Taketoshi Ogawa

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.

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.

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.

Pharmacological profiles of R-96544, the active form of a novel 5-HT2A receptor antagonist R-102444

We examined the pharmacology of (2R,4R)-4-hydroxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-96544), the active form of a novel 5-HT(2A) receptor antagonist, (2R,4R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-102444). R-96544 produced a concentration-dependent inhibition of platelet aggregation induced by serotonin (5-hydroxytryptamine, 5-HT) alone or in combination with ADP in platelets from humans, monkeys, cats, rabbits, rats and mice. An intravenous administration of R-96544 to rabbits significantly inhibited ex vivo platelet aggregation induced by 5-hydroxytryptamine (5-HT) combined with epinephrine. An oral administration of R-102444 to rats also resulted in significant inhibition of ex vivo platelet aggregation, whereas R-102444 was ineffective in an in vitro platelet aggregation assay. These antiplatelet effects of R-96544 and R-102444 were more potent than those of two other 5-HT(2A) receptor antagonists, sarpogrelate and its active metabolite (+/-)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol hydrochloride (M-1). A binding study using cat platelet membranes showed that R-96544 has high affinity for 5-HT(2A) receptors but no effect on non-serotonergic [3H]ketanserin-binding sites. R-96544 caused a parallel shift to the right of concentration-response curves for 5-HT in rat caudal artery contraction mediated by 5-HT(2A) receptors. Schild plot analysis gave a pA(2) value of 10.4 with a slope near unity (1.04). R-96544 also inhibited 5-HT(2A) receptor-mediated contraction of guinea pig trachea but not 5-HT(3) receptor-mediated contraction of guinea pig ileum and 5-HT(2B) receptor-mediated contraction of rat fundus preparation. R-96544 (i.v.) attenuated the pressor responses evoked by 5-HT (15 microg/kg, i.v.) but not by phenylephrine (5 microg/kg, i.v.) and angiotensin II (0.1 microg/kg, i.v.), after ganglionic blockade in anesthetized spontaneously hypertensive rats. These results show that R-96544, the active form of R-102444, is a novel 5-HT receptor antagonist with potent, competitive, and 5-HT(2A)-selective activity.

Antiplatelet and Antithrombotic Effects of Orbofiban, a New Orally Active GPIIb/IIIa Antagonist, in Guinea Pigs

Antiplatelet and antithrombotic effects of orbofiban, a new orally active glycoprotein IIb/IIIa antagonist, were evaluated in guinea pigs. SC-57101A (0.03-1 microM), the hydrochloride salt of the active form of orbofiban, inhibited in vitro ADP- and collagen-induced platelet aggregation in a concentration-dependent manner. Oral administration of orbofiban (3-30 mg/kg) resulted in dose-dependent inhibition of ADP- and collagen-induced platelet aggregation. The inhibition peaked at 1-2 hours postdose and then declined slowly. The agent also showed similar inhibition of platelet aggregation in guinea pigs with dietary-induced hypercholesterolemia. In contrast, the antiaggregatory effects of acetylsalicylic acid differed more widely between normal and hyperlipidemic animals compared to those of orbofiban. Plasma concentration of the active form, measured by a column-switching HPLC method, correlated well with the inhibition of platelet aggregation. Orbofiban (3-100 mg/kg, p.o.) caused dose-dependent inhibition of thrombus formation in an arteriovenous-shunt-thrombosis model. Orbofiban at high doses (> or =30 mg/kg) and acetylsalicylic acid (100 mg/kg) both prolonged cutaneous bleeding time measured by the template method. These results demonstrate that orbofiban is an orally active and potent inhibitor of platelet aggregation with an efficacy that correlates well with the plasma concentration of its active form.

Platelet α-granule secretion and its modification by SC-57101A, a GPIIb/IIIa antagonist

Abstract Platelet–agonist interaction results in aggregatory and secretory responses. While the activation of glycoprotein (GP) IIb/IIIa plays an essential role in platelet aggregation, its role in granule secretion is not clear. The present study was performed to examine the effect of 3-[[[[1-[4-(aminoiminomethyl) phenyl]-2-oxo-3 S -pyrrolidinyl]amino]carbonyl]amino]-propanoate monohydrochloride salt (SC-57101A), a GPIIb/IIIa antagonist, on platelet α-granule secretion responses to collagen, ADP, and thrombin receptor activating peptide (TRAP). Both SC-57101A and prostaglandin E 1 (PGE 1 ) inhibited collagen-, ADP-, and TRAP-induced platelet aggregation in a concentration-dependent manner. SC-57101A inhibited the collagen- and ADP-induced release of platelet-derived growth factor (PDGF) and β-thromboglobulin (β-TG) from platelets, but not TRAP-induced secretion of these granule contents. On the other hand, PGE 1 inhibited the release of PDGF and β-TG from platelets activated with all the agonists used. ADP and TRAP elicited P-selectin expression in the absence of platelet aggregation, while collagen produced no such reaction. SC-57101A only moderately inhibited P-selectin expression induced by ADP and had no inhibitory effect on that induced by TRAP. The inhibition of ADP-induced secretion of α-granule contents by SC-57101A was abolished when platelets were pretreated with aspirin. These results suggest that GPIIb/IIIa activation plays a minor role, if any, in α-granule secretion in human platelets.