Preclinical and Clinical Studies with Selective ...

Preclinical and Clinical Studies with Selective Reversible Direct P2Y12 Antagonists J.J.J. van Giezen, Ph.D.,1 and Robert G. Humphries, B.S.2

ABSTRACT

An important role for adenosine diphosphate (ADP)–induced platelet activation and aggregation was proposed more than 40 years ago. The clinical use of clopidogrel, a prodrug of an irreversible P2Y12 antagonist, has further proved the relevance of inhibiting signaling via the platelet-specific P2Y12 ADP receptor in the prevention of cardiovascular events. Pharmacological studies at AstraZeneca R&D Charnwood have identified direct, selective, and competitive P2Y12 antagonists, including cangrelor (also known as ARC69931MX), which is suitable for intravenous administration, and AZD6140, which is suitable for oral administration. In preclinical use, these compounds predictably and effectively inhibited platelet aggregation without significant increases in bleeding time. In clinical use, these compounds may have significant advantages over current antiplatelet agents. This article summarizes preclinical and clinical data on cangrelor and AZD6140 and discusses the potential of these compounds as novel antiplatelet therapies. KEYWORDS: AR-C69931MX, AZD6140, P2Y12 receptor, platelets, platelet antagonists

Objectives: On completion of this article, the reader should be able to (1) describe the pharmacology of cangrelor and AZD6140 and (2) cite some of the clinical data obtained with these two new antiplatelet agents. Accreditation: Tufts University School of Medicine (TUSM) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Credit: TUSM designates this educational activity for a maximum of 1 Category 1 credit toward the AMA Physicians Recognition Award. Each physician should claim only those credits that he/she actually spent in the educational activity.

A

major role for ADP in platelet activation, aggregation, and arterial thrombosis was proposed more than 40 years ago.1 In the ensuing decades, much effort has been devoted to gaining a deeper understanding of ADP-induced platelet activation and its role in responses to other platelet stimuli. The P2Y12,3 and P2Y124 receptors have been identified as important targets for antithrombotic therapy, and the antithrombotic efficacy of ticlopidine and clopidogrel have confirmed the importance of inhibiting signaling via the

platelet-specific P2Y12 ADP receptor. This overview summarizes the progress in developing direct, reversible antagonists for the P2Y12 receptor both as pharmacological tools and as potential new antiplatelet therapies.

DISCOVERY OF CANGRELOR, RELATED COMPOUNDS, AND AZD6140 The ability of adenosine triphosphate (ATP) to competitively antagonize ADP-induced platelet aggregation

The Platelet P2 Receptors: Biochemistry, Physiology, Pharmacology, and Clinical Aspects; Editor in Chief, Eberhard F. Mammen, M.D.; Guest Editors, Christian Gachet, M.D., Ph.D., and Marco Cattaneo, M.D. Seminars in Thrombosis and Hemostasis, volume 31, number 2, 2005. Address for correspondence and reprint requests: Dr. Johan J.J. van Giezen, AstraZeneca R&D Mo¨lndal, Department of Integrative Pharmacology, Pepparedsleden 1, S-431 83 Mo¨lndal, Sweden. E-mail: [email protected]. 1Associate Principle Scientist, AstraZeneca Mo¨lndal, Mo¨lndal, Sweden, 2AstraZeneca Charnwood, Loughborough, United Kingdom. Copyright # 2005 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. 0094-6176,p;2005,31,02,195,204,ftx,en;sth01047x.

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was first described almost three decades ago.5 This discovery led to the definition of the platelet-specific ADP receptor P2Y12 subtype (then named P2T), in which ADP is an agonist and ATP is a competitive antagonist.6 Because properties of ATP (e.g., its low potency at P2 receptors, poor stability) hampered its use as a pharmacological tool for in vitro and in vivo exploration of the P2Y12 receptor, efforts to identify ATP analogues that were more resistant to breakdown by ectonucleotidases and that displayed an increased affinity for the P2Y12 receptor began during the 1980s. Key observations relevant to structure-activity relationships (SAR) included the affinity-increasing properties of substituents in the 2 position of the adenine ring7,8 and stabilityincreasing properties of b,g-methylene substitutions in the triphosphate part of the molecule.9 These analogues provided a starting point for the AstraZeneca R&D Charnwood medicinal chemistry program that led to the discovery of the potent, selective P2Y12 receptor antagonists AR-C66096MX,10 AR-C67085MX,11 and, later, cangrelor (AR-C69931MX) (Fig. 1).12 FPL or ARL prefixes were used in early descriptions of these compounds and MX stands for the tetra sodium salt. All three compounds potently inhibit ADP-induced aggregation of human washed platelets and show high

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selectivity for the P2Y12 receptor over P2Y1 and P2X1 receptors, which are also present on platelets (Fig. 2). The inverse logarithmic concentration causing 50% inhibition (pIC50) for values of AR-C66096MX and ARC67085MX against ADP-induced (30 mM) aggregation of human washed platelets are 8.2 and 8.6, respectively. At high concentrations in a high-expression system, AR-C66096MX shows partial P2Y1 agonist properties,3 and AR-C67085MX shows partial P2Y11 agonist properties.13 These effects are less evident with cangrelor; therefore, it has become the compound of choice for ongoing investigations. Recent data suggesting potential interactions with the P2Y13 receptor14 require further investigation (see later). Because the triphosphate chain of these compounds results in a short half-life in vivo,15 they are highly suitable for intravenous administration. Efforts to find compounds for oral administration led first to the discovery of AR-C109318XX, the first selective and stable, nonphosphate P2Y12 antagonist. Further refinement to increase the oral bioavailability of this compound resulted in the discovery of the selective P2Y12 antagonist AZD6140, which is currently being developed as an oral antiplatelet agent. AR-C109318XX and AZD6140 belong to a new chemical class called cyclopentyl-triazolo-pyrimidines (CPTP) (Fig. 1).

Figure 1 Chemical structures of ATP, reversible P2Y12 antagonist cangrelor (¼ AR-C69931MX) in clinical development for intravenous administration, and cyclopentyl-triazolo-pyrimidines AR-109318XX and AZD6140.

DIRECT P2Y12 ANTAGONISTS/VAN GIEZEN, HUMPHRIES

Figure 2 A simplified representation of the P2-receptor model, for ATP/ADP-induced platelet activation, indication site of action of reversible P2Y12 antagonists cangrelor and AZD6140, and analogues of thienopyridines (updated from Humphries51).

PHARMACOLOGY OF CANGRELOR AND AZD6140 IN VITRO Effects of cangrelor on ADP-induced platelet function have been extensively tested in vitro. In a turbidometric assay using human washed platelets, cangrelor inhibited ADP-induced (30 mM) aggregation with a pIC50 of 9.4. In whole blood, the potency of cangrelor depends on the assay type. Using impedance aggregometry in heparinized blood (50% diluted blood in saline), the pIC50 was 9.2 with 3 mM ADP. Using citrated blood in the residual platelet count method,16 the pIC50 was 7.6 when aggregation was induced by 20 mM ADP (van Giezen, unpublished data, 2004). These differences may relate to the use of different ADP concentrations, the extent of blood dilution, and the anticoagulant used. The potency of cangrelor translates well to other species. The pIC50 was 9.2 for dog blood and 8.3 for rat blood using impedance aggregometry and heparinized blood. AZD6140 appeared somewhat less potent, with pIC50 values of 7.9 and 7.2 in the washed platelet and diluted whole blood impedance aggregometry assays, respectively. Additional screens show cangrelor and AZD6140 to have no significant affinity for other P2 receptors at concentrations > 30 mM and > 3 mM, respectively. However, a recent publication described cangrelor as a potent noncompetitive antagonist for the P2Y13 receptor; AR-C67085MX was shown to be approximately 150-fold less potent.14 These findings could not be

reproduced by a subsequent extended pharmacological characterization of the P2Y13 receptor (Greasley, unpublished data, 2004). Additional research is needed to fully understand the affinity of cangrelor for this receptor. Further experience with these compounds has also emphasized the importance of the assay conditions, particularly when conducting quantitative pharmacological studies for receptor characterization. For example, although cangrelor is a competitive P2Y12 antagonist under equilibrium conditions, apparent noncompetitive properties are evident under nonequilibrium conditions.17 This feature can be a problem in functional studies in platelets in which the tendency for spontaneous platelet activation associated with prolonged incubation times can make it difficult to achieve true equilibrium. The use of cangrelor as a pharmacological tool has helped to advance current understanding of the role of the different purine receptors on platelets. A review of the more than 50 publications in which cangrelor was used as a pharmacological tool is beyond the scope of this article, but a brief selection of these publications highlights the importance of cangrelor in describing the two pharmacologically distinct ADP receptors on platelets3; the role of P2Y12 in blocking the cyclic adenosine monophosphate–mediated active inhibitory process of disaggregation18; the relevance of activation of Rap1B via PI3K in P2Y12 signal transduction19,20; the presence

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of P2Y12 receptors on C6–2B glioma cells21; the role of P2Y12 in platelet shape change22; and aggregation induced by high shear,23 thrombin,24 collagen,25 or plasmin.26

EFFECT OF P2Y12 ANTAGONISTS IN EXPERIMENTAL ARTERIAL THROMBOSIS Cangrelor has been tested in several models of arterial thrombosis in vivo (Table 1). For example, in the wellestablished model of cyclic flow reductions (CFRs) in the femoral artery of anesthetized male beagles,12,27,28 thrombosis was induced by mechanical vessel damage by using a set of tweezers combined with a partial stenosis. This model allows increasing drug doses to be tested with parallel measurement of inhibition of ADPinduced platelet aggregation measured ex vivo and of tongue bleeding time. Data from this model show a significantly improved separation between antithrombotic effect and increase in bleeding time using the P2Y12 blockers clopidogrel, cangrelor, or AZD6140 over glycoprotein (GP) IIb/IIIa antagonists such as orbofiban (Fig. 3). In addition, although not statistically significant, a trend toward greater separation with the direct nonprodrug antagonists was observed. The values for the dose inducing a 3.5-fold increase in bleeding over the dose inducing 50% inhibition of the CFRs (BL3.5/ CFR50) were 5.7, 14, and 98 for clopidogrel, AZD6140, and cangrelor, respectively. The clinical relevance of the beneficial separation in this model with the reversible antagonists compared with clopidogrel remains to be determined. The substantial antithrombotic effect of cangrelor in dogs has been confirmed in a carotid artery thrombosis model in beagles.29 In this model, thrombosis was induced by an electrolytic injury of the vessel wall using a low current for 3 hours combined with partial stenosis. Infusion of cangrelor decreased the incidence of occlusion from 100% to 17% and decreased the thrombus weight by 83%. This effect was accompanied by a 3.4fold increase in buccal bleeding time and a 4.4-fold increase in tongue bleeding time. However, 1 hour after termination of the infusion, aggregation and bleeding parameters had returned to near basal levels, a finding that reflects the reversibility of the antagonism. Induction of a thrombus in rabbit mesenteric arteries by vessel wall puncture shows that, despite P2Y12 antagonism, plug formation was not influenced; that is, no increase in bleeding time or induction of rebleeding was observed.30 However, the formed thrombi were smaller, and less embolization was observed. The authors conclude that their data suggest that P2Y12 antagonism has the possibility to reduce the risk of downstream ischemia after a thrombotic event in diseased human arteries.

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Considered together, these observations highlight the benefit of P2Y12 antagonism in the prevention of thrombosis in preclinical models and suggest a possible benefit after a thrombotic event in humans. A recent publication also suggests a benefit in combination with thrombolytic therapy.31,32 In a canine coronary electronic injury thrombosis model, cangrelor was combined with thrombolytic therapy consisting of tissue plasminogen activator (t-PA) and heparin, started 30 minutes after stable thrombus formation on a background of aspirin. The combination with cangrelor prevented reocclusion and allowed maintenance of blood flow for the duration of the experiment. In addition, an increase in myocardial microvascular blood flow resulting in approximately 50% decreased infarct size was observed in the cangrelor group. This study showed a 1.8- to 2.8fold increase in bleeding time. These preclinical in vivo data show that high levels of platelet aggregation inhibition resulting in efficacious prevention of both arterial thrombosis and reocclusion after thrombolytic therapy can be achieved with reversible P2Y12 antagonists. CLINICAL EXPERIENCE WITH CANGRELOR The promising results obtained with cangrelor in preclinical studies supported the further evaluation of this compound in phase I and phase II clinical studies (partly reviewed in Storey33). Intravenous infusion of cangrelor was welltolerated in healthy volunteers, and cangrelor dose dependently inhibited ADP-induced platelet aggregation measured ex vivo at doses up to 4 mg/kg per minute.34 Despite abolishment of ADP-induced platelet aggregation, bleeding time increased by only 3.2 times or 2.9 times in men and women, respectively. The short half-life (mean 2.6 minutes) resulted in a rapid reversal of both the platelet-inhibitory effect and the effect on bleeding. Even at the highest dose tested, control levels for platelet aggregation and bleeding time were reached within 20 minutes after cessation of the infusion. The subsequent phase II program first assessed the safety, tolerability, and efficacy of cangrelor as adjunctive treatment to aspirin, heparin, and nitrates in an open multicenter ascending dose study in 39 acute coronary syndrome patients.15 This study showed dosedependent and predictable plasma levels that resulted in complete or nearly complete inhibition of 3 mM ADPinduced platelet aggregation in all patients at doses of 2 mg/kg per minute or higher (plasma concentration 70 nM). Bleeding times were increased 3- to 5-fold at 2 mg/kg per minute and by about 7-fold at 4 mg/kg per minute. Although trivial bleeding was common in these patients (22 of 39), no major or minor bleedings were

Inhibition of CRF versus

CFR in dog femoral artery

Occlusion time

Electrically damaged stenosed

infarct size

Coronary artery patency and

Table updated and modified from Humphries.51

thrombosis in dog

t-PA lysis of coronary artery

thrombus embolization

Occlusion time, rebleeding and

Mechanically damaged rabbit

mesenteric artery

Occlusion time and vessel patency

Electrically damaged, partially stenosed dog carotid artery

rabbit carotid artery

arterial/arterial shunt

Occlusion time

Prosthetic graft in dog femoral

prolongation of bleeding time

End Point

Cangrelor þ lytic therapy

Cangrelor

Cangrelor

AR-C67085MX

AR-C67085MX

cangrelor AZD6140

AR-C69931MX

P2Y12 Antagonist

Placebo þ lytic therapy

Placebo, clopidogrel

Vehicle

Aspirin

Aspirin

antagonists, aspirin

Clopidogrel, aGPIIb/IIIa

Comparator

and significant reduction in infarct size

Prevention of postlysis reocclusion

rebleeding but reduced embolization

No increase in bleeding time or

cessation of infusion

to control levels at 1 hour after

bleeding. Hemostasis had returned

> 80% reduction in occlusion incidence with small increase in

Aspirin no effect

occlusion compared with control.

Significant increase in time to

occlusion compared with aspirin

Significant increase in time to

antagonists

markedly improved antithrombotic/ antihemostatic separation for P2Y12

Wang et al31

Van Gestel et al30

Huang et al29

Leff et al27

Leff et al27

Humphries et al28

efficacy compared with clopidogrel and GPIIb/IIIa antagonists with

Leff et al,27

Ingall et al,12

References

compared with aspirin, equivalent

Greater antithrombotic efficacy

Main Findings

Summary of Effects of Intravenous Administration of Reversible P2Y12 Antagonists on Thrombosis in Preclinical In Vivo Models

Model

Table 1


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Figure 3 Effects of orbofiban, clopidogrel, cangrelor, and AZD6140 in a dog model of CFR in the femoral artery.12,27,28 CFRs are measured during a 30-minute period after increasing intravenous doses of study drug (n ¼ 5–6 dogs per treatment, mean standard error of the mean [s.e.m.]). Inhibition of ADP-induced (10 mM) platelet aggregation and tongue bleeding times are recorded at each dose interval. (PAI ¼ platelet aggregation inhibition)

recorded as defined by the thrombolysis in myocardial infarction (TIMI) criteria.35 A subsequent double-blind, randomized, placebo-controlled study in patients with unstable angina pectoris and non–Q-wave myocardial infarction36 and a double-blind placebo-controlled study in patients undergoing percutaneous transluminal coronary angioplasty (PTCA)37 further confirmed the safety profile of cangrelor. These studies in 91 and 200 patients, respectively, assessed the safety profile of cangrelor as adjunctive therapy to aspirin and either heparin or lowmolecular-weight heparin. In the study in patients with unstable angina pectoris, minor bleeding was slightly increased from 26% in the placebo-treated group to 38% in the cangrelor-treated group.36 In the study in patients undergoing PTCA, the incidence of bleeding was similar to that with placebo.37 Major bleeds were observed in 4 of 48 patients receiving the highest dose of cangrelor (4 mg/kg per minute) in the first part of this study.37 Only 2 of these bleeds were considered to be possibly related to the treatment and suggested a need for adjustment of the heparin dose. The other 2 major bleeds occurred subsequently, either after coronary artery

bypass grafting (CABG) or during repeat intervention and ReoPro administration. A clinical study to investigate cangrelor as an adjunct to fibrinolytic therapy for acute myocardial infarction in 101 patients suggested an increase in STsegment recovery without a concomitant increase in either non-CABG TIMI bleeding or major adverse clinical events during a 30-day follow-up.38 Although a trend toward increased TIMI grade 3 flow was observed, this effect was not statistically significant. The data suggest the feasibility of giving cangrelor as an adjunct to fibrinolytic therapy with possible improvements in reperfusion during fibrinolysis.

COMPARISON OF CANGRELOR WITH THIENOPYRIDINES The clinical data with the reversible direct P2Y12 antagonist cangrelor suggest that high levels of platelet inhibition can be consistently and rapidly obtained across patients.15 In this respect, cangrelor differs from the thienopyridine clopidogrel, which is a prodrug, the active metabolite of which binds irreversibly to the


P2Y12 receptor. Several recent articles demonstrated a wide range of platelet aggregation responses following clopidogrel dosing. Depending on the definition of platelet aggregation response and the concentration of agonist used, up to 30% of patients can be identified as nonresponders.39–41 After a 300-mg loading dose in

one study,42 66% of patients were nonresponders 2 hours after dosing and 24% were nonresponders after 5 days of subsequent maintenance therapy (75 mg). In other research, patients identified as nonresponders or poor responders after 5 days of treatment with clopidogrel did not significantly increase their response

Figure 4 Representation of combined data from the dog model of CFRs as shown in Figure 3 (n ¼ 5–6, mean s.e.m.). (A) After overcoming a threshold of about 30% inhibition of ADP-induced (10 mM) platelet aggregation, a nearly linear relationship exists between platelet inhibition and antithrombotic effect, independent of mechanism. Maximum antithrombotic effect is reached at 100% platelet inhibition. (B) A clear differentiation between prolongation of bleeding time at submaximal antithrombotic effect between treatment with GPIIb/IIIa and P2Y12 antagonists.

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when tested after 30 days of treatment.43 Clinical response seems to correlate with cytochrome P450 (CYP) 3A4 metabolic activity,44 an observation that suggests a possible risk of drug interactions with other CYP3A4 substrates such as atorvastatin.45,46 Increasing the clopidogrel loading dose to 600 mg may abolish this effect.47 The partial inhibition of the P2Y12 receptor by clopidogrel was confirmed by a study in healthy volunteers showing approximately 30% unoccupied P2Y12 receptors on platelets after 11 days of clopidogrel treatment.48 The same study demonstrated that blocking these remaining receptors ex vivo with cangrelor resulted in increased inhibition of 10 mM ADP-induced platelet aggregation. This profile was confirmed in a study that compared clopidogrel and cangrelor treatment in patients with ischemic heart disease.49 The results showed that, using 10 mM ADP, cangrelor infusion at 2 and 4 mg/mL per minute resulted in nearly complete platelet inhibition measured at 4 minutes after addition of ADP, whereas 4 to 7 days of clopidogrel treatment resulted in approximately 60% inhibition. Addition of cangrelor in vitro to blood from these clopidogreltreated patients again resulted in nearly complete inhibition. A study in 12 acute coronary syndrome patients provided additional evidence that cangrelor could overcome the incomplete inhibition produced by standard clopidogrel treatment.50 It could be questioned whether there is a need to aim for increased platelet inhibition or whether clopidogrel treatment already achieves maximal clinical benefit. Although clinical data are required to answer this question conclusively, preclinical data from the dog arterial thrombosis model (Fig. 3) suggest the possibility of better clinical outcome at higher levels of platelet inhibition. The body of data on platelet aggregation inhibition and inhibition of CFRs suggest that a threshold level of platelet inhibition must be achieved before significant antithrombotic activity can be measured (Fig. 4A). Using either GPIIb/IIIa or P2Y12 antagonists, more than 30% inhibition of platelet aggregation is needed to induce an antithrombotic effect. Thereafter, an almost linear relationship is observed, with maximum antithrombotic effect achieved at nearly complete platelet inhibition. The combined data from the dog arterial thrombosis model suggest that these effects are not associated with a similar increase in bleeding time for these antiplatelet therapies (Fig. 4B). The data suggest that significant increases in bleeding time are only observed at levels of P2Y12 antagonism that induce near maximum platelet inhibition. This effect differs from that observed with GPIIb/IIIa antagonists, for which a direct relationship between increased bleeding time and antithrombotic effect is observed. Although the current lack of studies linking the level of platelet inhibition in patients

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to either clinical outcome or to bleeding events hampers the interpretation of the animal data, the data provide a good platform for further development of cangrelor and AZD6140.

SUMMARY AND CONCLUSION The discovery of cangrelor has been of great value in understanding the role of ADP in platelet aggregation and in defining the roles of the P2Y1 and P2Y12 receptors on platelets. In addition, it has helped to define the central role of the ADP feedback mechanism in thrombosis. This role has been further confirmed by the clinical use of thienopyridines. The fact that intravenous administration of cangrelor to patients resulted in a uniformly high level of inhibition of platelet aggregation suggests the possibility for increased clinical benefit with this direct P2Y12 antagonist. The reversibility of effect with cangrelor suggests that patient management will be simplified in case of a bleeding event. Taken together, the experience with direct reversible P2Y12 antagonists provides considerable support for the further development of both cangrelor and AZD6140.

ACKNOWLEDGMENTS

The authors thank all colleagues at AstraZeneca R&D Charnwood and AstraZeneca R&D Mo¨lndal who have contributed in any way to the data and ideas presented in this article.

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