppressed infant mortality rates, have significantly lengthened overall life expectancy. Life expectancies in the 1950s ranged from thirty-five years in the most destitute countries, such as in sub-Saharan Africa, to fifty-one years in Latin America (United Nations Population Division, 2003). World-wide, life expectancy increased from age forty-five in the 1950s to age sixty-five at present (Cohen, 2003). Fertility rates and life expectancies of developing countries are fast catching up with the longer life spans and better health associated with industrial countries; the life expectancy in developing nations will be less than 10 years lower of that in the developed world by 2050, while world-wide life expectancy is predicted to reach age seventy-six by 2050(Cohen, 2003; Leeder, and et al., April 2004). This monumental and momentous achievement is double-

Plavix and Cardiovascular Disease edged, as it will multiply the size of the population at-risk for non-communicable diseases. Secondly, globalization, urbanization and industrialization have engendered the proliferation of lifestyle risk factors; as prevalence of cigarette smoking, obesity and physical inactivity has become more commonplace in developing countries, the diseases related to such unhealthy lifestyle habits have also penetrated their populations (Uemura, 1988). Ischemic heart disease (IHD), which is also known as coronary artery disease and refers to the heart problems caused by narrowed arteries, is the most lethal subset of CVD; each year the global mortality rate is estimated to be 7 million. This represents 13% of all male deaths and 12% of all female deaths (World Health Organization, 2000). IHD is thought to be the leading cause of death in the world, and moreover, is the leading cause of premature mortality and disability in developed countries (World Health Organization, 1999). Aside from ischemic heart disease with myocardial infarction (MI, or heart attack) and angina pectoris (chest pain), cardiovascular disease also encompasses several distinct - but not exclusive - conditions including hypertension (high blood pressure (HBP)), heart failure (HF), rheumatic heart disease, cerebrovascular disease (stroke) and congenital cardiovascular defects. These symptoms and events stem from a combination of genetics and lifestyle choices; risk factors for cardiovascular disease include family history as well as tobacco, high blood cholesterol (and other lipids), physical inactivity, overweight and obesity and Diabetes Mellitus(Office on Smoking and Health, 1989; American Heart Association, 2006; Center for Organization and Delivery Studies, 2001; Suk, 2003). The screening criteria for these primary risk factors is well-documented, and has greatly improved the identification of individuals at risk for developing one or more cardiovascular diseases. While the ideal therapy would be to prevent the onset of 5

Plavix and Cardiovascular Disease cardiovascular disease altogether by heeding the screening criteria and avoiding tobacco, reducing cholesterol levels, exercising and maintaining a healthy weight (Ardern, 2007), these CVD precursors have instead become increasingly prevalent in the United States and other countries (Pate, 1995; Wild, 2004). Cardiovascular disease is entrenched across ethic, geographic and socioeconomic lines, though the prevalence of risk factors certainly correlates with age, race and socioeconomic status (Stamler, 1993). The incidence of risk factors increases over successive age groups, while the onset of cardiovascular disease begins rising sharply at age forty and above, suggesting that there is a lag between developing risky lifestyle habits and conditions and contracting CVD (Khang, 2007). Race is another significant way to sort vulnerable populations. Blacks, 48.7% of which have two or more risk factors, and American Indians/Alaska Natives, at 46.7%, were at the highest risk, while Asians reported the lowest percentage (25.9%) of at-risk individuals. Socioeconomic measures turned out to present a significant gradient for CVD risk. In terms of education levels, one study found that people who had less than a high school diploma were much more likely to have multiple risk factors (52.5%) whereas 25.9% of college graduates had multiple risk factors. Looking at income, 52.5% of people with an annual household income of less than $10,000 had multiple risk factors, in comparison to 28.8% of those reporting a household income of more than $50,000. Studies investigating sex differences in CVD incidence and mortality have shown that while mortality rates were roughly equal at the start of the 20th century, male mortality from CVD has come to exceed female mortality in more recent times (Tamaki, 2006). The ecological basis for these risk factors and precursors has wide implications for the role of public health initiatives in curbing heart disease. However, it is unclear whether the â&#x20AC;&#x153;clusterâ&#x20AC;? effect of these risk factors has led to their correlation with

Plavix and Cardiovascular Disease this broad range of demographic measures, as opposed to true causal relationships. Therefore, the population distributions are of limited utility in targeting subgroups to treat or prevent CVD. Significant amounts of funding and expertise have instead been dedicated to studying and developing pharmaceutical and invasive therapies to treat cardiovascular disease and reduce the risk of cardiac events. Patients with Acute Coronary Syndromes (ACS), a subset of IHD, are diagnosed with either ST-Elevation Myocardial Infarction (STEMI), if they present with acute myocardial infarction with ST-segment elevation on an electrocardiogram, or UA/NSTEMI, if they have unstable angina (UA) and a non-ST-segment elevation myocardial infarction. Upon hospital admission, UA/NSTEMI is diagnosed three times as commonly as STEMI (Kasper, 2005). Out of the four identified pathophysiological processes that result in a reduced oxygen supply and/or an increased myocardial demand in oxygen and thus trigger UA/NSTEMI, the most frequent cause is plaque rupture or fissure with platelet aggregation (Kasper, 2005; Naghavi, 2003). The generally accepted treatment strategy for patients presenting with acute coronary syndromes combines both anti-ischemic treatment with antithrombotic therapy (Braunwald, 2003). The anti-ischemic component is applied immediately and comprised of both nitrates and beta blockers to slow the heart rate and reduce pain. The antithrombotic treatment, in recognition of the critical role of plaque rupture or erosion with superimposed nonocclusive blood clotting (Landmesser, 2004), is long term and primarily consists of combination therapy of aspirin and clopidogrel (The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001), which inhibit complementary pathways of platelet aggregation, with the addition of lowmolecular-weight heparin (LMWH) and intravenous GP IIb/IIIa inhibitors to target a

Plavix and Cardiovascular Disease common downstream receptor of the platelet pathway (Boersma, 2002). In addition to beginning these drug regimens, high-risk patients often undergo coronary arteriography followed by a coronary revascularization procedure, either coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI), and a long-term drug therapy to reduce the risk of future adverse cardiac events. Figure 1. Targets of Antithrombotic and Anticoagulant Drugs. This model indicates the points of action, indicted by the parallel lines, of various classes of drugs in the signaling pathways leading to platelet activation and aggregation. ADP denotes adenosine diphosphate; LMWHs low-molecular-weight heparins; UFH unfractionated heparin ((Nguyen, 2003)).

Atherothrombosis and the Role of Antiplatelet Therapy

Chapter 2 – Atherothrombosis and the Role of Antiplatelet Therapy .Atheroth rombosis: Plaque Rupture T riggers Clotting Atherosclerosis has long been thought of as a lengthy cumulative “response-to-injury” process, whereby endothelial dysfunction in response to initial minor trauma to the arteries ultimately results in yellowish plaques and arterial clogging (Ross, 1986; Ross, 1993). Certain arterial sites are subject to decreased fluid shear stress and increased blood turbulence; this characteristic set of changes in blood flow occurs primarily at arterial branch sites, bifurcations and areas of high curvature, making these locations particularly conducive to the development of lesions (Karsch, 1992; Ross, 1999). The resulting loss of elasticity in the artery’s vessel layers – so-called “hardening of an artery” – and the mechanical disruption of blood flow from a diseased artery can lead to everything from cerebral and myocardial infarctions to gangrene and organ failure; this intimate association of atheroschlerotic lesions and thrombotic conditions is thus termed atherothrombosis (Vorchheimer, 2006). Plaque rupture or erosion is the catalytic event leading to thrombus formation. The extent of plaque disruption, the degree of stenosis, and the physiochemical properties of the vessel surface exposed to circulating blood are three factors that heavily influence the amount of thrombus formation at the injury site (Cimminiello, 1999). The alterations in blood flow cause the endothelial tissue to display certain signal molecules that recruit platelets, T cells and monocytes to adhere to the exposed surface molecules. Platelet adhesion and subsequent activation is a critical step in triggering the generation of these lesions. The activated platelets release granules containing cytokines and growth factors that promote platelet aggregation and act in a feedback mechanism with thrombin to sustain 9

Atherothrombosis and the Role of Antiplatelet Therapy coagulation and amplify the inflammatory response. The levels of platelet deposition correlate positively with the degree of stenosis. Furthermore, platelet density is greatest at the top of the lesion and progressive narrowing of the artery prompts continued, plateletrich thrombus formation that, depending on the stability of the platelet aggregates, has the potential to lead to vessel occlusion or thrombogenic events (Vorchheimer, 2006). The cellular interactions in atherosclerosis share similar characteristics with those in other chronic inflammatory and fibroproliferative conditions such as cirrhosis and pulmonary fibrosis, but the presence of these lesions in the arteries â&#x20AC;&#x201C; rather than in the liver or in the lungs â&#x20AC;&#x201C; poses a unique set of consequences (Anthony, 1977; Kuhn, 1989). Atherothrombosis is one of the most important factors underlying the onset of acute coronary syndromes, and sudden death from cardiac arrest can most frequently be attributed to thrombi resulting from the disruption of coronary plaques (Badimon, 1999; Falk, 1995; Fuster, 1994; Langer, 1991).

.Aspirin an d Clopidogrel: Complementary Anticlotting Agents While invasive therapies are cost-effective for the highest-risk patients presenting with ACS, drug therapies aimed specifically at inhibiting platelet activation and aggregation are critical given the research showing that endothelial dysfunction and inflammation instigate atherosclerosis (Landmesser, 2004; Ross, 1999). The two most prominent irreversible antiplatelet drugs currently available are aspirin, also known as acetylsalicylic acid, and Plavix, a thienopyridine known generically as clopidogrel bisulfate. While aspirin has been known to inhibit prostaglandin and thromboxane synthesis since 1971 (Vane, 1971), the discovery of clopidogrel in 1989 was significant because it inhibits the adenosine diphosphate (ADP)

Atherothrombosis and the Role of Antiplatelet Therapy receptors to target a separate pathway for platelet aggregation (Bhukhanwala, 2006.). Aspirin and clopidogrel are usually prescribed in conjunction because their biological mechanisms combine synergistically to block platelet activation (Ibanez, 2006). Figure 2. Complementary Mechanisms of Aspirin and Clopidogrel. Aspirin and Clopidogrel inhibit different pathways to synergistically abrogate platelet function ((Blann, 2002)).

.Aspirin Targets TXA2-Mediated

Platelet Activation Aspirin acts upon the cyclooxygenase (COX) enzymes by irreversibly inhibiting COX-1, which normally activates prostaglandin production, and modifying COX-2 such that it produces an anti-inflammatory product rather than pro-inflammatory prostanoids. A comparison of aspirin to the selective COX-2 inhibitor, nimesulide, confirmed that COX-1 appears responsible for regulating platelet thromboxane A2 (TXA2) (Cullen, 1998). Furthermore, the effect of the selective inhibition of COX-2 on prostacyclin metabolite excretion demonstrates the importance of COX-2 in the process of prostacyclin synthesis (Catella-Lawson, 1999; Cullen, 1998). COX-2 and another enzyme, prostacyclin synthase, help endothelial cells convert arachidonic acid into prostacyclin (PGI2). A prostaglandin product, PGI2 also acts as a vasodilator and is an important component of endothelial regulation of platelet aggregation. PGI2 activates adenylyl cyclase, which elevates the intracellular concentration level of cAMP to inhibit platelet function (Tateson, 1977). Thus, the pro-inflammatory generation of TXA2 downstream of COX-1 in platelets and the anti-thrombotic activity of PGI2 downstream of

Atherothrombosis and the Role of Antiplatelet Therapy COX-2 in vessel walls are at odds with one another (Moncada, 2006). Aspirin suppresses the generation of platelet TXA2 and all events mediated by the TXA2 receptor, yet it also increases the risk of bleeding due to its non-specific binding to both the COX-1 and COX-2 enzymes (Collins, 1994b; Geiger, 1999). The dosage size of aspirin is important in clinical applications because of this hemostatic balance between the actions mediated by COX-1 and COX-2 for vascular hematosis, along with the different dosage-dependent response of COX in platelets versus in vessel walls (Burch, 1978). Multiple studies in the 1980s established that low-dosage aspirin permanently acetylates the platelet COX while sparing the vessel wall (De Caterina, 1985; Patrono, 1985). Aspirin is an effective antithrombotic at low doses (e.g. 75 mg per day) but can be a prothrombotic at higher doses; at lower levels the antithrombotic effects of TXA2 inhibition appear to outweigh the prothrombotic effects of PGI2 inhibition (Awtry, 2000). Measurements of cutaneous bleeding time showed that when higher doses of aspirin are used, it takes several hours to detect the prolongation of bleeding time -- the time lag allows vessel wall COX to recover and resume PGI2 production whereas the platelet COX enzymes are irreversibly inhibited (O'Grady, 1978). Hundreds of randomized clinical trials have proven the efficacy and safety of lowdose aspirin as an anti-platelet agent for the prevention of myocardial infarction, ischemic stroke and vascular death (Hennekens, 2002; Landolfi, 2004; Patrono, 2008; Sanmuganathan, 2001; Worrall, 2000a; Worrall, 2000b). However, the 110-year-old drug continues to incite debate about issues such as the uncertain threshold risk level for prophylactic use, the gender-related difference in efficacy, and the theory of aspirin â&#x20AC;&#x153;resistanceâ&#x20AC;? or failure to prevent the recurrence of vascular events (Altman, 2004; Berger, 2006; Patrono, 2008; Rohatgi, 2004).

Atherothrombosis and the Role of Antiplatelet Therapy .Clopidogrel Inhibits the ADP Receptor-Mediated Pathway to Platelet

Aggregation

There is a parallel pathway of platelet activation, which is mediated by the release of ADP from activated platelets, red blood cells and injured endothelial cells (Li, 2004). The subsequent activation of purinergic ADP receptors on platelets releases βγ and α factors; in turn, these factors activate various downstream pathways to affect platelet function. A widely studied target of platelet ADP stimulation had been the down-regulation of cyclic adenosine monophosphate (cAMP) levels. Early research suggested that the decrease in cytosolic levels of cAMP was a significant effector for the ADP receptors, as cAMP dephosphorylates the vasodilator-stimulated phosphoprotein (VASP), thereby repressing the inhibitory effect of pVASP on activating the aggregation and fibrinogen receptor (GP IIb/IIIa) (Hemmendinger, 1986; Ingall, 1999; Waldmann, 1987). These researchers postulated that impairing the dephosphorylation of pVASP would thus allow it to abrogate platelet aggregation. However, the flux in cAMP concentrations is not directly responsible for the platelet activation responses downstream of P2Y12 (Daniel, 1999; Yang, 2000). More recently, other signaling pathways important to platelet activation have been identified downstream of platelet ADP stimulation. In addition to decreasing cytosolic levels of cAMP, the clopidogrel-sensitive ADP receptor P2Y12 activates the PI3K, Akt, Rap1b and potassium channel signaling pathways (Kunapuli, 2003). The P2Y12 receptor is functionally responsible for potentiating dense granule secretion, activating the fibrinogen receptor and promoting thrombus formation (Dangelmaier, 2001; Jin, 1998a). Clopidogrel, by selectively and irreversibly inhibiting the ADP receptor P2Y12, prevents the activation of the downstream G-protein components. The exact details of

Atherothrombosis and the Role of Antiplatelet Therapy clopidogrelâ&#x20AC;&#x2122;s mechanism of action downstream of P2Y12 abrogation are still not clear although multiple signaling pathways are involved, some of which appear to synergize with P2Y1-mediated pathways (Geiger, 1999; Schwarz, 1999). Clopidogrel bisulfate actually evolved from an older antithrombotic compound, ticlopidine hydrochloride, first synthesized in 1974 by Maffrand and Elroy and launched for clinical trials in 1979 (Li, 2004; Maffrand, 1974). The two thienopyridine compounds are inactive in vitro and must be activated by in vivo metabolism, and while they do not share an active metabolite, both of the resulting metabolites are mechanistically similar and block the binding of ADP to the P2Y12 receptor (Quinn, 1999; Savi, 2001). It is worth noting that in the case of both ticlopidine and clopidogrel, their clinical efficacy as antiplatelet agents was established long before experiments had deciphered their molecular mechanisms of action. In fact, investigators had little idea how the drugs worked, though it was understood that ticlopidine was the first highly successful antiplatelet drug to be developed that did not target the cyclooxygenase pathway (Packham, 2005). Prior to the advent of thienopyridine therapies, the choice of drugs inhibiting platelet aggregation had been limited to aspirin and a small selection of agents that elevated cyclic AMP levels(Bruno, 1983; Mills, 1971). The preliminary clinical trials for ticlopidine, starting in 1979, did not produce any definite conclusions about its antagonist activity other than affirming that this novel agent did not achieve inhibition of platelet aggregation by targeting the same COX pathway as aspirin nor by inhibiting cyclic AMP-phosphodiesterases (Ashida, 1978; Bruno, 1983; Bruno, 1984). Tests on treated platelets revealed that ticlopidine had a permanent effect on the platelet surface membrane and that it abolished the primary wave of ADPinduced aggregation (Ashida, 1979; Ashida, 1979b; Ellis, 1981; O'Brien, 1978). Cattaneo et al.

Atherothrombosis and the Role of Antiplatelet Therapy later found in vivo studies that ticlopidine selectively inhibited ADP-induced platelet responses, along with collagen, an endoperoxide analogue and low levels of thrombin, but did not inhibit platelet aggregation mediated by epinephrine or high thrombin levels (Cattaneo, 1991). In hindsight, the evidence that ticlopidine inhibited the primary phase of platelet aggregation induced by ADP along with inhibiting aggregation triggered by other agonists, such as thrombin, collagen, arachidonic acid and prostaglandin endoperoxides, was a clear indication of the ADP receptor’s role in potentiating these other agonist responses (Ashida, 1979; Bruno, 1983; Packham, 2005). However, at the time, researchers were primarily focused on exploring the connection between ticlopidine’s effect on cAMP levels in platelets and the antiplatelet responses provoked by the drug (Bruno, 1983). It would not be until the 1990s that they ascertained that ticlopidine affects only one of two purinergic platelet ADP receptors, and not until 1998 that clopidogrel exclusively targets the P2Y12 receptor (Gachet, 1996; Gachet, 2001; Gachet, 1995; Hechler, 1998b). Aside from clinical applications, the use of ticlopidine and clopidogrel – along with other P2Y12 antagonists such as ARL 66096 -- has revealed much about the functional relationships between the ADP platelet receptors and the aggregation responses they stimulate (Daniel, 1998). All detailed discussion on the medical efficacy of clopidogrel hinges on an understanding of the drug’s mechanism of action in relation to the molecular activation and regulation of the thrombotic pathway. (For a full treatment of other therapeutic targets in thrombosis, see Appendix A.) Exploration of the reasoning behind clopidogrel administration and other current therapeutic strategies will require integrating our current understanding of the ADP-mediated pathways to platelet aggregation.

ADP Receptors and Platelet Activation

Chapter 3 â&#x20AC;&#x201C;ADP Receptors and Platelet Activation The critical role of adenosine diphosphate (ADP) in platelet aggregation has been recognized for well over four decades. ADP was in fact the first low molecular weight agent known to induce physiological responses in platelets though the resulting molecular mechanisms are still not yet fully understood (Gaarder, 1961; Hellem, 1960). Erythrocytes, endothelial cells, and platelet dense granules all release ADP, which is a primary platelet agonist along with thrombin, thromboxane and collagen (Gachet, 1996). ADP molecules released by dense granules upon the stimulation of platelets by other agonists such as thrombin or collagen interact with specific receptors on the platelet surface to activate different pathways, propagating signals for aggregation and further secretion (Cattaneo, 1997). There are multiple ADP-sensitive surface receptors, each with a distinct set of properties. The platelet surface has three purinoceptor subtypes, namely P2X1, P2Y1 and P2Y12 (Daniel, 1998; MacKenzie, 1996). The ionotropic P2X1 receptor is not implicated in ADP-induced platelet aggregation; it is instead responsible for regulating intracellular Ca2+ mobilization and is stimulated by both ADP and ATP (Hourani, 1991; Takano, 1999). Activation of the P2X1 receptor by binding to ADP controls the rapid influx of calcium into the platelet via an ion channel (Daniel, 1998; Kim, 1999). Both the P2Y1 and P2Y12 receptors, in contrast, are essential for ADP-mediated platelet aggregation (Jin, 1998a). While the P2Y receptors are both metabotropic receptors coupled to heterotrimeric G proteins, there are significant differences between their respective roles in activating platelets (Fredholm, 1994). Wang et al. demonstrated by quantifying mRNA levels on human platelets that even the expression levels vary among the

ADP Receptors and Platelet Activation

Figure 3. Nucleotide Receptor Signaling Pathways in Platelets. Adenosine diphosphate (ADP) activates the purinergic P2Y receptors P2Y1 and P2Y12, while adenosine triphosphate (ATP) activates the calcium ion channel receptor P2X1. Activation of the P2Y1 receptor, which is coupled to the Gq/phospholipase Cβ2 pathways, allows multiple proteins such as Src tyrosine kinase, p160ROCK, Erk2, RhoA, calcium and protein kinase C to regulate platelet function. P2Y1 primarily mediates platelet activation and platelet shape change. The P2Y12 receptor is coupled to the Gi pathway; the α subunit modulates cAMP levels, while the βγ dimer activates many downstream mediators of platelet function, including the G protein-gated inwardly rectifying potassium channel, PI3-K, Akt, Rap1 and Erk2. P2Y12 is necessary for sustained platelet aggregation but does not have a role in platelet shape change. The model demonstrates cross signaling between the pathways downstream of the P2Y receptors. The activation of P2X1 increases intracellular calcium levels and also activates ERK. Many of the signals converge on the “final common pathway” to platelet aggregation: the fibrinogen receptor ((Kahner, 2006)).

receptors, implying different mechanistic profiles (Wang, 2003b). The novel method of mRNA extraction and real-time PCR analysis enabled a highly sensitive mRNA quantification of all the P2 receptors present on platelets; the study verified that only three receptors have significant numbers of translationally-active mRNA transcripts, and determined the expression levels to be in the following order: P2Y12 >> P2X1 > P2Y1 (Wang, 2003b). A secondary assay of degradation rates found that P2X1 mRNAs degraded much faster than those encoding for the P2Y receptors, which suggests that P2X1 might have a

ADP Receptors and Platelet Activation relatively higher expression level in newly-released platelets (Wang, 2003b). These results corroborate the body of research indicating that P2Y1 has a higher affinity for ADP but a lower expression level than P2Y12. Storey et al. used whole blood single-platelet counting, an assay sensitive to microaggregation, to examine the various roles of the P2X1, P2Y1 and P2Y12 receptors in aggregation in the presence of antagonists for each of the receptors (Storey, 2000). P2Y1 is much more active in early aggregation; in fact, activation of P2Y1 alone is enough to induce primary platelet aggregation and is responsible for determining the maximal rate of platelet aggregation induced by ADP (Jarvis, 2000; Storey, 2000). Experimental evidence from the selective stimulation of the P2Y12 receptor by the antagonist AR-C69931MX, in contrast, showed that this receptor helped sustain and amplify the degree of ADP-induced aggregation (Storey, 2000). These results suggest that the P2Y12 receptor is essential for the sustained aggregation response provoked by ADP (Jarvis, 2000). Both the P2Y1 and P2Y12 receptors are G-protein coupled, yet activate different pathways (Daniel, 1998). Calcium mobilization and the activation of protein kinase C (PKC) are primary signaling events downstream of the P2Y1 receptor, whereas P2Y12 is important for downregulating cyclic AMP (cAMP) levels and activating phosphoinositide-3 kinase (PI3K). Research has made it increasingly clear, however, that there are fundamental overlaps in the relationship between the two receptors, including evidence of reciprocal cross-talk downstream of the receptors via src, extracellular signal-regulated kinase 2 (erk2) and calcium signaling pathways (Hardy, 2004; Kahner, 2006; Roger, 2004).

.Characte riz ation of the P2Y 1 receptor

ADP Receptors and Platelet Activation The P2Y1 receptor is a 373 amino acid protein, encoded by a single exon at a gene localized to chromosome 3q25.91 (Ayyanathan, 1996a; Ayyanathan, 1996b). P2Y1 receptors are found in multiple tissue types, including the central nervous system, cardiovascular and peripheral tissues (Ralevic, 1998; Webb, 1999). In fact, in addition to platelets, the heart, aortic endothelium, smooth muscle, brain and spinal cord, skeletal muscle, pancreas, spleen, testes, prostate and ovaries all express this receptor (Ayyanathan, 1996a; Communi, 2000; Daniel, 1998; Léon, 1996). Once bound by the ADP ligand on its external, free N- (amine) terminal, the internal, free C- (carboxyl) terminus of the P2Y1 receptor activates phospholipase C, and thus one of the main effects of the P2Y1 receptor is the intracellular mobilization of calcium ions (Fagura, 1998; Jin, 1998a). Both the elevation of cytosolic Ca2+ and ADP-induced platelet shape changes occur as a result of multiple sequential molecular interactions; in addition to these primary events, signaling along this receptor triggers platelet aggregation, TXA2 generation, adhesion to immobilized fibrinogen, procoagulant activity and the formation of thrombi under shear conditions (Jin, 1998a; Jin, 1998c; Murugappa, 2006). A number of molecules can activate or competitively inhibit the P2Y1 receptor. While ADP is its principal agonist, and appears to be the only agonist under physiological conditions, in vitro experiments identified 2-methylthio-ADP (2MeSADP) as the most potent agonist, followed by ADP, APDαS and ADPβS in decreasing order of potency (Hourani, 1991; Mills, 1996; von Kügelgen, 2000). Interestingly, ATP is a competitive antagonist of P2Y1 in platelets, as well as a weak and partial agonist in reconstituted or transfected systems, since the intrinsic efficiency of ATP is less than that of ADP and their relative activity levels depend on receptor density (Hechler, 1998a; Léon, 1997; Palmer, 1998; Waldo, 2004). Palmer et al. found that pre-treating cells to reduce the levels of functional P2Y1 receptors 19

ADP Receptors and Platelet Activation exacerbates this difference in binding efficiency, dramatically increasing ADP activity while concurrently abolishing the activity of ATP (Palmer, 1998). In nominal terms, platelets do express low levels of P2Y1 receptors â&#x20AC;&#x201C; 134 P2Y1 binding sites per platelet, plus or minus 8 -- relative to the 1000 to 2000 copies of thromboxane prostanoid (TP) receptor or thrombin protease-activated receptor-1 (PAR1) present on the platelet surface (Hechler, 2005; Savi, 1998). However, Kahner et al. recently suggested that scientific literature has not conclusively abandoned the possibility that ATP may physiologically stimulate the P2Y1 receptor, since ATP and ADP are released in equimolar concentrations from platelet granules (Gordon, 1986; Kahner, 2006). Pharmacological in vitro studies reported that the compounds A2P5P, A3P5P, A3P5PS, MRS2179 and MRS279 are all competitive antagonists of the P2Y1 receptor and in some cases can thus inhibit platelet function. In the late 1990s, further investigation by Nandanan et al. found that P2Y receptor agonist activity can be enhanced by producing compounds with specific deviations from the nucleotide structure (Baurand, 2001; Boyer, 1996; Boyer, 1998; Camaioni, 1998; Nandanan, 1999; Nandanan, 2000). Benzoyl-ATP antagonizes the P2Y1 receptor, though it is non-selective and is also an antagonist at the P2Y12 receptor (Vigne, 1999). While there has been extensive research on the agonists and antagonists at this receptor, and the primary steps of G-protein-coupled receptor signal transduction are well established, less is known about the complex signaling processes downstream of receptor activation. The stimulated receptor first undergoes a conformational change, allowing the transfer of a guanosine triphosphate (GTP) molecule in exchange for the guanosine

ADP Receptors and Platelet Activation diphosphate (GDP) molecule attached to the α subunit of the heterotrimeric Gq protein, which is coupled to the cytosolic side of the P2Y1 receptor (Nürnberg, 1995). Two Arg residues on the carboxyl terminus are essential for the Gq coupling (Ding, 2005). The conversion to GTP activates Gq, releasing the Gα subunit to travel along the intracellular surface of the plasma membrane, while the βγ subunits remain coupled to the receptor (Offermanns, 1997). Gαq proceeds to phosphorylate another enzyme embedded in the cytosolic side of the plasma membrane, the β2 isoform of phospholipase C, thereby activating it; the GDP-bound α subunit is then recycled back to rejoin the βγ subunits and await another signal (von Kügelgen, 2000). Meanwhile, the activation of phospholipase C (PLCβ2) causes a conformational shape change, allowing the enzyme to cleave a minor phospholipid, phosphatidylinositol (4,5)-bisphosphate (PI4,5P2), present on the plasma membrane bilayer into the secondary messengers diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) (Majerus, 1992). After the hydrolysis, both the catalytic enzyme PLCβ2 and the DAG product retain their hydrophobic character and remain tethered in the plasma membrane, whereas IP3, being soluble, travels through the cytoplasm to activate an IP3 receptor on the surface of the endoplasmic reticulum, which contains a high concentration of calcium ions (Berridge, 1993). The activation of the IP3 receptor allows the calcium ions to flood the cytoplasm from the ER; the signal transduction pathway continues as inactive protein kinase C, another membrane-bound protein on the cellular side, is activated by binding to diacylglycerol as well as two of the released calcium ions (Kiley, 1994). Intracellular calcium levels play a variety of important roles, as will be discussed further below.

ADP Receptors and Platelet Activation Active protein kinase C (PKC) can affect multiple physiological actions in a tissueand signal-dependent manner. PKC transfers a phosphoryl group to a cation channel on the membrane surface, thus affecting ion concentrations to create a localized site of depolarization that can then move electronically across the cell membrane to set off other signaling cascades. Active PKC can also activate yet another protein kinase to phosphorylate an inactive enzyme that is part of a biosynthetic pathway (Kiley, 1994). The calcium mobilization and activation of PKC as a result of P2Y1 signaling in platelets leads to platelet shape change and feeds into pathways for ADP-mediated platelet aggregation and generation of TXA2 (Jin, 1998b; Jin, 2002). The exact regulation of ion channels by the P2Y1 receptor is under dispute. Bogdanov et al. and others found examples of intracellular Ca2+ control over ion channels such as chloride and potassium; this has led some scientists to surmise that the receptorinduced activation of IP3 is responsible for changes in ion channel activity (Bogdanov, 1997; Mosbacher, 1998; Nörenberg, 1997). However, other research has implicated the activated G-protein βγ subunits or some other plasma membrane component as directly responsible for controlling potassium channel activity, by showing that the application of phospholipase and PKC inhibitors did not affect the outward current flow evoked by P2Y signaling (Ikeuchi, 1995). Gq activation also contributes to the activation of the small G proteins RhoA and Rac, via PKC, along with mitogen-activated protein (MAP) kinases and Src family kinases (Azim, 2000; Crittenden, 2004; Hardy, 2004; Kawasaki, 1998; Nemoto, 1992). The Gqactivated RhoA, a member of the Ras homologue (Rho) protein family, is known to bind

ADP Receptors and Platelet Activation with five protein kinases. RhoA also targets the p160 Rho-associated coiled-coil-containing protein kinase (p160ROCK) that signals downstream events to help initiate platelet shape changes (Leung, 1996; Paul, 1999b). The phosphorylation of myosin light chain (MLC), a 47,000 Da platelet protein, by MLC kinase has long been known to correlate with the initiation of platelet shape changes (Daniel, 1984). Platelet shape changes have received much attention as they are the first physiologically-measurable response of a platelet to an agonist (Paul, 1999b). Studies using platelet shape kinetics more recently found that MLC phosphorylation leading to platelet cytoskeletal rearrangement is mediated by calcium-independent pathways in addition to the P2Y1â&#x20AC;&#x201C;triggered calcium-dependent path. However, the absence of an increase in cytosolic Ca2+ concentration delays the characteristic rapid onset of platelet shape change and correspondingly, affects the timing and reduces the peak levels of MLC phosphorylation (Paul, 1999b). The platelet cytoskeleton first undergoes changes, including the disassembly of a microtubule ring, to transform the platelet from an inert discoid to an intermediate and transient spherical form (Deranleau, 1982). In the second phase of platelet shape change, the short pseudopods of this intermediate sphere gradually extend over a period of seven to eight seconds as actin filaments are polymerized into stress fibers and long filapodia (Bearer, 1995; Hantgan, 1984). Agonist-dependent phosphorylation of MLC contributes to both phases of platelet shape change. Addressing the identity of the calcium-independent pathway to MLC phosphorylation and subsequent shape change, Paul et al. thought RhoA/pRock160 could be involved in this calcium-insensitive pathway (Paul, 1999b). Studies performed with Gqâ&#x20AC;&#x201C; deficient mouse platelets shows that platelet shape change is not completely abolished, 23

ADP Receptors and Platelet Activation indicating that the Gq-coupled P2Y1 receptor is not entirely responsible for calciumindependent cytoskeletal rearrangement, though it is clearly implicated in the calciuminduced changes (Offermanns, 1997). The phosphorylation of platelet MLC kinase via the catalytic subunit of cyclic AMP-dependent protein kinase downregulates its activity, thereby increasing the amount of unphosphorylated platelet MLC and modulating actin-myosin interactions in platelets (Hathaway, 1981). DAG and calcium mobilization have been suggested to activate another signaling pathway, in parallel to PKC, which involves P2Y1 mediation. Members of the CalDAGGEF/RasGRP protein family have similar structural features to PKC that intimate an analogous integration of the DAG and calcium signals, including EF hands for binding calcium and DAG-binding C1 domains (Crittenden, 2004; Yamashita, 2000). Studies employing genetic ablation techniques on the mouse model showed that the gene product CalDAG-GEFI, which is expressed in the brain and blood, is necessary for integrindependent platelet aggregation (Crittenden, 2004). CalDAG-GEFI’s target, the small GTPase rap1, helps initiate activation of integrin-induced platelet signaling (Woulfe, 2002). Mice studies with Gαi-null and Gαq-null strains showed that rap1b activation was only reduced in Gαi-null strains, linking the P2Y12 receptor to the activation of rap1b (Woulfe, 2002). The CalDAG-GEF1-rap1b interaction is a cross-signaling event between the two P2Y platelet receptors. P2Y1 and not P2Y12 mediates the generation of ADP-induced p38 kinase-activating factor, though the exact contribution of this pathway to platelet activation has not yet been determined (Dangelmaier, 2000).

ADP Receptors and Platelet Activation The activation of Src family tyrosine kinases has long been linked to the P2Y1 receptor, but more recent debate has centered on whether P2Y1 is the only ADP receptor responsible for modulating this activity. Hardy et al. established in 2004 that the tyrosine kinases were activated downstream of P2Y1 but not P2Y12 (Hardy, 2004). They calculated the level of kinase activation specifically induced by P2Y1 by measuring the phosphorylation of the Y416 residue on these Src kinases. ADP-mediated Src tyrosine kinase activation appeared to be independent of P2Y12, but P2Y12 potentiates P2Y1-induced calcium mobilization via a pathway that is independent of cAMP but dependent on the activation of PI3-kinase. In 2006, however, Murugappan and Kunapuli used P2Y1 knockout mouse platelets to study the effect of ADP activation of the P2Y12 receptor on the tyrosine kinases. The results suggested that Src tyrosine activation can occur downstream of the P2Y12 receptor in platelets lacking P2Y1, implying that P2Y12 is also involved in Src activation (Murugappa, 2006). The inhibition of Gq activation results in the absence of downstream events – namely calcium mobilization and the activation of PKC – and thus prevents ‘inside-out’ signaling for fibrinogen (αIIbβ3) receptor activation (Quinton, 2002). Researchers have questioned whether PKC plays a role in (αIIbβ3) receptor signaling, as the application of PKC inhibitors did not affect levels of ADP-induced fibrinogen receptor activation, though it did abrogate calcium-induced platelet aggregation (Kahner, 2006; Tabuchi, 2003). PKC inhibition has also shown that PKC has a negative regulatory role on Erk2 phosphorylation (Garcia, 2005a; Garcia, 2005b). Part of this confusion is due to lingering uncertainty over which of the different isoforms of Src PTK to mediate Erk2 phosphorylation are targeted downstream of P2Y1 (Kahner, 2006). 25

ADP Receptors and Platelet Activation

.Characte riz ation of the P2Y 12 Receptor Of the three ADP-stimulated platelet receptors under discussion, the P2Y12 receptor was the last to be discovered and carefully studied. The identity of the P2Y12 receptor was not finally determined until 2001. Takasaki et al. used expression cloning and ligand screening techniques to establish that P2Y12 was a low-affinity receptor for ADP in platelets, enabling the cloning of P2Y12 from human and rat complementary DNA libraries (Takasaki, 2001). Savi et al. and others recognized P2Y12 as the target of clopidogrel and other antithrombotic drugs (Hollopeter, 2001; Savi, 2001). Foster et al. had previously isolated a human receptor, SP1999, which had similar properties to the purported third ADP receptor on platelets (Foster, 2001; Zhang, 2001). This research allowed them to verify that they had indeed cloned P2Y12 and establish that it was the target receptor of the antithrombotic drugs ticlopidine and clopidogrel (Foster, 2001). In contrast, both the P2X1 and P2Y1 receptors had been cloned by 1998 (Jin, 1998c; Sun, 1998). Part of the difficulty of cloning the P2Y12 receptor stemmed from the inability of researchers to purify the receptor protein. This failure was attributed to: (1) the unavailability of the reagents necessary to specifically label the receptor; and (2) intrinsic characteristics of the P2Y12 receptor in vivo â&#x20AC;&#x201C; namely, a low receptor density and its susceptibility to proteolysis (Puri, 1998). These properties also hampered the production of antibodies that would have aided attempts to clone the receptor via cDNA library screening (Puri, 1998). The receptor is now generally designated P2Y12 by the scientific community. Yet because of this convoluted history, this receptor has at various times and at some points simultaneously been referred to as P2T, P2TAC, P2T, tP2Y, P2YAC, P2YADP and P2Ycyc (Daniel, 1998; Hollopeter, 2001; Storey, 2000; von KĂźgelgen, 2000). P2Y12 is comprised of 26

ADP Receptors and Platelet Activation 343 amino acid residues and similar to P2Y1, forms a classic G protein-coupled receptor structure (Hollopeter, 2001). P2Y12 is in many respects a more attractive molecular target than either of the other platelet ADP receptors, as it has a much more limited distribution across human tissues than P2Y1 (Murugappa, 2006). P2Y12 receptors are expressed abundantly in platelets and only to a limited extent in the brain, whereas the P2Y1 receptor is expressed at a low level in platelets but is common to a diverse set of tissue systems (Boyer, 1993; Hollopeter, 2001; Vasiljev, 2003). In human platelets, P2Y12 mRNA is expressed in quantities twelve times greater than that of P2Y1 mRNA, which supports the finding that P2Y12 has more binding sites on platelets for ADP (Nurden, 1995; Wang, 2003b). P2Y12 is an easier pharmacodynamic target than P2Y1, a difference reflected in the lionâ&#x20AC;&#x2122;s share of research efforts being directed towards producing P2Y12 antagonists (Storey, 2001b). Pharmacological profiling showed that, similar to the P2Y1 receptor, ADP and 2MeSADP are both agonists of P2Y12. However, in contrast to P2Y1, which has a higher affinity for ADP over 2-MeSADP, the P2Y12 receptor has 100-fold higher affinity for 2MeSADP over ADP, and also displays other pharmacologically-unique characteristics (Hollopeter, 2001; Takasaki, 2001). There are four extracellular cysteine residues on the P2Y12 receptor molecule (Hollopeter, 2001). These reactive residues are important for binding; the active metabolite of clopidogrel was found to be a thiol, which binds covalently to one of these cysteine residues, and another thiol reagent, p-chloromercuriphenylsulfonic acid (pCMBS), antagonizes the P2Y12 receptor to disable ADP-induced P2Y12 activation (Hollopeter, 2001; Macfarlane, 1983; Savi, 2000). Research driven towards therapeutic ends has uncovered multiple agonists and antagonists at this receptor.

ADP Receptors and Platelet Activation Furthermore, the experimental use of pharmacological antagonists on the platelets of genetically engineered mouse models has yielded significant information about the functional roles of P2Y12. There was initially debate over the role of ATP at P2Y receptors. ADP was largely recognized as the natural agonist, but some studies reported ATP to be agonists in both native P2Y12-expressing cells, such as brain endothelial cells, as well as in transfected cells Simon, 2001; Takasaki, 2001; Unterberger, 2002; Zhang, 2001). This presented a huge discrepancy in the study of blood platelet activity, as analogues and derivatives of ATP were widely known to be antagonists of P2Y12 (Cusack, 1982a; Park, 1999). Kauffenstein et al. finally resolved this debate in 2004 using mouse platelets and a transfected cell system stably expressing P2Y12 receptors to show that ATP and its derivatives are true antagonists of P2Y12 (Kauffenstein, 2004). The weak and partial agonist activity of previous studies could have stemmed from diphosphate contamination and the cell ectonucleotidases acting on ATP to generate diphosphate nucleotide derivatives (Kauffenstein, 2004). This explanation was bolstered by Bodor et al.â&#x20AC;&#x2122;s experiments, which found ATP to be a low-affinity antagonist of P2Y12 (Bodor, 2003). His study took purified P2Y12 receptors and reconstituted them in proteoliposomes, thereby preventing the enzymatic breakdown or interconversion of ATP into other adenine nucleotides. Bodor et al. measured the stimulatory effects of ADP, 2MeSADP and ATP. As ATP is a competitive, albeit weak, antagonist of platelet aggregation induced by ADP, the active development of ATP derivatives has produced many P2Y antagonists. Benzoyl ATP (BeZATP) is a well-known non-specific antagonist of both platelet P2Y receptors (Vigne, 1999). Jantzen et al. and other researchers found 2-MeSAMP, a methylated version of the agonist 2-MeSADP, to be a selective antagonist at the Gi-coupled P2Y12 receptor (Hollopeter, 2001; Jantzen, 1999).

ADP Receptors and Platelet Activation Other purine compounds such as AR-C66096 and AR-C67085 were derived from ATP; AR69931MX in particular reversibly antagonizes the P2Y12 receptor and is a potential pharmaceutical agent, as will be discussed in the following section. The activation of P2Y12 potentiates platelet aggregation, mobilizes cytoplasmic calcium, stimulates the secretion of both alpha and dense granules via other agonists, leads to thromboxane A2 generation, phosphorylates various proteins and promotes platelet procoagulant activity to stabilize platelet aggregates (Fälker, 2004; Quinton, 2002; Reséndiz, 2003; Sage, 2000; Shankar, 2006a; Storey, 2000). Consistently, P2Y12 receptor-deficient mice showed impaired inhibition of adenylyl cyclase and compromised ADP-induced platelet aggregation as well as decreased thrombin-induced platelet aggregation, prolonged bleeding time and insensitivity to treatment with clopidogrel (Foster, 2001). Despite the array of specific biological processes affected by both P2Y12 and P2Y1, there are several pathways that are distinct to one and not the other. Unlike the P2Y1– mediated pathways, signaling via activation of the Gi-coupled receptor by ADP or epinephrine does not help induce platelet shape changes (Daniel, 1998; Jin, 1998a; Jin, 1998c). Studies utilized P2Y12 receptor antagonists, such as the above-mentioned ARC66096 and AR-C67085, to demonstrate that while P2Y12 is not essential for ADP-mediated platelet shape change, it is required for both ADP-induced and TXA2–mediated platelet aggregation (Daniel, 1998; Jin, 1998a; Jin, 1998c; Paul, 1999a). Studies of receptor specificity determined that the P2Y12 receptor, and not P2Y1, was the purinoreceptor responsible for potentiating thrombin-induced procoagulant activity by activating intracellular signals to produce and release thrombin upon stimulation by ADP (Dorsam, 2004a). The activation of Akt/protein kinase B and subsequent downstream events critical to platelet aggregation, 29

ADP Receptors and Platelet Activation dense granule secretion and thrombus stability is controlled by P2Y12 as well as a G12/13 pathway, but not by P2Y1 (Takasaki, 2001). P2Y12 is coupled specifically to the Gi2 protein, a member of the Gi family of Gproteins (Ohlmann, 1995). Downstream of receptor stimulation, the heterotrimeric Gi2 protein dissociates into the Gα and Gβγ subunits to activate different signaling pathways. The released Gαi2 protein inhibits adenylyl cyclase, resulting in depleted basal levels of cAMP in cells (Yang, 2002). The first attempts to differentiate the P2Y12 receptor from the related P2Y1 platelet receptor assumed the inhibition of adenylyl cyclase by P2Y12 was an important, if not the primary, signaling mechanism for this receptor to induce platelet aggregation. In reality, the reduction in cytosolic cAMP concentration triggered by Gαi2 appears to be essential but insufficient for platelet aggregation and ADP-induced activation of the fibrinogen receptor (Daniel, 1999; Yang, 2002). Furthermore, more recent studies concluded that the P2Y12–mediated inhibition of adenylyl cyclase and the resulting low cAMP levels are not directly responsible for the platelet responses induced by P2Y12 (Dangelmaier, 2001; Murugappa, 2006; Yang, 2002). The conclusions drawn by Yang et al. were of particular note; they used mouse platelets with null phenotypes for the α subunits of three platelet Gi proteins and for the prostacyclin (PGI2)-receptor, IP, to demonstrate that P2Y12 signals can inhibit the elevation in cAMP levels induced by PGI2 or forskolin, but cannot lower basal levels of cAMP in the platelet (Yang, 2002). While doing so, Yang et al. also established that the Gi-coupled surface receptors in platelets, namely P2Y12 and the epinephrine-stimulated alpha2A adrenergic receptor, have specific and non-redundant preferences for binding to certain types of Gi

ADP Receptors and Platelet Activation proteins. Furthermore, they found that cAMP levels are modulated by signaling between Gi2 and Gz and IP but not affected by the third G-protein Gi3. A primary effect of lowered cAMP concentrations after the inhibition of adenylyl cyclase via P2Y12 is the reduction in calcium removal from the cytoplasm by P2X1, suggesting that cAMP may play a significant role in ADP-mediated calcium signaling (Sage, 2000). Many signals are mediated by the βγ subunits of the Gi protein. The Gβγi2 dimer, acting independently of Gαi2, activates multiple cellular effectors including phosphoinositide-3-kinases (PI3K), Akt/protein kinase B, Rab1b, G protein-gated inwardly rectifying potassium channels (GIRKs) and Src family tyrosine kinases (Abrams, 1996; Clapham, 1997; Dorsam, 2004b; Hirsch, 2001; Kim, 2004; Schoenwaelder, 2007; Shankar, 2004; Shankar, 2006b; Woulfe, 2002). The directional relationships of these various effectors in the signaling pathways have been elucidated through treatment of platelets with proteinspecific inhibitors or by introducing genetic deficiencies in mouse models. The Gβγi2 dimer appears to directly activate GIRKs, Src tyrosine kinases and two isoforms of phosphoinositide-3-kinases (PI3K), while the other effectors are activated downstream of these molecules. GIRKS can be activated by binding to βγi2 subunits and inactivated by other G protein subunits, all in the cytosolic domain of the GIRK. This finding illuminated the possibility that GIRKs could have a role in G2Y12-induced signal transduction in addition to their established function of regulating cellular excitability in brain and heart membranes by controlling potassium ion conductance (Lei, 2000). Shankar et al. investigated the roles of GIRKs and Src tyrosine kinases in the

ADP Receptors and Platelet Activation signaling pathway towards P2Y12-potentiated TXA2 generation (Shankar, 2004; Shankar, 2006b). Treatment of human platelets with two structurally distinct pharmacological agents, SCH23390 and U50488H, which block GIRK channels from the outside-in revealed that GIRKs do mediate some of the functional responses instigated by P2Y12 receptor stimulation (Shankar, 2004). GIRKs exist as homo- or heterotetramers made up of four identical or dissimilar subunits, respectively. The human platelet expresses three subunit types, GIRK2, GIRK3 and GIRK4. Since the channel blockers SCH23390 and U50488H are known to affect different GIRK channel complexes depending on inhibitor concentration, it has been postulated that several different functional GIRK complexes might exist in human platelets and play distinct roles downstream of P2Y12 activation (Kuzhikandathil, 2002; Shankar, 2006b). Application of the channel blockers inhibited P2Y12-mediated potentiation of dense granule secretion, phosphorylation of Akt, and platelet aggregation mediated by low-dose thrombin, ADP, 2-MeSADP, and the TXA2 mimetic U46619. Shankar demonstrated that GIRKs do not directly antagonize the P2Y12 receptor, by showing that GIRK channel inhibitors do not prevent the inhibition of adenylyl cyclase (Shankar, 2004). Rather, GIRKS lay downstream of P2Y12 activation. Further delineation of this pathway by applying varying levels of GIRK inhibitors found that GIRK channels, in turn, coregulate the Src tyrosine kinases – along with Gβγi2 and Gq downstream of P2Y1 activation – to propagate cytosolic phospholipase A2 (cPLA2) generation and the generation of TXA2 (Shankar, 2006b). There appear to be two different populations of GIRK channels involved in ADP-induced platelet aggregation, one sensitive to inhibition by low levels of GIRK blockers that regulates ADP-induced TXA2 generation, and the other population -- sensitive to high concentrations of GIRK inhibitors -- that

ADP Receptors and Platelet Activation mediates P2Y-mediated platelet responses (Jin, 2002; Shankar, 2006b). Downstream of P2Y12 activation, PIK3 is another major functional effector in platelet physiology. Kauffenstein et al. found that treating mouse platelets with the PI3K inhibitors LY294002 and wortmannin completely eliminated the P2Y12-mediated partial platelet aggregation present in mouse platelets lacking P2Y1 receptors (Kauffenstein, 2001). The study also verified that the P2Y12-activated signaling pathway to stimulating the αIIbβ3 integrin is dependent on PIK3. In addition to its role in platelet aggregation, PIK3 has been implicated in the potentiation of platelet granule secretion. Dangelmaier et al. pinpointed the phosphoinositide-kinase 3 pathway as responsible for potentiating dense granule secretion. The resulting thrombin activates the PAR1 and PAR4 receptors to enhance collagen-induced thrombin generation in a feedback mechanism, thereby potentiating the generation of thrombin-induced TXA2 and further activating platelet aggregation along that pathway (Dangelmaier, 2001; Dorsam, 2004a; Shankar, 2006a). This crossover between receptormediated pathways explains why selective antagonism at the P2Y12 receptor can partially inhibit the platelet aggregation induced by other physiological agonists such as collagen, TXA2 and von Willebrand factor (Cattaneo, 1991; Heptinstall, 1995). An interesting facet on the role of PI3K in the P2Y12 signaling cascade is the research on the various isoforms of PI3K and their respective functions. Platelets express various isoforms of PI3K, but only PI3K p110 γ and PI3K p110 β are activated downstream of P2Y12 (Abrams, 1996; Hirsch, 2001; Jackson, 2005). Neither isoform is fully responsible for mediating the fibrinogen receptor activation and aggregation. The functional

ADP Receptors and Platelet Activation specifications of each isoform have only been elucidated in recent years. PI3K γ is primarily responsible for regulating the activation of the integrin αIIIaβ3 receptor through a non-catalytic signaling mechanism (Schoenwaelder, 2007). In mouse platelets lacking PI3K γ, Hirsch et al. observed defective fibrinogen receptor activation and impaired platelet aggregation, along with a reduction in the incidence of thromboembolic events induced by ADP (Hirsch, 2001). These responses were similar to those observed in Gαi2–deficient platelets as well as in platelets pretreated with P2Y12 antagonists. The PI3K γdeficient mouse platelets failed to exhibit the P2Y12-receptor dependent phosphorylation of Akt, suggesting that PI3K γ is an upstream regulator of the Akt pathway (Hirsch, 2001). PI3K γ was also linked to ADP-induced rap1b activation in another experiment with PI3K γ-deficient mice (Woulfe, 2002). Notably, however, the isolated PI3K γ-deficient mouse platelets did not exhibit full abrogation of P2Y12-induced platelet aggregation, but rather, characterized a “partial platelet aggregation defect”. This indicated the involvement of other Gi2-linked signaling pathways (Hirsch, 2001). PI3K β has a functionally distinct role in P2Y12 signal transduction. Jackson et al. demonstrated the importance of PI3K β’s regulation over integrin αIIIaβ3 adhesion in platelets by treating mouse platelets with a novel pharmacological inhibitor, TGX221 (Jackson, 2005). PI3K β appears to be critical to the formation of stable integrin αIIIaβ3 (GBIIbIIIa) adhesive contacts between platelets as well as to the sustained platelet aggregation induced by low (threshold concentration) doses of thrombin receptor activating peptide (TRAP), collagen and the TXA2 mimetic U4619. Pretreatment of mouse platelets with TGX221 results in inhibition and rapid reversal of ADP-induced platelet aggregation

ADP Receptors and Platelet Activation and prevents the stimulation of these Gi-dependent pathways to platelet aggregation by other agonists. Interestingly, this in vivo inhibition of PI3K β by TGX221 in mice does not prolong their bleeding times, despite abrogating their ability to form occlusive arterial thrombi (Jackson, 2005). These abnormalities in the thrombotic response are unique to platelet treatment with LY294002 and TGX221, and were not observed in PI3K γ-deficient mouse platelets, indicating that LY294002 and TGX221 both target PI3K β and not PI3K γ to achieve their antithrombotic effects. These results complement Kauffenstein et al.’s finding that PI3K inhibitors completely abolish the partial platelet aggregation due to P2Y12 activation, with LY294002 and wortmannin inhibiting the PI3K β and PI3K γ isoforms, respectively (Jackson, 2005; Kauffenstein, 2001). PI3K γ appears to be the only PI3K isoform responsible for potentiating agonist-induced dense granule release, whereas activated PI3K β is a principle effector for the Gi2-dependent production of phosphatidylinositol 3,4-bisphosphate (PI3,4P2); both isoforms appear to be involved in activating the Akt and rap1b pathways (Schoenwaelder, 2007). Other signaling molecules downstream of the GIRK channels and the PI3 kinases affect the functional platelet responses induced by P2Y12 stimulation. Akt, a serine/threonine kinase, is also known as protein kinase B (PKB), and is activated by a phosphorylation event downstream of PI3K (Kim, 2004). PI3K γ is suspected to be the PI3K isoform responsible for inducing Akt phosphorylation since PI3K γ-deficient mouse platelets are incapable of phosphorylating Akt (Hirsch, 2001). Additionally, the G12/13 pathway appears to potentiate the Gi-dependent phosphorylation of Akt via activation of Src kinases, though Gq does not

ADP Receptors and Platelet Activation play any role in Akt activation (Kim, 2006). The stimulation of PAR1 and PAR4 downstream of PLC can elicite rapid phosphorylation of Akt, even without PI3K activation via P2Y12, but ADP and Gi signaling is required to sustain Akt phosphorylation (Reséndiz, 2007). Though the exact functions of Akt have not yet been ascertained, aggregometry, flow cytometry and genetic studies have yielded some insights. Akt is implicated in regulating platelet aggregation, granule secretion and the formation of stable thrombi. The pharmacologic inhibition of Akt in normal platelets provokes deficient granule secretion and impaired activation of the integrin αIIbβ3 receptor (Reséndiz, 2007). Mouse platelets deficient in three alleles for isoforms of Akt (Akt1 +/-2-/-) have defective platelet aggregation and granule release, and cannot form stable thrombi in response to stimulation with ADP, U46619 (TXA2 mimetic) or AYPGQV, a PAR-4 peptide (Woulfe, 2004). Akt1-deficient mice exhibit longer bleeding times than wild-type mice and show impaired responses to the platelet agonists collagen and thrombin (Chen, 2004).

.Intrinsic Regulation of the ADP Receptor There are several important molecules that regulate the activity of ADP activation. Notably, ADP activation activity is amplified by the presence of epinephrine (EPI). EPI helps activate the Gαi protein, a ligand for the P2Y12 receptor. However, EPI alone is not sufficient to activate the ADP-mediated pathway to platelet aggregation. Chemokines also interact with the P2 receptors. In low levels of ADP, circulating chemokines can activate platelet aggregation by binding to the CXCR4 and CCR4 receptors on platelets (Abi-Younes, 2001; Gear, 2001; Kowalska, 2000). Some research has shown that chemokines such as

ADP Receptors and Platelet Activation macrophage-derived chemokine (MDC), thymus activation-regulated chemokine (TARC), and stromal cell-derived factor one (SDF-1), may be implicated in activating Gαi, and are involved in signaling through the P2Y1 receptor (Suttitanamongkol, 2001). Nitric oxide (NO), conversely, is a critical negative regulator of platelet function. Released not only by endothelial tissues but also secreted by both resting and activated platelets in a negative feedback loop, NO activates soluble guanylate cyclase (Zhou, 1995). Cyclic guanosine-3,’5’-monophsophate (cGMP), regulated by guanlyate cyclase, is the principal effector of nitric oxide’s antiplatelet activity and stimulates protein kinases (Moro, 1996). In clinical settings, some of the platelet hyperaggregability and the associated thrombosis disorders exhibited in many cardiovascular disease states have been traced to “platelet NO resistance,” the defective physiological response to the anti-aggregating efficiency of both endogenous NO and exogenous, pharmacologic NO-donors (Chirkov, 2001; Freedman, 1998; Rajendran, 2008; Weisbrod, 1997). The scavenging of NO by superoxide anion radicals (e.g. O2-) and the inactivation of soluble guanylate cyclase both contribute to platelet NO resistance (Brüne, 1990; Leo, 1997; Yao, 1993).

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents

Chapter 4 - Pre-Clinical Development of Plavix: Thieno pyri dine Deri vati ve s, En antiome r Se paration and the S anofi Patents The patents on Plavix are currently held by Sanofi-Aventis and Bristol Myers Squibb, and constitute a critical and profitable component of their CVD and thrombosis portfolios. To understand the recent dispute over the validity of the Plavix patents, it will first be necessary to review the history of the scientific discoveries pertaining to clopidogrel bisulfate, along with patent records and the subsequent marketing agreement with Bristol-Myers Squibb to bring the drug to market in the United States. This discussion of Sanofi’s development of clopidogrel bisulfate will focus on the facts considered most relevant to the District Court’s evaluation of the patent dispute between Sanofi and the generic challenger Apotex in 2007. Many of the more technical themes in the following section will be revisited in the next chapter; this section should help convey, in chronological order, the convoluted progression of chemical experimentation that led from Sanofi’s first therapeutic thienopyridine, ticlopidine, to the current standard in thienopyridines for antiplatelet therapy, clopidogrel. Aside from its implications for the patent litigation, this rich story – as told by the Sanofi scientists1 to the Southern District Court of New York, and augmented by other contemporary resources – stands on its own merits as a modern-day illustration of the complex and expensive process of pharmaceutical drug development, which is so often obscured behind corporate doors.

The following section relies heavily on several testimonies that were discussed in the Court findings. The first mention of each witness’s trial transcript will be fully cited, as follows: Testimony of Dr. Jean-Pierre Maffrand, Transcript of Preliminary Injunction Hearing dated Aug. 18 & 21, 2006 (hereafter denoted as “Maffrand PI Tr.”), as discussed in Sanofi-Synthelabo v. Apotex Inc., 488 F.Supp.2d 317 (S.D.N.Y. 2006). 1

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents .Thienopyridine Chemical Synthesis This Plavix story starts over 35 five years ago, in 1972, with the French Sanofi chemist Dr. Jean-Pierre Maffrand.2 He and his small team of chemists were charged with the goal of trying to develop a compound with better anti-inflammatory properties than the thencontemporary drug tinoridine, which was a thienopyridine with anti-inflammatory properties.3 Thienopyridines are a class of compounds so-named because of their characteristic chemical structure: a thiophene ring fused with a pyridine ring.

Figure 4. Chemical Structure of a Thienopyridine. A thienopyridine is a thiophene ring fused with a pyridine ring ((Nerenz, Grahn, and Jones, 1997)).

While Dr. Maffrand’s search to synthesize other antiinflammatory thienopyridines was ultimately unsuccessful, several of the thienopyridines synthesized by the Sanofi labs from 1972-1973 exhibited antiplatelet activity.4 These compounds, including one later named ticlopidine, appeared to help prevent platelet aggregation in blood. This property of certain thienopyridines was an important, if unexpected, discovery. Research had established the critical role that platelets played in potentiating some heart conditions and triggering acute events such as heart attacks and strokes. Dr. Maffrand and other Sanofi chemists decided to pursue this line of experimentation, in hopes of finding “[a] better drug than aspirin.”5 A salt form of this thienopyridine, ticlopidine hydrochloride, was initially synthesized by Maffrand and Eloy in

Testimony of Dr. Jean-Pierre Maffrand, Trial Transcript (hereafter denoted as “Maffrand T Tr.”), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 3 Maffrand T Tr. 1570-74 4 Maffrand T Tr. 1573-75 5 Maffrand T Tr. 1574-75 2

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents 1974, and J.J. Thebault verified its therapeutic potential in 1975 as the first drug in a new class of platelet inhibitors(Ashida, 1979; Féliste, 1987; Maffrand, 1974; Thebault, 1975). Figure 5. Chemical Structure of Ticlopidine . and 2-oxo-Ticlopidine. Ticlopidine is inactive . in vitro, and proceeds through hepatic metabolism to the inactive intermediate 2-oxoTiclopidine before another transformation yields . the active metabolite ((Yoneda, 2004)).

Early investigation on the safety and efficacy of the thienopyridine ticlopidine returned promising results. Sanofi obtained a patent for ticlopidine in 1977; marketing of the drug under the brand name “Ticlid” commenced in France the following year. Soon after the French launch, treatment with ticlopidine exposed some rare but life-threatening side effects associated with the drug: Ticlopidine was linked to the blood disorders neutropenia and thrombotic thrombocytopenic purpura (“TTP”).6 While major studies, such as one conducted by the New England Journal of Medicine in 1989, found that the medical benefits of ticlopidine hydrochloride were comparable to those with aspirin, its toxicity remained a problem (Hass, 1989). Ticlid was introduced into the United States in 1991, albeit with the FDA restriction that the drug was labeled with a “black box” warning for potentially fatal blood disorders.7 Clinicians were wary of the many adverse medical conditions triggered by ticlopidine, which included minor but prevalent gastrointestinal side effects and skin rashes along with the rare but fatal conditions such as neutropenia and bone marrow aplasia (Quinn, 1999). The severity of these side effects meant that all patients taking Ticlid had to be monitored for the manifestation of blood disorders, and prevented Ticlid from ever

Testimony of Schneller, Trial Transcript 763, 815-16 (hereafter denoted as “Schneller T Tr.”), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007); Maffrand T Tr. 1580-81. 7 Maffrand T Tr. 1581 6

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents securing a monopoly on the platelet aggregation market. The search was still on for a more effective antiplatelet drug. Meanwhile, Dr. Maffrand and his colleagues had not stopped work on developing other thienopyridine derivatives that would reduce the incidence of toxic side effects produced by the metabolism of ticlopidine while delivering better suppression of platelet aggregation (Quinn, 1999). Figure 6. Chirality and Drug Development. Stereoisomers are molecules that have the same chemical substituents, but different spatial arrangements. Different types of stereoisomers exist: diastereomers; cis-transisomers; and enantiomers, molecules that are nonsuperimposable mirror-images of each other. Enantiomers are thus defined by their chirality, or handedness (the relationship between the right and left hands is the most vivid example of chirality), and are the special category of stereoisomers most relevant to our discussion of clopidogrel bisulfate. The figure above shows the chirality of an amino acid. A racemate mixture is made up of equal amounts of both enantiomers and has no optical activity (e.g. does not rotate plane-polarized light), but a pure enantiomer either rotates the plane of light to the right (dextrorotatory enantiomer) or to the left (levorotatory enantiomer). While the active ingredients of drugs are frequently composed of the racemate of a pharmacologic compound, individual enantiomers can have specific, different physiological properties that make them better- or worse-suited as therapeutic agents in comparison to the other enantiomer or the racemic mixture. Aside from Plavix, examples of drugs made up of only one enantiomer include Nexium (Andersson, 2008), Lunesta (Drugs in RD, 2005) and Lexapro (Jacquot, 2007) (image from (Ames Research Center)).

Sanofi spent extensive resources focusing on several other thienopyridines prior to the development of clopidogrel. Several of these thienopyridines were chiral, but from Maffrandâ&#x20AC;&#x2122;s past experience, the greatest quantitative increase in therapeutic activity from separating a racemate into individual enantiomers appeared to be limited to an increase of just two-fold.8 Thus, modifying experimental compounds by replacing substituents was often

Maffrand T Tr. 1591-93; Testimony of Thomas K. Harden, Trial Transcript 2304-06 (hereafter denoted as â&#x20AC;&#x153;Harden T Tr.â&#x20AC;?), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 8

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents a more lucrative way to discover superior antiplatelet drugs than continuing research on an existing compound by isolating and retesting the individual enantiomers.9 .PCR 1033 and PCR 3349 It is worth describing two of the ticlopidine derivatives prepared by Sanofi prior to clopidogrel, since the process of preparing and testing these racemates and separating their enantiomers informed the methods later used in the process of developing clopidogrel bisulfate. In aggregate, the different experimental techniques used to resolve these enantiomers, as well as the variance in the antiplatelet and toxic activities exhibited by the individual enantiomers versus the racemate in each thienopyridine compound, underscores the intensive and highly unpredictable practice of empirical science that engendered clopidogrel bisulfate. The first of the two was synthesized at Sanofi in 1975.10 The compound, PCR 1033, is a methyl analog of ticlopidine. Unlike achiral ticlopidine, the methyl substitution -replacing one of the two hydrogen atoms on the bridge carbon linking the thienopyridine and phenyl groups â&#x20AC;&#x201C; makes the molecule stereospecific, with a chiral center at the bridge carbon. Sanofi first attempted to prepare a salt of PCR 1033 (the racemate) so that the compoundâ&#x20AC;&#x2122;s clinical properties could be tested. Chemists failed in their initial attempt to prepare PCR 1033 as a hydrochloride salt, but Sanofi eventually prepared the maleate salt of PCR 1033.11 The clinical tests established that PCR 1033 was unsuitable for further development â&#x20AC;&#x201C; despite being a more potent antiplatelet agent than ticlopidine, PCR 1033

Harden T Tr. 2304; Maffrand T Tr. 1592-94. Maffrand T Tr. 1584. 11 Maffrand T Tr. 1599-1600. 9

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents was also more toxic.12 Research on PCR 1033 was discontinued, but recognizing the potential for one of the enantiomers of PCR 1033 to have a better profile of antiplatelet activity to toxicity than the racemate or ticlopidine, Dr. Maffrand enlisted the help of another Sanofi chemist to separate the stereoisomers.13 Alain Badorc led the effort to separate the enantiomers.14 Unlike other isomers, the problem with enantiomers is that they have identical physical and chemical properties. This means the racemate cannot be directly resolved on the basis of properties such as solubility and melting point. Resolution of the racemate is a common problem encountered in the pharmaceutical industry whilst preparing biologically active compounds (Davankov, 1997). There are various methods for separating a racemic mixture, but it is hard to predict what technique will work for a given racemate (Davankov, 1997).15 Literature at the time was replete with descriptions of enantiomer separation using diastereomeric salt formation and asymmetric synthesis, along with various forms of chromatography (Armstrong, 1988). An expert witness testified in front of the District Court that at least ten different methods to resolve a racemate available to a chemist at 1987.16 While at least five of the methods would have been discounted by the expert witness as inappropriate to separate the enantiomers of PCR 4099, there was no way of predicting which technique would succeed because nothing in the prior art discussed relevant

Maffrand T Tr. 1587. Maffrand T Tr. 1587-88; Testimony of Alain Badorc, Trial Transcript 1807 (hereafter denoted as “Badorc T Tr.”), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 14 Badorc T Tr. 1807-09. 15 Testimony of Stephen G. Davies, Trial Transcript 1939 (hereafter denoted as “Davies T Tr.”), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 16 Davies T Tr. 1921. 12 13

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents procedures for resolving a chiral thienopyridine compound.17 Badorc successfully separated the enantiomers of PCR 1033 on his first attempt, using the technique of diastereomeric salt formation. The reagent used by Badorc in the original preparation was tartaric salt in ethanol.18 Badorc eventually revised this protocol to use hydrochloric acid, which created more stable enantiomeric salts of PCR 1033.19 The levorotatory and dextrorotary enantiomers were designated PCR 3071 and PCR 3072, respectively.20 Testing of the individual enantiomers showed that only one of the two enantiomers, PCR 3071, demonstrated antiplatelet activity. However, because PCR 3071 was shown to be more toxic than ticlopidine, development of these compounds was ultimately discontinued in 1981.21 Three years after the synthesis of PCR 1033, in 1878, Sanofi initiated development of another promising ticlopidine derivative, the ethyl analog PCR 3233.22 Following the protocol that had proven successful in the case of PCR 1033, the chemists first attempted to create a stable hydrochloride salt of PCR 3233 so that the analog could undergo clinical testing. However, the hydrochloric acid failed to react well with PCR 3233, an oily base; Badorc finally found success using nitric acid to form the nitrate salt of the ethyl analog, designated PCR 3549.23 While PCR 3549 exhibited greater potency than ticlopidine, it was also less well-tolerated. Again, Maffrand decided that it would be beneficial to see if any one of the enantiomers had a better pharmacologic profile than the racemate. Badorc began

Davies T Tr. 1939, 2023-24; Testimony of Robert A. McClelland, Trial Transcript 1127, 1230-32 (hereafter denoted as â&#x20AC;&#x153;McClelland T Tr.â&#x20AC;?), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 18 Badorc T Tr. 1808-09. 19 Badorc T Tr. 1810 20 Maffrand T Tr. 1598 21 Maffrand T Tr. 1598-99 22 Maffrand T Tr. 1601-02 23 Maffrand T Tr. 1604; Badorc T Tr. 1812. 17

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents attempting to resolve the enantiomers of PCR 3549 in November 1978, first trying the diastereomeric salt formation technique that had worked so well to resolve PCR 1033.24 Badorc eventually turned to chemical asymmetric synthesis after multiple failed efforts to generate a diastereomeric salt. Successful asymmetric synthesis begins with an enantiomerically pure precursor, which is then chemically modified to engineer the goal compound without disturbing the original stereochemical configuration (Yamada, 1976; Luo, 2008). Badorc synthesized the enantiomers of PCR 3549 and found that in contrast to the resolved enantiomers of PCR 1033, the levorotatory and dextrorotatory enantiomers of PCR 3549 respectively, designated as PCR 3620 and PCR 3621, had equal degrees of antiplatelet activity.25 This lack of an improvement over the racemate, combined with the racemate’s toxicity, led Sanofi to abandon further development of PCR 3549. .The Plavix Precursor: PCR 4099 and the ‘596 Patent Having exhausted the potential therapeutic application of methyl and ethyl analogs of ticlopidine, Sanofi began a more serious investigation of the other ticlopidine derivatives. Approximately 70 carboxylic acid, ester and amide analogs of ticlopidine, all in their racemic forms, were synthesized by Sanofi in the late 1970s and early 1980s.26 Some of the pharmacologic screening tests for antiplatelet activity, “designed to quickly screen a large number of candidate compounds in order to identify a manageable number of compounds [for] more sophisticated testing,” included the bleeding-time test, the silk-thread test, the

Maffrand T Tr. 1606; Badorc T Tr. 1813-14. Badorc T Tr. 1824, 1826; Maffrand T Tr. 1615-17. 26 Maffrand T Tr. Maffrand 1636-37, 1642. 24 25

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents collagen test and the ADP test.27 A few of these thienopyridine derivatives deserve special mention. The synthesis of carboxylic acid derivatives did not yield any compounds with antiplatelet activity. An ethyl ester analog of ticlopidine synthesized in 1980, however, did exhibit some antiplatelet aggregation activity during initial tests. The scientists prepared this compound, PCR 3935, as a hydrobromide salt after the failure to form a hydrochloride or bisulfate salt.28 However, Sanofi chemists regarded PCR 3935 with some suspicion, worrying that the ethyl ester substituent (O=C-O-CH2-CH3) made the compound susceptible to in vivo conversion to carboxylic acid.29 They alleviated this concern by synthesizing a similar analog – a thienopyridine with a methyl ester (O-C-O-CH3) substituent – in July 1980.30 While Sanofi was certainly not aware of it at the time, this chiral analog of ticlopidine, PCR 4099, was the precursor for the blockbuster drug Plavix. Maffrand and his colleagues prepared the racemic mixture of PCR 4099 as a hydrochloride salt and assayed for antiplatelet aggregation activity and toxicity. PCR 4099 performed well in these initial tests – not only was PCR 4099 a more potent inhibitor of platelet aggregation than ticlopidine, but PCR 4099 was also better tolerated.31 Sanofi began preparations to apply for French and U.S. patents for the class of thienopyridines modified with carboxylic acid, amide and ester substituents. Dr. Maffrand wrote an internal Sanofi memorandum, dated February 11, 1982, announcing that the patent

Maffrand T Tr. 1641; Testimony of Stephen R. Hanson, Trial Transcript 2226-7, 2232-33, 2238-39, 2243, 2246-67 (hereafter denoted as “Hanson T Tr.”) as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 28 Maffrand T Tr. 1640. 29 Maffrand T Tr. 1638-40. 30 Maffrand T Tr. 1638-40. 31 Maffrand T Tr. 1641. 27

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents would cover “PCR 4099 and its analogues.” 32 The note also mentioned PCR 4099 as one of the four “most interesting esters” based on the results of the pharmacological screening tests. In July 1983, Sanofi filed its application for a U.S. patent describing a genus of thienopyridine compounds that demonstrate antithrombotic and antiplatelet activity; the patent, “Thieno [3,2-c] Pyridine Derivatives and Their Therapeutic Application”, was issued on July 16, 1985 with an expiration date in July 2003 (Aubert, 1985). Maffrand, Aubert and Ferrand were credited as inventors. The U.S. patent 4,529,596, hereafter denoted “the ‘596 patent”, specified a general formula for the free base of thienopyridine derivatives. ‘596 made the additional claims that “[t]he invention relates both to each enantiomer and their mixture” and that the compounds described by the general formula could be treated with pharmaceutically acceptable acids or a mineral base to afford addition salts (Aubert, 1985). The patent also described twenty-one examples of such compounds, “to exemplify and to illustrate the different substituents which were claimed in the generic formula,”33 and reported the results of the aforementioned assays on pharmacological activity (Aubert, 1985). .Toxic Convulsions & the Decision to Resolve the Enantiomers of PCR 4099 The pharmacological activity data presented in the ‘596 patent showed that several of the other compounds performed as well as or better than PCR 4099 in various screening tests. However, the tests were a crude benchmark, primarily to measure the presence antiplatelet activity, and did not provide enough information to compare the relative potencies of the

Defendant’s Exhibition 632, as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007) (No. 02 CIV. 2255 (SHS)). 33 Maffrand PI Tr. 186. 32

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents compounds.34 Sanofi elected to pursue development of PCR 4099 along with some of the other compounds. The next stage, involving large-scale clinical testing of safety and efficacy, ultimately spanned seven years from 1980 to 1987 and cost “tens of millions of dollars.”35 The investigation on PCR 4099 encompassed over fifty separate tests on humans and animals in addition to a battery of metabolic, pharmacological, toxicological and pharmacokinetic studies. By 1985, Dr. Maffrand became aware of a pattern of troubling results from the toxicity studies conducted in rats, mice and baboons with PCR 4099.36 In every acute toxicity, dose-range finding and chronic oral toxicity study performed with PCR 4099, the scientists observed convulsions in the animals at certain dose ranges.37 A month-long oral toxicity study in baboons, for instance, reported that “[d]eath was preceded by convulsions and was most likely not accidental.”38 Further chronic toxicity studies determined the unquestionable link between PCR 4099 and convulsions. In 1987 a one-year study in baboons observed convulsions even at the lowest tested dose of PCR 4099, 25 mg per kg of body weight, and also found that the rate of convulsions was proportional to dosage.39 Sanofi was wary of having a second drug launch immediately followed by the revelation of rare but potentially lethal side effects – as had occurred with the launch of

See Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). Maffrand PI Tr. 143-144. 36 Maffrand T Tr. 1651. 37 Plaintiff’s Exhibits 114, 115, 116, 117, as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 38 Plaintiff’s Exhibit 113, as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 39 Testimony of Dr. Joseph V. Rodricks, Trial Transcript 2481-82 as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 34 35

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents ticlopidine in France.40 Thus, despite proceeding with the toxicity studies, Sanofi found these initial results worrisome enough that in November 1985 Dr. Maffrand made the decision to separate the enantiomers of PCR 4099 and test their individual therapeutic activity and toxicity.41 .Resolving the Racemate: Pharmacologic Implications Alain Badorc and Dr. Daniel Fréhel coordinated the effort to obtain pure samples of each enantiomer in the racemate mixture of PCR 4099. Badorc, the same scientist who had worked on PCR 1033 and PCR 3549, considered the methods used to successfully separate the enantiomers of those thienopyridine derivatives. Recognizing that PCR 4099 was “only barely basic and, therefore, only barely capable of carrying out a separation using diastereomeric salts,”42 Badorc decided against trying diastereomeric salt formation to resolve the racemate. There were more obstacles to obtaining the individual enantiomers of PCR 4099 than there had been with the prior thienopyridine derivatives. Figure 7. Chemical Structure of Clopidogrel. ((Pereillo, 2002)).

The methyl ester of PCR 4099 was reactive and rendered the molecule susceptible to racemization through various methods.43 Phenylglycine derivatives – which include the PCR 4099 compound – are generally susceptible to racemization. The presence of an ester molecule also often results in

Maffrand PI Tr. 144; Maffrand T Tr. 1651. Maffrand T Tr. 1651. 42 Badorc T Tr. 1829-30 (explaining that the weakness of PCR 4099 as a base would thus appear to rule out all but the strongest acids, few of which were commercially available, and it would still be unclear whether enough crystals would form to successfully resolve the compound); Also see Stephen M. Berge, et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 1 (1977) (reviewing the various considerations in choosing an acid. Diastereomer salt formation generally requires pairing weak bases with strong acids (low pKa), and strong bases with weaker acids (higher pKa)). 43 Badorc T Tr. 1832-33. 40 41

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents racemization. The ester group makes the chiral carbon atom susceptible to deprotonation via either an acid or a base; the subsequent protonation of the carbon can occur on either side, thereby racemizing the solution.44 According to his lab records, Badorc elected to attempt chemical asymmetric synthesis to engineer each enantiomer separately, but none of his synthetic reactions could produce a pure enantiomer. He began with the same reaction sequence that had successfully produced the enantiomers of PCR 3549, but the product racemized during the second step of the sequence.45 Badorc then attempted to apply diastereomeric salt formation to resolve an intermediate compound of PCR 4099, but could not convert the resolved enantiomers into PCR 40099, nor could he form diastereomeric salts with an acid precursor of PCR 4099. Finally, Badorc returned to the original method of indirect resolution via diastereomeric salt formation. Out of the thirty test tubes he prepared, each with varying chiral acids in various concentrations in various solvents, the only test tube that produced a precipitate â&#x20AC;&#x201C; albeit gummy and not crystalline â&#x20AC;&#x201C; was the tube containing camphorsulfonic acid and PCR 4099 in a 1:1 ratio in acetone.46 It took a further two months before Badorc finally calibrated the right concentration ratios of camphorsulfonic acid to produce crystals.47 Even then, much coaxing and crystal seeding was needed to produce enough crystals to resolve first the levorotatory enantiomer of PCR 4099 in the camphorsulfonate form, and then the dextrorotatory enantiomer, via the same procedure with the camphorsulfonate acid

Badorc T Tr. 1832; Davies T Tr. 1956-60, 1958-61, 2002-04. Badorc T Tr. 1836-37. 46 Badorc 1842-44. 47 Badorc T Tr. 1848-49. 44 45

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents of opposite chirality. 48 From that point, a simple reaction with a base (sodium bicarbonate, in this case) would release the free base of each enantiomer.49

Figure 8. The Original Sanofi Preparation of (+)-Clopidogrel. The method finally devised by Badorc to resolve the racemate PCR 4099 ((±)2 ) utilized chirally pure camphor-10-sulfonic acid in the acetone to create a diastereoisomer salt (19), which could then be recrystallized from the acetone to form clopidogrel ((+)2). Though not depicted, subsequent addition of the base sodium bicarbonate would react with this salt to form the free base of the R enantiomer ((Badorc, 1988; Li, 2004)).

.Bisulfate, an Unexpectedly-Acceptable Pharmaceutical Salt The chemical experimentation did not end with obtaining pure samples of the free base of each enantiomer. Badorc’s next challenge was to prepare each enantiomer as a pharmaceutically-acceptable salt so that they could undergo clinical testing. A thencontemporary review on pharmaceutical salts noted that besides the fundamental considerations – “cost of raw materials, ease of crystallization, and percent yield” – that often guide a chemist’s “empirical” choice of salt, the “the stability, hygroscopicity and flowability” of the resulting solid compound should also factor into the selection process (Berge, 1977).

48 49

Badorc T Tr. 1850-51. Badorc T Tr. 1952.

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents Based on Badorc’s previous experience with thienopyridines and the need to compare the pharmacological activity of each enantiomer with that of the racemate, he first chose to prepare the hydrochloride salt of each enantiomer of PCR 4099.50 Initial testing yielded a promising outcome: the d-enantiomer of PCR 4099 showed both more platelet inhibition and less toxicity than the l-enantiomer. They wanted to pursue further studies, but it was apparent that hydrochloric acid would not allow Sanofi to prepare large quantities of PCR 4099 in a stable tablet form – the resulting hydrochloride salt compounds were hygroscopic and unstable.51 The enantiomers obviously needed to be generated with a different acid, but there were many choices to choose from. One reference from the time listed 53 pharmaceutically-acceptable acids that were approved by the FDA as of 1974, and another 27 acids that had been used in pharmaceutical formulations in other countries (Berge, 1977). Again, Badorc did a screening test, with over twenty different acids in various solvents. In one case, the use of sulfuric acid – Badorc was intending to prepare the sulfate salt of clopidogrel – generated the bisulfate salt instead.52 The optimal pharmacologic properties of the bisulfate salt – stability, non-hygroscopicity, a high melting point and good solubility – were surprising.53 Prior art references of the time would not have matched sulfuric acid with clopidogrel because of the acid’s polarity; furthermore, bisulfate did not even show up on the FDA-approved list of pharmaceutical salts (Berge, 1977; Gould, 1986).

Ticlopidine was produced as ticlopidine hydrochloride, and the enantiomers of PCR 1033 had also been prepared as hydrochloride salts; Badorc T Tr. 1852-3. 51 Badorc T Tr. 1853; Testimony of Dr. Gilbert Banker, Trial Transcript 1338 (hereafter denoted as “Banker T Tr.”), as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 52 Badorc T Tr. 1797-98. 53 Badorc T Tr. 1855; Banker T Tr. 1357. 50

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents .One Active Enantiomer: Absolute Stereoselectivity of the ADP Receptor The finding that only one enantiomer was necessary to inhibit platelet aggregation was significant, as it established the absolute stereoselectivity of the ADP platelet receptor.54 The toxicological testing that accompanied the assays of antiplatelet activity compounded the positive news: the acute toxicity of clopidogrel was much lower than both the PCR 4099 racemate and the levorotary enantiomer.55 No convulsions were observed in animals that had been administered clopidogrel, whereas treatment with either PCR 4099 or the levorotary enantiomer produced similar incidences of convulsion.56 Despite clopidogrel’s clear advantage over PCR 4099, the decision to abandon Sanofi’s investment in testing and developing PCR 4099 in favor of the one active enantiomer was extremely difficult. Maffrand was hesitant to lose all the data from the testing on PCR 4099, which would have to be repeated anew on the preparation of one enamtiomer. Furthermore, Maffrand was not convinced that the inactive l-enantiomer was what “is causing the problems with PCR 4099 we are encountering today.”57 Pierre Simon, who then headed research and development at Sanofi, made the final decision on April 16, 1987 to stop work on PCR 4099 and turn all efforts to developing clopidogrel.58 The dual discovery that (+)-clopidogrel was entirely responsible for the antiplatelet affect of PCR 4099 and further more that (-)-clopidogrel was toxic gave Sanofi good reason to pursue a

Davies T Tr. 2014-15, 2018-19; Hanson T Tr. 2214, 2216, 2250-51; Harden T Tr. 2338. Maffrand T Tr. 1688-89; Testimony of Dr. Frédéric Lacheretz, Trial Transcript 2379 (hereafter denoted as “Lancheretz T Tr.”) as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 56 Lacheretz T Tr. 2377. 57 Maffrand PI Tr. 154; Defendant’s Exhibit 622, as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 58 Plaintiff’s Exhibit 57t, as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007). 54 55

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents new patent (Bouisset, 1991). Sanofi filed an application on February 12, 1988 for a patent (U.S. Patent 4,487,265) on clopidogrel bisulfate, concluding that the active ingredient was the bisulfate salt of the dextrorotatory enantiomer of MATTPCA (Badorc, 1988).

Figure 9. Comparison of Ticlopidine with Clopidogrel. The salt form of ticlopidine is formed with hydrochloric acid, whereas clopidogrel is prepared as a bisulfate salt ((Li, 2004)). Figure 10. Chemical Evolution of the Thienopyridines. Clopidogrel (2) is the (+)-enantiomer of the free base of the ticlopidine thienopyridine (1) with a methyl ester (O=C-OCH3) substituent on the bridge carbon ((Li, 2004)).

The patent examiner for ‘265 turned out to be the same individual – Bernard L. Denz – who had reviewed Sanofi’s ‘596 patent five years earlier.59 Denz initially rejected the application, criticizing that the claims within ‘265 appeared to be anticipated by the ‘596 patent, but offered Sanofi advice. Sanofi

Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007).

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents .Selective Synthesis of (+)-Clopidogrel

Figure 11. Sanofi and the Asymmetric Synthesis of (+)-Clopidogrel. Despite Badorc’s early attempts in the 1980s to resolve the PCR 4099 enantiomers via asymmetric synthesis, Sanofi did not develop successful methods to synthesize clopidogrel until the late 1990s. Figure A. The starting material for one such synthesis is a mandelic acid derivative, the chirally pure (R) form of (2-chloro-phenyl)-hydroxy-acetic acid (20), which is initially reacted to afford a methyl ester (21). The methyl ester is tosylated using toluene sulfonyl chloride to produce a toluenesulfonate ester (22). Thieno[3,2-c]pyridine (8) can then perform a nucleophilic SN2 attack to displace the tosylate group, thereby forming (+)-clopidogrel (2). Figure B. Another Sanofi process for synthesizing optically pure (+)-clopidogrel takes advantage of the chirality of an α-amino acid (phenylalanine with a chlorine substituent on the phenyl ring). The racemic α-amino acid (23) is resolved to isolate the (+)enantiomer ((S)-23). The addition of thionyl chloride and methanol generates the methyl ester (24), and treatment with (2-thienyl)-ethyl para-toluene-sulfonate (25) results in a SN2 displacement reaction to afford an amine (26). In the last step of the sequence, (26) is refluxed with paraformaldehyde in formic acid to encourage ring closure and produce (+)-clopidogrel (2) ((Bousquet, 1999; Li, 2004; Descamps, 1991)).

Pre-Clinical Development of Plavix: Thienopyridine Derivatives, Enantiomer Separation and the Sanofi Patents reapplied for the patent on January 9, 1989, having amended the claims to distinguish the limitations of the two patents and to also clarify the â&#x20AC;&#x153;markedâ&#x20AC;? difference in toxicity between the two enantiomers (Badorc, 1988). The patent was successfully issued to Sanofi on July 11, 1989 (Badorc, 1988). However, it took a significant period of research to develop a viable synthesis to selectively engineer the proper enantiomer (Defreyn, 1991; Descamps, 1991). The biologically active metabolite of the d-enantiomer selectively targeted the ADP receptor to inhibit platelet aggregation, and furthermore, had low toxicity; the l-enantiomer was both ineffective and toxic. For Plavix, a substantially pure, selective synthesis of the d-enantiomer was very important (Li, 2004). Subsequent research has produced new methods for the selective synthesis of the therapeutic clopidogrel enantiomer, and established new chemical processes for racemization and the conversion of one enantiomer to another (Castaldi, 2004; Valeriano, 2003). In the meantime, clopidogrel bisulfate entered clinical trials to test its efficacy at reducing adverse vascular outcomes in a variety of clinical settings.

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents

Chapter 5 - Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents .Clinical Indications of Plavix There is a wide range of clinical uses for clopidogrel and other antiplatelet agents in treating cardiovascular disease symptoms. Plavix is indicated for reducing thrombotic events in two main categories of cardiac conditions. In patients with a history of recent myocardial infarction (MI), recent stroke, or established peripheral artery disease, long-term use of Plavix helps prevent secondary vascular events. For patients with acute coronary syndrome (e.g. UA/NSTEMI), including the population undergoing percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), Plavix reduces the rate of endpoints of cardiovascular death, MI, stroke or refractory ischemia. Plavix is regarded as a basic component of secondary preventative care for patients hospitalized with NSTEMI/UA, MI and strokes, and is also a standard drug administered during PCI and cardiac catheterizations (Rosamond, 2007). .Prevalence of Prescribing Plavix The AWA/American Stroke Association’s “Get With the Guidelines – Stroke Program” catalogued the rates of antithrombotic prescription (Rosamond, 2007). In the first 48 hours upon presentation at the hospital with ACS, 93.9% of patients initiated an antithrombotic drug regimen, while 97.3% of patients were prescribed an antithrombotic at discharge. The ACS Registry Data found that within the set of acute medications administered within 24 hours of hospitalization, aspirin is most prevalent regimen, reaching 96% of inpatients, while 91% are put on β-blockers, 87% on heparin and 47% on glycoprotein IIb/IIIa inhibitors (Rosamond, 2007). Clopidogrel is more commonly prescribed at discharge than during 57

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents hospital stay. The same study noted that 74% of patients hospitalized with UA/NSTEMI are discharged with clopidogrel, whereas 95% are discharged with aspirin, 93% with Î˛-blockers, 63% with ACE inhibitors and 84% with cholesterol-lowering agents such as Lipitor (Rosamond, 2007).

.Plavix More Favorable th an Ticlopidine for Sec ondary Prevention .CAPRIE: Clopidogrel Comparable to Aspirin and Safer than Ticlopidine The CAPRIE trial60 was the largest clinical trial conducted on Plavix prior to FDA approval. CAPRIE was a major, randomized, blind clinical trial designed to assess the relative efficacy and safety of clopidogrel versus aspirin in preventing major vascular events in 19,185 patients with atherosclerotic vascular disease. The patients from the clinical subgroups of ischemic, cerebrovascular, cardiac and peripheral arterial disease were subjected to the same protocol, and either administered a long-term regimen of clopidogrel (75 mg once daily) or aspirin (325 mg once daily) (Gent, 1996). CAPRIE demonstrated that clopidogrel was super to aspirin at protecting these patients from ischemic stroke, myocardial infarction and death (New York Times, 1996; Gent, 1996). The relative risk reduction in the composite of the three primary adverse outcomes was 22.7% better with clopidogrel than with aspirin, though comparisons of outcomes in other clinical subgroups showed a smaller difference in benefit from clopidogrel (Gent, 1996). Overall, clopidogrel reduced the risk of adverse events by an additional 8.7% beyond the established 25% reduction attributed to aspirin (Gent, 1996). The steering committee noted that this was consistent with previous findings on ticlopidine, showing that thienopyridines have greater therapeutic value than aspirin to treat atherothrombosis (Gent, 1996). CAPRIE also established that Plavix has a relatively

Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents favorable tolerability profile; clopidogrel was at least as safe as medium-dose aspirin and safer than ticlopidine (Gent, 1996). Since CAPRIE, clopidogrel has been evaluated in several other large, randomized, placebo-controlled and double-blind, multi-center clinical trials for its benefits and tolerability in various settings. .CURE Trial for Dual Therapy Treatment of UA/NSTEMI The CURE trial61 investigators sought to establish a role for clopidogrel in addition to aspirin in the long-term prevention of secondary vascular episodes in patients with ACS without ST-segment elevation, who comprise a high risk group for major adverse events (The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001). The study compared primary outcomes and side effect outcomes in 12,562 patients, finding that the early and long-term use of clopidogrel with aspirin for up to a year was associated with a significant reduction in adverse cardiovascular events compared with that of aspirin alone (The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001). In evaluating the trial results, the investigators highlighted clopidogrelâ&#x20AC;&#x2122;s role in preventing ischemic coronary events such as MI, refractory ischemia, and ischemic strokes (The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001). Despite the increase in both minor and major bleeding episodes, clopidogrel did not increase the rate of hemorrhagic stroke or bleeding severe enough to require surgical intervention, and investigators equated the bleeding risk of clopidogrel with that of aspirin. CURE established that the combination therapy of clopidogrel and aspirin was as well-tolerated as aspirin taken alone (The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001).

Clopidogrel in Unstable Angina to Prevent Recurrent Events

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents .COMMIT and CLARITY-TIMI for Short-Term Treatment of STEMI In COMMIT62, patients presenting within 24 hours of onset of STEMI were randomized into a four-week course of either clopidogrel with aspirin or aspirin alone; the patients receiving the dual therapy had markedly lower death rates and a lower incidence of composite adverse cardiovascular outcomes (Chen, 2005). Figure 12. Risk Reduction Afforded by Clopidogrel and Aspirin. This figure shows the relative reductions in the risk of significant vascular events afforded by treatment with a single antiplatelet drug and the additional, incremental reduction from dual therapy treatment. (Data from the following metaanalyses of trials and individual, randomized clinical trials: ATT Antithrombotic Trialists' Collaboration; CHARISMA - Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance; COMMIT - Clopidogrel and Metoprolol in Myocardial Infarction Trial; CURE - Clopidogrel in Unstable Angina to Prevent Recurrent Events; and ISIS-2 - Second International Study of Infarct Survival (ISIS-2 (Second International Study Of Infarct Survival) Collaborative Group, 1988; Antithrombotic Trialistsâ&#x20AC;&#x2122; Collaboration, 2002; Bhatt, 2006; Chen, 2005; Collins, 1994a; Kapoor, 2008; Yusuf, 2001)).

In contrast to COMMIT, the CLARITY-TIMI 28 trial63 evaluated the role of clopidogrel in reducing adverse outcomes in patients presenting within 12 hours of STEMI who required coronary angiography (Sabatine, 2005b). CLARITY-TIMI 28 found that the patients treated with a loading dose and short week-long course of clopidogrel in addition to the standard therapy (e.g. aspirin, thrombolytic, and possibly heparin) had a significantly reduced rate of

62 63

Clopidogrel and Metorolol in Myocardial Infarction Trial Clopidogrel as Adjunctive Reperfusion Therapy â&#x20AC;&#x201C; Thrombolysis in Myocardial Infarction

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents occluded infarct-related artery, recurrent MI and death in comparison to patients treated with standard therapy alone (Sabatine, 2005a).

.Clinical Benefits of Plavix Treatment During and After Coron ary Stenting Procedures .CLASSICS: Clopidogrel Has a Favorable Tolerability Profile The CLASSICS trial64 primarily investigated the tolerability of clopidogrel in comparison to ticlopidine (Bertrand, 2000). CLASSICS was a two-part phase III Japanese study of a short course of clopidogrel and aspirin therapy in comparison with a short course of ticlopidine and aspirin therapy in patients undergoing intercoronary stenting (Bertrand, 2000). Within the former group, CLASSICS also investigated the benefit of a loading dose of clopidogrel. The results of the study were published in 2001, and corroborated the conclusions reached in CAPRIE. CLASSICS found clopidogrel to have a more favorable tolerability profile than ticlopidine, producing a lower incidence of major bleeding, neutropenia and other primary outcomes in addition to reducing the rate of minor adverse events such as blood abnormalities found in laboratory work-ups (Bertrand, 2000). .PCI-CURE: A Role for Clopidogrel During Stenting Studies conducting PCI with drug-eluting stents (as opposed to vein grafts) in addition to a CABG prescribed Plavix and found better patient outcomes than when just a CABG with completion angiography was performed. PCI-CURE was a study evaluating outcomes in the sub-group of PCI patients from the CURE trial, who were either treated with a long-term course of clopidogrel in addition to aspirin, or with aspirin alone, or with a short course of clopidogrel after PCI (Mehta, 2001). 2172 out of the 2658 patients who underwent PCI

Clopidogrel Aspirin Stent International Cooperative Study

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents received stents, and the clopidogrel recipients suffered a markedly lower rate of adverse cardiovascular events during and 8 months after PCI than did placebo recipients (Mehta, 2001). .PRONTO: A Loading Dose Before Coronary Stenting Use of thienopyridine therapy in the clinical setting of coronary artery stenting required separate clinical investigation. After CAPRIE showed that the second-generation thienopyridine had a much lower side effect profile than its predecessor ticlopidine, clopidogrel replaced ticlopidine as the drug of choice for preventing thrombosis after coronary stenting (Gent, 1996; Gurbel, 1999). However it was not clear whether either thienopyridine had a clear advantage during stenting procedures. Later clinical trials such as the PRONTO trial65 helped resolve the proper role of clopidogrel in coronary stenting (Gurbel, 2003a; Gurbel, 2003b). PRONTO demonstrated that a loading dose of 300 mg clopidogrel prior to the stenting procedure was more effective at reducing platelet activation than the administration of 75 mg clopidogrel at the time of the procedure (Gurbel, 2003b). The PRONTO trial, in conjunction with other studies, illuminated the onset and time course of platelet inhibition by clopidogrel. A single loading dose of 400 mg clopidogrel showed maximum inhibition within 2 to 5 hours, whereas 3 to 7 days of repeated doses of 75 mg clopidogrel were required to achieve the same level of inhibition (Gurbel, 2003b; Savcic, 1999).

Plavix Reduction of New Thrombus Occurrence

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents PRONTO helped establish the use of a loading dose prior to PCI, which was also investigated in CREDO66 â&#x20AC;&#x201C; the only large trial to evaluate the use of clopidogrel in treating patients in elective PCI (Steinhubl, 2002). CREDO compared the utility of a short onemonth course of dual therapy (clopidogrel with aspirin) followed by a long-term regimen of aspirin only (through the remainder of the year) with the outcomes in patients who were administered early and long-term clopidogrel with aspirin for one year after PCI (Steinhubl, 2002). The latter group experienced significantly lower rates of the composite end-points (stroke, MI and death) than patients in the group treated with only the short-term course of clopidogrel (Steinhubl, 2002). CREDO established the early and long-term use of clopidogrel to reduce adverse incidents in patients undergoing elective PCI.

.Plavix Not Indicate d For Primary Preven tion Plavix has shown fewer benefits in clinical studies investigating the potential role of clopidogrel treatment in high-risk patients with transient ischemic attack (TIA) or recent ischemic stroke, or in the primary prevention of adverse vascular events in patients with multiple risk factors or clinically evident CVD. .MATCH: Increases Bleeding Without Conferring Benefits in High-Risk

Ischemia MATCH trial67 investigated the use of clopidogrel in addition to standard long-term use of aspirin in high-risk patients with recent ischemic stroke or TIA (Diener, 2004). At the 18month follow up, MATCH found that the combined use of clopidogrel with aspirin not only resulted in a non-significant reduction in the composite primary outcomes (ischemic stroke,

66 67

Clopidogrel for Reduction of Events During Observation Management of Atherothrombosis with Clopidogrel in High-Risk Patients

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents MI, death and rehospitalization for acute ischemia), but also produced a significant increase in major and potentially fatal bleeding events (Diener, 2004). These results established that Plavix does not have a role in preventing ischemic vascular events or mortality, despite data from the earlier CURE trial, which suggested that clopidogrel did confer benefits in reducing adverse ischemic outcomes (Diener, 2004; The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001). .CHARISMA: No Role in Primary CVD Prevention CHARISMA indicated that Plavix should not be administered for primary prevention of cardiovascular disease. The CHARISMA trial,68 for patients at high risk of atherothrombotic events, showed that patients randomized to receive clopidogrel plus aspirin did not show a significant benefit over patients receiving aspirin alone (Bhatt, 2006). Critically, the subgroup of dual-therapy patients without documented cardiovascular disease had a significant increase in mortality as compared to the group treated with just aspirin (Bhatt, 2006). Overall, the dual-therapy patients had a nonsignificant reduction in adverse vascular events; CHARISMA concluded that clopidogrel plus aspirin therapy should not be used for primary prevention of CVD (Bhatt, 2006).

.Clopidog rel Metabolism an d Resis tance .In Vivo Metabolism of Clopidogrel The limiting factor to clopidogrelâ&#x20AC;&#x2122;s ability to abrogate the P2Y12 receptor has been attributed to the bioavailability of its active metabolite. Clopidogrel is a prodrug, a pharmaceutical substance that is inactive when administered and must be metabolized in vivo into its active

Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents metabolite. The inability of clopidogrel bisulfate to inhibit platelet activity in vitro was observed early during the pre-clinical development period at Sanofi. For some time, it was hypothesized that plasma components metabolized the drug, but neither the molecular structure nor the mechanism of metabolic transformation were understood. Eventually Savi et al. established that the activation steps occurred in the liver (Savi, 1992). Figure 13. Clopidogrel Metabolism In Vivo. Clopidogrel (2) is taken orally, and undergoes hepatic metabolism via the cytochrome P450 system. The multiple-step metabolic pathways for clopidogrel and for ticlopidine are identical; in contrast, the thirdgeneration thienopyridine prasugrel requires only one transformation by the cytochrome P450 system. The first 450 monooxygenasedependent metabolic step produces the intermediate form 2-oxo-clopidogrel (4), which is still inactive in vitro, and requires a second transformation, a hydrolysis, to produce the active metabolite (5) ((Li, 2004)).

The full reaction sequence from clopidogrel to the active metabolite was finally characterized in 1994 by Savi et al., and further elucidated as investigators explored the differential effects of the clopidogrel enantiomers. Only about 50% of ingested clopidogrel is absorbed through the gastrointestinal tract, but the circulating drug is rapidly and extensively biotransformed into its active metabolite in the liver (Caplain, 1999). Clopidogrel activation is regulated by the hepatic cytochrome P450-1A subfamily (Savi, 1994). The initial reaction preserves the chiral configuration of clopidogrel and produces the intermediate 2-oxo-clopidogrel; subsequent hydrolysis generates the active metabolite. The active metaboliteâ&#x20AC;&#x2122;s high instability contributed to the difficulty of the attempt to isolate and fully characterize the structure and stereochemistry of the compound (Pereillo,

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents 2002; Savi, 2000). The structure of active metabolite was finally determined in 2002 by Sanofi researchers, who incubated the 2-oxo-clopidogrel with human liver microsomes in vitro (Pereillo, 2002). The active metabolite is one of eight stereoisomers, but is the only isomer demonstrating biological activity, which suggests the absolute configuration necessary for interaction with the P2Y12 receptor (Pereillo, 2002; Savi, 2000). Elucidating the stereochemical configurations at certain chiral atoms helped ascertain the absolute chemical configuration necessary to produce antiplatelet aggregation activity: the active metabolite retained clopidogrelâ&#x20AC;&#x2122;s absolute S configuration at carbon 7, and had Z configuration at the carbon 3 â&#x20AC;&#x201C; carbon 16 double bond (Pereillo, 2002). The active metabolite structure is consistent with previous studies that suggested the presence of the thiol and carboxylic acid groups. The thiol group, in fact, is what reacts with a putative cysteine residue on platelet P2Y12 receptors to effect platelet abrogation (Savi, 2001). Figure 14. Purported Chemical Structure of the Active Metabolite of Clopidogrel. The absolute configuration of the active metabolite is essential for its interaction with the P2Y12 receptor to abrogate platelet aggregation. The structure is consistent with prior studies showing functions for a carboxylic acid and thiol group. The active metabolite retains S configuration at carbon 7 (from its precursor, the prodrug clopidogrel) but has Z conformation at the carbon 3 â&#x20AC;&#x201C; carbon 16 double bond ((Pereillo, 2002)).

Figure 15. Mechanism of Action of the Metabolite. The highly labile, active metabolite of clopidogrel is thioreactive, and irreversibly reacts with a thiol group of a cysteine amino acid on the P2Y12 receptor((Savi, 2001)).

.Hepatic Function and

Clopidogrel Metabolism The active clopidogrel metabolite undergoes rapid, 66

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents subsequent biotransformations to an inactive acid metabolite, SR 26344, and the liver is responsible for eliminating this and other inactivated metabolites of clopidogrel (Slugg, 2000). Cirrhotic liver disease has variable and unpredictable effects on the pharmacodynamics and pharmacokinetics of drugs, especially those that are highly proteinbound or eliminated in the bile or via oxidative pathways in the liver (Morgan, 1995). The importance of the hepatic enzyme metabolism is underscored by studies at the turn of the century exploring the effect of liver function on the pharmacokinetics and pharmacodynamics of clopidogrel (Slugg, 2000). Cirrhosis and other liver conditions slightly impairing hepatic function did not appear to affect the pharmacokinetic properties or antiplatelet activity of clopidogrel (Slugg, 2000). The study laid emphasis on the risk of clopidogrel treatment in patients with severe hepatic impairment, who are prone to have severe bleeding diatheses (Slugg, 2000). .Genetic Polymorphisms and Variable Clopidogrel Resistance The clinical success of Plavix has illuminated subtleties in the interaction of the active metabolite with the P2Y12 receptor to inhibit platelet aggregation. Its clinical failure has prompted an even larger research response. Clinical studies on clopidogrel resistance in patients with cardiovascular disease have revealed how both the ADP-receptor-mediated pathway and clopidogrelâ&#x20AC;&#x2122;s binding activity are complicated by genetic variation between individuals. Initial experiments on individuals treated with similar loading dosages of clopidogrel suggested that genetic factors could account for the varying degrees of platelet inhibition they exhibited (JĂ¤remo, 2002). Researchers identified P2Y12 gene sequence variations among individuals that prevent ADP-induced platelet aggregation in healthy subjects (Fontana, 2003), and 67

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents showed how the more severe phenotype of defective platelet function could result from congenitally defective P2Y receptors (Cattaneo, 2005). Various polymorphisms in the gene for the P2Y12 receptor have also been investigated as a possible source of observed resistance to clopidogrel treatment. Investigators of one such study did not observe any direct association between the single P2Y12 gene mutation and responsiveness to antiplatelet treatment, which suggests that the interindividual variability in resistance to clopidogrel and aspirin is multi-factoral and not due to a single gene mutation (Lev, 2007). In contrast, however, interindividual variability of platelet inhibition after clopidogrel administration does correlate with interindividual differences in CYP 34A metabolic activity (Lau, 2004b). Multiple studies verified that certain polymorphisms in the gene for the CYP 34A enzyme contribute to differences in metabolizing clopidogrel (Angiolillo, 2006; Lau, 2004b; Suh, 2006). The link between gene sequence variation and variability in individual responsiveness to clopidogrel has had important ramifications in the clinical setting, because the presence of a drug-resistant subpopulation of patients (due to altered cytochrome metabolism or other factors) has been correlated with an increased risk of recurrent cardiovascular events (Fefer, 2007; Matetzky, 2004). .Cytochrome-Dependent Drug Interactions A subtler phenomenon related to the in vivo metabolism of clopidogrel was the suggestion in early studies of a drug-drug interaction between clopidogrel and the statin drug atorvastatin (a substrate for CYP34A). The pharmacologic administration of lipophilic statins, which are also metabolized through CYP 3A4, appeared to attenuate clopidogrelâ&#x20AC;&#x2122;s antiplatelet activity by competitively inhibiting the activation of clopidogrel (Lau, 2003). This generated great debate in the medical literature since a large segment of patients with CVD may be treated 68

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents with both clopidogrel and a statin such as atorvastatin (Lau, 2004a; Steinhubl, 2006; Tafreshi, 2006). However, additional clinical studies clarified that despite the ex vivo interaction, concurrent treatment with atorvastatin does not reduce clopidogrelâ&#x20AC;&#x2122;s ability to abrogate platelet aggregation and the resulting therapeutic benefit from antiplatelet therapy (Saw, 2003; Saw, 2007; Vinholt, 2005). Atorvastatin and pravastatin also did not affect platelet inhibition by aspirin or clopidogrel treatment in patients with coronary stent thrombosis (Wenaweser, 2007). When atorvastatin was administered concomitantly with clopidogrel for five weeks in patients diagnosed with ACS, atorvastatin did not diminish clopidogrel potency, nor did clopidogrel affect atorvastatinâ&#x20AC;&#x2122;s therapeutic efficacy (Mitsios, 2004; Mitsios, 2005). The matter of the allegedly interfering interaction of statin-clopidogrel has largely been debuked and does not appear to necessitate further consideration in clinical studies. .Dual Resistance to Aspirin and Clopidogrel More recently, Lev et al. characterized the role of dual drug resistance in a study tabulating aspirin and clopidogrel resistance in patients undergoing PCI (Lev, 2006). Aspirin-resistant patients, as a group, were more likely to be resistant to clopidogrel; resistance to both approved long-term antithrombotic therapies highlights their unique risk of incurring thrombotic complications after PCI (Lev, 2006; Lev, 2007). This finding led the investigators to urge point-of-care to prevent adverse thrombotic outcomes in the subpopulation of patients resistant to these two standard oral antiplatelet therapies (Lau, 2004). Lau et al. proposed a temporary solution, to increase platelet inhibition in patients resistant to clopidogrel by administering rifaplin (a CYP 34A activator) in addition to clopidogrel (Lau, 2004b). Nonetheless, studies similar to those linking clopidogrel resistance and recurrent cardiovascular events have established a similar correlation between resistance to dual

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents antiplatelet therapy and poor outcomes. Cuisset et al. demonstrated that a population of patients with ACS who were low responders to dual antiplatelet therapy had a greater incidence of recurrent adverse cardiovascular outcomes after stenting (Cuisset, 2006; Cuisset, 2007). The discovery of this set of patients with dual drug resistance and the current absence of a therapeutic alternative for oral antiplatelet therapy adds momentum to the push for developing novel therapies that will completely abrogate P2Y12-mediated platelet activity.

.Beyon d Clopidogrel: The Search for Therapeutic Antagonis ts A t and Downstre am of the P2Y 1 2 Receptor The thienopyridine clopidogrel, having displaced its predecessor ticlopidine, has been a bulwark in the armamentarium of antiplatelet agents. Yet while Plavix is the current â&#x20AC;&#x153;gold standardâ&#x20AC;? for antiplatelet therapy, drugmakers have not been able to resolve several worrisome issues surrounding the drug(Johnson, 2007). In sum, the incidence of bleeding caused by clopidogrel, though comparable to aspirin, still poses a concern during invasive procedures (Plosker, 2007). Medical literature has widely documented the issue of interpatient variability in platelet responsiveness to treatment with clopidogrel (O'Donoghue, 2006). Some populations do not respond to this antiplatelet therapy, which is troubling especially in light of the documented association between clopidogrel resistance and aspirin resistance (Wang, 2006). The clinical management of clopidogrel resistance and nonresponsiveness has been challenging (Plosker, 2007). The possibility of developing or discovering an even more efficacious antagonist of the platelet P2Y12 receptor has thus been a tempting carrot for pharmaceutical companies and medical researchers. .Prasugrel, the Third-Generation Thienopyridine A promising antithrombotic agent that specifically inhibits the P2Y12 receptor is the third70

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents generation thienopyridine CS-747 (prasugrel), which is currently under development by Eli Lilly (Sugidachi, 2006). Phase I studies showed that prasugrel induced greater and more consistent anti-platelet effects than either placebo or clopidogrel (Brandt, 2007; Jakubowski, 2005; Eli Lilly, 2005). A study comparing the degree of platelet inhibition in stable aspirintreated patients with coronary artery disease found that prasugrel achieved consistently higher inhibition of platelet aggregation than clopidogrel, and prasugrel administration elicited a lower proportion of pharmacodynamic non-responders compared with the approved dose of clopidogrel (Jernberg, 2006). Researchers have suggested that prasugrelâ&#x20AC;&#x2122;s ability to treat patients who are normally resistant to Plavix may be due to differences in active metabolite formation. Prasugrel proceeds through one step to form its active component, primarily through cytochrome CYP2B6 and the P450 (CYP3A) cytochrome mentioned above. There are two sequential steps required to activate clopidogrel, one of which is dependent on CYP3A alone. Prasugrel levels were not affected by the addition of the potent CYP3A inhibitor ketoconazole, while ketoconazoleâ&#x20AC;&#x2122;s inhibition of CYP3A4 and CYP3A5 thus disturbed the formation of Figure 16. Platelet P2 Receptor Model. A representation of P2Y and P2X receptor molecules on the platelet surface with a simplified model of ATP- and ADP-induced platelet aggregation and the drugs (cangrelor and AZD6140 directly, and the thienopyridines ticlopidine and clopidogrel via an active metabolite) that antagonize the P2Y12 receptor ((van Giezen, 2005)).

clopidogrelâ&#x20AC;&#x2122;s active metabolite and compromised its ability to inhibit platelet activity in

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents patients (Farid, 2007). The positive Phase I results allowed Lilly and its partner company in development, Daiichi Sankyo, to proceed onto further studies (Goodall, 2006a). The randomized, double-blind and dose-ranging Phase II trial, JUMBO-TIMI 26,69 measured the safety profile of prasugrel in comparison with clopidogrel in patients undergoing PCI (Wiviott, 2005). Clinically significant bleeding events at 30 days marked the primary end point of the trial, while myocardial infarction, recurrent ischemia and clinical target vessel thrombosis were secondary end points. Patients on the prasugrel regimen fared similarly to patients treated with clopidogrel; the rates of hemorrhagic complications were very low and similar for both groups, and prasugrel-treated patients suffered fewer incidences of both the primary and secondary end point events (Wiviott, 2005). The findings from JUMBO-TIMI 26 allowed prasugrel to advance to the next stage: large, Phase III clinical trials to assess efficacy in addition to safety. While early results appear promising, the pharmaceutical success of prasugrel hinges on the outcome of the Phase III trials with the TRITON-TIMI 38 study (Wiviott, 2006). This study is currently underway to compare the efficacies of prasugrel versus clopidogrel in treating almost 13,000 patients who have acute coronary syndrome and are undergoing PCI to open blocked heart arteries (Wiviott, 2006). In the meantime, research has continued on the pharmacodynamics and pharmacokinetics of prasugrel. Earlier this year, studies measuring the extent of platelet aggregation in blood samples taken from healthy patients established prasugrelâ&#x20AC;&#x2122;s effectiveness at blocking procoagulant and pro-inflammatory platelet responses (Judge, 2008). Judge et al. compared the platelet aggregation produced by preincubating the blood samples with the active metabolite of prasugrel, R-138727, versus with

Joint Utilization of Medications to Block Platelets Optimally â&#x20AC;&#x201C; Thrombolysis In Myocardial Infarction 26

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents cangrelor (AR-C66931MX). Flow cytometry measurements revealed that R-138727 instigated concentration-dependent inhibition of platelet aggregation and blocked the inhibition of VASP phosphorylation. In addition, R-138727 inhibited platelet procoagulant activity, as measured by annexin V binding and microparticle formation, and abrogated proinflammatory responses, as instanced by reduced release of sCD40 ligand and lack of platelet-leukocyte co-aggregate formation. A radioligand binding assay with the agonist (33)P 2-MeSADP demonstrated the extent of P2Y12 receptor antagonism in both resting and stimulated platelets using prasugrel or cangrelor in comparison to clopidogrel. Critically, prasugrel outperformed clopidogrel in this regard; at R-138727 30 micromol/L and cangrelor 1 micromol/L, both substances completely abolished (33)P 2-MeSADP binding to the P2Y12 receptor, whereas administration of clopidogrel could only partially inhibit (33)P 2MeSADP binding. The platelets did exhibit calcium mobilization following agonist stimulation when in the presence of either R-138727 or cangrelor. However, platelet stimulation and granule secretion did not reveal any additional P2Y12 receptors available for (33)P 2-MeSADP binding. Eli Lilly announced on January 4, 2008 that the NDA application to approve prasugrel had been submitted to the FDA on December 26, 2007 (Eli Lilly, 2008). If approved, the company plans to market prasugrel under the trade name â&#x20AC;&#x2DC;Effientâ&#x20AC;&#x2122; (Eli Lilly, 2008). .Cangelor, A Promising ATP Derivative The search has not been limited to thienopyridine derivatives. Many efforts in the late 1990s and early years of the 21st century were focused on deducing the potential therapeutic benefits of the ATP analogue, AR-69931MX (Daniel, 1998; Humphries, 1995; Ingall, 73

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents 1999; Jin, 1998a; Jin, 1998c). The history of the discovery of AR-69931MX is worth delving into because it exemplifies the search for ATP analogues that could be of pharmacologic utility. ATP itself was a poor compound for exploring P2Y12 receptor properties, as ATP is prone to breakdown by ectonucleotidases and has a relatively low affinity for the P2 receptors. The emphasis on creating a more stable molecule that would specifically target P2Y12 influenced the direction of medicinal chemistry research in the 1980s, and garnered key insights into structure-activity relationships (SAR) of various substitutions to the chemical structure of ATP(Cusack, 1982a; Cusack, 1982b; Welford, 1986). Substituents in the 2 position of the adenine ring of ATP increased affinity and specificity for the P2Y12 receptor (Cusack, 1982a; Cusack, 1982b). βγ-methylene substitutions in the triphosphate part of ATP increased stability. As several AstraZeneca scientists recounted, these initial studies formed the foundation for the pharmaceutical company’s R&D Charnwood medicinal chemistry program’s forays (van Giezen, 2005). Astra Zeneca eventually discovered the compounds AR-C66096MX and ARC67085MX, followed by AR-C69931MX; the use of the ARL prefix, for ADP Receptor Ligand, was common in early descriptions of these ATP analogue compounds, and MX was the convention representing the tetrasodium salt. These two purine derivatives were potent and selective P2Y12 antagonists that reduced platelet aggregation in a concentrationdependent manner (Humphries, 1994; Humphries, 1995). However, the first two antagonists seemed to interact with other P2Y receptors at high concentrations in a high-expression system. Under these conditions, AR-C66096MX appeared to be a partial agonist of P2Y1, while AR-C67085MX exhibited partial P2Y11 agonist properties (Communi, 1999; Fagura,

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents

Figure 17. Chemical Structure of P2Y12 Antagonists Currently In Development. Chemical structure of ATP (adenosine triphosphate, a weak P2Y12 antagonist) in comparison to the structures of various ATP derivatives: AR-C69931MX (Cangrelor, an intravenous, reversible P2Y12 inhibitor in clinical development), and the two cyclopentyl-triazolo-pyrimidines, AR-C109318XX and AZD6140 ((van Giezen, 2005)).

1998). The findings of non-specific interactions rendered these two P2Y12 antagonists unsuitable for further investigation as possible pharmaceutical agents. However, the third purine derivative, AR-C 69931MX (soon to be named cangrelor), did not show these agonist properties and remained in contention. In 2001 and 2002, multiple researchers conducted safety profile and tolerability studies that gave positive assessments of the potent, rapid-acting, intravenous antiplatelet drug AR-69931MX (cangrelor) in patients with acute coronary syndromes and with UA and non-Q-wave MI (Jacobsson, 2002; Storey, 2001a; Wang, 2003a). Cangrelor was indicated for intravenous administration because of its short pharmacodynamic half-life, due to the presence of the triphosphate chain (Storey, 2001a). The drug, under development by Astra Zeneca, successfully entered Phase II and Phase III clinical trials for the potential treatment

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents of UA and as an extremely fast-acting parenteral antithrombotic agent (Chattaraj, 2001). The direct, reversible mechanism of cangrelorâ&#x20AC;&#x2122;s inhibition of P2Y12 receptors complements the activity of the irreversible pro-drug clopidogrel, which has high levels of platelet nonresponsiveness. One such study in healthy volunteers demonstrated that 30% of platelet P2Y12 receptors were unoccupied after 11 days of clopidogrel administration (Jarvis, 2000). Subsequently, treating the platelets with cangrelor ex vivo increased the inhibition of ADPinduced platelet aggregation. Astra Zeneca also began investigating derivatives of the cangrelor molecule in hopes of creating an oral antithrombotic. AR-C109318XX was the first nonphosphate P2Y12 antagonist found to be selective as well as stable. Scientists tweaked the AR-C109318XX compound to increase oral bioavailability, and produced yet another P2Y12 agonist, AZD6140 (van Giezen, 2005). Specifically, AstraZeneca generated AZD6140 by removing a triphosphate side chain, converting the ribose to a carbocycle and modifying the pyrine into a trizolopyridine. Both compounds represented a new chemical class of platelet inhibitors called cyclopentyl-triazolo-pyrimidines (CPTP). AZD6140 showed particular promise because it was orally-active as well as being a potent, reversible P2Y12 receptor antagonist (van Giezen, 2005). Neither AR-69931MX nor AZD6140 require metabolic activation, a valued property since some platelet resistance to clopidogrel inhibition has been linked to variance in cytochrome P450 (CYP) 3A4 metabolic activity (Lau, 2004). However, the development of AZD6140 was abruptly halted in 2007 when Serebruany, a noted clinical researcher in the combination therapy of aspirin and clopidogrel, and others discovered that this experimental compound caused dyspnoea (Serebruany, 2007). This finding shed doubt on the safety of the parent compound and related 76

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents derivatives such as AR-69931MX. Nonetheless, the STEP-AMI70 angiographic trial conducted by Greenbaum et al., which was published in the same year as Serebruanyâ&#x20AC;&#x2122;s article, concluded that congrelor has great patency in combination with the standard treatment alteplase (tissue plasminogen activator (t-PA)) and can potentially be used in conjunction with fibrinolysis to treat acute MI (Greenbaum, 2007). A year earlier, Greenbaum et al. had completed another trial studying the efficacy versus bleeding risks of cangrelor in patients undergoing PCI. This study, again, suggested that cangrelor outperformed the glycoprotein IIb/IIIa receptor antagonist abciximab as a short-acting and reversible antiplatelet agent (Greenbaum, 2006). Cangrelor achieved a shorter prolongation in bleeding time after the application of drug infusion was halted, while not increasing the rate of adverse cardiac risks seen with abciximab treatment. In any case, two Phase III clinical trials with a combined planned enrollment of 15,400 patients began in 2006 and are still underway. CHAMPIONPCI is comparing the efficacy and safety profile of cangrelor to clopidogrel in subjects requiring PCI, while CHAMPION-PLATFORM is considering whether cangrelor, in addition to usual care, is superior to usual care alone in patients requiring PCI (Greenbaum, 2006; Mitchell, 2006). The trial enrollment is expected to continue through 2008 (Mitchell, 2006). .Specifying Targets Downstream of P2Y12 As the complexities of the molecular signaling pathways downstream of P2Y12 receptor activation have been elucidated, therapeutic research efforts have increasingly considered targeting P2Y12 effectors rather than the receptor itself. This could potentially create a more sophisticated pharmaceutical arsenal, with specific protein inhibitors to exert therapeutic

Safety, Tolerability, and Effect on Patency in Acute Myocardial Infarction

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents antithrombotic effects whilst leaving the remaining pathways downstream of P2Y12 intact to prevent undesirable side effects such as prolongation of bleeding time. One promising line of research is directed at PAR1 in human platelets. Thrombininduced activation of platelets has long been recognized as an essential part of the hemostasis cascade, despite a dearth of knowledge to outline the exact signaling pathways mediating this process. In a break-through study, Voss et al. established that PAR1 couples with Gi/o to activate PI3K, which is responsible for regulating platelet aggregation and activation of the integrin ÎąIIbÎ˛3, and for potentiating the PAR1-mediated increase in intraplatelet calcium concentration (Voss, 2007).

Figure 18. Model of Thrombin Receptors and P2Y12. Further delineation of the cross-signaling between pathways downstream of the thrombin receptors PAR1 and PAR4, as well as downstream of the ADP-activated P2Y12 receptor, suggest that new therapies targeting the PAR receptors may allow a more specific inhibition of platelet aggregation without disrupting intrinsic haemostatic function. PAR1 activates Gi/o, G q, and G12/13, while PAR4 activates G1 and G12/13. The downstream secretion of dense granule releases ADP, which then agonizes the P2Y12 receptor in a feedback loop. Furthermore, the calcium mobilization signaled by PAR4 via phospholipase C converges with signaling downstream of P2Y12 to stimulate platelet aggregation ((Holinstat, 2006; Holinstat, 2007)).

PAR1 can potentiate these downstream signals regardless of whether the P2Y12 receptor has been stimulated by ADP, since it activates Gi independently through coupling. This finding by Voss et al. built on previous experiments that had judged the independence of the PAR and P2Y receptors by examining whether the protease-activated receptors could inhibit adenylyl cyclase in the absence of ADP. Such studies had concluded that both PAR1 and 78

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents PAR4 rely on ADP to activate Gi signaling, yet that thrombin and thrombin receptoractivating peptides can promote platelet aggregation independent of Gi pathways (Kim, 2002). PAR1’s direct activation of Gi, bypassing the need for ADP stimulation of the Gi– coupled P2Y12 receptor, provides support for continuing efforts to evaluate whether inhibition of the PAR1-mediated Gi/phosphoinositide-3 kinase signaling axis can be as effective as clopidogrel while potentially avoiding the unwanted side effect of excess bleeding (Voss, 2007). Further research may also be influenced by a more complete model of how the thrombin receptors PAR1 and PAR4 differentially function to activate platelets in conjunction with G-protein-linked signaling and the ADP receptors. Holinstat et al. first presented such a model in 2006: upon thrombin-induced activation following vascular injury, PAR1 signals through Gq, Gi and G12, while PAR4 signals through Gq and G12 (Coughlin, 1999; Covic, 2000; Holinstat, 2006; Kahn, 1998). These combined signaling pathways activate GP IIbIIIa and rap1, while also inducing platelet aggregation and the secretion of α granules and dense granules. The model proposes that PAR1 directly activates Gi and consequently does not rely on P2Y12 receptor activation, whereas PAR4 signaling converges with P2Y12 downstream signaling, perhaps at the point of rap1b activation (Holinstat, 2006; van der Meijden, 2005; Woulfe, 2004). Holinstat’s data was consistent with other findings that activation of P2Y12 was important for rap1 activation, though inhibition of P2Y12 does not fully abrogate rap1 activation, implicating that another mechanism is present which partially activates rap1. PAR1 may partially regulate the intra-platelet calcium mobilization mediated by PAR4, which appears to play a role in P2Y12 activation (Covic, 2000).

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents One of Holinstatâ&#x20AC;&#x2122;s more significant contributions to the existing literature was his observation that the multiple receptor activation pathways following thrombin are not redundant: PAR4-mediated signaling is to some extent regulated by P2Y12, whereas PAR1 signaling is independent of P2Y12 activation (Holinstat, 2006). Part of the antiplatelet effect achieved by abolishing P2Y12 activation by ADP is purported to be due to the subsequent attenuation of PAR4 signaling (van der Meijden, 2005). In contrast to Voss et al., Holinstat suggested that specifically targeting the PAR4 signaling pathway is a promising therapeutic approach, as it might result in a better side effect profile than existing P2Y12 antagonists such as clopidogrel (Holinstat, 2006). More research would be required to ascertain the identity of the effectors in the pathway downstream of PAR4 and P2Y12 synergy; these molecules could prove to be powerful targets for intervention (Graff, 2004; Holinstat, 2006). .Conclusions on the Pharmaceutical Horizon for Plavix & Potential

Competitors There is no question that Plavix remains the dominant player in the prescription drug category for antiplatelet therapies. Its broad range of indications and proven effectiveness in over 50 million patients will help the drug retain competitiveness in the coming years after recent blows to profits. Sanofi and BMS already suffered a recent blow to profits while fending off the pre-expiration generic foray into the branded Plavix market from the Canadian manufacturer Apotex, and have invested huge resources successfully defending the Plavix patents in court. Plavix will most likely be out-priced by the entry of generic competitors after the patent expiration in November 2011, nevertheless, the outlook for antiplatelet agents such as Plavix looks extremely promising. In 2007, an analyst from Credit Suisse reported 20%

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents growth in Plavix prescriptions, an increase from the 12% growth recorded the previous year (Johnson, 2007). Analysts have predicted that the market for Plavix could double to reach $11 billion by 2011, barring any further pre-expiration generic competition (Johnson, 2007). Yet the high price of Plavix, the severe bleeding that can accompany prolonged use, and the irreversible inhibition of the platelet receptors– which means that the body requires a five-day period to completely regenerate new platelets and flush the effects of clopidogrel bisulfate out of the system once prescription use is discontinued – and variable resistance to the drug all leave room for other pharmaceutical products to potentially pilfer a part of the antiplatelet market (Johnson, 2007). Any novel agent is unlikely to entirely displace Plavix, which along with aspirin has a firm monopoly due to polytherapeutic indications (Fareed, 2008). Any newcomer must hurdle the same immense regulatory barrier of conducting an individual trial for each indication that Plavix has acquired over its decade in the U.S. market. Ultimately, when reviewing the emerging therapeutics described above – prasugrel and cangrelor – which may be approved in time to challenge Plavix, we return to the question that plagued the Sanofi researchers during the arduous development of clopidogrel bisulfate itself. Increased potency so often comes alongside increased bleeding and other side effects that are themselves inherent derivatives of the drug’s potency. Is the increased benefit or lower mortality worth the risk of such side effects? Thus far, in prasugrel’s case, the balance appears to tip slightly in its favor. The third-generation thienopyridine certainly wins over its grandparent ticlopidine, and appears to have inched ahead of clopidogrel in terms of its efficacy-to-tolerance ratio. Part of prasugrel’s greater reduction in adverse cardiovascular outcomes may be due to its different path of metabolism, and prasugrel’s resulting ability to overcome platelet resistance to clopidogrel. It is conceivable that prasugrel could successfully

Clinical and Metabolic Studies on Plavix and Novel Antiplatelet Agents treat the segment of the population with dual resistance to clopidogrel and aspirin. Nonetheless, the inextricable association between the ADP-mediated signaling pathways to platelet aggregation and those to support intrinsic hemostatic function suggests that there is an ceiling to the level of therapeutic platelet inhibition that can be obtained by specifically antagonizing the P2Y12 receptor before the risk of bleeding complications outweighs the benefits conferred by preventing platelet aggregation.

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute

Chapter 6 - From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute .Forging the S anofi and BMS Pharmaceutical Partnership The seeds for Bristol-Myers Squibb’s eventual partnership with Sanofi to develop clopidogrel bisulfate were sewn in 1992 when Bristol-Myers Squibb (BMS) underwent a dramatic shift in top management. Kenneth Weg was promoted to President of World-Wide Pharmaceuticals, and a new President, Charles Heimbold, stepped to the helm of the company, one of the three largest pharmaceutical companies in America.71 Heimbold, according to Weg, laid out two main objectives for the executive team: (1) in 5 years, to double sales and double profits; (2) to intensify research efforts and evaluate the current pipeline, divided into two parts: (a) what products were losing exclusivity and which ones would continue to be profitable, and (b) what BMS could do to strengthen its franchises. In regards to the former, the analysis was that BMS was “closer to zero-percent annual growth,” Weg recalled in an interview.72 For the latter, it was clear that “in 1992-93, BMS was flying along, doing exceedingly well,” said Leon Rosenberg, then-President of the Pharmaceutical Research Institute. The company had just developed the chemotherapy drug Taxol and the statin Pravachol, and was launching the antibiotic Cefprozil as well as the second-generation AIDs drug Zerit.73 Essentially, said Rosenberg, “the late stage pipeline looked very attractive. What we began to realize was that it was going to be a tough act to follow: the discovery pipeline was not promising.”

Interviews with Kenneth Weg, then-President of World-Wide Pharmaceuticals, Bristol-Myers Squibb, November 15, 2007; December 3, 2007. 72 Weg, 2007. 73 Interview with Leon Rosenberg, then-President of the Pharmaceutical Research Institute, Bristol-Myers Squibb, October 26, 2007. 71

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute Franchises could help shore up the BMS pipeline; Weg outlined what the company was looking for in a franchise: clearly marketed products, expertise, a strong pipeline, scientific and clinical know-how, as well as a history of licensing.74 The priority therapeutic areas for franchises included the cardiovascular, metabolic, oncology, CNS disorders and infectious diseases & virology segments. BMS’s cardiovascular portfolio was at particular risk – the group was going to lose exclusivity on a high percentage of drugs in the near horizon, including the blood pressure and heart treatment drug Capoten, whose patent would expire in 1993.75 Weg believed that the day of beta-blockers and ace inhibitors was coming to an end. Though he saw a future in Angiotensin II Receptor Blockers (ARB) that had to be taken only once daily, the sole ARB in development at BMS had to be taken twice a day, which “wasn’t going to do it.”76 At that point, Sanofi (then known as Elf Sanofi, a unit of the giant governmentowned oil company Societe Nationale Elf Aquitaine) came and “presented an interesting offer,” said Weg.77 Sanofi had the seemingly opposite problem from BMS: a promising pipeline, but no foothold or sales force in the United States. Sanofi had specifically sought out BMS for its experience in developing and marketing the cardiovascular drugs Capoten and Pravachol (Freudenheim, 1993). Sanofi proposed a 50-50 collaboration, which would unite Sanofi and BMS in a global co-development and co-marketing partnership for two promising heart drugs in a mature stage of the Sanofi pipeline: an ARB (Avapro) to treat hypertension; and the anticlotting drug (clopidogrel) for the prevention of heart attacks and

Weg, 2007. Weg, 2007. 76 Weg, 2007. 77 Weg, 2007. 74 75

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute strokes (Freudenheim, 1993; Yvon Bastien).78 BMS would pay Sanofi for part of the research already incurred, and they would split the expenditures for future clinical endeavors.79 Rosenberg described the first executive meeting wherein Ken presented the possibility of partnering with Sanofi for the two drugs. “There was a lot of skepticism on the part of other people in the organization about doing such a deal,” Rosenberg said. “[The concern] was not in terms of ‘control’… though any sort of partnership like [this] is very complicated when not in total control: who decides the timing, prioritization, findings...” But rather, Rosenberg remembered concerns being framed in terms of company targets: “Does BMS need another hypertensive drug? We were doing well with our two ace inhibitors. Is there room in the marketplace for another aspirin?”80 Rosenberg and Weg “did some serious homework” to answer these questions, and ultimately returned to a subsequent board meeting to answer these questions with a resounding “yes” – on all counts.81 “We should do the deal,” Rosenberg recalls telling the board, justifying the decision by pointing out that the pharmacologic and clinical data collected thus far on both drugs looked promising and their indications would complement the existing the BMS portfolio.82 BMS did not have any second-generation hypertension drugs, and Avapro would fill that gap. Likewise, Plavix appeared to operate along a wholly different mechanism than aspirin to abrogate platelet function, and could thus carve out a significant niche of the antiplatelet market.

Weg, 2007; Rosenberg, 2007. Weg, 2007. 80 Rosenberg, 2007. 81 Rosenberg, 2007. 82 Rosenberg, 2007. 78 79

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute The partnership between Sanofi and BMS was cemented in 1993 (Freudenheim, 1993). The deal between the two companies generated a behemoth in the pharmaceutical industry; together, Sanofi and BMS held 4.49% of the $200 billion global drug market in 1993, displacing the former No. 1 drug maker, Merck & Company, with a 4.29% share (Freudenheim, 1993). Weg credited the initiation of several novel organizational strategies that enabled their productivity to reach full potential. For instance, BMS set up an alliance team of people whose full-time jobs were to liaise with their counterparts at Sanofi – “something,” Weg added, “which is pretty standard in the industry now, and also made it very productive.”83 Rosenberg noted the weighty consideration that took place prior to BMS’s decision to enter the collaboration. “If it had not been for very strong partnering between Ken and myself, BMS probably wouldn’t have done this deal. And as the saying goes, the rest is history…” Plavix flew through clinical studies, and the conclusion of the CAPRIE trial in 1996 proved the drug’s safety and efficacy sufficiently to enable clopidogrel to be registered with the U.S. Food and Drug Administration (FDA). Plavix was approved for sale in the United States by the FDA in November 1997 and quickly became a best-selling drug under the partnership (New York Times, 1997; News, 1997). Initially, Plavix was indicated for the reduction of thrombotic events (e.g. heart attacks or strokes) in patients who had previously suffered those events or who had arterial disease or acute coronary syndrome. Initial predictions of Plavix sales estimated that the drug would bring in $300 –million to $1 billion a year (News, 1997). Some physicians voiced doubts that “clopidogrel would beat aspirin,” and as one analyst noted, “it’s a tough go convincing physicians to not use aspirin…Aspirin

Weg, 2007.

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute has been studied in so many trials. It clearly works” (News, 1997). The FDA refused to allow Plavix to be marketed as “better than aspirin,” calling Plavix’s 8.7% benefit over aspirin in the clinical trial too “marginal” to conclude that clopidogrel was superior (New York Times, 1997). However, as Sanofi and BMS collaborated on further clinical trials, Plavix gained additional roles. Medical research showed that clopidogrel provided additional benefit and potency to antiplatelet activity when prescribed alongside aspirin (The Clopidogrel in Unstable Angina to Prevent Recurrent Events Trial Investigators, 2001), and later that it could also be taken with antihypertensive drugs to prevent cerebral infarction (Coniglio, 2001). It is now also commonly administered during PCI stenting procedures to open blocked arteries. Just recently, in 2006, Plavix gained a new indication to treat the most severe heart attacks (FDA Consumer, 2006; Pharma, 2006b; Bristol-Myers Squibb, 2006b). By all accounts, Plavix was a major bolster to company profitability and, together with Avapro, resuscitated the flailing cardiovascular segment of BMS’s drug portfolio. As Weg emphasized, “the partnership certainly rebuilt the company’s ability to compete in the cardiovascular market, and allowed [our] existing sales force to continue relationships with physicians.” Initially limited to developing and marketing Avapro and Plavix, the collaboration soon expanded to other cooperative development efforts. The partnership, as Weg recalls was legally very complicated – essentially a “series of Joint Ventures” that dictated, depending on where in the world the products were marketed, which company would credit sales to its P&L statement.84

Weg, 2007.

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute Plavix is currently prescribed to over 48 million Americans (Neumeister, 2007). IMS Health, a leading provider of market research services and sales management to life science and pharmaceutical companies, ranked Plavix – with $5.9 billion in global sales in 2005 – as the second-best-selling drug in the world behind the cholesterol agent Lipitor (Carreyrou, 2006). In 2005 Plavix made up a full fifth of revenues at Bristol-Myers Squibb (Phillips, 2006). The importance of Plavix to the company cannot be overestimated; and the fear of losing this blockbuster appeared to have been equally significant in magnitude. David Balto, a prominent attorney in antitrust and intellectual property litigation, commented that “conversations with investment bankers who cover these [pharmaceutical companies]” clarified that “the pressure these companies feel…to achieve some level of peace or resolution…when they feel they might lose patent coverage on an important drug is enormous – [Plavix] really matters a lot to BMS.”85 The following section will illustrate just how much BMS was willing to stake to save the Plavix monopoly. .The Generic Threat to Plavix Plavix was such a success that it triggered interest from several generic companies. While Apotex and the two pioneer companies have been embroiled in recent news, a long sequence of events precipitated the agreement between Sanofi/BMS and Apotex and the ensuing criminal investigation and patent battle (Goodall, 2006b). The generic challenge to Plavix came from the Apotex – the largest drug company in Canada, whose reputation for being an “aggressive and litigious” generic manufacturer was enabled by private ownership and the CEO’s ability to take such risks unbeholden to investors and unencumbered by financial disclosures (Shuchman, 2006). Apotex beat out another generic firm to file the first

Interview with David Balto, Partner, Robins, Kaplan, Miller & Ciresi L.L.P., April 21, 2008.

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute ANDA (#76-274) for FDA approval to market a generic version of Plavix in November, 2001.86 Since the patents protecting Plavix were not due to expire until 2011, Apotex had to justify the application with Paragraph IV certification (Shuchman, 2006). The certification alleged that the most recent patent, which differentiated between the enantiomers of clopidogrel bisulfate, was invalid and thus that protection from generic entry would expire (along with the original patent) in 2003 (Paine, 2003). As required by law, Apotex notified Sanofi and BMS of the impending ANDA application. Sanofi and BMS promptly responded by suing Apotex for patent infringement, thus triggering a mandatory 30-month hold on the ANDA application (Carey, 2006b). A trial to contest patent validity was set for 2006. However the mandatory 30-month stay on FDA approval of the ANDA application expired in May of 2005 without any legal resolution of the patent dispute. The FDA finally approved Apotex’s ANDA on January 20, 2006 since the patent litigation had not yet been decided (Karst, 2008). The generic firm, however, did not proceed directly to market. Instead, the firm courted attempts by Sanofi and BMS to settle preemptively. While many analysts felt that “there was a good chance Apotex would lose” the battle over patent validity, BMS “couldn’t take a chance on the courts’ deciding otherwise,” and had lawyers contact Apotex to initiate settlement negotiations (Shuchman, 2006). As will be discussed extensively in the following chapter, there was an established precedent of innovator-generic settlements in the pharmaceutical industry, and prior settlement agreements, when successfully executed, had resulted in delaying the entry of generic competition for multiple brand-name drugs.

Sanofi-Synthelabo v. Apotex. 488 F.Supp.2d 317 (S.D.N.Y. 2006).

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute BMS had a lot riding on the success of such a reverse payment agreement. The company had reported $3.8 billion in Plavix sales for 2005 alone. To put this figure in context, revenues from Plavix comprised about 20% of the company’s total revenue (Phillips, 2006; Martinez, 2006). Sanofi and BMS announced on March 21 that they had reached a tentative agreement, pursuant to several conditions, with Apotex (BMS, 2006). The proposed agreement would have settled the pending patent infringement lawsuit between the two parties. The Court suspended the commencement date of the trial, originally scheduled for June 2006, to pend resolution of the settlement.87 While the main thrust of the tentative agreement was clear -- to allow BMS and Sanofi to retain their exclusive right to sell Plavix until 2011 without competition from Apotex – the provisions of the agreement were more complicated. The parties engineered stipulations to cover the possibilities of failed litigation and of an at-risk launch in addition to the scenario of settlement approval. The main clause would have Sanofi and BMS grant Apotex an exclusive, royalty-bearing license to sell its generic clopidogrel bisulfate product for a sixmonth period starting on September 17, 2011. Apotex, in turn, would agree to not sell its product until the license became effective. The license could be effective earlier if a third party were to prove the ‘265 patent invalid or unenforceable. The generosity of the terms proffered to Apotex might be indicative of the extent to which Sanofi and BMS depended on the profitability of Plavix (Rubenstein, 2007; Herskovits, 2006.; Shuchman, 2006). The entire agreement hinged on antitrust review and approval by the Federal Trade Commission (FTC) and state attorneys general (Bristol-Myers Squibb, 2006a). The civil review was necessary due to a court order after a previous antitrust violation by BMS

Sanofi-Synthelabo v. Apotex. 488 F.Supp.2d 317 (S.D.N.Y. 2006).

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute (Pechersky, 2007). Despite recognition by BMS that â&#x20AC;&#x153;there is a significant risk that [the] required antitrust clearance will not be obtained,â&#x20AC;? the agreement contained stipulations to pay Apotex in the event that the proposed settlement was rejected as well as in the event of the finalization of the settlement (BMS, 2006). If the settlement were rejected, both parties planned on pursuing the patent infringement litigation. The press release also acknowledged that since the FDA had granted Apotex approval for its generic version of Plavix, the reinstatement of litigation would allow Apotex to launch its product at-risk. .A Sweetheart Deal, Turned Sour In a blow to negotiations, the state attorneys general rejected the first proposed settlement agreement (Bristol-Myers Squibb, 2006g). The FTC opposed the clause in the initial draft agreement wherein BMS would not launch an authorized generic version of Plavix in 2011, and would instead grant Apotex that exclusive license to sell generic clopidogrel bisulfate for a six-month period effective prior to patent expiration (Carreyrou, 2006). Meredyth Smith Andrus, an assistant attorney general involved with reviewing the original settlement, affirmed that the companies had submitted a revised version of the settlement for review (Martinez, 2006). According to the WSJ, there was no deadline set for when the FTC and state attorneys general would have to reach a decision, though reviews of this nature typically took up to two and a half months. A news release issued by BMS on June 25, 2006 included one of the main amendments to the previously announced settlement: the Apotex license was revised to become effective on June 1, 2011, rather than on the previously-chosen date of September 17, 2011 (Bristol-Myers Squibb, 2006g). BMS confirmed that the governmental review was underway, but cautioned, â&#x20AC;&#x153;there is no assurance that the revised agreement will address all the risks of the FTC and state attorneys general and there remains

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute a significant risk that antitrust clearance will not be obtained” (Bristol-Myers Squibb, 2006g). The three parties modified the proposed settlement, commonly referred to as the ‘May agreement’ because it was signed and dated on May 26, 2006 (Bristol Myers Squibb, 2006i). The revised settlement deleted the clause granting an exclusive license to Apotex for that 6 month-period (Bristol Myers Squibb, 2006i). The second draft of the agreement retained a monetary transfer, whereby Sanofi and BMS would give up to $40 million to Apotex. This payment was ostensibly to compensate Apotex for the generic firm’s aggressive investment in building an inventory of clopidogrel bisulfate product (Bristol Myers Squibb, 2006i). The press, however, regarded the payment to Apotex as an exchange for the generic company’s promise to delay the launch until the official patent expiration date in June 2011 (Carreyrou, 2006e). While that payment might appear exorbitant, the offered amount followed a long string of such reverse payments between innovator and generic firms in recent U.S. history. Assuming that Plavix sales held steady for the five years, the settlement was expected to protect at least $30 billion in revenue for Sanofi and BMS (Carreyrou, 2006f). .The BMS Decision to Settle with Apotex: Dumb or Desperate? The punitive terms of the agreement left many confused as to why CEO Dolan had decided to enter into such an arrangement. “I find it difficult to fathom…No one will negotiate with [Apotex CEO] Barry Sherman,” said Balto, referring to the company’s extremely litigious reputation.88 The face amount of the payment was mere token in comparison to the potential savings in revenues for the innovator companies, but the other provisions of the settlement

Balto, 2008.

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute appeared heavily lopsided in Apotex’s favor. Aaron Barkoff, a lawyer focusing on pharmaceutical patent litigation who also reports on developments in Hatch-Waxman litigation and pharmaceutical patents in Orange Book Blog, said in an interview that he’d never “seen a settlement agreement where the innovator gives up so much,” and was at a loss to explain why BMS had decided to enter into such an unbalanced agreement.89 The settlement had restrictive limitations on the legal action that Sanofi and BMS could take. One clause forced the two companies to wait five business days after a generic launch of clopidogrel before pursuing a preliminary injunction (Bristol Myers Squibb, 2006i). Furthermore, the contract forfeited the two firms’ right, under the Hatch-Waxman Act (35 U.S.C.§284), to assess triple damages from Apotex if they were victorious in the patent case. In place of those rewards, Sanofi-Aventis and BMS could claim damages only up to half of the net sales of generic Plavix (Bristol Myers Squibb, 2006i). On a tip-off from the FTC to the Justice Department, agents from the Federal Bureau of Investigation (FBI) raided the Manhattan headquarters of BMS on July 27, 2006 (Carreyrou, 2006). The raid was part of a larger criminal investigation into the deal struck between BMS, Sanofi and Apotex to delay the generic launch (Carreyrou, 2006f). The criminal investigation did not bode well for the settlement struck between the three companies. According to the WSJ, both Sanofi and BMS had been informed of the investigation but declined to comment further. BMS had a checkered history of anticompetitive actions. Aside from the consent decree in 2003 to settle charges of blocking generic entry for three drugs, BMS was also on probation for accounting fraud (Los Angeles

Interview with Aaron F. Barkoff, Partner at McDonnell, Boehnen, Hulbert & Berghoff LLP, April 21, 2008.

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute Times, 2005; Shuchman, 2006) In 2004, the company had entered into an agreement with the Securities and Exchange Commission to settle charges of accounting fraud (Carreyrou, 2006c). BMS paid a $150 million fine to settle the charge of “channel-stuffing,” a practice whereby a pharmaceutical company gives incentives to drug wholesalers to purchase larger quantities of their products than demand necessitated (Los Angeles Times, 2005; Davies, 2005). The channel-stuffing scheme, which was used to meet quarterly sales targets, had resulted in BMS overstating their revenues by $2.5 billion for the years 1999-2001 (Davies, 2005). A permanent injunction was placed on BMS to prevent further violations of specific accounting provisions under federal securities laws (Los Angeles Times, 2005). The accounting restatements and federal investigation had dragged into June 2005, when the Justice Department finally entered into a “deferred-prosecution” agreement with BMS to settle the scandal (Lublin, 2005). The consent order stipulated that the then-chairman of BMS, Peter Dolan, give his position up to another member of the board, though he remained CEO (Los Angeles Times, 2005; Lublin, 2005). The consent order also forced BMS to comply with a two-year probation period against other acts of criminal conspiracy before the federal indictment would be dropped (Los Angeles Times, 2005; Davies, 2005; Lublin, 2005). The former federal judge Frederick B. Lacey, also involved the SEC-mandated supervision of the company’s accounting practices, was appointed by the U.S. state attorney for New Jersey to monitor BMS under the consent agreement for federal oversight of corporate governance (Los Angeles Times, 2005). Dolan indicated the company’s willingness to cooperate in full with the criminal investigation during a meeting previously scheduled to discuss second-quarter earnings (Carreyrou, 2006h). Later in the same discussion, a senior BMS spokesperson voiced the

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute company’s belief that “all of its conduct relating to the proposed Plavix settlement has been entirely appropriate and coordinated throughout with senior outside counsel" (Carreyrou, 2006h). The agents, acting on behalf of the antitrust division of the Justice Department, searched multiple offices including Dolan’s and left with batches of documents. At the time of the raid it was not apparent what the FBI agents were attempting to find, though any attempt to mislead the government during the review of the settlement could lead to prosecution"(Carreyrou, 2006g). Furthermore, since Sanofi/BMS and Apotex were rivals, any disclosure of pricing information or other confidential and competitive information during the negotiations could be interpreted as criminal collusion (Carreyrou, 2006f). On July 28, 2006, just one day after the government raid, the state attorneys general announced its decision to reject the deal to delay generic entry by Apotex (Bristol-Myers Squibb, 2006d). The move was characterized as a signal of “an increasing regulatory crackdown on such agreements” (Carreyrou, 2006e). While the FTC’s separate decision had not yet been announced at the time of article publication, the agency had opposed similarly structured agreements in the past to protect consumer interests. Balto, a former FTC antitrust enforcer, was quoted in the article as saying that the proposed settlement would have “led to substantial consumer harm, costing at least $1 billion a year,” by delaying the entry of a lower-costing generic version of Plavix (Bristol-Myers Squibb, 2006d; Carreyrou, 2006e). The article also cited a 2002 FTC survey showing that branded-drug makers win only a quarter of patent-litigation cases; this finding supports the idea that settlements tend to benefit the branded-drug makers at the expense of the generic companies (Carreyrou, 2006e). The idea of government interference in regulating pay-for-delay settlements will figure prominently in the policy discussion to follow, but that event represents only one

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute chapter of the Plavix story. .Apotex on the Attack: The Launch of Generic Plavix At-Risk of Legal Action After regulatory denial of the settlement, public attention turned to the possibility of an atrisk launch by Apotex. The first hint public announcement insinuating an imminent launch came from Medco Health Solutions Inc. during a conference call with financial analysts on Friday afternoon, August 4 (Carreyrou, 2006a). Medco is the largest pharmacy benefits manager company in America; Hoover’s reports that Medco administers some 550 million prescriptions a year, and helps health plans contain their pharmaceutical drug costs by negotiating discounts, processing claims and designing drug formularies (Hoover’s, 2008). The chief executive of Medco, David Snow, announced during the conference call that it was increasing earnings expectations, in part based on the assumption that a generic version of Plavix would soon be available (Carreyrou, 2006b). Another Medco executive added that the launch could occur within the month. Pharmacy benefit managers such as Medco garner higher profit margins on generic drugs than on the pricier brand-name drugs, as the full cost savings is not always passed on to the health plans or consumers, and the news of a potential launch of generic Plavix thus translated into higher share prices for the company that afternoon (Carreyrou, 2006a). Journalists honed in on the launch insinuated by Medco remarks, as Apotex was legally permitted to risk litigation and release their stores of generic Plavix under the terms of the agreement. On August 8, 2006, just weeks after U.S. legal authorities had blocked the proposed settlement, Apotex unleashed their generic copy of Plavix (clopidogrel bisulfate) on an atrisk basis (Pharma, 2006a; Bowe, 2006; Bruser, 2006; News, 2006a; Nowak, 2006; Zehr, 2006). The Apotex CEO, Barry Sherman, had called it “the biggest launch ever,” in an 96

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute interview on the eve of the launch (Shuchman, 2006). Indeed. This launch of the generic 75 mg tablets flooded pharmaceutical distribution channels, with immediate repercussions for Plavix sales and prescription rates. Major purchasers of Plavix, from wholesalers to pharmaceuticals benefit managers, took advantage of the discounted rate to build stockpiles of up to six months worth of generic Plavix (Jarvis, 2006). One customer reportedly placed a $75 million dollar order for the drug (Shuchman, 2006). The Dow Jones reported that Medco Health Solutions, the pharmacy benefit manager responsible for distributing 24% of Plavix prescriptions in America, would begin supplying generic Plavix (Goodall, 2006b). Despite the rumors of a pending at-risk launch, the event still surprised many in the pharmaceutical world and left Sanofi and BMS in a compromised position (Goodall, 2006b; Kennedy, 2006). Sanofi immediately attempted to file for a legal injunction after Apotex launched its version of clopidogrel bisulfate on August 8. However, BMS had filed its 10Q Quarterly Report with the SEC on the same day as the generic launch, enclosing the two proposed settlement agreements as Exhibit 99.1 and 99.2 (Bristol Myers Squibb, 2006i). The District Court dismissed Sanofiâ&#x20AC;&#x2122;s first motion on the grounds that it breached the clause in the second agreement obliging Sanofi to give five days notice to Apotex prior to moving for a legal restraining order. The two companies remained bound to the clauses in the failed settlement that outlined permissible courses of action in the event of Regulatory Denial. In addition to the term dictating that Sanofi and BMS could not file an injunction to halt generic sales for at least five business days post-launch, the agreement stipulated that they forego their right to collecting triple damages from Apotex if victorious in defending the patents in court (Jarvis, 2006). Instead, damages were limited to 50% of net sales of generic Plavix. Furthermore, if Apotex won the patent dispute, BMS and Sanofi were liable to pay a

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute large bond to compensate Apotex for lost sales. Sanofi thus gave notice in accordance with the provisions of the settlement. Exactly five business days later, on August 14, 2006, Sanofi and BMS proceeded to file a motion in front of a New York District Court to preliminarily enjoin Apotex from further distribution of its generic version of Plavix in the United States (Bristol-Myers Squibb, 2006c; Thompson, 2006a). Sanofi also requested that the Court mandate a recall of all generic Plavix already manufactured and distributed. The District Court relied on the established four-factor test for preliminary injunction relief to make the decision: (1) that Sanofi had a reasonable likelihood of success on the existing merits; (2) irreparable harm if an injunction were not granted; (3) a balance of hardships tipping in its favor; and (4) the injunction’s impact on the public interest(Judge Sidney H. Stein, 2006.). Stein, in addressing the validity of the Plavix patents, appeared to side with Sanofi. The legal document noted that Apotex had admitted to the infringement of the ‘265 patent. Apotex conceded that the subject of its ANDA, the generic Plavix product, did in fact infringe claim 3 of Sanofi’s‘265 patent, which claims “[h]ydrogen sulfate of the dextrorotatory isomer of methyl alpha-5 (4,5,6,7-tetrahydro (3,2-c) thieno pyridyl) (2chlorophenyl)-acetate,” known as clopidogrel bisulfate. Apotex cited three allegations against the validity of the ‘265 patent, and moreover, contested that the patent was unenforceable for multiple reasons: First, Apotex purported that the patent was invalid on the grounds of anticipation. Apotex argued that a prior Sanofi patent anticipated the discovery of clopidogrel bisulfate, pursuant to 35 U.S.C. § 102(b). Specifically, U.S. Patent No. 4,529,596 or “the ‘596 patent”), covered the thienopyridines, a genus of novel chemical compounds of which clopidogrel bisulfate is a member. The ‘596 patent pre-dated the ‘265 patent by four 98

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute years. In a related argument presuming this anticipation, Apotex claimed the subject matter described in the ‘265 patent would have been obvious at the time of the invention to a person of ordinary skill in the field, pursuant to 35 U.S.C. § 103. Third, Apotex asserted that the judicial doctrine of obviousness-type double patenting invalidated the ‘265 patent. Judge Sidney H. Stein handed down an opinion on August 31, 2006, three weeks after the launch of generic Plavix. The Court rejected the anticipation, obviousness, and obviousness-type double patenting invalidity defenses, and concluded that Sanofi was likely to triumph in defending the ‘265 patent against Apotex’s claims of invalidity. In addition, the Court opinion stated that Sanofi would suffer irreparable harm if Apotex were allowed to continue distribution of its generic version of clopidogrel bisulfate. On these grounds, the Court gave a partial victory to Sanofi and BMS by granting preliminary injunctive relief to block further sales of generic Plavix, but rejected their request to order a recall of any generic drugs already in circulation.90 The District Court warned that the permanence of the injunction would hinge on the ultimate decision reached in the patent infringement trial set to commence in January 2007. .Injunction, In Limbo Judge Stein’s analysis soothed supporters of Big Pharma, but also harkened to the long legal battle still ahead. In the wake of the injunction, the criminal probe continued through the fall. The antitrust division was investigating whether Sanofi and BMS had forged a side deal with Apotex that had been hidden from government regulators (Carreyrou, 2006c). Apotex alleged that the second, revised agreement had contained a breakup fee payable to the generic, which BMS had failed to disclose in the settlement as presented to

See Sanofi-Synthelabo v. Apotex Inc., 02 Civ. 2255 SHS (S.D.N.Y. 2006).

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute regulators and as reprinted in its Quarterly Report (Bristol Myers Squibb, 2006i; Carreyrou, 2006c). Allegedly, there was also a second unwritten term of the settlement, wherein Sanofi and BMS promised to not launch an authorized generic (Bristol Myers Squibb, 2006i; Carreyrou, 2006c; Decker, 2006). CEO Dolan was forced to step down in September 2006, in large part because of his perceived mishandling of the Plavix debacle (PBR Staff Writer, 2007; Carey, 2006). In addition to the probe into corporate governance, BMS announced on October 26 that the scope of the probe had widened to include an investigation of the company’s conduct in relation to the 2004 consent order with the SEC (Bristol-Myers Squibb, 2006j). A pharmaceutical analyst at Deutsche Bank, Barbara Ryan, told the WSJ, “whenever we see a government investigation expanded, it’s not a good thing, and because this company is already on probation, it’s even more troubling” (Carreyrou, 2006c). Public scrutiny was not contained to the criminal investigations at BMS; the business world paid close attention to the ramifications that the short-lived generic launch would have on the company’s profitability. In the year to August 2006, before the launch, Plavix was the third-best-selling drug in the world behind Lipitor and the acid-reflux drug Nexium (IMS Health, 2006). Plavix commanded a large segment of the booming cardiovascular drug market, which totaled $74.9 billion for the year to August 2006. The launch depressed Plavix sales immediately, and had a lasting impact, forcing BMS and Sanofi to offer rebates on branded Plavix (Carreyrou, 2006d). Branded Plavix prices slipped down to $148 for a month’s supply, in comparison to about $124 for the generic version marketed by Apotex (Carreyrou, 2006d). On a positive note, the first reports of revenues from generic and branded clopidogrel in the United States surfaced in October, indicating that the 100

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute preliminary injunction had helped staunch the hemorrhaging loss in revenues for BMS (Carreyrou, 2006c). The overall number of U.S. prescriptions of branded and generic Plavix increased by 14% in the third quarter, with Apotexâ&#x20AC;&#x2122;s generic version seizing 70% to 75% of the U.S. market (Carreyrou, 2006c). The company reported that sales of Plavix in the alliance with Sanofi fell 36% to $630 million in the third quarter, which was a smaller drop in revenue than analysts had predicted, perhaps because of the overall increase in prescriptions (Bristol-Myers Squibb, 2006j; Carreyrou, 2006c). The hit to sales was concentrated in the United States; Plavix sales in the U.S. fell 43%, to $474 million, as compared to the third quarter of 2005 (Bristol-Myers Squibb, 2006j). BMS net profits in the third quarter plummeted by 65% to $338 million, with the drop attributed to the residual glut of generic Plavix along with the loss of patent exclusivity over two other products: the cholesterol drug, Pravachol, and the chemotherapy agent, Taxol (Bristol-Myers Squibb, 2006j). BMS executives mentioned that the company was emphasizing consumer promotion to bolster Plavix prescription rates, while also attempting to negotiate rebates with managed care plans to recover sale volumes of the branded drug (Iskowitz, 2006). At the Merrill Lynch Global Pharmaceutical, Biotech & Medtech Conference that fall, a BMS executive announced, "We decided to continue to focus on Plavix full steam" (Iskowitz, 2006). Apotex had also decided to focus on Plavix full steam; the company had appealed the preliminary injunction and had also motioned to stay the preliminary injunction, pending the success of the appeal (Service, 2007). The United States Court of Appeals for the Federal Circuit rejected their motion for a stay in September, but expedited the court date for the appeal of the preliminary injunction (Bristol-Myers Squibb, 2006e).

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute The Federal Circuit of the U.S. Court of Appeals finally denied Apotex’s appeal against the preliminary injunction on December 8, 2006 (Bristol-Myers Squibb, 2006f). In the Rehearing, the Court upheld the reasoning behind the District Court’s decision to grant the injunction, and maintained that Apotex had not provided compelling arguments to suggest that the Sanofi patent was invalid (Globe and Mail, 2006; Dj, 2006). Following on the heels of the U.S. Court of Appeals decision to uphold the injunction preventing Apotex from continuing to market clopidogrel in the states, Canada’s Federal Court of Appeals proffered a similar victory to Sanofi and BMS on December 28, 2006 (Hunt, 2006; Izmirlieva, 2006; News, 2006b). This injunction across the two biggest markets in North America provided a respite, albeit temporary, for the two companies (Thompson, 2006b; Thompson, 2006c), and essentially paused the debates on patent validity until the start of trial in the United States in January 2007. The reprieve came at an especially opportune moment for BMS, which had been more heavily dependent on Plavix revenues and also more directly impacted by the generic launch in North American markets. Since the preliminary court injunctions did not require Apotex to withdraw any generic clopidogrel bisulfate already released into the markets, news articles at the time lamented that the extent of losses on the Plavix brand would not become evident until BMS posted its full-year 2006 results (Izmirlieva, 2006). For the fourth quarter of 2006, sales of Plavix at BMS dropped by 62% year-on-year, to $343 million from $906 million in 2005 (PBR Staff Writer, 2007). Sanofi, anticipating some losses from the temporary availability of generic clopidogrel, had downgraded its expected growth in earnings per share for 2006 from 12% to 2%. Stockpiles of generic Plavix in American distribution channels from the brief launch in August 2006 did in fact outlast the analyst

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute predictions of a six-month supply of generic Plavix, but were eventually depleted through 2007 (Izmirlieva, 2007). In March 2007, data from Wolters Kluwer showed that 400,000 U.S. prescriptions were filled with generic Plavix, while 1.8 million prescriptions were dispensed with the brand-name drug (Rubenstein, 2007). These preemptive tactics on the part of Sanofi and the extensive worries within BMS underscored their recognition of the extent of potential damage from the botched Plavix settlement.

.Taking Plavix to Trial On January 22, 2007, the American trial began to debate the validity of the â&#x20AC;&#x2DC;265 patent and the right of Apotex to enter the market with generic clopidogrel. The Court presided over the trial without a jury from January 22 through February 15, 2007, and also considered the information that had been presented during the evidentiary hearing for the preliminary injunction, held in August 2006.91 On June 22, 2007, Judge Stein of the Southern District Court of New York handed down a ruling to uphold the Sanofi patents; the Court issued a permanent injunction to forbid Apotex from re-entering the market with its generic version of clopidogrel bisulfate, however, the treble damages that would have been awarded to Sanofi were nullified because of their preemptive out-of-court settlement forged in 2006. This section will explore the specific intellectual property issues over the validity of the clopidogrel bisulfate patents that were discussed in the patent litigation trial. Apotex declared that the â&#x20AC;&#x2DC;265 patent claiming the right enantiomer of clopidogrel bisulfate was invalid (1) by anticipation; (2) by obviousness; and (3) by the judicial doctrine of

Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y. Jun 19, 2007).

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute obviousness-type double patenting.92 In the latter allegation, which relies on proving that the prior art patent was obvious, Apotex essentially charged that the most recent patent for Plavix is invalid because it is an instance of double-patenting, or “patent layering” wherein Sanofi and BMS first patented racemate clopidogrel bisulfate, and then obtained a patent on the biologically active enantiomer from the racemic mixture to extend the time before patent expiration (Reuters, 2007; Neumeister, 2007). I will weave together the court discussion and findings on each of these charges separately, and show the value of the court’s process of deliberation and decision for informing future litigation. Prior to plunging into the debate, it is worth nothing that in such trials concerning patent validity in a case of patent infringement, the patentee typically has the burden of responsibility to prove that the allegations are false, while the respondent (infringer) has the “heavy burden”93 of providing sufficient evidence to prove the patent invalid. Sufficient evidence – “clear and convincing evidence,”94 is a much higher bar for the infringer to hurdle than the standard burden (preponderance of evidence, or >51%) in non-patent litigation.95 This allocation of legal burden, pursuant to section 45 of the Patent Act, enables courts to give patents the presumption of validity. We will return to the presumption of validity in the following section on antitrust issues, but it suffices to note that there is dispute over the strength of the presumption of validity in patent litigation – as Barkoff pointed out,

Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y., Jun 19, 2007). See Am. Hoist & Derrick Co. v. Sowa & Sons, Inc. 725 F.2d 1350, 1359 (Fed. Cir. 1984) (quoting Radio Corp. of Am. V. Radio Eng’g Labs., 54 S.Ct. 752, 79 L.Ed. 163 (1934)). 94 See Sanofi-Synthelabo Canada Inc. v. Apotex, 2005 FC 390, 39 C.P.R. 4th 202, [2005] R.P.C. 855 (F.C.) (March 21, 2005) at para. 10, citing Merck Frosst Canada Inc. v. Canada (Minister of National Health and Welfare) 1994, 55 C.P.R. (3d) 302 (F.C.A.) at p. 319. 95 Barkoff, 2008. 92 93

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute “someone has filed a certiorari petition challenging the burden of ‘clear and convincing evidence’…saying the burden of proof is too high.”96

.Anticipation The charge of patent invalidity by “anticipation” is also commonly described as invalidity based upon lack of novelty, and “requires that the same invention…was known or used by others before it was invented by the patentee”.97 As a legal standard, the District Court established that a patent is held to be invalid due to anticipation when “each and every limitation is found either expressly or inherently in a single prior art reference.”98 The District Court, in affirming the earlier decision to grant a preliminary injunction, rejected Apotex’s arguments alleging anticipation: (1) that the ‘265 patent was anticipated by the ‘596 patent or its Canadian counterpart, the ‘875 patent; (2) that the ‘596 patent enabled a person of ordinary skill in the art to practice clopidogrel bisulfate without undue experimentation.99 Claim 3 of the patent-in-suit, ‘265 patent100, had four key limitations: (1) the bisulfate salt of (2) the dextrorotary enantiomer of (3) the MATTPCA compound (4) substantially separated from the levorotatory isomer (Badorc, 1988). In contrast, the ‘596 patent disclosed a genus of potentially therapeutic compounds. The District Court disagreed with Apotex’s contention that Claim 2, when examined with the limitations proposed in Claims 1 and 8, would disclose clopidogrel bisulfate to a person with ordinary skill in the art. Several of the ‘596 patent’s claims are relevant for the discussion to follow (Aubert, 1985). Claim 2 of the

Barkoff, 2008. See Hoover Group, Inc. v. Custom Metalcraft, Inc. 66 F.3d 299, 302 (Fed. Cir. 1995). 98 See Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y., Jun 19, 2007) (citing, e.g., Celeritas Techs. Ltd. V. Rockwell Int’l Corp., 150 F.3d 1354, 1361 (Fed. Cir. 1998)). 99 See Amgen Inc. v. Hoechst Marion Roussel, Inc., 314 F.3d 1313, 1355 (Fed. Cir. 2003). 100 Claim 3 of the ‘265 patent reads: “Hydrogen sulfate of the dextro-rotatory isomer of methyl alpha-5 (4,5,6,7tetrahydro (3,2-c) thienopyridl) (2-chlorophenyl)-acetate substantially separated from the levo-rotatory isomer.” 96 97

105

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute ‘596 patent recited the free base, MATTPCA, without describing either of the enantiomers or any salt; the District Court reasoned that Claim 2 obviously did not expressly describe clopidogrel or its bisulfate salt, but did acquiesce that it was a “more difficult question” to determine whether the missing limitations were inherently described elsewhere in the ‘596 patent. Claim 1 of the patent disclosed a generic formula for the compounds, which included “their addition salts with pharmaceutically acceptable mineral or organic acids…including both enantiomeric forms or their mixtures”.101 Claim 8 described “a therapeutic composition having blood-platelet aggregation inhibiting activities and anti-thrombotic activities,” and specified that the effective ingredient could be a compound of claim 1, either one of the two enantiomers or their mixture, in the claimed form or as an addition salt.102 .A Genus Cannot Anticipate a Species without a “Pattern of Preferences” In Sanofi v. Apotex, the District Court referenced two historical cases where a previous patent claiming a generic formula was found to anticipate a species covered in a more recent patent; in both In re Petering103 and In re Schaumann104, the generic formula demonstrated a “pattern of preferences” leading to the species. Furthermore, the court noted that those two cases propose that a genus covered in a patent might be small enough that the one disclosure is equivalent to the description of each species within the genus. In contrast, the Federal Circuit distinguished the prior art specification in the Plavix case as not showing such a “pattern of preferences”. The ‘596 patent identified twenty-one exemplary thienopyridine derivatives, and did not disclose a preference for MATTPCA. Furthermore, the Court noted

‘596 patent at col. 13, II. 8-19. ‘596 patent at col. 14, II. 5-11. 103 301 F.2d 676 (C.C.P.A. 1962). 104 572 F.2d 312 (C.C.P.A. 1978). 101 102

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute that the prior art specification gave multiple examples of these compounds in various forms: as a free base, as an oxalate, a sodium salt, and multiple variants of the compounds with hydrochloride salts and hydrobromide salts in addition to bisulfate salts. Out of the twentyone non-limiting examples of compounds, three were hydrochloride salts and four were bisulfate salts; the example containing the racemate of clopidogrel was presented as a hydrochloride salt.105 Expert witnesses had testified that the generic formula claimed in ‘596 could have lead to millions of compounds if all the possible combinations of products and salts were considered.106 The Court rejected Apotex’s claim that the disclosures in the ‘596 patent were limiting enough that, when read by a person of ordinary skill in the art, would “describe a dramatically smaller genus of nine preferred compounds, including clopidogrel bisulfate.”107 In Sanofi v. Apotex, the Court reasoned that the prior patent covering a genus must disclose a “pattern of preferences” for the species specified in the later patent to constitute anticipation. This framework has broader ramifications as a legal precedent for future pharmaceutical intellectual property cases involving a prior art specification and the potential for anticipation (Peterson, 2007).

.Obviousness Apotex charged that based on the disclosure of the prior art patent, it would have been obvious for a person of ordinary skill in the art to produce clopidogrel bisulfate. Apotex

Snyder T Tr. 236; Maffrand PI Tr. 139. Maffrand Tr. 1771; Davies Tr. 1914; as discussed in Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y., Jun 19, 2007). 107 Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y., Jun 19, 2007). 105 106

107

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute argued that the Plavix patents were “no different” than in In re Adamson108, where the PTO found the claims on a levorotary enantiomer and its addition salts to be obvious given the disclosures in two prior art references (Peterson, 2007). One prior claim in Adamson had disclosed a genus of compounds described by the same formula, without specifying the racemate or enantiomeric status. The other prior art reference had described that the racemic mixtures could be separated into the pure enantiomers, which frequently have very distinct physiological properties from each other and the racemate. The Court disagreed with this parallel drawn by Apotex. The evidence documenting Sanofi’s four years and the “tens of millions” of dollars the company had spent on developing the racemate PCR 4099, all before discontinuing that line of research to attempt to obtain the individual enantiomers of PCR 4099, convinced the Court that separating the enantiomers of PCR 4099 would not have been obvious to a person of ordinary skill at that time, contrary to what Apotex alleged. The Court also called attention to scientific testimony showing that the bisulfate salt of clopidogrel – distinct from every other salt formulation of clopidogrel that Sanofi attempted to develop – had an unexpectedly favorable pharmacologic profile. .A Disclosure Describing the Racemate Does Not Render the D- and L-Enantiomers Obvious – Especially When Results of Separation Are Not Predicted The District Court concluded that “whether or not it may have been ‘obvious to try’ separating the enantiomers of PCR 4099” and then secondarily choose to form the dextrorotary enantiomer as a bisulfate salt, “the wide range of possible outcomes and relative

108

275 F.2d 952, 954 (C.C.P.A. 1960).

108

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute unlikelihood that the resulting compound would exhibit the maximal increase in antiplatelet aggregation and the absence of neurotoxicity makes clopidogrel bisulfate non-obvious.”109 This case has already been referred to in another pharmaceutical patent litigation case, Bayer Schering Pharma AG v. Barr Labs, Inc.110, which was decided on March 3, 2008. In Bayer Schering, the Court refused to grant Barr’s requests to strike the internal studies on the development path of the drug drospirenone and bar a Bayer scientist’s testimony as irrelevant. The Court noted that those pieces of evidence could be “probative of how the person of ordinary skill in the art would read the published results”. The Bayer Court thus followed the framework established in Sanofi for judging obviousness based in part on the innovation’s path of development, noting, “the Sanofi case is instructive in this regard”. The Court recognized the potential of the evidence uncovered in exploring the innovation’s path of development to substantiate an obviousness defense, yet maintained that the Court still had to decide how much weight to accord such evidence in the Bayer case.

.Obviousness-Type Double Patenting: A Narrower Interpretation The judicial doctrine of obviousness-type double patenting is necessarily a narrower inquiry than the test for obviousness. This criterion is only used in the case where a Court is reviewing a situation in which the claims for two patents are drawn to inventions so alike that one is obvious in view of the other, and that the claims were made to effectively extend the life of the patent, which would have otherwise been terminated at the first expiration date.111 The conclusion that Sanofi had successfully defended its patents against charges of

Sanofi-Synthelabo v. Apotex Inc., 492 F.Supp.2d 353 (S.D.N.Y., Jun 19, 2007). Bayer Schering Pharma AG v. Barr Labs., Inc., 2008 U.S. Dist. LEXIS 15917 (D.N.J. Mar. 3, 2008). 111 See 35 § U.S.C.A. 103; Patents 291k120 k (Patents for Same Invention). 109 110

109

From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute anticipation and obviousness effectively blew the wind out of the sails in Apotex’s third allegation. The Court succinctly noted that the question of obviousness-type double patenting was “subsumed by the broader statutory inquiry pursuant to 35 U.S.C. § 103”, and dismissed the allegation. .Plavix as a Legal Precedent The District Court’s decision to uphold the ‘265 patent and slap a permanent injunction on Apotex has been widely referenced in legal reviews addressing permanent injunction and the exclusionary rights of patents after a Supreme Court case in 2006 (Diessel, 2007; Schoenhard, 2008; Beckerman-Roda, 2007). This case, eBay, Inc. v. MercExchange, L.L.C. 112

, was more remarkable for the minority opinion than for the unanimous decision of the

Supreme Court to uphold the standard four-part test as a litmus for granting permanent injunctions for valid patent infringement.113 Justice Kennedy penned the four-justice minority concurrence, deriding the use of injunctive relief “as a bargaining tool” and further arguing that “legal damages may well be sufficient to compensate for the infringement and an injunction may not serve the public interest.”114 The Supreme Court was emphatic that no rule existed to delineate that a permanent injunction “automatically follows” a determination that a (presumed to be valid) patent has been infringed. In the wake of eBay v. MercExchange, Sanofi has been cited as an example of a case where the Court relied on the traditional fourfactor test and ultimately did issue a permanent injunction in a patent case, whereas in many

eBay, Inc. v. MercExchange, L.L.C. (eBay III), 126 S. Ct. 1837 (2006). See eBay, Inc. v. MercExchange, L.L.C. (eBay III), 126 S. Ct. 1839 (2006) ("(1) that [the plaintiff] has suffered an irreparable injury; (2) that remedies available at law, such as monetary damages, are inadequate to compensate for that injury; (3) that, considering the balance of hardships between the plaintiff and defendant, a remedy in equity is warranted; and (4) that the public interest would not be disserved by a permanent injunction."). 114 See eBay III, 126 S. Ct. at 1842 (Kennedy, J., concurring). 112 113

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute other cases, District Courts denied granting injunctions or even reneged the granting of injunctive relief (Schoenhard, 2008).

.The Permanent Injunction: A H ard Pill f or Apotex to Swallow Apotex appealed the decision. Chief Executive Barry Sherman said winning the appeal would allow Apotex to “once again make possible billions of dollars in savings for the public” (Associated Press, 2007a). Despite Apotex’s immediate request, a period of dormancy followed the June 22nd ruling. Plavix was lauded “Best in Brand Resilience” in a feature on pharmaceutical brands of the year in 2007; the article noted the rebound of brand-name Plavix back onto pharmacy shelves, and a $120 million direct-to-consumer ad initiative that helped increase sales (Armstrong, 2008). Talk of a generic version of Plavix flared up again this year. The appeal by Apotex reached the U.S. Court of Appeals for the Federal Circuit, and oral hearings for the case began in March (Apotex, 2008). While the Court of Appeals has not yet handed down their decision, Barkoff said he “expects the Court to affirm” the previous decision.115 However, the generic threat has expanded beyond Apotex. On January 14th, 2008, the FDA approved an ANDA for another company; the Indian generic drug manufacturer Dr. Reddy’s Laboratories was awarded the ANDA for a generic version of Plavix. Subsequently on January 23rd, Dr. Reddy’s entered into a stipulated order116 with Sanofi that will allow the generic firm to manufacture and market generic Plavix with 10 days written notice, and to import generic Plavix without giving notice if the Federal Circuit rules in favor of Apotex’s appeal (Karst, 2008; Lund, 2008). Sanofi’s lawsuits against two other ANDA applicants for

115 116

Barkoff, 2008. See Sanofi-Synthelabo v. Dr. Reddy’s Laboratories, 02 Civ. 3672 (SHS) (S.D.N.Y. January 23, 2008).

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute infringing the ‘265 patent – Teva Pharmaceuticals and Cobalt Pharmaceuticals – remain in limbo pending the Federal Circuit decision (Lund, 2008).117 In what appears to be the most recent twist to the Plavix saga, Apotex filed a petition on February 13th to stay the FDA approval and assert its claim to the remaining days of its 180-day period of marketing exclusivity under the Hatch-Waxman Act. This petition has been characterized as a “long-shot”, at best, by various legal commentators (Karst, 2008). “Apotex’s petition has no chance at all of success,” Barkoff said.118 “In my mind, they’re losing a lot of credibility by filing that.” A Dr. Reddy’s spokesperson was also skeptical of the Apotex petition, saying that Dr. Reddy’s counsel felt there was no legal basis for the petition (Lund, 2008). Lawyers for Apotex requested for the FDA to stay all other ANDA applications for generic Plavix currently in progress, since Apotex only used 24 days of the 180-days of generic market exclusivity granted by the FDA in 2006 (Karst, 2008). The petition rationalized the FDA’s obligation to recognize the 156 remaining days of exclusivity by noting the “anomaly” that would be produced if Apotex, which was “the first to file a Paragraph IV certification challenging the validity or the enforceability of the ‘265 patent – conduct that Congress clearly intended to encourage,” ends up being the last to market (Apotex, 2008). Apotex disclosed that the U.S. Court of Appeals for the Federal Circuit was due to commence oral hearings on March 3rd of this year to reconsider the lower court ruling, which upheld the validity of the ‘265 patent. Apotex contended that if it wins this

See Sanofi-Aventis v. Teva Pharmaceuticals, No. 07-1521 (Fed. Cir. Nov. 21, 2007); Sanofi-Aventis v. Cobalt Pharmaceuticals, No. 07-1522 (Fed. Cir. Dec. 6,2007) (staying cases pending decision in Sanofi-Synthelabo v. Apotex, Inc., No. 07-1438 (Fed. Cir.)). 118 Barkoff, 2008. 117

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute appeal, the court will issue a mandate lifting the injunction – at the earliest, 21 days after the release of the opinion, though it could be longer if Sanofi requests a rehearing (Apotex, 2008). The petition thus insisted that other ANDA filers such as Dr. Reddy’s will be able to enter the market immediately after the Federal Circuit ruling, while Apotex will have to wait for the mandate. Apotex further argued this will irreparably harm the company and belie the purpose of the 180-day exclusivity provision (Apotex, 2008). In its arguments, Apotex appealed to fundamental Congressional intentions: “Granting Apotex a full 180 days of marketing exclusivity if it succeeds in its patent challenge would best effectuate congressional intent and policy underlying the Hatch-Waxman amendments in general and the exclusivity provision in particular…’to make available more low cost generic drugs.’”119 Apotex is “going to take some reputational damage from the FDA by filing such a ridiculous argument,” said Barkoff, crisply discounting the entire Apotex petition.120 In summary, the story of the Plavix brand and the subtleties of the recent generic challenge are salient on many levels. The long, expensive, and semi-empirical pharmaceutical development process that eventually engendered this blockbuster drug within the Sanofi laboratories is a modern-day illustration of the steep costs of drug innovation – which is intrinsically entangled with the high prices and strong patent protection associated with the branded pharmaceutical industry. The push by Apotex to obtain FDA approval to market a generic version of Plavix, which began in 2003 despite the fact that the listed (and later proven valid) patent was not due to expire until November 2011, shows how HatchWaxman has fostered the growth of litigious generic drug manufacturers – with firms such

See H.R. Rep. No. 98-857, pt. 1, at 14 (1984), reprinted in 1984 U.S.C.C.A.N. 2647, 2647. (quoting main purpose of Hatch-Waxman Amendments within the Apotex petition to stay further ANDA approvals). 120 Barkoff, 2008. 119

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From a Profitable Pharmaceutical Partnership to the Plavix Patent Dispute as Teva and Barr Labs among the ranks of Apotex. This increase in pharmaceutical litigation and the resulting propensity to engage in collusive dealings suggests that the Hatch-Waxman pilings may not be able to withstand this strain; the following section will address policy implications of the Plavix case, specifically in regards to the attempted pay-for-delay settlement agreement between Sanofi, BMS and Apotex. Finally, the decision of the District Court to grant a preliminary injunction, later upheld in the form of permanent injunctive relief, has repercussions for future cases discussing potential patent infringement. Sanofi v. Apotex established that a genus claimed in a prior art patent cannot anticipate one of the enclosed species without describing a pattern of preferences for that species. An additional legal precedent offered by Sanofi v. Apotex concerns the obviousness of enantiomer separation, which is a particularly significant issue for patents in the pharmaceutical industry. The District Court found that the disclosure of a racemate mixture does not render the individual enantiomers obvious, especially when the method of separation is not transparent and the value and activity of each enantiomer is unexpected. Finally, the framework of reasoning that the District Court took to evaluate the intricate and technical arguments proposed by each side – relying on the evidence documenting Sanofi’s experimental methodology and financial investment in developing the subject matter claimed in the patent-in-suit – has been alluded to in subsequent trials discussing pharmaceutical patent validity.

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Implications for “Pay-For-Delay” Policy

Chapter 7 - Implications for “Pay-For-Delay” Policy The Federal Trade Commission (FTC) and the media have used Plavix as a case study to focus public attention on loopholes within existing patent laws which allow companies to extend monopoly and delay entry of generics (Bhukhanwala, 2006). Underpinning this tension, the financial motivation for pioneer drug manufacturers remains, as always, to preserve monopoly (Rubenstein, 2007). They recognize that brand-name market share plummets immediately upon generic entry. Prices of brand-name drugs can never recover to previous levels, as BMS found in the months following the unleashed supply of generic clopidogrel from Apotex (Rubenstein, 2007; Bhukhanwala, 2006; Shuchman, 2006). The motivation on the part of pioneer pharmaceuticals to preserve profits abuts the public demand for broad and cheap consumer access to drugs. Seeking to lower costs, insurance companies and public programs such as Medicaid and Medicare use their clout – Medicare and Medicaid together provide pharmaceutical benefits for 88 million Americans – to contract with generic drug providers and apply downwards pricing pressure on brand-name pharmaceuticals (Carey, 2006b; Center on Budget and Policy Priorities, 2006; Jarvis, 2006; Bhukhanwala, 2006).

.The Pay-For-Delay Problem While many potential policy questions arise naturally from the complexity of the Plavix case, I plan to focus on the topic of “pay-for-delay” settlements in the U.S. pharmaceutical industry. “Pay-for-delay” settlements, alternatively referred to as pharmaceutical “sweetheart deals,”121 “reverse payments”122 and “exclusion(ary)

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Implications for “Pay-For-Delay” Policy payments,”123 pose a puzzle of immense and current importance to antitrust enforcement authorities (Hemphill, 2006; Hubbard, 2007). The pay-for-delay settlement arises as a way for a pharmaceutical innovator who holds a patent on a branded drug, and its rival, a generic drug maker seeking to market a generic version of that drug prior to patent expiration, to circumvent the costly litigation necessary to establish the patent’s (in)validity or (lack of) infringement by the proposed generic product. Though the terms of the agreement vary, the settlement essentially involves a payment made by the branded manufacturer to the generic manufacturer to ensure that the generic product remains off the market. People representing a spectrum of interest groups have criticized pharmaceutical legislation, primarily the HatchWaxman Act, for exacerbating the antitrust-patent conflict, encouraging excess litigation and thus spurring collusive settlement agreements. Case after case of pharmaceutical maneuvers have fueled the debate on whether the Act “encouraged competition as intended, or, instead facilitated the use of anticompetitive strategies to delay the marketing of generics” (Consumer Reports, 2001). A report by the Antitrust Division of the New York State Attorney General’s Office characterized the debate over reverse payments as “undoubtedly the most contentious antitrust issue pertaining to pharmaceuticals in the last few years” (Hubbard, 2007). The Federal Trade Commission charged that such settlements cost American consumers billions of dollars (Associated Press, 2007b). What are the boundaries of feasible antitrust regulation and enforcement on pay-for-delay settlement?

See, e.g., Schering-Plough Corp., 402 F.3d at 1068 (labeling the transaction a “reverse payment” because the plaintiff pays the defendant, contrary to the normal settlement where the plaintiff receives a lump sum payment from the defendant). 123 “Exclusion payment” including both the situation where the defendant (e.g. generic pharmaceutical firm) is either paid to not enter the market, or is paid to exit the market(Hovenkamp, Janis, and Lemley, 2002). See, e.g., In re Cardizem CD Antitrust Litig., F.3d 332 F.2d 896 (6th Circ. 2003); also In re Terazosin Hydrochloride Antitrust Litig., 164 F. Supp. 2d 1340, 1343 (S.D. Fla. 20) (prior to appeal to Eleventh Circuit court, which overturned this district court decision finding the agreement per se illegal). 122

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Implications for “Pay-For-Delay” Policy .Problem Statement: The policy discussion herein is addressed to the U.S. antitrust enforcement agency responsible for supervising the pharmaceutical industry, the Federal Trade Commission. The FTC monitors anticompetitive conduct such as collusion, monopolization and unlawful restraint and is the primary opponent of “reverse payment” settlement agreements in court. The Commission holds additional authority as “an advocate for competition in specific industries, filing comments before state legislatures and regulatory bodies about proposed legislation or regulation and how these proposals can best be adapted to permit competition to flourish” (Balto, 2000). In line with the FTC’s multiple capacities, my policy question is two-fold, and addresses (1) regulatory as well as (2) legislative control over pharmaceutical market behavior and the innovator firms’ propensity to “pay-for-delay.” The first question applies to the Federal Trade Commission and its relationship with the Courts; where have the Commission and the Courts differed in drawing the line of permissible competitive practices, and whose reasoning is more sound? I will propose recommendations for the FTC as it continues to seek certiorari from the Supreme Court to obtain a per se presumption of illegality for pharmaceutical patent litigation settlements involving a payment from the patentee to delay generic entry. The latter policy concern looks at the causal issue: should the Hatch-Waxman regulatory framework be amended to ablate these incentives for collusion? Again, I will review current legislative proceedings and make recommendations for the Commission’s future endorsement of Congressional actions.

.The Commission v. the United States Cou rt S ystem

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Implications for “Pay-For-Delay” Policy The Federal Trade Commission (FTC) found that in 2006, 14 out of 28 completed preexpiration patent challenges settlements involved reverse payments; just two years prior, in 2004, none of the settlements included reverse payment agreements (Federal Trade Commission, 2007). In fact, between 2000 and 2004, as a result of FTC oversight, no payfor-delay agreements were brokered and several generic entered the market prior to patent expiration (Shuchman, 2006). The recent spike in drug companies making deals to delay generics by paying off manufacturers has in part been attributed to the discrepancy in how the justice officials and antitrust authorities view such settlement agreements. As FTC commissioner Jon Leibowitz noted, “these settlements are becoming very common, and it’s not surprising, because the courts seem to have given their imprimatur to some extent” (Shuchman, 2006). Leibowitz is not alone in noting the inconsistency of the court decisions in antitrust cases involving reverse payments.124 There is a large body of legal precedents both allowing and rejecting settlements that were similar to the proposed Plavix agreement. Successful monopoly extension through “collusive dealing” (Anthony, 2000) has been investigated in The Commission v. Schering Plough and Upsher-Smith, as well as in The Commission v. Abbott-Geneva (Balto, 2000; Soehnge, 2003). A successful “exclusivity” settlement was also engineered between a pioneer pharmaceutical and generic manufacturer in Pharmachemie B.V. v. Barr Labs (Balto, 2000). More recently and prominently, two Supreme Court cases pitched the Commission against pharmaceutical companies involved in pay-for-delay settlements in

Compare In re Cardizem CD Antitrust Litig., 332 F.3d 896, 908 (6th Cir. 2003) (“[T]he …agreement to pay Andrx $40 million per year not to bring its generic product to market and compete with Cardizem CD…is a naked, horizontal restraint of trade that is per se illegal because it is presumed to have the effect of reducing competition in the market for Cardizem CD and its generic equivalents to the detriment of consumers…”) with Valley Drug, 344 F.3d at 1310-11 (“[W]e do not think that a payment from the patentee to the alleged infringer should be automatically condemned under the antitrust laws…”). 124

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Implications for “Pay-For-Delay” Policy 2006. In both cases, the Supreme Court denied certiorari to the FTC’s appeal against lowercircuit rulings that the reverse payments were not anticompetitive. These cases deserve more detailed scrutiny. They demonstrate the aggressive stance of the FTC in pursuing reverse payment settlements, and the Supreme Court’s implicit approval of these pay-for-delay deals. In both cases reviewed by the Supreme Court, ScheringPlough v. Federal Trade Commission125 and In re Tamoxifen Citrate Antitrust Litigation,126 the Supreme Court declined to further review the FTC’s charge that the payments were anticompetitive. The Supreme Court’s toleration of settlements including reverse payment in these two decisions has subsequently fostered an explosion in subsequent innovator-generic agreements in spite of the FTC’s efforts to keep them at bay (Hubbard, 2007), in effect, opening up ‘a Pandora’s box’127 of anticompetitive settlements. .Pay-For-Delay in Schering-Plough The Schering-Plough128 case arose after Schering-Plough entered into two settlement agreements involving payments to the generic firm to delay generic entry for an extended release formulation of the widely prescribed potassium chloride supplement, K-Dur 20. The Commission sued, charging that the payments were anticompetitive because they were made in exchange for preventing imminent generic launches and thereby harmed consumers by withholding the cheaper, generic alternative to K-Dur 20.

402 F.3d 1056 (11th Cir. 2005), cert denied, ___ U.S. ___, 126 S. Ct. 2929 (2006). 429 F.3d 370 (2d Cir. 2005). 127 See Supplemental Brief for the Petitioner at 2, Shering-Plough Corp., 126 S. Ct. 2929 (No. 05-273), 2006 WL 1647529, as discussed in (Ponsoldt, 2006). 128 Schering Plough v. Federal Trade Commission 402 F.3d 1056 (11th Cir. 2005), cert denied, ___ U.S. ___, 126 S. Ct. 2929 (2006). 125 126

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Implications for “Pay-For-Delay” Policy The Court of Appeals for the Eleventh Circuit vacated the FTC findings, instead applying the framework outlined in Valley Drug Co. v. Geneva Pharma129 to examine: (i) the scope of the patent’s exclusionary effect; (ii) whether or not, and to what extent, the agreement exceeded that scope; and (iii) the resulting anticompetitive effects. Under this analysis, the Court found that the branded company could claim the full exclusionary effect of the patent since the FTC had not proved the patent to be invalid or not infringed. Furthermore, presuming patent validity, the Court of Appeals declared there was insufficient evidence to argue (1) that the payments were exchanged for delayed generic entry, and (2) that the payments had an anticompetitive effect. The FTC appealed to the Supreme Court for certiorari, challenging the decision that the payment was not anticompetitive, and also alleging that the Court of Appeals had failed to recognize the facts presented by the FTC. The Eleventh Circuit has largely ruled in favor of the pharmaceutical agreements over the complaints of the Federal Trade Commission, in marked contrast to the Sixth Circuit, which has found reverse payments to be per se illegal. 130 The FTC cited this disparity in opinion between courts as a major reason that the case warranted review, and wrote in the petition that “the economic implications of the court of appeals’ ruling, which invites collusive agreements between branded drug companies and generic challengers, are staggering,”131 given that American consumers spend well over a hundred billion dollars each year on prescription drugs (Kaiser Family Foundation, 2005). An amicus brief filed by the states supported the FTC’s petition for review. However, the Supreme Court asked the United

344 F.3d 1294 (11th Cir. 2003). In re Cardizem CD Antitrust Litigation, 332 F.3d 896, 908 (6th Cir. 2003), cert denied, 544 U.S. 1049 (2005). 131 See Petition for a Writ of Certiori, as discussed in Federal Trade Commission v. Schering-Plough Corporation, 402 F.3d 1056 (11th Circuit 2005), cert denied, ___ U.S. ___, 126 S. Ct. 2929 (2006). 129 130

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Implications for “Pay-For-Delay” Policy States for its view. The Solicitor General, on behalf of the United Sates,132 submitted an amicus brief recommending that the Supreme Court deny the petition for certiorari, and the Supreme Court acquiesced to this advice, thus upholding the lower court’s ruling to allow the reverse payment agreements. .Pay-For-Delay in Tamoxifen In contrast to the FTC’s direct involvement in Schering-Plough, the Tamoxifen133 case was initiated by consumer action against the pharmaceutical companies. At issue was an agreement forged between patent-holder Zeneca and the generic contender Barr Laboratories to settle patent litigation on the cancer drug Tamoxifen; the settlement was crafted while the case was on appeal after a District Court had found the patent invalid. In the agreement, the parties agreed to vacate the District Court decision that the patent was invalid. Zeneca would pay Barr $21 million and grant a non-exclusive license to market branded Tamoxifen in exchange for Barr agreeing to delay entry of the generic version until patent expiration. The plaintiffs charged that the payment clause within the settlement agreement was anticompetitive on several grounds: (1) the amount of the payment was termed “excessive,” allegedly exceeding the generic drug’s projected revenues; (2) the timing was suspect, since the two firms entered into the settlement after the District Court found the patent invalid; and (3) the payment ultimately potentiated the delay of generic entry and the agreement to vacate the District Court decision. However, the District court dismissed the case, finding no evidence of suspicious or anticompetitive conduct in the settlement. The

The United States Solicitor General is appointed to argue on behalf of the government of the United States in front of the Supreme Court, and typically represents the views of the executive branch, e.g. currently those of the Bush administration; Barkoff, 2008. 133 In re Tamoxifen Citrate Antitrust Litigation, 466 F.3d 187 (2d Circuit 2005). 132

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Implications for “Pay-For-Delay” Policy Court of Appeals for the Second Circuit upheld this decision, affirming that the settlement should be considered legal based on (1) policy favoring settlement, (2) a patent’s inherent presumption of validity, and (3) the lower court’s framework of evaluating the settlement as within the scope of the patent. In addition, the Second Circuit opined that an allegation of “excessive payment” alone was not sufficient to constitute a valid antitrust claim. Similar to Schering-Plough, the plaintiffs filed a petition for certiorari, asking for the Supreme Court’s clarification on the following question: “Whether the federal antitrust laws prohibit a brand name drug patent holder and a prospective generic competitor from settling patent infringement litigation by agreeing that the generic manufacturer will not challenge the validity of the patent or market its own version of the drug until the expiration of the patent, in exchange for a substantial payment from the patent holder.”134 Again, as in Schering-Plough, the Supreme Court requested to hear the views of the United States, and again, the Solicitor General submitted an amicus brief suggesting that the Supreme Court ought to deny certiorari. The brief argued that though the Court of Appeals had not fully scrutinized the settlement in question, this was outweighed by the problem that the Tamoxifen case was “not a good vehicle for resolving the question presented,” as the factual setting was “unlikely to recur” and the FTC’s allegations of antitrust were not wellfounded (Hubbard, 2007). Again, the Supreme Court followed the lead of the amicus brief and turned down the request for certiorari. Twice stung by the Supreme Court’s refusal to review the lower District rulings that allowed reverse payments, the FTC has redoubled its efforts to effect change via regulatory movements, especially since the bills for legislative action have been stalled by a concerted

134

In re Tamoxifen Citrate Antitrust Litigation, 466 F.3d 187 (2d Circuit 2005).

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Implications for “Pay-For-Delay” Policy push by innovator and generic pharmaceutical industry lobbyists to protect their right “to settle patent litigation as they please.”135

.Plavix an d Pay-For- Delay The distinct events and opinions of the Plavix pay-for-delay settlement can be projected against the milieu of most relevant legal analyses of the “reverse payment” phenomenon along with selected previous court decisions. From a policy and legal standpoint, the regulatory setting of the proposed Plavix settlement was unique in that the companies were required to obtain regulatory approval from the FTC and state attorneys general prior to the consummation of the agreement. Balto suggested that in antitrust circles, the proposed Plavix deal will not be memorable since it was never litigated.136 Nonetheless, we can use the story of the settlement, and the consequences once the deal was dissolved – a generic “at-risk” launch and a long litigation trial that ultimately proved the patent valid – to frame the policy problem of pay-for-delay settlements, and to help propose a solution. The attempted Plavix settlement agreement was an aberration among pharmaceutical deals of its kind. It stands as a surprising case not because of the attempted pay-for-delay settlement, but because Apotex called Sanofi’s bluff -- launching generic clopidogrel bisulfate at-risk of heavy legal and financial retribution from Sanofi. In one sense, the debacle surrounding the Plavix deal serves, perversely, to support a presumption of per se illegality around such pay-for-delay settlements.

135 136

Barkoff, 2008. Balto, 2008.

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Implications for “Pay-For-Delay” Policy The FTC commissioner Leibowitz echoed this idea in 2006, making a distinction between most pay-for-delay agreements and the release of generic Plavix after the proposed settlement was dissolved. “This isn’t a harbinger of more competition to come. It’s the exception that says if we don’t change the rules, every other time what we’re going to see is reasonable companies…making payments to keep generic out of the market and generics [companies] accepting those payments because they make more money by staying out of the market than by competing. For the most part, consumers are going to be left…footing the bill, while pharmaceutical companies make these anticompetitive agreements that keep drugs out of the market”(Shuchman, 2006). Yet, conversely, the proposed Plavix settlement and the chaos that ensued after its dissolution by antitrust enforcement authorities can also support the opposite argument, against the per se illegality of reverse payments. As both Balto and Barkoff suggested, the propensity of a pharmaceutical innovator to settle should not be viewed in the justice system as an innovator’s internal reflection on the relative weakness of its patent. The converse may be true, and certainly holds for the Plavix case: “there’s an argument that if patent is strong, the company will [want to settle] for predictability [of revenues] and certainty.”137 Settlement affords branded companies the chance to dispose of litigation with a neat and predictable sum of money; for generic companies, the “pay-for-delay” scheme can compensate them for taking the risk of challenging the patent prior to entry. The Plavix example – wherein the two-week launch of generic clopidogrel was essentially “a gift to consumers” and at least in hindsight, there was no anticompetitive conduct because the patent was held valid – demonstrates that a settlement involving such a reverse payment ought not imply nefarious objectives or a weak patent in the involved parties.

137

Barkoff, 2008.

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Implications for “Pay-For-Delay” Policy .Congress and Courts: Calibrating the Reach of Antitrust Enforcement Despite recent failures, the FTC has not ceased its efforts to bring a reverse payment case to the Supreme Court. In fact, the Commission “is really dead set on having the Supreme Court review a pay-for-delay case,” said Barkoff. Both Barkoff and Balto, on separate occasions, raised the FTC’s current strategy of attempting to engineer a ‘division split’ within the United States Court system, which would make their case more likely to receive Supreme Court review. In Balto’s opinion, the FTC has little chance of obtaining certiorari, since courts up to this point are very wary of denying companies the right to settle. “The FTC’s course here is a very difficult one,” said Balto. “Any judge trying to write a decision here has to rely on past decisions, and the past decisions are well put together.” Another problem with proscribing reverse payment settlements arises from the litigation perspective. Balto urged to “look at how the courts function: settlements are essential…otherwise courts would be overwhelmed.” Balto pointed out that settlements are crucial to keep the judicial system moving, and are thus far more commonplace than trials or hearing.138 Nonetheless, I recommend that in the regulatory realm, the Federal Trade Commission should continue to pursue certiorari with the Supreme Court, as the current conditions of ambiguity around the legality of pay-for-delay settlements is only producing more litigation, and at the extreme, the potential for explosive situation such as the at-risk launch of generic Plavix. However, the Commission needs to reevaluate the intersection of patent law and antitrust concerns that are specific to the pharmaceutical industry. The circumstances of anticompetitive behavior in the pharmaceutical industry necessarily demand a different judicial treatment than run-of-the-mill antitrust cases in other industries.

138

Balto, 2008.

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Implications for “Pay-For-Delay” Policy The essential question of any antitrust violation, as Balto explained, is to explain what would have happened ‘but for’ the allegedly anticompetitive act. The ‘but for’ analysis “is a black and white rule,” said Balto, but in the case of the pharmaceuticals, this presents a paradox: the relevant alternative to the settlement is to litigate the patent, the long patent litigation being precisely what the settlement is designed to avoid. Thus far in antitrust cases on agreements forged to preemptively settle patent litigation, “these parties have really eschewed trying to demonstrate what would have happened during the patent litigation,” Balto said, which poses a problem for the Courts as they struggle to determine what the ‘but for’ would have been. “As a practical matter, you don’t necessarily want to come down with a separate patent litigation… So unless there’s an obvious sham, the courts won’t say a patent is invalid.”139 This presumption of patent validity in courts leads to an obvious predicament, and Courts have largely ignored this inconsistency in favor of presuming the validity of a patent during antitrust litigation, even in the case where a lower court has marked the patent invalid.140 In light of this analysis, I believe that the FTC might have more success in regulating settlements with pay-for-delay clauses if they encourage Courts to take a more moderate stance in appraising such settlements. I recommend that they take heed of the legal commentators advocating Courts to apply rule-of-reason rather than making a blanket presumption of antitrust infringement (Hemphill, 2006; Ponsoldt, and Ehrenclou, 2006). This approach of reviewing each case would not presume settlement legality nor would it abide by a rule of per se illegality (Hemphill, 2006). This approach entails applying a

139 140

Balto, 2008. In re Tamoxifen Citrate Antitrust Litigation, 466 F.3d 187 (2d Circuit 2005).

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Implications for “Pay-For-Delay” Policy rebuttable presumption of unlawfulness when a patentee pays an alleged infringer to preserve the patentee’s monopoly during the course of patent infringement litigation (Ponsoldt, 2006). “Reverse payments should not be made per se illegal,” said Barkoff, though qualifying his statement with the disclaimer that his legal practice does not focus on antitrust litigation.141 “I think that any analysis of the antitrust case has to look at the underlying weakness or strength of the patent, at least in order to gage the antitrust implications of the settlement…[Courts] can’t just stop at the presumption of validity.” Under this regime, a proposed settlement such as the one negotiated between Sanofi/BMS and Apotex might have been presumed unlawful because of the reverse payment. However, Sanofi could then have appealed this presumption by demonstrating its ability to prove patent validity. In hindsight, the attempted settlement would not be anticompetitive, as Sanofi held a valid patent and was thus justified in maintaining exclusivity.

.Arguments Justifying P ay-For- Delay Are these pay-for-delay settlements really exploiting provisions of the Hatch-Waxman Act? As the FTC moves forward in attempting Supreme Court review on this issue, the Commission should note the compelling arguments for the permission of such settlements within the legal constructs of Hatch-Waxman legislation that have been proposed in various court opinions. Scott Hemphill, a legal professor at the New York University School of Law, characterized “the promotion and delay of litigation” as “central preoccupations of the regulatory regime” in the Hatch-Waxman Act (Hemphill, 2006). Let us characterize various

141

Bartoff, 2008.

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Implications for “Pay-For-Delay” Policy justifications for a court’s willingness to approve sweetheart deals and condone the allocative harm they perpetrate. .1.

A Judicial Intuition to Promote Settlement

Hemphill suggested that the common judicial reflex favoring settlement is intensified by the properties inherent to pharmaceutical litigation (Hemphill, 2006). Patent policy in the United States generally reflects a preference for innovation, at the expense of competition and immediate consumer access, as well as a preference for settlement (Hemphill, 2006). Furthermore, pharmaceuticals have been associated with a need for strong patents that are “strong, stand alone, and confer almost total control over subsequent uses of the product” (Kaiser Family Foundation, 2005). The judicial impulse to favor settlement over prolonged litigation is reacting to the highly scientific and technical subtleties of pharmaceutical cases, combined with the large expense of litigation. Such courts value the ability of the parties to best represent their own interests, recognizing the antitrust liability though they discount the weight of consumer or “public” interest in this balance. In fact, “Courts don’t think there’s a public interest in the settlement. [That idea] makes judges uncomfortable,” said Balto.142 One view aired during a 1902 Supreme Court case has resonated through subsequent legal decisions in the last century; addressing a license agreement that sought to settle copious litigation on the validity of multiple pharmaceutical patents, the Court opined that settling patent litigation is “a legitimate and desirable result in itself.”143 In re Ciproflaxacin Hydrochloride Antitrust Litigation,144 the court worried that rules restricting settlements would attenuate

Balto, 2008. Bement v. Nat’l Harrow Co., 186 U.S. 70, 93 (1902). 144 363 F. Supp. 2d 514, 529 (E.D.N.Y. 205). 142 143

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Implications for “Pay-For-Delay” Policy desirable settlements. Professor Robert Mnookin’s testimony,145 which emphasized the economical benefits conferred by settlements in avoiding undue litigation expense, helped sway the decision in Schering-Plough. At times, the FTC has weighed in with a moderate opinion on settlements. “To be sure, settlements are usually a good thing,” Commissioner Leibowitz said in a 2006 speech (Leibowitz, 2006). “From my perspective – and the perspective of many – America is too litigious a society. Too often people say, ‘I’ll see you in court,’ rather than ‘let’s work this out.’” He tempered this comment, however, cautioning that continued acceptance of reverse payments would shift the incentives for generic drug firms away from market entry. “The goal in filing an ANDA won’t be to work hard to be first to market. Instead, it will be to work hard to position yourself to be first to settle” (Leibowitz, 2006).

.2.

Distortion of the Incentives to Innovate and Challenge Patents

A second argument cites the incentive distortions produced by restricting pay-for-delay settlements. Courts have recognized both the potential negative effect on the patentee’s incentive to innovate, and the corollary restrictions on the infringer’s incentive to challenge patents.146 As can be imagined, industry groups are proponents of this view as well. Bill Tauzin, the chairman and CEO of the Pharmaceutical Research and Manufacturers of America, echoed, “A total ban on settlements in which the brand name company gives something of value to the generic could stop proconsumer settlements, reduce the value of patents, and reduce incentives for innovation” (Lamb, 2007).

No. 9297, 2002 WL 1488085, ¶ 384 (F.T.C. June 27, 2002). Asahi Glass Co v. Pentech Pharm., Inc., 289 F. Supp. 2d 986, 994 (N.D. I11. 2003) (Quoting Posner, J.: “A ban on reverse-payments would reduce the incentive to challenge patents by reducing the challenger’s settlement options should he be sued for infringement…”) 145 146

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Implications for “Pay-For-Delay” Policy The prohibition of all settlements involving such a reverse payment results in certain settlements being unequivocally unattainable, which presents its own conundrum. If every pharmaceutical patent confrontation instigated by an ANDA-IV application must be fully litigated without the option of preliminary settlement, this may deplete the supply of innovative entrants (e.g. generic drug manufacturers).

.3. The risk of legal extrapolation from pharmaceutical cases to general pay-for-delay situations While the settlement is a Pareto improvement for both parties involved, the agreement typically brings less consumer benefit then the litigation would – if taken to conclusion. However, claiming a general public interest on behalf of consumers in these pharmaceutical cases “makes judges uncomfortable,” said Balto,147 who pointed out that the antitrust agencies and FTC disagree and actively try to argue for the public’s interest. For the Courts, however, not only is it dubious whether the public interest ought to be entitled to an “expected outcome of the avoided litigation” (Hemphill, 2006), but there is also the risk that the same general right could be claimed to prevent settlements in other industries.148 .4.

Reverse Payments are a “Natural By-Product” of Hatch-Waxman Regulation The views described above essentially argue that the expected allocative losses from a payfor-delay settlement ought to be tolerated, as they are merely a consequence of existing legislation. Some Courts have in essence justified these sweetheart deals. By recognizing that generic firms face very little risk yet can reap high rewards in challenging patents under the

Balto, 2008. See, e.g., Cipro, 363 F. Supp. 2d at 529 (worrying that limitations on settlement rule “could not logically be limited to drug patents, and would work a revolution in patent law”). 147 148

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Implications for “Pay-For-Delay” Policy current regulatory framework of Hatch-Waxman legislation, whereas the branded pharmaceutical manufacturers do not gain from generic entry but could lose a sizable fortune along with patent monopoly, Courts have determined that “[r]everse payments are a natural by-product of the Hatch-Waxman process.”149 In fact, this argument was used successfully against the FTC in Schering-Plough.

.The Commission an d Congress I will first provide a brief summary of the U.S. laws relevant to patents and to the competing interests of pioneer versus generic drug manufacturers, before evaluating the Plavix proceedings in the context of the policy question. I will then present policy recommendations on how the Federal Trade Commission can proceed to address these issues in guiding future Congressional action to amend the regulatory framework of the pharmaceutical industry in the United States. .History of Pharmaceutical Regulation in the United States

)The Hatch-Waxman Act Prior to the 1962 Amendment of the Federal Food, Drug and Cosmetic Act, drug regulation was limited to addressing safety concerns (Mossinghoff, 1999). The United States had not attempted to systematically regulate the pharmaceutical industry or the intellectual property rights of medicinal products. The Amendments, made under Public Law 87-781 in 1962, instituted a formal drug approval process for generic equivalents of pioneer drugs, and revolutionized patent protection and monopoly rights.150 This landmark legislation remained

149 150

Schering-Plough v. FTC, 402 F.3d 1056, 1074 (11th Cir. 2005). Drug Amendments Of 1962 To The Federal Food, Drug And Cosmetic Act Of 1938. Section 1.C.

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Implications for “Pay-For-Delay” Policy largely untouched until 1984, when Congress made significant amendments to the Food, Drug and Cosmetic Act (FDCA) that would change the way that both new and generic drugs would be approved and regulated. As part of the FDCA package, Congress passed the Drug Price Competition and Patent Restoration Act (Public Law 98-417), primarily to address the perceived imbalance between the interests of pioneer drug developers to protect patents, and the considerable need of American consumers for lower-cost generic versions of these pioneer prescription drugs.151 This definitive legislation, popularly referred to as ‘Hatch-Waxman’ for its authors in Congress, simplified the approval process for generic entry and extended patent protection for pioneer drug manufacturers to allow them to cope with the significant regulatory barriers on the path to FDA approval(Soehnge, 2003). In 1984, Senator Orrin Hatch (R-Utah) said the following about his cosponsored bill: “The public receives the best of both worlds – cheaper drugs today and better drugs tomorrow” (Epstein, 2004). Does this still ring true today amidst the noise of costly litigation, coercive arrangements between innovator and generic drug manufacturers and regulatory antitrust interference? Measured by the growth of the generics industry and the change in prescription rates, the Hatch-Waxman Act has been a tremendous success. In 1984, generic drugs accounted for only 18.6% of U.S. prescriptions (Frank, 2007). In 2007, a full 63% of U.S. prescriptions were written for generic products (Frank, 2007). In large part, this jump can be attributed to the decrease in regulatory burden promulgated by Hatch-Waxman. Prior to the Act, obtaining FDA approval for a pharmaceutical product was essentially the same whether

Hatch-Waxman Act refers to the Drug Price Competition and Patent Term Restoration Act of 1984, Pub. L. No. 98-417, 98 Stat. 1595 (primarily codified at 21 U.S.C. § 355 (2000 & Supp. IV 2004). 151

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Implications for “Pay-For-Delay” Policy or not the proposed drug was equivalent to existing, approved drugs in the market or the first of its kind. Each application for a new drug product had to include the submission of results from an expensive battery of preclinical (animal) and clinical (human) trials showing the drug’s efficacy and safety. This presented an enormous barrier to generic manufacturers, few of which had the depth of R&D and the financial capabilities to hurdle these regulatory obstacles. One of the critical tenants of the Hatch-Waxman legislation was to streamline the approval process for generic versions. Section 505(j) 21 U.S.C. § 355(j), outlined the process whereby generic drug manufacturers can file Abbreviated New Drug Applications (ANDAs) to seek FDA approval of the generic (Mossinghoff, 1999). As its name suggests, an ANDA is a shortened version of the NDA required by a pioneer company wishing to produce and market a new drug for a particular use. It is no longer necessary for generic manufacturers to perform costly tests to prove the safety and efficacy of a drug that has previously been NDA-approved (Dimasi, 2003). The law152 allows the generic to bypass those tests and obtain ANDA approval by merely establishing bioequivalence and pharmaceutical equivalence of the pioneer drug and the proposed generic copy.153 Proving bioequivalence, which normally requires less than $1 million, is far less expensive than conducting NDA

21 U.S.C. § 355(j) (2) (A), (8)(B) (2000). Bioequivalence is defined between two pharmaceutically equivalent agents where in vivo rates and absorption levels are not statistically different when administered to patients at the same molar dose under similar experimental conditions(21 U.S.C. § 355(j) (8) (B) (2000)). Pharmaceutical equivalence, as defined by the FDA, is drug products containing the same active ingredient(s), same dosage form and route of administration, and identical strength or concentration(Center for Drug Evaluation and Research, 2007).) Generic drugs that are both pharmaceutically equivalent and bioequivalent are thus bestowed with the label of ‘therapeutic equivalence’ from the FDA, allowing them to be marketed as substitutes for their reference-listed counterparts(Center for Drug Evaluation and Research, 2007). Pharmaceutically equivalent drugs may, however, differ in shape, scoring, excipients / inactive fillers (e.g. colors, flavors, preservatives), the release mechanism, and labeling(Center for Drug Evaluation and Research, 2007). 152 153

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Implications for “Pay-For-Delay” Policy clinical trials.154 The Act also redefined the financial incentives for both innovator and generic firms. As multiple courts argued in their application of Hatch-Waxman, the combination of patent laws and food and drug legislation struck “a balance between two competing policy interests: (1) inducing pioneering research and development of new drugs and (2) enabling competitors to bring low-cost, generic copies of those drugs to market.”155

)Evaluating Hatch-Waxman as a Balance of Patent/Antitrust Law Hatch-Waxman can also be viewed as an industry-specific regulatory regime attempting to navigate the intersection between antitrust and intellectual property law.156 One judge, in a statement whose logic has since been enshrined as classic, asserted his opinion on the lack of conflict at this juncture between antitrust and intellectual property: “A patent, under the statute, is property. Nowhere in any statute is a patent described as a monopoly…That the property right represented by a patent, like other property rights, may be used in a scheme violating antitrust laws creates no conflict between laws establishing any of those property rights and the antitrust laws…It is but an obfuscation to refer to a patent as ‘the patent monopoly’ or to describe a patent as an ‘exception to the general rule against monopolies’.”157 In contrast, many others find the methods taken to achieve these two legal realms inherently in conflict: antitrust law strives for allocative or “static” efficiency, distributing resources to avoid the distortion produced by monopolies wherein consumers faced with high prices

See Requirements for Submission of In Vivo Bioequivalence Data; Proposed Rule, 68 Fed. Reg. 61,640, 61,645 (Oct. 29, 2003). 155 Quoted in Andrx Pharm, Inc. v. Biovail Corp., 276 F.3d 1368, 1371 (Fed. Cir. 2002); Also quoted in AAIPharma Inc. v. Thompson, 296 F.3d 227, 230 (4th Cir. 2002). 156 See, e.g., (Hovenkamp, Janis, and Lemley, 2002), §1.3 . 157 See Carl Schenck, A.G. v. Nortron Corp., 713 F.2d 782 (Fed. Cir. 1983) (Judge Markey’s opinion). 154

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Implications for “Pay-For-Delay” Policy must choose less desirable substitutes.158 In championing dynamic efficiency, patent law conversely can encourage high prices and even permit monopoly as a method of rewarding innovation.159 Pharmaceutical pioneers pay a high price for innovation. A recent report in the Journal of Health Economics estimated the average research and development costs per new drug to be $407 million out-of-pocket in year 2000 dollars when the expenditures for failed compounds were factored in; the costs of conducting the clinical trials necessary for FDA approval comprised over half of that staggering bill (DiMasi, 2003). Competitive, profitpreserving practices often generate dynamic benefits alongside allocative distortions. Experts have taken differing views on how to appraise the patent-antitrust paradox and on how to find the optimal balance of legislation for the pharmaceutical industry.

)Hatch-Waxman Eases Generic Entry – Making It Too Easy? The Hatch-Waxman Act fulfilled its primary goal of redistributing the risks of generic litigation, but detractors suggest that the Act favored generic companies by granting them the right to challenge the validity of an existing patent without incurring the financial risk of damages from patent infringement (Behrendt, 2002; Soehnge, 2003). A review of the protocol for obtaining FDA approval for a proposed generic drug will segue into a conversation on some of the more contentious statutes in Hatch-Waxman and their significance in mediating the industry-specific antitrust-patent axis of policy. For

See, e.g., Richard A. Posner, Antitrust Law 9-32 (2nd Ed., 2001); Daniel A. Crane, Exit Payments in Settlement of Patent Infringement Lawsuits: Antitrust Rules and Economic Applications, 54 Fla. L. Rev. 747 (2002). 159 See, e.g.,Keith Leffer and Cristopher Leffler, Efficiency Trade-Offs in Patent Litigation Settlements: Analysis Gone Astray?, 39 University of San Francisco Law Review, (2004); Louis Kaplow, The Patent-Antitrust Intersection: A Reappraisal, 97 Harvard Law Review, 1813, 1822 (1984) (“profit plays a central role, because it serves a as a reward – and in turn, an incentive – for the inventive activity that produces the benefits of the patent system.”). 158

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Implications for “Pay-For-Delay” Policy every patent relating to the NDA-approved drug, the ANDA applicant must include the pertinent patent certifications and explain why its ANDA does not infringe on any valid claims of existing patents. Two types of certifications can be submitted: (1) a “paragraph” certification160 can declare one of four explanations - that the patent in question (paragraph I) has not been filed, (paragraph II) has expired, (paragraph III) will expire, or (paragraph IV) has not expired but is invalid or will not be infringed; (2) the other type of certification is a “section eight” statement, explaining that “the method of use patent does not claim” the use for which the ANDA was submitted.161 ANDAs directed at marketing a generic drug after patent expiration, which claim either Paragraph I, II or III certification, have not garnered much attention. For our purposes, the only pertinent type of ANDA applications are those seeking to secure generic entry prior to patent expiration via Paragraph IV certification (hereafter referred to as ANDA-IVs). ANDA-IVs are effectively challenging existing patents held by pioneer pharmaceutical companies. Accordingly, there are significantly more regulations concerning the process of submitting an ANDA under paragraph IV certification. Despite extra regulation, filing ANDA-IVs almost invariably results in charges of infringement, followed by a lengthy litigation and/or settlement process. An ANDA-IV has “important legal ramifications. It automatically creates a cause of action for patent infringement.”162

21 U.S.C. § 355(j)(2)(A)(vii). 21 U.S.C. § 355(j)(2)(A)(viii). 162 Mylan Pharms., Inc. v. Shalala, 81 F. Supp. 2d 30, 32 (D.D.C. 2000). 160 161

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Implications for “Pay-For-Delay” Policy

)Legal Ramifications of the ANDA-IV The topic of paragraph IV certifications was highly contentious during the creation of the Act, as research industry advocates pressed legislators to create a window of time between when the FDA rules that a given patent is in fact invalid or not infringed by an ANDA application, and when the generic drug is officially approved for market entry (Mossinghoff, 1999; Soehnge, 2003). As the law stands now, any FDA-approved drug containing a novel active ingredient is protected from ANDA-IV applications for the first four years after NDA approval.163 Furthermore, whenever a generic files an ANDA-IV application, the generic company must notify the patent owner and NDA holder via a detailed (2)(b)(i) notice. The pioneer then has 45 days to file an infringement action.164 During this period, the FDA does not take any action on the generic application. If the pioneer does not make a timely suit against the ANDA-IV applicant, FDA approval “shall be made effective immediately”.165 If the infringement suit is filed within the forty-five-day period, however, by law the NDA holder (pioneer company) can enjoy up to another thirty months of market exclusivity before generic entry is approved.166 The suspension of the generic application is lifted at the earlier of (1) thirty-months, or (2) resolution in a district court, determining that the patent in question is invalid or not infringed.167 The thirty-month stay significantly alters the incentives between pioneer and generic companies; the thirty-month stay has been likened to an automatic preliminary injunction – which courts would shy from issuing in normal patent

See 21 C.F.R. § 314.10(a) (2006) defines these novel products as drugs containing no “active moiety” previously approved in another NDA; 21 U.S.C. § 355(j)(5)(F)(ii) (Supp. III 2003) regulates the protection period following NDA approval of such drugs. 164 21 U.S.C. § 355(j)(2)(B)(iii) (Sup. IV 2004); 21 C.F.R. § 314.95(f) (2007). 165 21 U.S.C. § 355(j)(5)(B)(iii). 166 21 U.S.C. § 355(j)(5)(B)(ii); 21 C.F.R. § 314.95(f). 167 21 U.S.C. § 355(j)(5)(B)(iii)(I). 163

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Implications for “Pay-For-Delay” Policy litigation for other industries – on the generic pharmaceutical product. In practice, the “thirty-month” stay decreed by Hatch-Waxman can last for more than three years. Up until the Medicaid Modernization Act in 2003, in fact, a common maneuver utilized by pharmaceutical companies involved stringing together a succession of thirty-month stays to block generic entry indefinitely.168 Since the introduction of Hatch-Waxman, ANDA-IV applications have become the standard route through which generic companies can form a formidable challenge to the monopoly of brand-name drugs still under patent protection. Data collected from the FTC indicated that more than 200 pre-expiration challenges were filed between 1984 and 2003, at an increasing rate that has continued to rise (Federal Trade Commission, 2002; Timothy Muris, 2003). ANDA-IV applications represent only a small fraction of total generic filings. The same FTC study noted that ninety-four percent of ANDA applications between 1984 and 2000 did not attempt to obtain Paragraph IV certification (Federal Trade Commission, 2002). The calculated choice of a generic company to mount such a patent challenge is in large part based on financial incentives; ANDA-IVs are predominantly filed for blockbuster drugs, where the relatively low chance of winning the patent challenge is balanced by the potential for extraordinary high returns. In contrast to the relatively low rate of ANDA applications specifying Paragraph IV certification, nine out of the ten best-selling drugs in 2000 (as reported by the New York Times) attracted generic challenges, and a different version of the tenth was eventually challenged by an ANDA-IV application (Center for Drug

This method relied on the single drug product in question being covered by multiple patents. The pioneer could list new patent information during the thirty-month stay, thereby requiring the generic firm to file another ANDA certification addressing the new patent information. The pioneer could sue, triggering another thirty-month stay. If the pioneer then listed new patent information, the cycle would start yet again. 168

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Implications for “Pay-For-Delay” Policy Evaluation and Research, 2008; Pear, 2001). Generic versions of at least four of these best sellers – Paxil, Prilosec, Prozac, and Zocor – made it into the market prior to patent expiration (Biotech Week, 2003; Worldwide Biotech, 2006; Dickinson, 2000; Sherrid, 2000; Tesoriero, 2006). The contention that “with respect to the most important new drugs, litigation is the norm, not the exception,” has become increasingly true.169 The (2)(b)(i) notice provided to the pioneer must include a detailed opinion explaining the reasoning behind the ANDA-IV filer’s assertions of patent invalidity or infringement.170 Generics have contended that the patent in question was invalid for various reasons. Several successful cases against the patent’s validity alleged it was inherently anticipated by prior art,171 was an obvious-type double patent,172 was procured using inequitable conduct,173 or was invalid because clinical testing prior to approval violated the public use bar.174 Alternatively, generics have also successfully claimed that their version of

See (Hemphill, 2006); But cf. (Epstein, and Kuhlik, 2004) (asserting “[W]hatever the dramatic tales in individual cases, litigation is the exception and not the norm. In the vast majority of cases – approximately 95 percent of the time – generics are content to wait until patent expiration to begin commercial sales (although recent trends point toward more patent challenges).”). 170 21 U.S.C. § 355(j)(2)(B)(i)-(ii); 21 C.F.R. §§314.50(i), 314.94(a)(12) (2002) (describing the required elements of patent certification in an ANDA application). 171 In SmithKline Beecham v. Apotex Corp., 403 F.3d 1331, 1334 (Fed. Cir. 2005); In KSR Int'l v. Teleflex, 550 U.S. ___ (2007) (upholding the TSM test in the Opinion of the Court. Courts have typically applied the “Teaching, Suggestion or Motivation” (“TSM”) test to evaluate invalidity charges of anticipation based on prior art. The TSM test calls an invention obvious and unpatentable if and only if the prior art references include some teaching, suggestion or motivation that would lead to the invention in question. The prevalence of combined inventions for drug formulations make the TSM test especially relevant in pharmaceutical patent litigation(Aaron F. Barkoff, 2007).). 172 See, e.g., In re Metoprolol Succinate Patent Litig., Case No. 06-1254 (Fed. Cir., July 23, 2007) (Gajarsa, J.; Schall, J. dissenting-in-part). 173 See, e.g., In re Ciprofloxacin Hydrochloride Antitrust Litig., 363 F. Supp. 2d 514, 530 (E.D.N.Y. 2005); In Purdue Pharma LP v. Endo Pharmaceuticals Inc., 04-1189, 04-1347 and 04-1357, U.S. Court of Appeals for the Federal Circuit. (invalidating the Purdue patents for the controlled release pain medication, OxyContin, and judging the patents unenforceable based on Purdue’s inequitable conduct during the prosecution case. Perdue misrepresented their scientific findings on the effectiveness of OxyContin in front of the U.S. Patent and Trademark Office(Stephen Albainy-Jenei, 2005). ). 174 The public use bar, pursuant to 35 U.S.C. § 102(b), prohibits public use of the patented object prior to obtaining a patent. While certain experimental uses are excluded, the patent was invalidated on the argument that the public use bar had been violated in clinical trials. In SmithKline Beecham v. Apotex Corp., 403 F.3d 169

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Implications for “Pay-For-Delay” Policy the drug is bioequivalent but does not infringe on existing patents, for example by devising a different method of achieving a time-release mechanism.175 Another important, if controversial, law in Section 505(j)(5)(B)(iv) grants 180 days of market exclusivity to the generic drug maker that first files a successful ANDA against the patent holder of a brand-name drug (Behrendt, 2002).176 This effectively generates a duopoly in the marketplace for the 180-day duration, shared by the innovator and the new generic entry (Hemphill, 2006).177 After the period of generic exclusivity, other generic players are free to enter pending approval of their own ANDA applications. .Legislation to Amend Hatch-Waxman Ensuing legislation attempted to correct some of the imbalances generated by the HatchWaxman Act. In 1993, an amendment to The Patent and Trademark Office Authorization Act clarified when generic drug applications would infringe on patents and when patents could be extended (Soehnge, 2003). 2003 brought new corrections to the Hatch-Waxman scheme, with new FDA regulations and the passage of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003.178 This act was most notable for introducing a prescription drug benefit to the national Medicare plan, but Title XI of the Medicare Modernization Act had important repercussions for the pharmaceutical industry.

1306, 1308 (Fed. Cir. 2005), vacated on rehearing en banc, 403 F.3d 1328 (Fed. Cir. 2005), affirmed on other grounds, 403 F.3d 1331 (Fed. Cir. 2005). 175 The Complaint Counsel’s Trial Brief at 17-18 described the generic party’s argument that its product had a different composition and viscosity than that claimed by the innovator’s patent for the drug K-Dur. In re Schering-Plough Corp., No. 9297 (F.T.C. Jan. 23, 2002), 2002 WL 14488085. 176 See 21 U.S.C. § 355(j)(5)(B)(iv) (2000 & Supp. 2003) It should be noted that the Medicare Act in 2003 brought important limitations onto the 180-day exclusivity period, as will be discussed further below. MMA of 2003: Pub. L. No. 108-173, Title XI, subtitles A-B, 117 Stat. 2066, 2448-64 (codified at 21 U.S.C. § 355 (Supp. III 2003). 178

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Implications for “Pay-For-Delay” Policy Federal regulations passed in June of 2003 eliminated the loophole in the HatchWaxman Act that had previously allowed pharmaceutical innovators to infinitely delay generic entry by perpetuating a series of consecutive thirty-month stays (U.S. Food and Drug Administration, 2003). Prior to the Act, each new patent entry on a contended drug required the ANDA-IV applicant to file a new notice of Paragraph IV certification. Since filing an ANDA-IV is a technical act of infringement,179 the pioneer could instigate an infringement lawsuit against the generic company for each separate notification. Each lawsuit would trigger the FDA to issue a thirty-month stay-of-effectiveness on the ANDA approval. Pharmaceutical pioneers had thus exploited the system, creating new patent entries ranging from “double patents” to “intermediates” to “product by process patents” for the brandname drugs listed in the FDA Orange Book that were being challenged by ANDA applications (Srivastava, 2004). In 2003, the law was amended so that only one thirty-month stay could be issued per ANDA application. The new regulations also restricted pioneer patent-listing practices to curb this form of anticompetitive behavior. 180 Title XI for ‘Access to Affordable Pharmaceuticals’, inter alia, clarified the protocol for obtaining and forfeiting the 180-day exclusivity period for the first generic manufacturer to file a successful ANDA application.181 The 180-day period of market exclusivity is an extremely powerful incentive for generic manufacturers to file ANDA applications, and the

See 35 U.S.C. § 271(e)(2)(A); 21 U.S.C. § 355(j)(5)(B)(iii) (Under Hatch-Waxman, filing an ANDA “for a drug claimed in a patent or the use of which is claimed under a patent” is a technical act of infringement pursuant to the patent statute). 180 See Applications for FDA Approval to Market a New Drug: Patent Listing Requirements and Application of 30-Month Stays on Approval of Abbreviated New Drug Applications Certifying That a Patent Claiming a Drug is Invalid or Will Not Be Infringed, 67 Fed. Reg. 65, 448-56 (Oct. 24, 2002) (describing rationale for forbidding more than one 30-month stay in 65, 455-56, and for limiting NDA patent listing protocol in 65, 449-53). 181 See Medicare Prescription Drug, Improvement, and Modernization Act of 2003, Pub. L. No. 108-173, § 1102(a)(1), 117 Stat. 2066, 2457-58 (2003). 179

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Implications for “Pay-For-Delay” Policy idea of rewarding the first successful ANDA filer was in fact designed to stimulate prompt and plentiful patent challenges. Barkoff illustrated how this 6-month clause instigates situations such as the battle for generic Plavix in spite of a relatively strong patent. “In a lot of these cases, the market for generic drugs, especially for a first filer, is worth so much money that even if the case law is pretty clear, it’s worth a shot. You really never know what a District Court is going to do, and even with the Federal Circuit it’s somewhat unpredictable,” said Barkoff.182 In the case of Plavix, where the U.S. market was worth 3.8 billion dollars in 2005, Barkoff calculated that “six months of that [generic exclusivity] is worth 1.9 billion,” since for the drug price does not drop much with the first generic entry. “Apotex stood to gain at least $1 billion…and probably paid its patent attorneys $5 million. The potential return on investment is huge.”183 The amendment to allow for the forfeiture of first-file exclusivity was triggered by the number of cases where pioneer pharmaceutical companies had successfully negotiated settlements with generic companies that took advantage of the 180-market exclusivity conferred by paragraph IV certifications (Weissman, 2002; Pacific Research Institute for Public Policy). A “statutory bottleneck” is created when the first ANDA filer neither marketed its product nor procured a judicial determination that the patent at issue was invalid or not infringed, leaving the FDA powerless to grant ANDA-IV approval to subsequent filers and thus preventing generic entry for the life of the patent. This statutory bottleneck used to play an important role in the incentives scheme for sweetheart deals

182 183

Barkoff, 2008. Barkoff, 2008.

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Implications for “Pay-For-Delay” Policy negotiated between innovators and generics, as will be discussed further below. The 2003 amendment effectively eliminated this loophole. Another revision passed through this Act requires that any settlement agreement of a drug-patent dispute must be submitted to federal antitrust authorities for review. The 2003 amendments also gave the FTC broader rulemaking powers in order to carry out the provisions of the subtitles. .Current Proposed Legislation and Future Directions For the most part, the provisions of Hatch-Waxman have prevailed. Legislative attempts to address the legal status of such payments in settlement agreements have been introduced by various congressmen. In January of 2007, Senator Kohl (D-Wis) and nine other co-sponsors introduced the “Preserve Access to Affordable Generics Act” (S.316, H.R. 1432) in the first session of the 110th Congress;184 in February it was placed on the Senate legislative calendar. The proposed bill, to amend the Clayton Act, forbids patent settlements wherein an ANDA filer (1) “agrees not to research, develop, manufacture, market, or sell the ANDA product for any period of time” and (2) “receives anything of value.” The bill excludes settlement agreements that are limited to the ANDA filer’s right to market its generic prior to patent expiration. Further measures of the bill amend the terms of the 180-day exclusivity period, clarifying that a FTC or court finding that an agreement violates this Act results in forfeiture of the exclusivity period, thereby allowing other ANDA applications to proceed.

184seeking

to amend the Clayton Act, 155 U.S.C. § 12 et seq. The contents of the bill were previously introduced in July 2006 to the 109th Congress as S.3582.

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Implications for “Pay-For-Delay” Policy Another, similar bill was recently introduced into the House of Representatives. The “Protecting Consumer Access to Generic Drugs Act of 2007” (H.R. 1902) is essentially identical except for the provision specifying that an unlawful reverse payment violates Section 5 of the Federal Trade Commission Act rather than violating the Clayton Act. Aside from legislative action barring reverse payments, other proposed legislation has addressed the issue of “authorized generics” and citizen petitions. Lawsuits challenging the introduction of “authorized generics” in court have, to this date been unsuccessful. The “Fair Prescription Drug Competition Act” (S.438, H.R. 806),185 if enacted, will close the loophole currently allowing pioneer companies to circumvent the loss of market share by introducing their own, authorized generic entry prior to generic entry from another company. The bill prohibits the drug manufacturer with an NDA-approved drug, upon which an ANDA-IV application has been filed, from “manufactur[ing], market[ing], sell[ing], or distribut[ing] an authorized generic drug, directly or indirectly, or authoriz[ing] any other person to manufacture, market, sell, or distribute an authorized generic drug” until expiration of the 180 day exclusivity period. Perhaps not surprisingly, the proposed bills have stalled in Congress, due in no small part to the vigorous lobbying efforts of the pharmaceutical industry (Associated Press, 2007b). The New York Times reported in November 2007 that neither bill had made it to the floor for a vote, and remained in consideration by committees. Senator Hatch, who fathered the original Hatch-Waxman legislation, and other committee members voiced reservations about passing Kohl’s bill in its original form (Associated Press, 2007b). The content of his

185

Previously introduced in the 109th Congress as S.3695 and H.R. 5993.

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Implications for “Pay-For-Delay” Policy bill aside, Senator Kohl recognized the power of the drug lobby, which spent an estimated $38.8 million between June 30, 2006 and July 1, 2007 on senate legislation and other issues, according to the Associated Press (Associated Press, 2007b). However, Kohl maintained the stance of the FTC, which had pushed Congress to take action on the bills on the premise that forbidding such settlements could save American consumers billions of dollars; he was quoted as saying, “If we can just get this to a vote, it will be pretty hard for people to vote against it. A vote against this is a vote against consumers” (Associated Press, 2007b). .Recommendations to the Commission and Congress: This discussion has interesting implications on the jurisdiction of antitrust enforcement to monitor these “pay-for-delay” transactions. In their endorsement of Congressional amendments to close remaining loopholes in Hatch-Waxman, the Commission should continue to emphasize the distinction between proscribing settlements involving any reverse payment, and a moderated prohibition on specific instances of clearly-collusive pay-for-delay settlements. The FTC must be able to effectively communicate that distinction to defuse the powerful pharmaceutical innovator and generic lobbies, who argue that such legislation will leave the industry unable to settle. Balto reinforced this view, referring to a period from 2004 to 2006: “Basically everyone thought that reverse payments were per se illegal. Generic firms still entered the market, and still made settlements. Going and prohibiting reverse payments of some type will not deter settlements and will not deter generic challenge, nor would it change the incentives [for generics] significantly.”186

186

Balto, 2008.

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Appendix A

Appendix Aâ&#x20AC;&#x201C; Therapeutic Targets in the Molecular Regulation of Platelets and Thrombosis The process of blood clotting deserves to be examined in detail, as it will not only provide the ancillary background necessary for understanding the discussions about Plavix but will also serve as a way to introduce the full portfolio of antithrombotic drugs and their specific biochemical mechanisms. Appendix A will review current knowledge on platelets and other key cellular and molecular components of the coagulation system. As the critical factors are identified, their descriptions will be accompanied by a description of the specific ways in which modern drugs antagonize these pathways to reduce inflammation and inhibit platelet function.

.Coagulation Casc ade The pathway of blood coagulation has primarily been studied in the context of hemostasis, which is largely defined as the arrest of bleeding or blood flow via one of several routes: (1) the blood vessel walls closing temporarily in normal vasoconstriction; (2) the presence of an abnormal obstruction such as a atherosclerotic plaque; (3) coagulation; or (4) as a result of a surgical procedure such as ligation. There are four major steps in hemostasis and the process of forming a clot and dissolving it after tissue repair to restore normal blood flow. In the first step of hemostasis, vascular constriction limits blood flow to the injury site. Next, the coagulation cascade is activated, which is responsible for inducing the formation of a loose platelet plug on the endothelial lining of blood vessels. Then, a fibrin mesh assembles and traps the plug, thereby forming a clot (thrombus) and allowing tissue repair to take place. In the final step of the hemocoagulation cascade, plasmin helps to dissolve the clot and restore blood flow. 146

Appendix A There is a critical distinction to be made between the intrinsic and extrinsic pathways to forming a fibrin clot. Trauma activates the extrinsic pathway by releasing the integral membrane glycoprotein tissue factor (TF), which complexes with factor VII and initiates the production of thrombin to stimulate the blood-clotting cascade. The intrinsic pathway, conversely, is not activated by a vascular injury. Rather, the intrinsic pathway can be triggered by contact between the vessel wall and either bacteria or lipoprotein particles, such as in the case of the atherosclerosis. This contact or the rupturing of the endothelial lining of blood vessels exposes anionic surfaces, thereby triggering surface molecules, including kininogen and kallikrein, to activate factor XII (Hageman factor) and thus start the coagulation cascade. Intrinsic and extrinsic pathways converge in a final common pathway from activating prothrombin and then fibrinogen to producing the final cross-linked fibrin clot. Though the thrombotic events instigated by atherosclerotic lesions are initiated via the intrinsic pathway, the extrinsic pathway is still considered important as it describes clotting under normal physiological conditions in the process of hemostasis following a vascular injury. Since the risk in many anticoagulant drugs is precisely the same as their therapeutic purpose – that they inhibit or dampen the body’s clotting mechanisms regardless of whether the clot was initiated via the intrinsic or extrinsic pathway – it is worthwhile to take a closer look at hemostasis, the body’s mechanism to prevent uncontrolled hemorrhage with a balanced system of prothrombotic and antithrombotic factors.

.Hemostas is – The Coagulation Cascade and Platelet Plug Formation .Platelets "Even scientists watching the platelet scene for many years are bewildered by what they are witnessing, new actors (proteins, enzymes, phospholipids) appearing every time they care to look and only a few disappearing again behind the curtains. The actors seem, however, to obey certain rules: they usually become quite excited when 147

Appendix A Ca2+ appears, and relax whenever cyclic AMP makes an entry. This makes understanding the plot a little easier" (Vermylen, 1983). Platelets, as a key component of a clot, have long been a focus of research into cardiovascular disease. However, the importance of antiplatelet therapies has become more pronounced as platelets were found to not only be a mechanistically integral component to attach proteins and cells in the clotting process, but also to play a dynamic role in mediating coagulation and inflammation processes by the release of various factors upon platelet activation (Vorchheimer, 2006). Platelets, briefly defined, are anucleate cell fragments that are formed from megakaryocytes. Megakaryocytes are large cells produced in bone marrow; the cytoplasm of each megakaryocyte splits into multiple compartments as the cell ages, and its plasma membrane eventually ruptures and each fragment reforms to produce several disk-shaped thrombocytes, or platelets, that survive in the bloodstream for eight to ten days (Willoughby, 2002). While platelets retain most of the biological mechanisms intrinsic to nucleate cells – including signal transduction pathways, cytoskeletal components and metabolic enzymes – the lack of a nucleus means that platelets are incapable of altering gene transcription to regulate cellular processes (Maguire, 2003). The protein synthesis capabilities of platelets are limited to expressing megakaryocyte-derived mRNA. Platelets store many of the molecules require to respond to external stimuli in secretory granules, whose contents are released upon activation; other molecules that mediate inflammatory and mitogenic endothelial cell functions are present in the cytoplasm or on the platelet membrane (Davì, 2007; Holmsen, 1989). These various compounds propagate platelet activation and aggregation during hemostasis; their specific roles will be discussed below. As stated before, the activation of a platelet triggers the release of that platelet’s granule contents, marking the point in the 148

Appendix A hemostatic pathway where ADP and the ADP receptors become involved. Granules are comprised of the nucleotide ADP, the eicosenoid TXA2, serotonin, phospholipids, lipoproteins, chemokines such as stromal-derived factor-1 (SDF-1), macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC)), as well as the adhesive proteins Von Willebrand Factor (glycoprotein), fibrinogen and P-selectin. Other protein-derived mediators are stored in the cytoplasm and only displayed on the platelet surface after activation. The primary example is the trimeric transmembrane protein CD40. Experimental evidence has shown that while CD40 is constitutively present on B cells, it is also expressed in T cells, macrophages, monocytes and endothelial cells(BĂźchner, 2003; Henn, 1998). In fact, CD40 is expressed within seconds of platelet activation in vitro, and directly affects thrombus formation in vivo by triggering an inflammatory response on endothelial cells of the vessel wall (Henn, 1998; Hermann, 2001). The CD40 ligand, once on the plasma membrane, cleaves itself within a few hours to release a soluble fragment. In the extracellular environment, soluble CD40 ligand binds to CD40 on endothelial cells to activate pathways increasing the production of reactive oxygen species to limit endothelial cell migration, and upregulating the expression of tissue factor, chemokines and adhesion molecules (Henn, 1998; Slupsky, 1998; Urbich, 2002). The blockade of vascular endothelial growth factor (VEGF-) induced endothelial migration by CD40 ligand disrupts endothelial regeneration after plaque erosion; high levels of CD40L have therefore been suspected to contribute to acute coronary events (SchĂśnbeck, 2001; Urbich, 2002). An important therapeutic affect of clopidogrel, as noted in an experiment by Hermann et al. in 2001, is that it completely inhibits the ADP-induced expression of CD40L in platelets (Hermann, 2001). It is worthwhile to delve into the complexities of platelet adhesion, activation and

149

Appendix A subsequent aggregation and recruitment. In a nonhemostatic situation, platelets do not adhere to or signal other cells as they flow through the blood vessels (Samara, 2003). After tissue trauma and vascular injury, the damaged endothelial lining must interact with platelets and form a strong attachment to allow the platelets to escape the large shear force generated by the bloodstream. Damage to a blood vessel exposes multiple factors in the subendothelium that catalyze the interactions between the endothelium and passing platelets. These components of the endothelial matrix, including collagen, von Willebrand factor (vWF), laminin, fibronectin and thrombospondin, enable a damaged vessel to recruit platelets out of the bloodstream within seconds after injury (Ferguson, 2000). The platelet, accordingly, has multiple adhesion and signaling molecules on its surface to respond to these compounds. The stimulation of platelet receptors is primarily responsible for triggering (1) the activation of internal signaling pathways to stimulate further platelet activation and the release of platelet granule contents, and (2) an increase in the capacity of the plateletâ&#x20AC;&#x2122;s adhesive properties, leading to thrombus formation (Freedman, 2005). The platelet receptors with therapeutic importance will be introduced in the order of their appearance in the timeline of platelet adhesion, platelet activation and subsequent aggregation (or thrombus formation). To start, there are two main families of adhesive and signaling molecules, the 7-transmembrane receptor (otherwise known as G-Protein Coupled Receptors or GPCR) family and the integrins. The 7-transmembrane family includes the protease-activated receptors (PAR) for thrombin, ADP receptors, lipid receptors, chemokine receptors and prostaglandin receptors, making it the major agonist-stimulated family; half of modern medicinal drugs target a receptor in this family (Filmore, 2004). 7-transmembrane

150

Appendix A

Figure 19. The Molecular Regulation of Platelet Activation: Agonists, Receptors and Downstream Effectors. Panels (A), (B), and (C) refer to three main outside-in signaling pathways for platelets, mediated by the agonists (A) thromboxane A2 (TXA2), (B) adenosine diphosphate (ADP), and (C) thrombin. TXA2 is synthesized via the cyclooxygenase (COX) pathway; aspirin treatment irreversibly inhibits the COX pathway and thus prevents TXA2 production and subsequent platelet activation. ADP, released from platelets and red blood cells, agonizes the P2Y1 and P2Y12 receptors, which couple with the Gq and Gi protein pathways, respectively. Both P2Y receptors are necessary for effecting ADP-mediated platelet aggregation, but P2Y12 – which is the target of clopidogrel and other thienopyridine antagonists – is primarily responsible for amplifying and sustaining this ADP-mediated activation. Thrombin is generated from both platelets and endothelial tissues at sites of vascular injury; thrombin is the most potent platelet activator and also regulates fibrin clot generation. Thrombin activates the PAR1 and PAR4 receptors, which are coupled with G12/13, Gq, and Gi proteins, and thus converges with ADP-mediated signaling. Panel (D), in contrast, depicts the platelet’s insideout signaling pathways. The release of granule contents, which contain agonists such as thrombin and ADP, further potentiates platelet activation, while other pathways induce the activation of the glycoprotein IIb/IIIa receptor to bind fibrinogen and von Willebrand factor, which allows stable platelet-to-platelet adhesion. In the figure, TXAS denotes thromboxane synthase, PGH2 prostaglandin H2, and PLA2 phospholipase A2 ((Davì, 2007)).

151

Appendix A receptors are characterized by their namesake – these complexes are integral membrane proteins that have seven helical domains spanning the plasma membrane -- and are stimulated by the binding of an extracellular agonist, which causes the transmembrane components to undergo a conformational change that activates an internal signaling pathway (Foord, 2005; Strange, 2008). Integrins, in contrast, are transmembrane molecules important for adhesion as well as for signaling. These integrins are composed of noncovalently associated heterodimers of α- and β-subunits, which exist in either low- or high-affinity states. Since the affinity state is controlled by phosphorylation events in their cytoplasmic domains, these integrin receptors are regulated by inside-out signaling in addition to outside-in signaling. There are two significant integrin receptors: Glycoprotein (GP) Ib/IIa (α2β1), which binds reversibly to collagen via vWF to initiate platelet “rolling” under high flow conditions and is the primary receptor involved in platelet adhesion, and Glycoprotein (GP) IIb/IIIa (αIIbβ3), which plays a prominent role in aggregation after platelet activation (Kulkarni, 2000; Savage, 1996). Integrin αIIbβ3 primarily mediates platelet-to-platelet interactions by binding to fibrinogen, and in high flow conditions ultimately arrests the platelet at the site of vessel damage.

)Platelet Adhesion: The platelet receptor GP Ib/IIa (the α2β1 integrin) is the primary platelet surface molecule responsible for platelet agglutination on blood vessel walls, as it interacts with vWF bound to the collagen on damaged endothelial cell surfaces and subsequently causes the platelet to exit the blood stream and adhere to this matrix. Out of all the subendothelial factors mentioned above, collagen has the strongest affinity for the platelet, but its interaction with the platelet’s

152

Appendix A Gp Ib/IIa receptor is very tenuous. vWF, which stabilizes the GP Ib/IX/V receptor complex interaction, thereby plays an important role in mediating the adhesion of platelets to the collagen on the exposed endothelial surface (Vorchheimer, 2006). The binding to both vWF and collagen also instigates the platelet to change shape from a disk to become more spherical, facilitating the platelet’s ability to “roll” along the endothelial surface, tethered by these transient interactions -- strong enough to overcome high shear forces and slow the platelet’s movement, but not sufficient to arrest the cell entirely (Savage, 1996). The initial conformational change triggered by the GP Ib/IX/V receptor complex stimulates internal signaling pathways to further increase the surface area of the platelet in contact with components of the endothelial lining and to transform the GP IIb/IIIa receptor from the low-affinity state into a state of high affinity for binding its ligands (Freedman, 2005).

()Therapeutic Targ ets Along the Collag en-vWF-GP Ib Axis: The α2β1 integrin, since it is primarily responsible for collagen adhesion to the platelet, and other collagen receptors including the signaling receptor GP VI and the GP Ib/IX/V receptor complex, are all attractive as potential targets for inhibiting platelets at an early stage of activation (Clemetson, 2007a). Collagen-inhibiting compounds such as caffeic acid phenythyl ester (CAPE) are also being investigated as a novel approach to existing antithrombotic therapies (Hsiao, 2007). Many pharmaceutical research efforts have been focused on exploiting the central role of the GP Ib/IX/V receptor complex in both platelet adhesion and aggregation to produce antithrombotic drugs targeting interactions along the collagen-vWF-GP Ib axis (Deckmyn, 2005; Mousa, 2002). Among the successes, several anti-von Willebrand factor monoclonal antibodies have been developed to specifically disrupt the binding activity between vWf and the GP Ib/IX/V receptor complex

153

Appendix A (Kageyama, 1997; Wu, 2002; Yamamoto, 1998). Snake venom has proved to be a useful resource of antithrombotic agents (Clemetson, 2007b). Isolation processes have yielded derivatives such as agkistin that are successful antagonists of the interactions between endogenous vWF and endothelial GP Ib (Morita, 2004; Yeh, 2000). Other snake venom toxins acting along this axis include jararhagin, jaracetin and the jararaca GP Ib-BP molecule (Marsh, 2005). Through proteolytic cleavage, the metalloproteinase jararhagin disables vWF binding to the platelet GP Ib-IX complex and impairs platelet adhesion to collagen via the integrin α2β1 (Kamiguti, 1996; Kamiguti, 1997b). Jararhagin also blocks the phosphorylation of the platelet protein pleckstrin to selectively abolish the secretion-dependent phase of collagen-induced platelet aggregation (Kamiguti, 1997a). In contrast, jararaca GP Ib-BP binds specifically to the platelet GP Ib receptor and competitively inhibits vWF adhesion (Fujimura, 1995; Kawasaki, 1996). Snake venom extraction has also yielded multiple agonists of the GB Ib integrin; while these compounds are not directly useful for antithrombotic therapies, they have been important pharmacologic tools to further research on platelet integrin function. Alboluxin, a snake C-type lectin purified from Trimeresurus albolabris venom, agonizes both GP Ib and GP VI, inducing full platelet activation (Du, 2002). Stimulation of GP VI is implicated in platelet actvation, as glycoprotein VI is the major collagen signaling receptor on platelets, and mediates collagen adhesion-induced activation of GP IIb/IIIa along with the αIIbβ3 integrin (Goto, 2002; Nakamura, 1999). However, the snake lectin proteins can have very different mechanisms of action. Bilinixin, another snake C-type lectin from Agkistrodon bilineatus venom, acts upon the integrin α2β1 but not on GP VI nor on GP IIb/IIIa to promote platelet agglutination without eliciting full platelet activation with shape change 154

Appendix A (Du, 2001). Conversely, the snake C-type lectin-like protein Stejnulxin, derived from Trimeresurus stejnegeri venom, specifically agonizes the platelet membrane GP VI to induce platelet aggregation (Lee, 2003).

..Platelet Activation: The second step of platelet activation is primarily mediated by the stimulation of various platelet surface receptors. These receptors fulfill two distinct roles, one to perpetuate the cycle of platelet activation, and the other to activate the plateletâ&#x20AC;&#x2122;s adhesive properties for thrombus formation (Freedman, 2005). To achieve the former, internal signaling pathways downstream of these receptors lead to platelet granule secretion to release inflammatory and platelet agonists to promote further platelet activation. For the latter, other signaling pathways activate surface receptors for adhesive molecules such as fibrinogen, thus potentiating platelet-to-platelet aggregation. The enzyme thrombin is a critical mediator of platelet activation and plays a large role in subsequent thrombus formation. The molecular regulation of thrombin is controlled by the essential cofactor enzyme tissue factor (tf), a transmembrane receptor which is present on exposed endothelial surfaces and on platelets (Siddiqui, 2002; Zillmann, 2001). When exposed to blood, tissue factor forms a high-affinity active complex with Factor VII to form Factor VIIa; this complex, along with Factor IX, activates the serine endopeptidase Factor X, which cleaves prothrombin in two sites to generate active thrombin (Camerer, 1996; Eilertsen, 2004). Prothrombin is a 72,000-dalton single-chain protein; cleavage in two sites by Factor VIIa generates the two-chain active thrombin molecule, which consists of a small Îą-chain and a catalytic Î˛- chain held together by a single disulfide bond (Bode, 1997).

155

Appendix A Quiescent platelets contain tissue factor pre-mRNA transcripts; once the platelets are activated, the release of CDC2-like kinase (Clk)1 initiates splicing the transcripts into mature RNA and thereby increases tf expression (Schwertz, 2006). The upregulation of plateletderived spliced tf transcripts has been associated with prothrombotic activity (Gerotziafas, 2004; Panes, 2007; Schwertz, 2006). The potent clot-promoting activity of thrombin was first recognized by Wright and Minot in a paper titled “the viscous metamorphosis of blood platelets” in 1917 (Wright, 1917). Thrombin, also known as the active form of Factor II, is a serine protease that is the most potent platelet agonist, and in addition to potentiating the physiological stimulation of platelets, also interacts with multiple other compounds. White et al. estimated in 1981 that a single platelet has about 33,000 binding sites for thrombin, 31,000 of which have a lowaffinity for thrombin while the remaining 2,000 are high-affinity binding sites (White, 1981). This analysis was somewhat deceptive because the only platelet surface molecule with which thrombin physically binds is the glycoprotein Ib receptor; there are two other receptors activated by thrombin in a non-binding and irreversible substrate-enzyme interaction (Lundblad, 2005). Thrombin activates the protease-activated receptor-1 (PAR1) and PAR4 surface receptors on platelets to initiate a G-protein signal transduction cascade (Hung, 1992b). The stimulation of PAR proceeds by a unique mechanism, whereby thrombin cleaves the receptor molecule’s N-terminus, forming a ligand to the receptor (Coughlin, 1999; Vu, 1991). The platelet PAR1 and PAR4 receptors promote the release of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urinokinasetype plasminogen activator (ut-PA), platelet derived growth factor A (PDGF-A) chain, tissue factor and the inflammatory interleukins (ILs) IL-1 and IL-6 (Naldini, 2000; Naldini, 2002;

156

Appendix A Wu, 2005). For some time, there was dispute over the physiological role and importance of PAR4, as PAR1 could facilitate secretion and aggregation without PAR4 (Kahn, 1998; Kahn, 1999). PAR4 has more recently been implicated in the initiation of thrombin generation and development of clot elasticity (Vretenbrant, 2007). The PAR receptors are also coupled with effectors of the Gq and Giâ&#x20AC;&#x201C;linked pathways downstream of the platelet agonist ADP (Hung, 1992a). These receptors feed into signaling pathway that release platelet granule contents to trigger a critical feedback mechanism of amplifying platelet activation and aggregation, fueling the growing thrombus (Trejo, 2003). Thrombin exerts its prothrombotic affects along multiple pathways besides PAR activation. Thrombin binds to the GB Ib/IX/V complex in addition to activating both PAR1 and PAR4 to achieve Gi-independent activation of Rap1b (Lova, 2004). The combination of GP Ib/IX/V stimulation, PAR activation and thrombin activation induces calcium signaling to mediate several events to promote platelet activation: the degranulation of platelet storage vesicles, which releases the primary platelet agonist ADP and serotonin along with other molecules, the generation of TXA2, and the upregulation of the number of GP IIb/IIIa receptors present on the platelet surface (Vorchheimer, 2006). Thrombinâ&#x20AC;&#x2122;s serine proteolytic activity also promotes fibrin clot formation and activates Factors XI, VIII, and V in the coagulation cascade. The structural features of thrombin help explain its ability to efficiently interact with multiple different molecules in a specific manner. Thrombin has a fibrinogen recognition site and heparin binding site, along with multiple insertion loops, allowing the protease to not only activate three platelet surface receptors, but also to catalyze the conversion of fibrinogen into fibrin and to activate multiple factors downstream of thrombin in the coagulation cascade (De Cristofaro, 157

Appendix A 2003). The two coagulation cascade factors initially activated by thrombin are Factor V and Factor XI, another serine protease that activates Factor IX in an amplifying feedback pathway to generate more thrombin (Hoffman, 1995; Monroe, 1996). Platelet activation and the resulting intrinsic signaling engenders a burst of thrombin large enough to catalyze the cleavage of soluble fibrinogen into insoluble fibrin molecules (Hoffman, 1995; Wolberg, 2008). This potentiates the generation of the fibrin mesh clot over the platelet plug, as discussed further in the section on the third stage of hemostasis.

.Therapeutic Uti lity of Antithrombotics Several pharmacologic anticoagulants have been developed which target circulating thrombin molecules. Bivalirubin, which binds specifically and directly to the thrombin catalytic site, is a reversible thrombin inhibitor marketed as Hirulog and Angiomax (Shammas, 2005). In drug trials bivalirubin has performed successfully, in comparison to treatment with unfractionated heparin, and produces less major in-hospital bleeding than heparin or heparin combined with Gp IIb/IIIa inhibitors (Lincoff, 2003). The Randomized Evaluation in PCI Linking Angiomax to Reduced Clinical Events (REPLACE)-2 trial found good outcomes, and the study indicated bivalirubin along with a provisional Gp IIb/IIIa in elective PCI procedures (Maroo, 2004). In contrast, the search for therapies based on antagonizing the thrombin â&#x20AC;&#x201C; PAR interactions have not produced fruitful dividends, and this failure has been attributed to the ubiquitous nature of the PAR signaling pathways (Lundblad, 2005). .ADP Receptors: This topic is addressed fully in Chapter 3.

158

Appendix A

)Platelet Aggregation and Recruitment: The glycoprotein (GP) IIb/IIIa (αIIbβ3) receptor is expressed only on platelets and is the most prevalent platelet integrin, properties consistent with its role as the principal mediator of the primary, reversible phase of platelet aggregation (Phillips, 1988). GP IIb/IIIa acts as a bidirectional conduit to transmit signaling between the platelet cytoplasm and external plasma. Thrombus formation is primarily dictated by the platelet aggregation resulting from bivalent binding of fibrinogen to GP IIb/IIIa complexes. In fact, blocking eighty percent of GP IIb/IIIa surface receptors is sufficient to fully inhibit aggregation, regardless of which upstream pathways were agonized to promote platelet aggregation (Coller, 1991). In unstimulated platelets, the majority of GP IIb/IIIa molecules are in the inactive state where the membrane-proximal and transmembrane regions of the cytoplasmic tails are in contact with each other; this resting-state complex, with the integrin dimers clasped in a “handshake”, functions as a low-affinity adhesion receptor (Naik, 1997a; Naik, 1997b). The four adhesive proteins fibrinogen, fibronectin, vWF and vitronectin all contain the RGD (Arg-Gly-Asp) amino acid sequence and are all targets of the GP IIb/IIIa receptor (Phillips, 1988). Prior to platelet activation, the receptor selectively recognizes and binds only with surface-bound fibrinogen; it is this interaction which provides the catalyst for irreversible adhesion and a series of signal transduction events (Savage, 1991; Savage, 1992). The extensive flattening and other changes in platelet morphology as a result of adhesion are precisely regulated by integrin signaling and are important to ensure successful platelet thrombogenesis in rapid blood flow conditions (Kuwahara, 2002). When platelets are activated, a network of signaling events affect the multiple proteins bound to the 159

Appendix A cytoplasmic tails of the integrin subunits. The internal signaling pathways activated by the platelet agonists ADP, thrombin, collagen, vWF and thromboxane A2 all converge to regulate GP IIb/IIIa activation (Brass, 1997). GP IIb/IIIa is commonly referred to as the final common pathway for platelet aggregation. The process of inside-out signaling is sonamed because the cytoplasmic tail-binding proteins mediate the unclasping of the complex, the conformational signal responsible for activating the integrin’s extracellular domains (Ma, 2007; Smyth, 1993; Vinogradova, 2002). The resulting conformational change in the extracellular domains of the integrin facilitates the high-affinity binding of soluble plasma fibrinogen (Naik, 1997a). The binding sites for fibronectin, vitronectin and von Willebrand factor in addition to fibrinogen on activated GP IIb/IIIa receptors is consistant with the observed homology of certain domains of the αIIb dimer with the α-subunits of fibronectin and vitronectin receptors (Poncz, 1987). Fibrinogen is the extracellular adhesion molecule responsible for forming bridges between platelets, but also for modulating outside-in signaling via integrin αIIbβ3. Binding of the fibrinogen dimer to the GP IIb/IIIa receptor is in itself a reversible process; however, it triggers GP IIb/IIIa-mediated signaling (outside-in signaling) that irreversibly stabilizes aggregation by promoting integrin clustering and recruiting a network of cytoskeletal proteins (Freedman, 2005). Aside from inducing cytoskeletal reorganization, the outside-in activation of GP IIb/IIIa also results in calcium mobilization, tyrosine phosphorylation and the activation of phosphoinositide metabolism.

()Therapeutic Interventions at th e Final Common Pathway of Plat elet Aggregation Platelet integrin αIIbβ3’s pivitol role in platelet aggregation is underscored by the multiple

160

Appendix A intravaneous and oral ÎąIIbÎ˛3 receptor antagonists developed for clinical utilities(Mousa, 2002). There are three small molecules, namely abciximab, eptifibatide and tirofiban, which abrogate the fibrinogen GP IIb/IIIa receptor and have been approved for intravenous use in patients with acute coronary syndromes (ACS) or undergoing percutaneous coronary interventions (PCI) (Boersma, 2002; Boersma, 2002b). Multiple clinical trials have demonstrated the dose-dependent antithrombotic action of the chimeric human/mouse monoclonal antibody abciximab (marketed as ReoPro), which is a long-lasting, noncompetitive blocker of the GP IIb/IIIa while also binds vitronectin and Mac-1 receptors (Adgey, 1998; Ghaffari, 1998; Ibbotson, 2003; Lincoff, 2000; Teirstein, 1999). These trials found a role for intravenous abciximab therapy for patients presenting with ACS and those undergoing PCI (Teirstein, 1999). The cyclic heptapeptide eptifibatide (Integrelin) and the tyrosine-derived nonpeptide tirofiban (Aggrastat), in contrast to abciximab, are lowmolecular-weight molecules that act rapidly and specifically on the GP IIb/IIIa receptor but have pharmacodynamic half-lives of only 2 hours (Leclerc, 2002). In their evaluation of the randomized clinical trial results described above, the researchers Chew and Bhatt emphasized that caution should be taken in the application of platelet GP IIb/IIIa inhibitors, citing the importance of choosing high-risk patients, early invasive strategies and optimal dosing patterns to optimize efficacy while reducing the unanticipated effects of potent GP IIb/IIIa blockade (Chew, 2002). Nonetheless, the search for potent yet safe pharmacologic molecules continues. Snake venom proteins containing the same RGD amino acid sequence present in fibrinogen and other adhesive proteins have been proven to interfere with platelet aggregation by

161

Appendix A reversibly inhibiting the GP IIb/IIIa receptor (Marsh, 2005). Some of the more notable disintegrins acting upon this integrin receptor include trigramin from Trimeresuru gramineus venom, which abolishes vWF- GP IIb/IIIa interactions, and rhodostomin from Calloselasma rhodostoma, which obstructs fibrinogen adhesion to GP IIb/IIIa (Huang, 1987; Huang, 1989). Two other venom-derived disintegrins, both of which interfere with fibrinogen binding, are halysin (Agkistrodon halys) and triflavin (Trimeresurus flavoviridis) (Huang, 1991a; Huang, 1991b). Antithrombotic success with the rapid-acting injectable αIIbβ3 receptor antagonists such as Abciximab, Eptifibatide and Aggrastat for acute therapy has not yet been duplicated in studies of oral αIIbβ3 receptor antagonists such as sibrafiban for long-term or chronic use (Cannon, 1998; Quinn, 1998). In some ways, GP IIb/IIIa inhibitors have thus far failed to live up to the expectations that they could prevent platelet aggregation and thrombosis while not disturbing the hemostatic mechanisms for depositing a monolayer of platelets on cover exposed/injured vessel surfaces (Leclerc, 2002). All the clinical trials thus far with oral αIIbβ3 receptor antagonists have had disappointing outcomes, with either no clinical benefit or increased thrombotic events (Mousa, 2002). A major study of Sibrafiban versus aspirin to Yield Maximum Protection from ischemic Heart events post-acute corONarY syndromes (SYMPHONY), sponsored by Roche, found that two doses of sibrafiban did not confer any additional benefit over aspirin treatment in preventing secondary ischaemic events, and furthermore, high doses of sibrafan resulted in more bleeding (SYMPHONY Investigators, 2000). Enrollment in the second SYMPHONY trial, which compared low-dose sibrafiban and aspirin combination therapy, high-dose sibrafiban alone and aspirin alone, was prematurely halted as soon as the results of the first SYMPHONY study were available (Second SYMPHONY Investigators, 2001). Consistent with the disappointing findings of 162

Appendix A the first trial, analysis of the preliminary results for the second SYMPHONY detected increased mortality amongst those in the combination therapy and the high-dose sibrafiban groups in comparison to those treated with aspirin. Likewise, the oral antagonist xemilofiban was a disappointment in the Evaluation of oral Xemilofiban in Controlling Thrombotic Events (EXCITE) trial, and the compound orbifiban did not exceed placebo outcomes in the Orbofiban in Patients with Unstable coronary Syndromes (OPUS) trial (Drugs In RD, 2003; Gowda, 2004; Scirica, 2006). Searle, who had sponsored both compounds, terminated further clinical investigation for both of these oral αIIbβ3 receptor antagonists, though VDDI Pharmaceuticals began a new study with modified dosage schedules and more narrow patient selection (Drugs in RD, 2003). Ultimately, the attempts to develop platelet αIIbβ3 receptor antagonists collectively underscore the major benefit and drawbacks to this line of therapy. The αIIb/β3 integrin, as the final common pathway in platelet aggregation, is an obvious target of antithrombotic therapy. On the flipside, any antagonist of this receptor causes profound platelet inhibition regardless of the platelet agonist. Platelet αIIbβ3 receptor blockade thus disrupts natural hemostasis mechanisms alongside the various cardiovascular and cerebrovascular thromboembolic disorders, and many novel drug candidates with promising antithrombotic profiles have been discontinued for promoting excess bleeding (Doggrell, 2001). Furthermore, the greatest proven contribution of parenteral GP IIb/IIIa blockade has been in preventing ischaemic events in patients undergoing PCI. While these GP IIb/IIIa antagonists have proved to be very successful at prevent cardiac events during the unstable phase of acute coronary syndromes, large clinical studies showed that the injectable antagonists failed to prevent secondary cardiac events in the longer term (Mousa, 2002). The 163

Appendix A clinical failure of oral GP IIb/IIIa antagonists largely squashed the hope that chronic GP IIb/IIIa blockade could extend these potent antiplatelet properties to protect patients from cardiovascular events after the stabilization of ACS. The clinical utility of αIIbβ3 receptor antagonists remains limited to short-term and intravenous acute therapy, while long-term antithrombotic prevention is primarily mediated by targeting aspirin and ticlopidine.

.Hemostas is: Fibrin Clotting In the third step of hemostasis, a fibrin mesh clot forms to trap the platelet plug. The abovementioned proteolytic enzyme thrombin converts fibrinogen to fibrin by cleaving four arginine-glycine peptide bonds in fibrinogen’s central globular region. The structure of the fibrinogen molecule can be characterized by its subunits, the A and B fibrinopeptides and the fibrin monomers. Fibrin monomers assemble periodically via 23 nm-long repeats into fibrin through linkage of α-subunit knobs into corresponding γ-subunit holes to form protofibrils, as well as β-β subunit binding between protofibrils. The quantity of thrombin produced during the propagation phase after platelet activation is large enough to activate the transglutaminase Factor VIII. Protransglutaminase is transformed by thrombin to form active Factor VIII, also known as the essential clotting factor. This catalytic enzyme helps cross-links the fibrin fibers after polymerization, which improves the elasticity of individual fibers and enhances overall clot viscoelasticity and stability (Glover, 1975). Specifically, factor VIII catalyzes the formation of covalent longitudinal isopeptide bonds between the side chains of glutamine and lysine residues in the γ-chains of fibrin as well as amide bonds between the γ-chains and specific lysine residues of the Aα-chains that stabilize the soft fibrin clot (Sobel, 1996; Weisel, 1993).

164

Appendix A

()Therapeutic Defi brinogenation There have been various efforts to investigate the possibility of therapeutic defibrination, or defibrinogenation, for clinical applications. This line of therapy, attacking plasma fibrinogen rather than interfering with the platelet surface adhesion molecules, is distinct from the afore-mentioned pharmaceutical disintegrins, platelet αIIb/β3 receptor blockade, and signaling receptor antagonists. Most recently, studies have explored the anticoagulant properties of the fibrinogenase family of snake venom enzymes that degrade specific fibrinogen chains (Swenson, 2005). The fibrin(ogen)olytic proteins are distributed into two classes, the metalloproteinases and serine proteases. Despite differing in their mechanism of cleaving action and specificity for certain amino acid sequences on fibrinogen, both classes of fibrin(ogen)olytic enzymes perform the same function of dissolving fibrin-rich clots and preventing further clot formation. Zinc metalloproteinases such as fibrolase, purified from Agkistrodon contortrix venom, and basiliscusfibrases 1, 2 and 3, all from the venom of Crotalus basiliscus, targeted a specific Lys-Leu bond on the α chain of fibrinogen (Retzios, 1994). Seeing the therapeutic promise in fibrolase, investigators produced the biochemical alfimeprase, a recombinant, truncated version of fibrolase which has the same direct proteolytic activity on the α chain (Toombs, 2001). In preclinical trials, alfimeprase application proved to be 6-fold more rapid at lysing clots than the current standard therapy of plasminogen activators; alfimeprase has also performed well in Phase I and Phase II clinical studies (Deitcher, 2006). While alfimeprase failed to meet primary endpoints in Phase III clinical trials, and clinical trials in progress have been halted, it has been suggested that alfimeprase still has therapeutic antithrombotic

165

Appendix A potential, pending further investigation and drug refinement (Shah, 2007). Other fibrin-clot dissolving venom agents include the serine protease afaacytin, isolated from Cerastes cerastes venom, Vipera lebetina fibrinogenases (VIF), and atroxase from Crotalus atrox (Gasmi, 1997; Laraba-Djebari, 1995; Marrakchi, 1998). Vipera lebetina venom contained lebatase, a directacting fibrolytic enzyme that appeared to attenuate ADP-induced platelet aggregation, in addition to the serine proteases degrading fibrinogen (Siigur, 1998; Siigur, 2001). In contrast to fibrolase, axtroxase cleaves both the Îą and Î˛ chains nonspecifically, resulting in extensive hydrolysis of fibrinogen (Willis, 1988). These compounds, however, have not been successfully pursued for medical purposes despite their fibrin clot-dissolving properties. Another anticoagulation method involved the use of a snake venom thrombin-like enzyme (SVTLE) to remove plasma fibrinogen from circulation. Reid and Chan published the first account reviewing the possibility of isolating such an anticoagulant agent from snake venom in 1968 (Reid, 1968). SVTLEs achieve their anticoagulatory affect by the rapid, progressive removal of fibrinogen out of the circulatory system. The thrombin-like enzymes convert intravascular plasma fibrinogen into microclots of cleaved fibrin molecules in organ tissues, which are subsequently removed by intrinsic enzymatic processes (Regoeczi, 1966). Since then, several fibrinolytic SVTLE compounds have been approved for pharmaceutical use. The active, fibrinolytic fraction of Ancistrodon rhodostoma snake venom is marketed by Knoll as Ancrod (Esnouf, 1967). There have been few well-designed clinical trials to evaluate possible therapeutic indications for the difibrinogenating agent Ancrod. Of note, Sherman et al. conducted a randomized, placebo-controlled Stroke Treatment with Ancrod Trial (STAT), and concluded that treatment with Ancrod within three hours had a favorable riskbenefit profile for the treatment of acute ischemic stroke (Sherman, 2000). Several reviews

166

Appendix A of stroke therapies in 2002 noted the successful clinical results of Ancrod, especially in the context of how few of the 20th century clinical trials testing novel compounds have produced positive results for acute stroke outcomes (Gladstone, 2002; Madhavan, 2002). Nonetheless, the standard treatment of intravenous recombinant tissue plasminogen factor (t-PA) produced a higher rate of positive clinical outcomes than intravaneous Ancrod at the threemonth endpoint (The National Institute of Neurological Disorders And Stroke Rt-PA Stroke Study Group, 1995; Madhavan, 2002). Furthermore, a larger clinical investigation in 2006 found that when given within 6 hours of stroke onset, Ancrod performed no better than placebo at producing functional success at three months; the European Stroke Treatment with Ancron Trial recommended that Ancrod not be used after three hours of acute ischemic stroke (Hennerici, 2006). Further clinical investigations into Ancrod and many of the other venom-based agents appear to have stagnated after that point, and rt-PA remains the only approved specific treatment for acute ischemic stroke.

.Hemostas is: Clot Diss olution Fibrin eventually encourages the production of the lytic enzyme, plasmin, to elicit the fourth and final step of hemostasis. Tissue-type plasminogen activators synthesized by endothelial cells convert the proenzyme plasminogen into active, serine proteinase molecules, e.g. plasmin (Stassen, 2004). Plasmin dissolves the blood clot by digesting the fibrin components, and normal blood flow resumes (Arnout, 2006). Plasminogen activators are thus used in clinical settings to promote clot dissolution.

167

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