Atsuhiro Sugidachi

The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties.

CS‐747 is a novel antiplatelet agent that generates an active metabolite, R‐99224, in vivo. CS‐747 itself was totally inactive in vitro. This study examined in vivo pharmacological profiles of CS‐747 after single oral administration to rats. Orally administered CS‐747 (0.3–10 mg kg−1) partially but significantly decreased [3H]‐2‐methylthio‐ADP binding to rat platelets. CS‐747 (3 mg kg−1, p.o.) treatment neutralized ADP‐induced decreases of cyclic AMP concentrations induced by prostaglandin E1, suggesting that metabolites of CS‐747 interfere with Gi‐linked P2T receptor. CS‐747 (0.3 and 3 mg kg−1, p.o.) markedly inhibited ex vivo washed platelet aggregation in response to ADP but not to thrombin. CS‐747 also exhibited a marked inhibition of ADP‐induced ex vivo platelet aggregation in PRP with a rapid onset (<0.5 h) and long duration (>3 days) of action (ED50 at 4 h=1.2 mg kg−1). R‐99224 (IC50=45 μM) inhibited in vitro PRP aggregation in a concentration‐related manner. CS‐747 prevented thrombus formation in a dose‐related manner with an ED50 value of 0.68 mg kg−1. CS‐747 was more potent than clopidogrel (6.2 mg kg−1) and ticlopidine (>300 mg kg−1). CS‐747, clopidogrel, and ticlopidine prolonged the bleeding time. The order of potency of these agents in this activity was the same as that in antiaggregatory and antithrombotic activities. These findings indicate that CS‐747 is an orally active and a potent antiplatelet and antithrombotic agent with a rapid onset and long duration of action, and warrants clinical evaluations of the agent.

Patients with poor responsiveness to thienopyridine treatment or with diabetes have lower levels of circulating active metabolite, but their platelets respond normally to active metabolite added ex vivo

OBJECTIVES We evaluated the prevalence and mechanism of poor responsiveness to clopidogrel and prasugrel in coronary artery disease patients with and without diabetes. BACKGROUND Low platelet inhibition by clopidogrel is associated with ischemic clinical events. A higher 600-mg loading dose (LD) has been advocated to increase responsiveness to clopidogrel. METHODS In this study, 110 aspirin-treated patients were randomized to double-blind treatment with clopidogrel 600 mg LD/75 mg maintenance dose (MD) for 28 days or prasugrel 60 mg LD/10 mg MD for 28 days. Pharmacodynamic (PD) response was evaluated by light transmission aggregometry and vasodilator-stimulated phosphoprotein phosphorylation. The PD poor responsiveness was defined with 4 definitions previously associated with worse clinical outcomes. Active metabolites (AM) of clopidogrel and prasugrel were measured. Clopidogrel AM was added ex vivo. RESULTS The proportion of patients with poor responsiveness was greater in the clopidogrel group for all definitions at all time points from 1 h to 29 days. Poor responders had significantly lower plasma AM levels compared with responders. Patients with diabetes were over-represented in the poor-responder groups and had significantly lower levels of AM. Platelets of both poor responders and diabetic patients responded fully to AM added ex vivo. CONCLUSIONS Prasugrel treatment results in significantly fewer PD poor responders compared with clopidogrel after a 600-mg clopidogrel LD and during MD. The mechanism of incomplete platelet inhibition in clopidogrel poor-responder groups and in diabetic patients is lower plasma levels of its AM and not differences in platelet P2Y(12) receptor function.

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrel's active metabolite.

Background and methods: Prasugrel is a novel orally active thienopyridine prodrug with potent and long‐lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y12 receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated.Results: Oral administration of prasugrel to rats resulted in dose‐related and time‐related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5′‐diphosphate (ADP) (10 μm) with an IC50 value of 1.8 μm. Clopidogrel AM similarly inhibited platelet aggregation with an IC50 value of 2.4 μm. Similar results were also observed for ADP‐induced (10 μm) decreases in prostaglandin E1‐stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities.Conclusions: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.

Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type Gi-linked P2T antagonist, CS-747

CS‐747 is a novel thienopyridine‐type platelet ADP inhibitor which lacks in vitro activity. This study examined pharmacological profiles of R‐99224, a hepatic metabolite of CS‐747. R‐99224 produced a concentration‐dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03 – 1 μg ml−1), which was relatively specific to ADP compared to collagen and thrombin. R‐99224 (0.1 – 3 μg ml−1) also elicited a similar inhibition of ADP‐induced aggregation in rat platelets. The inhibition by R‐99224 (10 μg ml−1) persisted even after platelets were washed three times. Intravenous injection of R‐99224 (0.1 – 3 mg kg−1) to rats resulted in a dose‐dependent inhibition of ex vivo ADP‐induced platelet aggregation. R‐99224 (0.1 – 100 μM) decreased binding of [3H]‐2‐methylthio‐ADP ([3H]‐2‐MeS‐ADP), a stable ligand for platelet ADP receptors, to washed human platelets. The inhibition by R‐99224 reached a plateau at a concentration of 3 μM (1.4 μg ml−1), but complete inhibition was not achieved even at the highest concentration used (100 μM). R‐99224 (10 μM) in combination with ARL‐66096 (0.3 μM), an ATP analogue‐type Gi‐linked P2T receptor antagonist, produced no additional inhibition of [3H]‐2‐MeS‐ADP binding. In contrast, [3H]‐2‐MeS‐ADP binding was completely abolished by R‐99224 (10 μM) in combination with A3P5PS (300 μM), a selective P2Y1 antagonist, suggesting that R‐99224 selectively binds to the Gi‐linked P2T receptor. R‐99224 (0.01 – 3 μg ml−1) inhibited ADP‐induced [125I]‐fibrinogen binding to human platelets in a concentration‐dependent manner. R‐99224 (0.1 – 1 μg ml−1) also inhibited the ADP‐induced decrease in cyclic AMP levels in PGE1‐stimulated platelets, whereas the agent did not affect ADP (10 μM)‐induced Ca2+ mobilization. These findings suggest that R‐99224 is a selective and irreversible antagonist of Gi‐linked P2T receptors and that R‐99224 is a responsible molecule for in vivo actions of CS‐747.

The reversible P2Y12 antagonist cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to produce irreversible inhibition of platelet function

Summary.  Background: Agents that act as antagonists at P2Y12 ADP receptors on platelets are in use (clopidogrel), and in development for use (cangrelor and prasugrel), in patients with cardiovascular disease. Cangrelor is a direct‐acting reversible antagonist being developed for short‐term infusion; clopidogrel and prasugrel are oral prodrugs that provide irreversible inhibition via transient formation of active metabolites. At the cessation of cangrelor infusion, patients are likely to receive clopidogrel or prasugrel as a means of maintaining antiplatelet therapy. Objectives: To apply an experimental in vitro approach to investigate the possibility that cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to inhibit ADP‐mediated platelet function. Methods: The effects of cangrelor and the active metabolites of clopidogrel (C‐AM) and prasugrel (P‐AM) on platelet function were assessed by ADP‐induced platelet P‐selectin expression in whole blood. The method involved rapid removal of the antagonists by dilution, and measurement of residual platelet inhibition. Results: Cangrelor, C‐AM and P‐AM markedly inhibited P‐selectin expression. The effect of cangrelor, but not of C‐AM and P‐AM, was reversible following antagonist removal. Preincubation of blood with cangrelor prior to addition of C‐AM or P‐AM reduced the ability of metabolites to irreversibly antagonize P2Y12. Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor. Conclusions: Cangrelor influences the ability of the active metabolites of clopidogrel or prasugrel to inhibit platelet function irreversibly. Careful consideration should be given to the timing of administration of an oral P2Y12 antagonist following cangrelor infusion.

Impaired responsiveness to the platelet P2Y12 receptor antagonist clopidogrel in patients with type 2 diabetes and coronary artery disease.

BACKGROUND Several studies have shown that patients with diabetes mellitus (DM) exhibit an impaired response to clopidogrel. This may contribute to their increased risk of recurrent atherothrombotic events, despite the use of dual-antiplatelet therapy. The mechanisms for impaired clopidogrel response in DM patients have not been fully elucidated. OBJECTIVES The aim of this study was to explore the mechanisms for impaired clopidogrel-mediated platelet inhibition in patients with DM using a comprehensive methodological approach embracing both pharmacokinetic (PK) and pharmacodynamic (PD) assessments as well as ex vivo and in vitro investigations. METHODS Patients (DM, n = 30; non-DM, n = 30) with stable coronary artery disease taking aspirin 81 mg/day and P2Y12 antagonist naive were enrolled. Blood was collected before and at various times (0.5, 1, 2, 4, 6, and 24 h) after a 600-mg loading dose of clopidogrel. PD assessments included vasodilator-stimulated phosphoprotein, light transmission aggregometry, and VerifyNow P2Y12 ex vivo, before and after dosing and following in vitro incubation with escalating concentrations (1, 3, and 10 μM) of clopidogrels active metabolite (Clop-AM). Exposure to Clop-AM was also determined. RESULTS PD assessments consistently showed that during the overall 24-h study time course, residual platelet reactivity was higher in DM patients compared with non-DM patients. In vitro incubation with Clop-AM revealed altered functional status of the P2Y12 signaling pathway in DM platelets as measured by vasodilator-stimulated phosphoprotein, but not with other PD assays. Clop-AM exposure was ∼40% lower in DM patients than in non-DM patients. CONCLUSIONS The present study suggests that among DM patients, impaired P2Y12 inhibition mediated by clopidogrel is largely attributable to attenuation of clopidogrels PK profile. This is characterized by lower plasma levels of Clop-AM over the sampling time course in DM patients compared with non-DM patients and only modestly attributed to altered functional status of the P2Y12 signaling pathway.

Relationship between degree of P2Y12 receptor blockade and inhibition of P2Y12-mediated platelet function

The thienopyridine P2Y12 receptor antagonists clopidogrel and prasugrel prevent arterial thrombosis and are routinely used following percutaneous coronary intervention. However, the optimal level of P2Y12 blockade to effectively inhibit platelet function is unknown. These studies utilised the active metabolite of prasugrel (R-138727) to achieve a range of P2Y12 blockade in vitro and assessed several aspects of platelet function. Blood from healthy volunteers was incubated with R-138727 (0-10 microM). P2Y12 receptor number was assessed using a 33P-2MeSADP binding assay. Platelet aggregation (PA) was measured by optical aggregometry with ADP 2-20 microM. VASP phosphorylation, annexin V binding, microparticle formation and P-selectin expression were assessed by flow cytometry. Increasing numbers of unblocked receptors were required for a sustained aggregation response with decreasing concentrations of ADP. A P2Y12 receptor blockade of 60-80% resulted in strong inhibition of final PA response, P-selectin expression, microparticle formation and vasodilator-stimulated phosphoprotein (VASP). PA induced by ADP 2 microM and P-selectin expression were particularly sensitive to low levels of receptor blockade whereas the VASP phosphorylation assay was relatively insensitive, requiring 60% receptor blockade to achieve substantial inhibition. Different assays varied in their ability to discriminate particular ranges of P2Y12 blockade and 80% or greater P2Y12 receptor blockade is required for consistently strong inhibition of several aspects of platelet function. These data guide the interpretation of results from different assays used to monitor the effects of P2Y12 receptor antagonists.

Stereoselective inhibition of human platelet aggregation by R-138727, the active metabolite of CS-747 (Prasugrel, LY640315), a novel P2Y12 receptor inhibitor

CS-747 (Prasugrel, LY640315) is a thienopyridine antiplatelet prodrug that is metabolized to the thiol-containing active metabolite R-138727,which binds to and irreversibly inhibits the platelet P2Y12ADP receptor. R-138727 is composed of 4 stereo-isomers, (R, S)-, (R, R)-, (S, S)-, and (S, R)-isomers (the first letter for the configuration of a chiral center at the sulfur-bearing position and the second for that at the benzylic position). In the present study, we determined the stereoselectivity of P2Y12 antagonist effects by assessing the antagonism of the [3H]-2-MeS-ADP that binds to human P2Y12 receptors expressed in Chinese hamster ovary cells as an affinity assay, and by the inhibition of ADP-induced aggregation of washed human platelets as a functional assay. R-138727 and its 2 components, R-99224, a mixture of (R, S)- and (S, R)-isomers and R-100364, a mixture of (R, R)- and (S, S)-isomers, inhibited [3H]-2-MeS-ADP binding and platelet aggregation. The rank order of potency of these compounds were identical in both assays: R-99224>R-138727>> R-100364. Inhibition of ADP-induced platelet aggregation by R-138727 and R-99224 was concentration- and time-related. In experiments using the 4 single stereo-isomers, all isomers inhibited ADP-induced platelet aggregation, but the (R, S)-isomer was found to be the most potent, followed by the (R, R)-isomer. These in vitro studies indicate that R- 138727 is an effective antagonist of P2Y12 and potent inhibitor of ADP-induced platelet aggregation, and that these antiplatelet activities of R-138727 are largely dependent on its (R, S)-isomer. This suggests that the (R)-configuration of the reactive thiol group of the active metabolite of CS-747 is critical for P2Y12 and platelet inhibitory activities.

The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses.

The aim of these studies was to investigate the extent of platelet P2Y12 receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y12 receptor antagonism was determined using a radioligand binding assay (33P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 µmol/L and cangrelor 1 µmol/L completely inhibited 33P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y12 receptors. Prasugrels active metabolite effectively blocks the P2Y12 receptor with the highest concentrations tested yielding complete inhibition of P2Y12-mediated amplification of several important platelet responses.

Regulation of Functionally Active P2Y12 ADP Receptors by Thrombin in Human Smooth Muscle Cells and the Presence of P2Y12 in Carotid Artery Lesions

Objective—The platelet P2Y12 ADP receptor is a well-known target of thienopyridine-type antiplatelet drugs. This study is the first to describe increased transcriptional expression of a functionally active P2Y12 in response to thrombin in human vascular smooth muscle cells (SMC). Methods and Results—On exposure to thrombin, P2Y12 mRNA was transiently increased, whereas total protein and cell surface expression of P2Y12 were markedly increased within 6 hours and remained elevated over 24 hours. This effect was mediated by activation of nuclear factor &kgr;B. Preincubation with thrombin significantly enhanced the efficacy of the P2Y receptor agonist 2-methylthio-ADP to induce interleukin 6 expression and SMC mitogenesis. Effects induced by 2-methylthio-ADP were prevented by RNA interference-mediated knockdown of P2Y12 and a selective P2Y12-antagonist R-138727, the active metabolite of prasugrel. In addition, positive P2Y12 immunostaining was shown in SMC of human carotid artery plaques and was found to colocalize with tissue factor, the rate-limiting factor of thrombin formation in vivo. Conclusion—These data suggest that the P2Y12 receptor not only is central to ADP-induced platelet activation but also may mediate platelet-independent responses, specifically under conditions of enhanced thrombin formation, such as local vessel injury and atherosclerotic plaque rupture.