`Enhancement by Aspirin in Nonhuman Primates
`Laurence A. Harker, Ulla M. Marzec, Andrew B. Kelly, Nicolas R. F. Chronos, I. Birgitta
`Sundell, Stephen R. Hanson and J.-M. Herbert
`
`Circulation
`
`1998;98:2461-2469Circulation.
`
`doi: 10.1161/01.CIR.98.22.2461
`is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
`Copyright © 1998 American Heart Association, Inc. All rights reserved.
`Print ISSN: 0009-7322. Online ISSN: 1524-4539
`
`The online version of this article, along with updated information and services, is located on the
`World Wide Web at:
`http://circ.ahajournals.org/content/98/22/2461
`
`Permissions:
` Requests for permissions to reproduce figures, tables, or portions of articles originally published
` can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialCirculation
`
`in
`Office. Once the online version of the published article for which permission is being requested is located,
`click Request Permissions in the middle column of the Web page under Services. Further information about
`document.
`Permissions and Rights Question and Answer
`this process is available in the
`
`Reprints:
` Information about reprints can be found online at:
`http://www.lww.com/reprints
`
`Subscriptions:
` Information about subscribing to
`http://circ.ahajournals.org//subscriptions/
`
`
`is online at:
`
`Circulation
`
`Downloaded from
`
`
`
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 1 of 10
`
`
`
`Clopidogrel Inhibition of Stent, Graft, and Vascular
`Thrombogenesis With Antithrombotic Enhancement by
`Aspirin in Nonhuman Primates
`Laurence A. Harker, MD; Ulla M. Marzec, MS; Andrew B. Kelly, DVM; Nicolas R.F. Chronos, MD;
`I. Birgitta Sundell, PhD; Stephen R. Hanson, PhD; J.-M. Herbert, PhD
`
`Background—A recent study showed that clopidogrel reduces thrombo-occlusive complications in patients with
`symptomatic atherosclerosis more effectively than aspirin.
`Methods and Results—The effects of clopidogrel and aspirin have been compared, singly and in combination, for
`measurements of 111In-labeled platelets and 125I-labeled fibrin deposition in baboon models of arterial thrombosis and
`related to platelet aggregation and expression of activation epitopes induced by ADP, collagen, and thrombin receptor
`agonist peptide (TRAP) and to template bleeding times (BTs). Low-dose oral clopidogrel (0.2 mg 䡠 kg⫺1 䡠 d⫺1) produced
`cumulative (1) intermediate decreases in 111In-platelet and 125I-fibrin deposition for segments of prosthetic vascular graft,
`deployed endovascular metallic stents, and endarterectomized aorta (P⬍0.009 in all cases); (2) elimination of
`ADP-induced platelet aggregation (P⬍0.001); (3) modest inhibition of collagen-induced platelet aggregation (P⬍0.01);
`(4) no reduction in TRAP-induced platelet aggregation; and (5) minimal prolongation of BTs (P⫽0.03). High-dose oral
`clopidogrel (ⱖ2 mg/kg) produced the same effects within 3 hours. The effects of clopidogrel dissipated over 5 to 6 days.
`Aspirin 10 mg 䡠 kg⫺1 䡠 d⫺1 alone did not decrease 111In-platelet and 125I-fibrin deposition on segments of vascular graft
`but detectably decreased 111In-platelet and 125I-fibrin accumulation on stents (P⬍0.01), minimally inhibited ADP- and
`collagen-induced platelet aggregation (P⬍0.05 in both cases), and minimally prolonged BTs (P⫽0.004). Within 3 hours
`of aspirin administration,
`the antithrombotic effects of acute high-dose or chronic low-dose clopidogrel were
`substantially enhanced, and BTs were modestly prolonged without inhibiting platelet aggregation induced by TRAP
`(P⬍0.001 in all cases compared with clopidogrel alone).
`Conclusions—Clopidogrel produces irreversible, dose-dependent, intermediate reduction in thrombosis that is substan-
`tially enhanced by the addition of aspirin. The effects of combining aspirin and clopidogrel need to be evaluated in
`patients at risk of vascular thrombosis. (Circulation. 1998;98:2461-2469.)
`Key Words: clopidogrel 䡲 thrombus 䡲 stents 䡲 aspirin
`
`Heart attacks, strokes, and peripheral arterial occlusion are
`
`generally caused by thrombo-occlusive episodes in stenot-
`ic atherosclerotic arteries.1 Ruptured atherosclerotic plaques
`initiate complex interactions among damaged atheromatous
`vascular structures and highly reactive platelets and coagulation
`proteins, resulting in the formation of tissue factor–dependent
`vascular thrombosis. Tissue factor–initiated thrombotic occlu-
`sion also complicates interventional procedures used in the
`management of patients with symptomatic atherosclerotic vas-
`cular disease, including thrombolytic reperfusion for acute cor-
`onary thrombosis, angioplasty, various types of atherectomy,
`deployment of endovascular stents, endarterectomy, and implan-
`tation of small-caliber vascular grafts.2,3
`Large-scale, randomized, placebo-controlled trials have estab-
`lished that daily oral aspirin therapy decreases the relative risk of
`
`vaso-occlusive episodes in patients with symptomatic athero-
`sclerotic disease by 20% to 25%.1,4 The benefits of aspirin are
`attributable to its irreversible interruption of thromboxane A2
`(TxA2) generation by platelets.5 Controlled clinical trials have
`also established that oral
`ticlopidine therapy decreases the
`relative risk of vaso-occlusive events in symptomatic atheroscle-
`rotic patients by 30% to 35%.1,4,6–9 Ticlopidine, a thienopyridine
`requiring hepatic modification in vivo to exhibit antiplatelet
`effects, selectively inhibits ADP-dependent platelet aggregation
`that is cumulative over 8 to 10 days.10–12 Recent clinical studies
`demonstrate that adding aspirin to ticlopidine therapy markedly
`reduces thrombo-occlusive events associated with the deploy-
`ment of coronary stents.13–16 Unfortunately, ticlopidine therapy
`has a number of troublesome adverse effects, including revers-
`ible neutropenia, diarrhea, and idiosyncratic cutaneous rashes.
`
`Received February 9, 1998; revision received June 5, 1998; accepted July 2, 1998.
`From the Division of Hematology and Oncology, Department of Medicine, and Yerkes Regional Primate Research Center, Emory University School
`of Medicine, Atlanta, Ga, and Sanofi Recherche, Toulouse, France (J.-M.H.).
`Correspondence to Laurence A. Harker, MD, Blomeyer Professor and Director, Division of Hematology and Oncology, Emory University School of
`Medicine, 1639 Pierce Dr, WMB Room 1003, Atlanta, GA 30322. E-mail lharker@emory.edu
`© 1998 American Heart Association, Inc.
`Circulation is available at http://www.circulationaha.org
`
`Downloaded from
`
`2461
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 2 of 10
`
`
`
`2462
`
`Clopidogrel Inhibits Thrombogenesis
`
`is
`is a ticlopidine-like thienopyridine that
`Clopidogrel
`severalfold more potent and is free of the adverse effects
`plaguing ticlopidine therapy.17,18 Like ticlopidine, clopidogrel
`is devoid of direct antiplatelet effects and must undergo
`hepatic metabolic modification to exhibit selective inhibition
`of ADP-induced platelet aggregation.22 Clopidogrel acts by
`irreversibly inactivating platelet ADP receptor–initiated sig-
`naling in a dose-dependent manner.10,20,21 In a recently re-
`ported large-scale, randomized, controlled clinical trial, clo-
`pidogrel was shown to be significantly more effective than
`and at least as safe as aspirin in decreasing arterial thrombo-
`occlusive episodes in patients with symptomatic atheroscle-
`rotic disease.22 Clinical trials reporting that ticlopidine plus
`aspirin markedly reduces thrombotic occlusion of coronary
`stents suggest that adding aspirin to clopidogrel therapy may
`enhance its antithrombotic efficacy.13–16
`Clopidogrel and aspirin each inhibit platelet recruitment by
`interrupting ADP- and TxA2-mediated platelet activation,
`respectively. Presumably, the relative contributions of ADP-
`and TxA2-dependent platelet recruitment varies with different
`clinical thrombotic processes. Accordingly, the present study
`was designed to measure the relative antithrombotic effects of
`administering clopidogrel and aspirin, singly and in combi-
`nation, for 3 different thrombosis models in baboons, ie,
`graft, stent, and vascular thrombosis models. The relative
`antihemostatic and antithrombotic effects of oral clopidogrel,
`clopidogrel with aspirin, clopidogrel with heparin, or the
`combination of clopidogrel, aspirin, and heparin were
`determined.23
`
`Methods
`Baboon Model of Thrombosis and Hemostasis
`Twenty-four normal juvenile male baboons weighing 9 to 11.5 kg
`and bearing chronic exteriorized arteriovenous (AV) femoral shunts
`were used in these studies. Before experimentation, all animals were
`observed to be disease-free for at least 3 months. All procedures
`were approved by the Institutional Animal Care and Use Committee
`(Emory University) in compliance with National Institutes of Health
`guidelines (Guide for the Care and Use of Laboratory Animals,
`1985), Public Health Service policy, the Animal Welfare Act, and
`related university polices. In these models, quantitative, reproduc-
`ible, nonoccluding, platelet-rich thrombi were formed on deployed
`endovascular metallic stents, segments of vascular graft, and seg-
`ments of endarterectomized baboon aorta interposed in the AV
`shunts under physiological flow conditions for 60 minutes without
`systemic anticoagulation. The forming thrombus incorporated circu-
`lating prelabeled autologous 111In-labeled platelets and homologous
`125I-labeled fibrin(ogen). These chronic AV shunts, per se, do not
`detectably activate platelets or coagulation.24,25
`Thrombogenic Devices
`Nonocclusive thrombi were formed over 60 minutes in the exterior-
`ized AV shunts of awake animals by deployment of metallic
`endovascular stents, interposing 2-cm-long, 4-mm-ID thrombogenic
`segments of uncrimped Dacron vascular grafts or incorporating
`segments of endarterectomized homologous aorta in established AV
`shunts while blood flow was controlled at 100 mL/min.
`Stent thrombosis was produced by deployment of stainless steel
`endovascular stents (3.5 mm) in the exteriorized chronic AV shunt.
`The stainless steel stents, a gift from Johnson & Johnson Interven-
`tional Systems, Warren, NJ, were mounted on sterile water–filled
`noncompliant Duralyn coronary angioplasty balloons (Cordis Corp).
`The stents were manually crimped onto the deflated balloon and
`inserted into a 3.3-mm-ID, 20-cm-long segment of silicone rubber
`
`tubing (Technical Products Inc). The balloon was inflated 3 times to
`a pressure of 10 atm to achieve maximal apposition of the stent struts
`with the tubing wall. The shunt tubing was then filled with sterile
`saline to remove potentially confounding air bubbles from the
`surface of the stent and to facilitate interpositioning of stent-
`containing segments in the exteriorized chronic AV femoral shunt.
`Graft thrombosis was produced by segments of Dacron vascular
`grafts (Bioknit, C.R. Bard, Inc) rendered impervious to blood
`leakage by external wrapping in Parafilm (American Can Co) and
`5.3-mm-ID “heat-shrinkable” Teflon tubing. Connections were con-
`structed to ensure that the devices were isodiametric and suitable for
`incorporation into the AV shunts.
`Endarterectomy thrombosis was produced by endarterectomizing
`fresh baboon aorta (5- to 6-mm ID) obtained from other donor
`animals,
`flushed with saline, and divided into 4-cm lengths.
`Branches were ligated, and specimens were stored in normal saline.
`For endarterectomies, the aortic segments were inverted and the
`intima and inner media were removed for a distance of 1 cm in the
`central portion of the vessel by sharp dissection. After completion of
`the endarterectomy, each segment was returned to its normal
`configuration and cannulated with 1-cm lengths of heat-shrinkable
`Teflon tubing (Small Parts, Inc) attached to segments of 4-mm-ID
`silicone rubber medical
`tubing (Dow Corning, Inc). The aortic
`segments were encased with heat-shrinkable Teflon tubing and each
`end was carefully sealed by heating, but direct heat to tissues was
`avoided. The resultant configuration maintained stable geometry,
`with a smooth transition from vessel to tubing.
`
`Clopidogrel Dosing
`Clopidogrel was administered orally at 7 doses spanning more than
`2 orders of magnitude, ie, 0.1, 0.2, 0.5, 2, 5, 10, and 20 mg 䡠 kg⫺1 䡠
`d⫺1. Antithrombotic and antihemostatic effects of high-dose clopi-
`dogrel for stent and graft thrombosis were obtained 3 hours after
`dosing with 2 to 20 mg/kg. Because the effects of low-dose
`clopidogrel were cumulative, doses of 0.1, 0.2, and 0.5 mg/kg
`clopidogrel were administered daily for 6 days to determine full
`dose-response effects (see below). Thrombus formation was mea-
`sured in the following sequence on different days during the
`subsequent 2 weeks, assessing the effects of (1) clopidogrel 0.2 mg 䡠
`kg⫺1 䡠 d⫺1 alone, (2) clopidogrel 0.2 mg 䡠 kg⫺1 䡠 d⫺1 with heparin 100
`IU/kg bolus and 100 IU/kg infused over 1 hour, (3) clopidogrel 0.2
`䡠 d⫺1 plus aspirin 10 mg/kg administered 2 hours
`mg 䡠 kg⫺1
`previously, and (4) clopidogrel 0.2 mg 䡠 kg⫺1 䡠 d⫺1 in combination
`with aspirin 10 mg/kg and heparin 100 IU/kg infused over 1 hour.
`Treatments were discontinued for 1 week to permit the antiplatelet
`effects of clopidogrel and the enhancing effects of aspirin to dissipate
`before other studies were begun.
`
`Measurements of Thrombus Formation
`Autologous platelets were labeled with 1 mCi [111In]indium oxine
`(111In) as previously described24,26 and reinjected at least 1 hour
`before the thrombogenic devices were interposed. 125I-labeled ba-
`boon fibrinogen (5 Ci), purified and labeled as described previous-
`ly,27 was injected intravenously 10 minutes before thrombogenic
`devices were introduced. Images of the segments containing vascular
`graft, deployed stents, or endarterectomized aorta were acquired
`separately with a General Electric 400T MaxiCamera, stored, and
`analyzed with a Medical Data Systems A3 image processing system
`(Medtronic) interfaced with the camera, by routines already de-
`scribed.27 The total numbers of deposited platelets in regions of
`interest were calculated by dividing the deposited platelet activity
`(counts/min) by the circulating blood activity (counts 䡠 min⫺1 䡠 mL⫺1)
`and multiplying by the circulating platelet count (platelets/mL).
`Fibrin was determined after completion of the experiments by
`removal of the thrombogenic segments for counting 125I-fibrin
`radioactivity 30 days later when the 111In activity had decayed. Total
`fibrin accumulated was calculated by dividing the deposited 125I
`activity (counts/min) by the clottable fibrinogen activity (counts 䡠
`min⫺1 䡠 mL⫺1) and multiplying by the plasma fibrinogen level
`(mg/mL).26,27
`
`Downloaded from
`
`
`
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 3 of 10
`
`
`
`Harker et al
`
`December 1, 1998
`
`2463
`
`Effects of Clopidogrel on Ex Vivo Platelet
`TABLE 1.
`Aggregation and Bleeding Time
`
`Agonist Concentration Producing
`Half-Maximal Platelet AC50
`
`Oral Dose,
`mg/kg
`
`Baseline
`0.1 after 6 d
`
`ADP,
`mol/L
`5.1⫾2.8
`⬎100
`
`0.2 after 6 d
`
`⬎100
`
`0.5 after 6 d
`
`⬎100
`
`2.0 after 3 h
`
`20 after 3 h
`
`20⫾13
`P⬍0.0001
`⬎100
`
`Collagen,
`g/mL
`2.2⫾1.0
`8.4⫾4.9
`P⫽0.008
`7.2⫾4.0
`P⫽0.001
`9.5⫾8.8
`P⫽0.003
`7.5⫾4.9
`P⫽0.0004
`12⫾6.0
`P⬍0.0001
`(P⫽0.37)
`
`TRAP,
`mol/L
`38⫾25
`31⫾24
`P⬎0.5
`45⫾23
`P⬎0.4
`43⫾27
`P⬎0.7
`59⫾36
`P⬎0.2
`74⫾54
`P⫽0.06
`(P⫽0.7)
`
`Bleeding
`Time, min
`3.1⫾0.7
`6.9⫾5.3
`P⬍0.01
`7.7⫾7.4
`P⫽0.03
`16⫾11
`P⫽0.0002
`24⫾9.5
`P⫽0.0001
`⬎30
`P⬍0.0001
`
`volar surface of the forearm as previously described in nonhuman
`primates.28,31
`
`Statistical Analysis of Data
`Data were presented as mean⫾SD. Student’s t test for paired or
`unpaired data was used when data were normally distributed.
`Otherwise, Mann-Whitney nonparametric analysis was used. Facto-
`rial ANOVA and ANCOVA were used. A value of Pⱕ0.05 was
`considered to be the estimate of statistical significance.
`Results
`Baboon Models of Graft, Stent, and
`Vascular Thrombosis
`Segments of Dacron vascular graft interposed in exteriorized
`AV shunts flowing at 100 mL/min induced rapid platelet
`deposition that reached plateau levels by 60 minutes (Figure
`1, top). Neither aspirin 10 mg/kg PO 2 hours previously nor
`heparin 100 IU/kg IV bolus and 100 IU/kg infused over 60
`minutes significantly reduced thrombus formation (Figure 1,
`top; P⬎0.2).
`Deployed metallic endovascular stents in AV shunts flow-
`ing at 100 mL/min also produced thrombus that reached
`plateau values by ⬇60 minutes (Figure 1, bottom). Whereas
`heparin had no effect on stent thrombosis (P⬎0.2), oral
`aspirin 10 mg/kg modestly decreased platelet accumulation
`111In-platelet deposition decreased from
`on stents,
`ie,
`2.56⫾0.96⫻109 to 1.76⫾0.57⫻109 platelets (P⬍0.01; Figure
`1, bottom).
`Control endarterectomized aortic segments produced sub-
`stantial
`thrombus by 60 minutes,
`ie, platelet deposition
`averaged 1.6⫾0.39⫻109 platelets/cm, and mean fibrin accu-
`mulation was 1.1⫾0.3 mg/cm.
`Baseline platelet concentrations averaged 285⫾60⫻103/
`L, and control mean plasma fibrinogen concentration was
`2.95⫾0.27 mg/mL. Baseline template BT averaged 3.3⫾0.8
`minutes. During baseline thrombus formation on thrombo-
`genic devices, platelets remaining in the circulation did not
`change aggregatory responsiveness or express activation
`
`Figure 1. Platelet deposition in formation of graft and stent
`thrombus. Autologous 111In-platelets accumulate rapidly on seg-
`ments of Dacron vascular graft (top) or deployed endovascular
`metallic stents (bottom) interposed in exteriorized chronic AV
`shunts flowing at 100 mL/min in nonanticoagulated baboons.
`Plateau levels of thrombotic accumulation are achieved by ⬇60
`minutes. For graft thrombosis, platelet deposition is not signifi-
`cantly reduced by heparin (100 IU/kg bolus and 100 IU/kg
`infused during 60 minutes) or aspirin (10 mg/kg PO 2 hours
`before study). Although heparin fails to decrease stent thrombo-
`sis, aspirin therapy produces intermediate reduction in platelet
`deposition.
`
`Laboratory Studies
`Platelet counts, erythrocyte counts, and total leukocyte counts were
`performed on whole blood collected in 2 mg/mL disodium EDTA
`with a Serono Baker model 9000 whole-blood analyzer.28 Blood
`samples for testing platelet hemostatic function were collected in
`citrate (3.2% for platelet aggregation studies and 3.8% for flow
`cytometric determination of P-selectin and ligand-induced binding
`site [LIBS] expression).
`Platelet aggregation was determined within 1 hour of drawing
`blood with a Chrono-Log aggregometer by recording the increase in
`light
`transmission through a stirred suspension of platelet-rich
`plasma (PRP) maintained at 37°C. PRP and platelet-poor plasma
`(PPP) were prepared by differential centrifugation, as previously
`described.29 The platelet count in the PRP was adjusted to 300⫻
`103/L. Percent aggregation was calculated linearly between the
`optical densities of PPP and PRP. ADP (Sigma Chemical Co),
`collagen (Nycomed Arzenmittel), and thrombin receptor agonist
`peptide (TRAP1–6) (Peninsula Laboratories) were added at doses
`spanning the range of responsiveness. The results were plotted and
`expressed as the agonist concentration that induced half-maximal
`aggregation (AC50).29 The appearance of activated platelets in the
`peripheral blood was evaluated by flow cytometry using fluoresce-
`inated monoclonal antibodies against neoantigens expressed on
`membrane surfaces of activated platelets, including conformationally
`altered integrin ␣IIb3 (or glycoprotein IIb/IIIa), LIBS (a gift from Dr
`E. Plow, Cleveland, Ohio),30 and the secretory granule membrane,
`P-selectin, a gift from Biogen Inc, Cambridge, Mass.30
`Template bleeding time (BT) measurements were performed at
`baseline and at 60 minutes. BT testing was carried out on the shaved
`
`Downloaded from
`
`
`
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 4 of 10
`
`
`
`2464
`
`Clopidogrel Inhibits Thrombogenesis
`
`TABLE 2. Antithrombotic Effects of Clopidogrel, Heparin, and Aspirin
`
`Baseline
`Three hours after high-dose clopidogrel
`Clopidogrel 2.0 mg/kg
`
`Clopidogrel 2.0 mg/kg⫹aspirin 10 mg/kg
`
`Clopidogrel 20 mg/kg
`
`Clopidogrel 20 mg/kg⫹aspirin 10 mg/kg
`
`Six days after low-dose clopidogrel
`Clopidogrel 0.2 mg/kg
`
`Clopidogrel 0.2 mg 䡠 kg⫺1 䡠 d⫺1⫹aspirin 10 mg/kg
`
`Clopidogrel 0.2 mg 䡠 kg⫺1 䡠 d⫺1⫹heparin 100 IU/kg
`
`Clopidogrel⫹aspirin⫹heparin
`
`1.2⫾0.77 (8)
`1.7⫾0.42 (6)
`3.16⫾0.84 (6)
`P1⫽0.0094
`P1⫽0.007
`P1⫽0.03
`0.57⫾0.6 (7)
`1.85⫾0.28 (6)
`2.07⫾0.41 (6)
`P1⫽0.0006
`P1⫽0.013
`P1⫽0.0001
`P2⫽0.1
`P2⫽0.48
`P2⫽0.02
`0.64⫾0.56 (7)
`1.22⫾0.6 (5)
`2.45⫾0.38 (6)
`P1⫽0.0006
`P1⫽0.002
`P1⫽0.0007
`P2⫽0.14
`P2⫽0.15
`P2⫽0.1
`0.44⫾0.44 (6)
`1.39⫾0.33 (5)
`2.01⫾1.03 (5)
`P1⫽0.0004
`P1⫽0.003
`P1⫽0.0003
`P2⫽0.05
`P2⫽0.21
`P2⫽0.09
`Numbers in parentheses are numbers of observations. P1 indicates control vs treatment; P2, clopidogrel vs combinations.
`
`Graft Thrombosis
`
`Stent Thrombosis
`
`Platelets⫻109
`4.36⫾1.21 (27)
`
`Fibrin, mg
`3.06⫾1.03 (13)
`
`Platelets⫻109
`2.56⫾0.96 (7)
`
`Fibrin, mg
`0.56⫾0.27 (6)
`
`2.68⫾0.64
`P⫽0.03
`2.66⫾0.31
`P⫽0.024
`2.28⫾0.05
`P⫽0.007
`2.05⫾0.41
`P⫽0.003
`
`2.32⫾0.36
`P⫽0.25
`2.39⫾0.66
`P⫽0.31
`1.83⫾0.50
`P⫽0.07
`1.48⫾0.48
`P⫽0.02
`
`0.66⫾0.26
`P⫽0.01
`0.39⫾0.16
`P⫽0.006
`0.55⫾0.23
`P⫽0.008
`0.19⫾0.01
`P⫽0.003
`
`Bleeding
`Time, min
`3.1⫾0.7 (14)
`
`24⫾9.5
`
`⬎30
`
`⬎30
`
`⬎30
`
`7.7⫾74 (14)
`P1⫽0.03
`16⫾11 (8)
`P1⫽0.0002
`P2⫽0.05
`10⫾8.6 (8)
`P1⫽0.004
`P2⫽0.45
`22⫾8.4 (7)
`P1⫽0.0001
`P2⫽0.0008
`
`0.21⫾0.07
`P⫽0.07
`0.15⫾0.07
`P⫽0.04
`0.16⫾0.05
`P⫽0.04
`0.08⫾0.07
`P⫽0.02
`
`0.34⫾.06 (8)
`P1⫽0.04
`0.13⫾0.05 (7)
`P1⫽0.002
`P2⫽0.0001
`0.18⫾0.06 (7)
`P1⫽0.004
`P2⫽0.0002
`0.11⫾0.07 (6)
`P1⫽0.003
`P2⫽0.001
`
`epitopes, ie, ex vivo platelet aggregation induced by ADP,
`collagen, or TRAP was unaltered, and there was no signifi-
`cant flow cytometric expression of P-selectin or LIBS.
`
`Antithrombotic and Antihemostatic Effects
`of Clopidogrel
`In vitro, clopidogrel exhibited no direct inhibitory effects on
`baboon platelet aggregation or LIBS expression induced by
`ADP, collagen, or TRAP (P⬎0.5).
`Oral dosing of clopidogrel at 0.1 mg/kg for 6 days
`abolished ADP-induced platelet aggregation (undetectable
`aggregation despite the addition of ⬎100 mol/L ADP;
`Table 1). This dosing regimen also modestly inhibited platelet
`aggregation induced by collagen, as shown by the increased
`concentration of collagen required to produce half-maximal
`aggregation, ie, from 2.2⫾1.0 to 8.4⫾4.9 g/mL (P⫽0.008),
`and prolonged the BT to 6.9⫾5.3 minutes (Table 1; P⬍0.01).
`However, neither TRAP-induced platelet aggregation nor
`thrombus formation on segments of vascular graft or de-
`ployed endovascular stents was significantly decreased
`(P⬎0.2 in all cases).
`Increasing the dose of clopidogrel to 0.2 mg/kg for 6 days
`significantly reduced platelet and fibrin accumulation on vascu-
`lar grafts and stents (Table 2; P⬍0.01 in all cases), prolonged the
`BT to 7.7⫾7.4 minutes (Tables 1, 2, and 3; P⬍0.01 compared
`with baseline values) without additional inhibition of platelet
`aggregation and expression of activation epitopes induced by
`
`ADP or collagen, and produced no reduction in TRAP-induced
`platelet activation (Tables 1 and 3).
`Within 3 hours, increasing clopidogrel dosing 10-fold to 2
`mg/kg significantly decreased platelet deposition (Figure 2;
`Table 2; P⬍0.01 in both cases) but not fibrin accumulation
`(Table 2; P⬎0.1 in both cases) for both vascular grafts and
`stents. ADP-induced aggregation was markedly inhibited
`(AC50 20⫾13 mol/L; P⬍0.0001 compared with baseline),
`collagen-induced aggregation was inhibited beyond that pro-
`duced after 6 days of 0.1 mg 䡠 kg⫺1 䡠 d⫺1 clopidogrel (Table 1),
`and BT was prolonged to 24⫾9.5 minutes (P⬍0.0001 com-
`pared with baseline). TRAP-induced platelet aggregation was
`not reduced (Table 1).
`When the dose of clopidogrel was increased another
`10-fold to 20 mg/kg, the accumulation of both platelets and
`fibrin on segments of vascular graft and stents was signifi-
`cantly decreased within 3 hours of oral administration (Table
`2; Figure 2; P⬍0.02 in all cases). ADP-induced aggregation
`was abolished, and BT was increased to ⬎30 minutes.
`TRAP-induced platelet aggregation was minimally inhibited
`(Table 1; P⬍0.06 compared with baseline), and no additional
`inhibition of collagen-induced aggregation was produced
`(Table 1; P⬎0.4). The reduction in thrombosis and prolon-
`gation of the BT resulting from high-dose clopidogrel grad-
`ually dissipated over 6 days, after therapy was discontinued.
`Thus, high-dose clopidogrel selectively and irreversibly abol-
`ished ADP-dependent platelet activation within 3 hours
`(Tables 1 and 3).
`
`Downloaded from
`
`
`
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 5 of 10
`
`
`
`Harker et al
`
`December 1, 1998
`
`2465
`
`TABLE 3. Cumulative Effects of Low-Dose Clopidogrel on Platelet Function and
`Thrombus Formation
`
`Stent thrombosis
`Platelet deposition⫻109
`P
`Fibrin accumulation, mg
`P
`Graft thrombosis
`Platelet deposition⫻109
`P
`Fibrin accumulation (mg)
`P
`BT, min
`P
`Platelet aggregation (AC50)
`ADP, mol/L
`P
`Collagen AC50, g/mL
`P
`TRAP1–6 AC50, mol/L
`P
`Expression of activation epitopes
`LIBS
`LIBS after ADP
`P
`LIBS after TRAP1–6
`
`P-Selectin
`P
`P-Selectin after ADP/epitopes
`P
`
`Baseline
`
`2.56⫾0.96 (7)
`
`0.56⫾0.27 (6)
`
`4.36⫾1.21 (27)
`
`3.06⫾1.03 (13)
`
`3.1⫾0.7 (14)
`
`5.1⫾2.8 (18)
`
`2.2⫾1.0 (16)
`
`38⫾25 (18)
`
`1440⫾476 (16)
`20 500⫾8900 (17)
`
`7570⫾7210 (9)
`
`352⫾339 (17)
`
`2650⫾720 (17)
`
`Numbers in parentheses are numbers of observations.
`
`Day 3
`(Clopidogrel 0.2
`mg 䡠 kg⫺1 䡠 d⫺1)
`
`Day 6
`(Clopidogrel 0.2
`mg 䡠 kg⫺1 䡠 d⫺1)
`
`1.82⫾1.30 (3)
`0.3
`0.42⫾0.08 (3)
`0.42
`
`3.52⫾1.42 (3)
`0.3
`2.34⫾0.23 (3)
`0.3
`4⫾0.5 (3)
`0.05
`
`⬎100
`0.0001
`7.2⫾3.4 (10)
`0.0001
`35.6⫾20.8 (10)
`0.8
`
`1890⫾540 (7)
`6250⫾3400 (7)
`0.0035
`2100⫾820 (7)
`0.099
`162⫾28 (7)
`0.092
`1640⫾384 (7)
`0.0022
`
`1.20⫾0.77 (8)
`0.009
`0.34⫾0.21 (8)
`0.04
`
`3.16⫾0.84 (6)
`0.03
`1.7⫾0.42 (6)
`0.0007
`7.7⫾7.4 (14)
`0.03
`
`⬎100
`0.0001
`7.2⫾4.0 (10)
`0.00001
`45⫾23 (10)
`0.5
`
`1200⫾192 (7)
`7100⫾4800 (7)
`0.0012
`2710⫾1270 (4)
`0.19
`164⫾54
`0.033
`1110⫾270 (7)
`0.0001
`
`The dose-response effects of clopidogrel for platelet accu-
`mulation in graft and stent thrombosis are displayed in Figure
`3 and documented quantitatively in Table 2. Although clopi-
`dogrel has a steep antithrombotic dose-response at ⱕ0.2
`mg/kg, the dose-response relationship is relatively flat for 10-
`to 100-fold increased dosing, indicating that the antithrom-
`botic effects of clopidogrel remained intermediate, despite
`large doses of drug. By contrast, the BT was progressively
`prolonged to ⬎30 minutes by high-dose clopidogrel.
`The overall extent to which clopidogrel reduced thrombus
`formation was substantially greater for stent thrombosis than
`for graft thrombosis (Figure 3 and Table 2), implying that
`ADP-mediated platelet recruitment was quantitatively more
`important for thrombus forming on stents than for thrombus
`forming on segments of vascular graft.
`The effects of clopidogrel on the formation of thrombus
`at sites of arterial endarterectomy are shown in Figure 4.
`Dosing clopidogrel at 0.2 mg 䡠 kg⫺1 䡠 d⫺1 for 6 days
`substantially decreased platelet and fibrin accumulation on
`
`segments of endarterectomized aorta (P⬍0.001). Increas-
`ing the dose of clopidogrel to 5 mg/kg for 3 days abolished
`thrombus forming on endarterectomized aorta (Figure 4;
`P⬍0.0001).
`
`Antithrombotic and Antihemostatic Effects of
`Combining Aspirin and Clopidogrel
`Combining aspirin 10 mg/kg and clopidogrel significantly en-
`hanced the reduction in platelet and fibrin deposition produced
`within 3 hours by high-dose clopidogrel (20 mg/kg), as shown in
`Figure 5 and Table 2 for stent thrombosis (P⬍0.01 in both cases)
`and in Figure 6 and Table 2 for graft thrombosis (P⫽0.001 in
`both cases). Although platelet deposition was significantly re-
`duced by 2 mg/kg clopidogrel after 3 hours, fibrin accumulation
`was not significantly decreased (Table 2; P⬎0.07), although the
`BT remained maximally prolonged at ⬎30 minutes
`Single-dose aspirin therapy (10 mg/kg) also enhanced the
`reduction in platelet and fibrin deposition after 6 days of
`low-dose clopidogrel (0.2 mg 䡠 kg⫺1 䡠 d⫺1) for stent and graft
`
`Downloaded from
`
`
`
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 6 of 10
`
`
`
`2466
`
`Clopidogrel Inhibits Thrombogenesis
`
`Figure 4. Dose-response effects of clopidogrel for vascular
`thrombosis. Platelet deposition is largely interrupted by dosing
`clopidogrel at 0.2 mg 䡠 kg⫺1 䡠 d⫺1 for 6 days and is completely
`abolished by dosing 5 mg 䡠 kg⫺1 䡠 d⫺1. Number of observations
`is shown in parentheses.
`
`10 mg 䡠 kg⫺1 䡠 d⫺1 was administered concurrently throughout
`the 6-day period of low-dose clopidogrel therapy (0.2 mg 䡠
`kg⫺1 䡠 d⫺1), antithrombotic effects were not observed earlier or
`more intensely than the effects produced by adding a single
`dose of aspirin after 6 days of low-dose clopidogrel therapy
`(Figure 8). Reductions in platelet and fibrin accumulation
`remained incomplete after 3 days of concurrent clopidogrel
`and aspirin therapy, compared with the effects on day 6 for
`stent thrombosis (Table 3 and Figure 7; P⬍0.05 in both
`cases) and graft
`thrombosis (Figure 8; Tables 2 and 3;
`P⬍0.05 in all cases). ADP- and collagen-induced platelet
`aggregation and ADP-induced P-selectin and LIBS expres-
`sion were fully abnormal after 3 days of concurrent 10 mg 䡠
`kg⫺1
`䡠 d⫺1 aspirin and 0.2 mg 䡠 kg⫺1
`䡠 d⫺1 clopidogrel
`administration (Table 3).
`
`Effects of Combining Heparin With Clopidogrel
`Intravenous heparin therapy (100 IU/kg bolus and 100 IU/kg
`over 60 minutes) after 6 days of oral clopidogrel did not
`reduce 111In-platelet deposition on stents or vascular graft
`thromboses (Table 2; P⬎0.1 in both cases). Although 125I-
`fibrin accumulation was not reduced on vascular grafts by the
`addition of heparin to clopidogrel (P⫽0.15), 125I-fibrin accu-
`mulation was decreased on stents by the combination of
`clopidogrel plus heparin (P⫽0.0002). Importantly, the en-
`hanced antithrombotic effects produced by addition of either
`aspirin or heparin to clopidogrel were not further augmented
`for vascular graft thrombosis and stent thrombosis by addi-
`tion of both aspirin and heparin to clopidogrel (Table 2;
`P⬎0.2 in all cases).
`The addition of heparin after 6 days of clopidogrel 0.2 mg 䡠
`kg⫺1 䡠 d⫺1 did not detectably prolong the BT beyond that
`produced by clopidogrel alone (Table 3; P⬎0.4). Similarly,
`platelet aggregation and expression of P-selectin and LIBS
`induced by either collagen or TRAP were not increased by
`combining heparin with clopidogrel (P⬍0.05 in all cases).
`
`Antithrombotic Benefits Versus Prolongation of
`Template BTs
`Antithrombotic efficacy was compared with changes in the
`BT by relating 111In-platelet deposition on segments of
`vascular graft (a measure of thrombus formation) and tem-
`
`Figure 2. Antithrombotic effects after 3 hours of high-dose clo-
`pidogrel. Oral clopidogrel at doses of 2 and 20 mg/kg decrease
`platelet and fibrin deposition on segments of vascular graft to
`comparable intermediate levels (top) within 3 hours of adminis-
`tration. High-dose clopidogrel similarly produces comparable
`intermediate antithrombotic effects on deployed endovascular
`stents (bottom) within 3 hours after oral administration.
`
`thrombosis (Figures 7 and 8; Table 2; P⬍0.01 for all cases),
`with a BT of 16⫾11 minutes (Table 2). Thus, by reduction of
`the chronic clopidogrel dosing to 0.2 mg 䡠 kg⫺1
`䡠 d⫺1,
`antithrombotic efficacy was retained while the prolongation
`of template BTs was minimized (Figure 9). When oral aspirin
`
`Figure 3. Dose-response effects of clopidogrel on platelet dep-
`osition for graft and stent thromboses. Six days of oral clopi-
`dogrel decreases platelet deposition in log-linear dose-
`dependent manner for graft and stent thromboses that plateaus
`at intermediate levels for doses ⬎0.2 mg 䡠 kg⫺1 䡠 d⫺1. Within 3
`hours of dosing clopidogrel at 2.0 and 20 mg/kg, platelet depo-
`sition is inhibited to maximal extent achievable with this therapy.
`Arrow denotes baseline deposition.
`
`Downloaded from
`
`
`
` by guest on May 15, 2013http://circ.ahajournals.org/
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`Ex. 1019, p. 7 of 10
`
`
`
`Harker et al
`
`December 1, 1998
`
`2467
`
`Figure 5. Platelet deposition in stent thrombosis after 3 hours
`of high-dose clopidogrel plus aspirin. Platelet deposition is sub-
`stantially reduced within 3 hours after oral dosing of clopidogrel
`at 2.0 mg/kg (left) or 20 mg/kg (right). Addition of aspirin 10
`mg 䡠 kg⫺1 䡠 d⫺1 further decreases platelet deposition (left and
`right) in formation of stent thrombosis.
`
`plate BT (generally a measure of overall platelet hemostatic
`function). The minimal regimen producing maximal interrup-
`tion of graft thrombosis achievable by this therapy, ie, 0.2
`mg 䡠 kg⫺1 䡠 d⫺1 clopidogrel plus 10 mg/kg aspirin, prolonged
`the BT to 16⫾11 minute

Accessing this document will incur an additional charge of $.
After purchase, you can access this document again without charge.
Accept $ ChargeStill Working On It
This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.
Give it another minute or two to complete, and then try the refresh button.
A few More Minutes ... Still Working
It can take up to 5 minutes for us to download a document if the court servers are running slowly.
Thank you for your continued patience.

This document could not be displayed.
We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.
You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.
Set your membership
status to view this document.
With a Docket Alarm membership, you'll
get a whole lot more, including:
- Up-to-date information for this case.
- Email alerts whenever there is an update.
- Full text search for other cases.
- Get email alerts whenever a new case matches your search.

One Moment Please
The filing “” is large (MB) and is being downloaded.
Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!
If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document
We are unable to display this document, it may be under a court ordered seal.
If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.
Access Government Site