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In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.

Background New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y 12 may be used in combination with glycoprotein ( GP ) II b‐ III a antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y 12 and GPII b‐ III a inhibition on platelet function and procoagulant activity. Methods and Results Healthy donor blood was treated with the active metabolite of prasugrel (R‐138727 5 μmol/L), GPIIb‐IIIa antagonists (abciximab 3 μg/mL or eptifibatide 0.9 μg/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between R‐138727 and GPIIb‐IIIa antagonists were observed. R‐138727 and the GPIIb‐IIIa antagonists had additive inhibitory effects on collagen‐stimulated platelet aggregation and on the collagen plus ADP–stimulated level of activated platelet surface GPIIb‐IIIa. R‐138727 and abciximab each inhibited collagen plus ADP–stimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADP–stimulated prothrombin cleavage. Addition of R‐138727 reduced prothrombin cleavage in eptifibatide‐treated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment. Conclusions The complementary effects of abciximab and R‐138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo.

Arterial thrombosis in the rabbit was established as a novel model to evaluate the effects of antithrombotic agents. Endothelial injury was produced by applying electrical stimulation to the femoral artery. The process of primary endothelial injury, and subsequent platelet activation and fibrin formation were confirmed by electron microscopy. In this model, vessel occlusion occurred within 30 min after stimulation without changes in heart rate and blood pressure. Using this model, several agents were evaluated for their antithrombotic activities: aspirin (30 mg/kg, p.o.), ticlopidine (10-100 mg/kg, p.o.), heparin (300 unit/kg, i.v.), PPACK (10-33 micrograms/kg/min, i.v.), WEB-2347 (1 mg/kg, p.o.) and nicardipine (10 micrograms/kg, i.v.). Fifty per cent decrease in vessel temperature (T1/2), assessed by a thermic probe, averaged 15.1 +/- 1.2 (n = 11, p.o.) and 15.6 +/- 1.9 min (n = 8, i.v.) in the vehicle groups, and this was significantly prolonged by aspirin (23.0 +/- 2.6 min), ticlopidine at a dose of 100 mg/kg (24.6 +/- 2.5 min), heparin (27.2 +/- 2.8 min) and PPACK at a dose of 33 micrograms/kg (30.0 min). However, WEB-2347 and nicardipine were without effect. The effect of aprosulate, a new class of polyanion with anticoagulant activity, was further examined. Aprosulate (1-30 mg/kg, i.v.) inhibited thrombus formation in a dose-dependent manner. These results show that acute occlusive thrombus can be readily and reproducibly formed in the rabbit femoral artery and suggest that this thrombus formation depends on the activation of both platelets and blood coagulation. The merit of this model lies in its simplicity for evaluating the antithrombotic effects of antiplatelet and anticoagulant agents and is therefore expected to be extensively used in the future.

We evaluated the effects of prasugrel, a third-generation thienopyridyl prodrug, on P2Y12 receptors, adenosine 5'-diphosphate (ADP)-induced platelet aggregation, and myocardial infarction (MI) in rats. Oral administration of prasugrel (0.3-3 mg/kg) resulted in the dose-related inhibition of washed platelet aggregation induced by ADP (1-10 μM). Ex vivo [H]-2-MeS-ADP binding to platelet P2Y12 receptors was also inhibited by prasugrel in a similar dose range. The antiaggregatory effects of prasugrel correlated strongly with P2Y12 blockade with correlation coefficients of 0.85-0.92, suggesting that the antiaggregatory activity of prasugrel largely reflected P2Y12 blockade achieved in vivo. We further examined the effects of the in vivo P2Y12 inhibition by prasugrel (1-10 mg/kg, po) on MI induced by thrombotic coronary artery occlusion in rats. In surviving rats, infarct size at 24 hours after photoirradiation was evaluated. In the vehicle group, necrosis area/total left ventricular area was 37.9% ± 6.8% (mean ± SE, n = 7). At all prasugrel doses tested (n = 7 for each dose), necrosis area/total left ventricular area was significantly smaller than that in the vehicle group: 14.4% ± 4.0% for 1 mg/kg (P < 0.01), 19.8% ± 4.5% for 3 mg/kg (P < 0.05), and 14.8% ± 3.6% for 10 mg/kg (P < 0.01). At the highest administered dose of prasugrel (10 mg/kg), blood pressure and heart rate were unchanged. Arrhythmia was observed in 5 of 7 animals in the vehicle group at 24 hours after irradiation; in contrast, no arrhythmia was found in the group treated with prasugrel (10 mg/kg). Taken together, these results demonstrate that prasugrel is a selective P2Y12 inhibitor in vivo, providing effective inhibition of platelet aggregation and MI in rats.

The VerifyNow® P2Y12 (VN-P2Y12) test reports thienopyridine-mediated platelet inhibition relative to a "BASE" channel, potentially eliminating the need for predrug patient assessment, by activating platelets through a P2Y(12)-independent pathway. The original formulation of the BASE channel used a protease activated receptor-1 (PAR-1) peptide as agonist. However, more potent P2Y(12) antagonism required more complete activation of platelet thrombin receptors for the BASE measurement in order to negate any contribution of the P2Y(12) receptor. Accordingly, the current BASE channel formulation consists of both PAR-1 and protease activated receptor-4 (PAR-4) activating peptides to facilitate a higher degree of platelet activation. The aim of this study was to compare the performance of PAR-1 versus PAR-1/PAR-4 activating peptides as the BASE channel formulation using prasugrel's active metabolite, R-138727, in vitro to achieve high-grade P2Y(12) inhibition. Blood samples from 20 healthy donors were spiked in vitro with R-138727 at concentrations that include plasma levels achieved following prasugrel administration and were incubated for 30 minutes at 37°C. All samples were run in triplicate using both the PAR-1 and the PAR-1/PAR-4 BASE formulation in the VN-P2Y12 test device. The data confirmed the sensitivity of the original BASE formulation to high-grade P2Y(12) inhibition as reflected in the concentration-dependent decrease in values. Incorporation of PAR-4 activating peptide eliminated the effect of P2Y(12) blockade at all concentrations of R-138727. Thus, the use of PAR-1/PAR-4 in the BASE channel of the VN-P2Y12 cartridge addresses the impact of high grade P2Y(12) blockade and may allow more accurate reporting of "% inhibition" in patients treated with more effective P2Y(12) antagonists.

SummaryThienopyridines and other agents target the platelet P2Y12 receptor and inhibit several platelet activities mediated by adenosine diphosphate (ADP). The measurement of vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation, expressed as platelet reactivity index (PRI), mirrors the degree of P2Y12 receptor inhibition and can detect the well-known variable response to clopidogrel. The commercially available VASP assay uses flow cytometry (FC) and requires that the test be run within 48 hours of blood collection. A new ELISA VASP assay offers the advantages of using more widely available technology and the potential to freeze and store samples before analysis. The objectives of the present study were to compare the performance of the ELISA and FC methods and to describe the relative flexibility of the ELISA-based assay. Human blood samples encompassing a wide range of levels of P2Y12 blockade achieved in vitro by preincubation with P2Y12 antagonists or in vivo from patients treated with clopidogrel were included, reflecting the wide spread of values reported in clinical studies. The correlation between the PRI measured by ELISA and FC was highly significant (r=0.95, pNote: All work done at BioCytex, Marseille, France and Daiichi Sankyo, Tokyo, Japan.

We compared plasma 11-dhTXB2 and TXB2 for their ability to reflect in vivo TX formation in rabbits treated with AA and CS-518, a TX synthase inhibitor. The average plasma level of TXB2 in rabbits was much higher than that of 11-dhTXB2, probably because of artificial formation of TXB2 during blood sampling. CS-518 (1 mg/kg, p.o.) caused a long-lasting suppression of the 11-dhTXB2 level, and its inhibitory effect on 11-dhTXB2 was much more extensive than that on TXB2. AA-infusion for 5 min resulted in transient and remarkable increases of both TXs, and prevention of such increases by CS-518 pretreatment (1 mg/kg, i.v.) was shown: inhibitions of 11-dhTXB2 and TXB2 were 85% and 40%, respectively. The inhibitory effect caused by CS-518, which was more clearly observed on plasma 11-dhTXB2 than on TXB2, was due to not only the completely inhibited levels without artificial formation but also the durable high levels based on the long half-life of 11-dhTXB2 in AA-infused rabbits. CS-518 injection during sustained AA-infusion also resulted in a 2-fold faster disappearance of plasma 11-dhTXB2 than was seen without CS-518, despite its long half-life. Considering the absence of artificial formation, the long half-life, and the good response to change of TX formation, plasma 11-dhTXB2 is superior to TXB2 as an index for monitoring in vivo TX synthase activity and its pharmacological modification with AA and CS-518.

[Ca(2+)](i) and pH(i) were measured in unstimulated and thrombin-stimulated platelets from young (3 months) and aged (24 months) rats in the presence and absence of external Ca(2+). There was no difference in the basal level of [Ca(2+)](i) between young and aged rat platelets in the presence of external Ca(2+). On the other hand, thrombin-induced Ca(2+) mobilization was markedly increased in aged rat platelets compared to young rat platelets in the presence of external Ca(2+). This difference in Ca(2+) mobilization was more pronounced in the absence of external Ca(2+), suggesting that Ca(2+) release from intracellular storages was enhanced in aged rat platelets. In contrast to what was observed for Ca(2+), the basal level of pH(i) in aged rat platelets was higher than that in young rat platelets whereas thrombin-induced pH(i) increases (ΔpH) were similar in both groups in the presence and absence of external Ca(2+). These results indicate that age-associated changes occur in thrombin-induced Ca(2+) release from intracellular storages in rat platelets. These changes seem to be related to those in basal pH(i) levels.

The in vivo effects of a new synthetic inhibitor of blood coagulation, aprosulate sodium, were investigated. Intravenous bolus injection of aprosulate or standard heparin in rats produced an immediate prolongation of the APTT which were characterized by a moderate dose-dependency and long-lasting duration when compared with those of standard heparin. Standard heparin inhibited plasma factor Xa activity, but aprosulate did not even at the highest dose used. Both agents inhibited thrombus formation in a dose-dependent manner in an arterio-venous shunt model. At antithrombotic doses, standard heparin prolonged the bleeding time measured by the tail transection method, but aprosulate did not. The present results suggest that aprosulate has promising in vivo profile as an anticoagulant and antithrombotic agent.

We previously reported that thrombin-induced Ca(2+) mobilization was enhanced in aged rat platelets. Since Ca(2+) mobilization in platelets is believed to be closely associated with platelet activation, we examined Ca(2+) mobilization and the aggregatory response to ADP and thrombin in young (3 months) and aged (24 months) rat platelets. Blood levels of fibrinogen and Ca(2+) in aged rats were higher than those in young rats. ADP-induced platelet aggregation in aged rats was significantly enhanced in platelet rich plasma and in washed platelet suspension, suggesting that age-associated hyperaggregability to ADP is attributable to changes in platelets themselves. On the other hand, thrombin (0.03-0.3 unit/ml)-induced aggregation in washed platelet suspension from aged rats was comparable to that from young rats. But, thrombin (0.3 unit/ml)-induced intracellular Ca(2+)mobilization was enhanced in aged rat platelets in the presence of extracellular Ca(2+). Likewise, ADP-induced Ca(2+) mobilization was enhanced in aged rat platelets. These results suggest that enhanced Ca(2+) mobilization in aged rat platelets is associated with hyperaggregability to ADP but not to thrombin.

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 κB. 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.

SummaryThe 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 μM). P2Y12 receptor number was assessed using a 33P-2MeSADP binding assay. Platelet aggregation (PA) was measured by optical aggregometry with ADP 2–20 μM. 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 μM 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.

To develop a novel and effective anticoagulant with potent and selective factor Xa (FXa) inhibitory activity, a new series of cinnamyl derivatives with enhanced lipophilicity and prodrug forms were synthesized and their biological activities were evaluated. As a result, we found that cinnamyl derivative (N-{4-[1-(acetimidoyl)piperidin-4-yloxy]-3-carbamoylphenyl}-N-[(Z)-3-(3-amidinophenyl)-2-fluoro-2-propenyl]sulfamoyl)acetic acid dihydrochloride (26d, R-142086) with a fluorine atom on the double bond exhibited potent anticoagulant activity and no mutagenic potential. Moreover, orally administered R-142086 exhibited potent anti-FXa activity and anticoagulant activity in dogs.

To develop a potent and oral anticoagulant, a series of compounds with cinnamyl moiety was synthesized and their factor Xa (FXa) inhibitory activities were examined. As a result, some cinnamyl derivatives showed potent FXa inhibitory activities in vitro. Among them, compounds with substituent at the 3-position on the central benzene ring represented by (N-{4-[1-(acetimidoyl)piperidin-4-yloxy]-3-chlorophenyl}-N-[(E)-3-(3-amidinophenyl)-2-propenyl]sulfamoyl)acetic acid dihydrochloride (45b) and (N-{4-[1-(acetimidoyl)piperidin-4-yloxy]-3-carbamoylphenyl}-N-[(E)-3-(3-amidinophenyl)-2-propenyl]sulfamoyl)acetic acid dihydrochloride (45j) exhibited potent FXa inhibitory activities with IC50 values of less than 10 nM in vitro. These compounds also showed potent anticoagulant activities both in vitro and ex vivo. Furthermore, these compounds exhibited no lethal toxicity (30 mg/kg, i.v.).

Prasugrel is an orally available thienopyridyl prodrug with more potent in vivo antiplatelet effects compared to clopidogrel. In the present study, we examined the effects of prasugrel in rat models of cerebral and peripheral arterial occlusive diseases. Cerebral arterial thrombosis was induced by photochemical irradiation of the middle cerebral artery. Prasugrel (3 and 10 mg/kg) dose-relatedly and significantly reduced thrombus-mediated cerebral infarction 24 h after the irradiation. The effect of prasugrel was further examined in an embolic infarction model. Four h after an oral administration of prasugrel, non-occlusive thrombus formation in the right common carotid artery was initiated. In this model, prasugrel (0.3-3 mg/kg) reduced incidence, total area, and total number of cerebral infarcts in a dose-related manner 24 h after the vascular injury. Clopidogrel (10 or 30 mg/kg) was less potent than prasugrel at the doses tested on these thrombotic and embolic infarctions. Finally, the effect of prasugrel on lauric acid-induced peripheral arterial occlusive diseases was evaluated. After injection of lauric acid into the femoral artery, the lesions were scored for the following 10 days as they gradually progressed from the toe throughout the leg. Prasugrel (0.03-3 mg/kg/day) administered from the day before the lauric acid injection for 11 successive days inhibited the progression of the disease in a dose-related manner. Clopidogrel (3-30 mg/kg/day) showed similar effect but its effect was less potent than prasugrel. These results suggest that prasugrel could be a useful drug for preventing thromboembolic diseases including cerebral infarction and peripheral arterial occlusive diseases.

Agents that act as antagonists at P2Y(12) 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.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.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.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 P2Y(12). Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor.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 P2Y(12) antagonist following cangrelor infusion.

Allergic inflammation was induced by injecting an antigen solution into an air pouch made on the dorsum of immunized rats with the antigen azobenzene-arsonate-conjugated acetyl bovine serum albumin. In this model, leukocyte infiltration into the pouch fluid was prominent 4–8 h after the antigen challenge. Most of the infiltrated leukocytes were neutrophils. Administration of the platelet-activating factor (PAF) antagonists such as CV-3988 and L-652,731 into the air pouch 15 min before and at the time of the antigen challenge failed to suppress leukocyte infiltration at 8 h. However, when the PAF antagonist was injected into an air pouch 4 h after the antigen challenge, neutrophil infiltration at 8 h was suppressed in a dose-dependent manner. Combined treatment with the 5-lipoxygenase inhibitor AA861 and the PAF antagonist did not potentiate the effect of the PAF antagonist, suggesting that participation of leukotriene B4 in neutrophil infiltration in this model is negligible. Eosinophil infiltration was very weak at 8 h, and the PAF antagonist showed no significant effect. At 8 h, the PAF level in the serum of the immunized rats was significantly higher than that of the nonimmunized rats. Intravenous administration of the PAF antagonist 15 min before the antigen challenge suppressed leukocyte infiltration more effectively than local administration into the pouch. These results indicate that PAF plays a significant role in neutrophil infiltration in allergic inflammation.

A series of cinnamylindoline derivatives were synthesized, and their factor Xa (FXa) inhibitory activities and selectivity over trypsin were evaluated. Among them, some novel derivatives showed potent FXa inhibitory activities and good selectivity over trypsin. Especially, (E)-2-{5-[1-(acetimidoyl)piperidin-4-yloxy]-2-[2-(5-amidino-2-hydroxyphenyl)ethen-1-yl]indolin-1-ylsulfonyl}acetic acid (22f) having 2-hydroxycinnamyl moiety exhibited the most potent FXa inhibitory activity in vitro. Furthermore, 22f also exhibited potent anticoagulant activities in vitro.

Prasugrel is a novel orally active thienopyridine with faster, higher and more reliable inhibition of platelet aggregation than clopidogrel reflecting its metabolism in vivo to an active metabolite with selective P2Y(12) antagonistic activity. Several lines of evidence support the contention that prasugrel provides selective P2Y(12) receptor antagonistic activity. To date, however, direct evidence of P2Y(12) specific action by prasugrel in vivo is limited. In the present study, effects of prasugrel on ex vivo platelet aggregation were examined in wild type (WT) and P2Y(12)(-/-) mice. In WT mice, prasugrel showed platelet inhibition that was 8.2 times more potent than clopidogrel. In P2Y(12)(-/-) mice, ADP induced platelet aggregation was minimal, and its extent was similar to that in prasugrel-treated WT mice. In addition, no further inhibition of platelet aggregation was observed after administration of prasugrel to P2Y(12)(-/-) mice. Furthermore, prasugrel-treated WT mice showed similar aggregation patterns using collagen- and murine PAR-4 agonist peptide to those of P2Y(12)(-/-) mice treated with vehicle or prasugrel. Overall, these results clearly provide additional in vivo evidence that prasugrel has selective P2Y(12) antagonistic activity.

Background and methods Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y(12) 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 microm) with an IC(50) value of 1.8 microm. Clopidogrel AM similarly inhibited platelet aggregation with an IC(50) value of 2.4 microm. Similar results were also observed for ADP-induced (10 microm) decreases in prostaglandin E(1)-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.

A series of bisamidine derivatives each having a ring structure in the center of the molecule was synthesized and their Factor Xa (FXa) inhibitory activities were evaluated. Among them, some indoline derivatives showed potent inhibitory activities in vitro. In particular, (R)-18a having an (R)-configuration at the 2-position of the indoline ring exhibited the most potent FXa inhibitory activity in vitro, more potent than DX-9065a. Furthermore, (R)-18a exhibited more potent anticoagulant activity than DX-9065a. We also succeeded in obtaining an X-ray crystal structure of FXa bound with (R)-18a.

SummaryCS-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 P2Y12 ADP receptor. R-138727 is composed of 4 stereoisomers, (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.

R-102444 is a prodrug that is metabolized into R-96544, a potent and selective 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. The effects of R-102444 on peripheral vascular disease were examined using two different rat models: one induced by lauric acid and the other by ergotamine plus epinephrine. R-96544 (0.3-30 nM) relaxed the 5-HT (3 microM)-precontracted rat caudal artery in a concentration-dependent manner. The intravenous administration of R-96544 (0.3-3 microg/kg) to anesthetized rats inhibited the pressor response to 5-HT (50 microg/kg i.v.) dose dependently. The oral administration of R-102444 (1 mg/kg) to rats resulted in a marked inhibition of platelet aggregation induced by 5-HT plus ADP, and statistically significant inhibition was still evident 8 h after the dosing. In contrast, sarpogrelate, at a dose of 100 mg/kg p.o., produced only a moderate antiplatelet effect. Oral administration of R-102444 (1 mg/kg/day, o.d.) significantly prevented the progression of peripheral vascular lesion induced by the injection of lauric acid into a rat femoral artery, whereas sarpogrelate (100 mg/kg/day) showed only a minimal effect. Both 5-day treatments with R-102444 (1-30 mg/kg/day p.o., o.d.), one commenced 1 h before the injection of epinephrine plus ergotamine and one just after injection, resulted in the prevention of rat tail gangrene in a dose-dependent manner, whereas sarpogrelate (100 mg/kg) produced a minimal protection in this model. Based on these results, we conclude that 5-HT2A receptor activation is involved in peripheral vascular disease in the rat and that R-102444 is a useful oral agent for the investigation of diseases involving 5-HT2A receptor activation.

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.

1. 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. 2. R-99224 produced a concentration-dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03 - 1 microg ml(-1)), which was relatively specific to ADP compared to collagen and thrombin. 3. R-99224 (0.1 - 3 microg ml(-1)) also elicited a similar inhibition of ADP-induced aggregation in rat platelets. The inhibition by R-99224 (10 microg 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. 4. R-99224 (0.1 - 100 microM) decreased binding of [(3)H]-2-methylthio-ADP ([(3)H]-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 microM (1.4 microg ml(-1)), but complete inhibition was not achieved even at the highest concentration used (100 microM). 5. R-99224 (10 microM) in combination with ARL-66096 (0.3 microM), an ATP analogue-type G(i)-linked P2T receptor antagonist, produced no additional inhibition of [(3)H]-2-MeS-ADP binding. In contrast, [(3)H]-2-MeS-ADP binding was completely abolished by R-99224 (10 microM) in combination with A3P5PS (300 microM), a selective P2Y(1) antagonist, suggesting that R-99224 selectively binds to the G(i)-linked P2T receptor. 6. R-99224 (0.01 - 3 microg ml(-1)) inhibited ADP-induced [(125)I]-fibrinogen binding to human platelets in a concentration-dependent manner. R-99224 (0.1 - 1 microg ml(-1)) also inhibited the ADP-induced decrease in cyclic AMP levels in PGE(1)-stimulated platelets, whereas the agent did not affect ADP (10 microM)-induced Ca(2+) mobilization. 7. These findings suggest that R-99224 is a selective and irreversible antagonist of G(i)-linked P2T receptors and that R-99224 is a responsible molecule for in vivo actions of CS-747.

A series of 12-(2-phenylethyl)phenoxy]ethylpyrrolidine derivatives were synthesized, and their affinity for serotonin-2 (5-HT2) and dopamine-2 (D2) receptors was examined. Among them, compound 17, (2R,4R)-4-hydroxy-2-[2-[2-[2-(3-methoxyphenyl)ethyl]phenoxylethyl] -1-methylpyrrolidine hydrochloride, showed high 5-HT2 receptor affinity in vitro. This compound was a more potent inhibitor of ex vivo 5-HT-induced platelet aggregation than compound 3, which was previously shown to be more potent than ketanserin (1) and sarpogrelate (2a). However, compound 17 produced gastric irritation in rats. Therefore, we carried out a further derivatization of 17, and compound 45 (R-102444), a lauryl ester prodrug of compound 17, was found to be a promising candidate as an antithrombotic agent. Oral administration of R-102444 produced a marked inhibition of 5-HT-induced ex vivo platelet aggregation, and R-102444 did not cause any gastric irritation. The antiaggregatory effects of R-102444 were more potent than those of sarpogrelate (2a) and its active metabolite, M-1 (2b). In addition, R-102444 exhibited more potent antithrombotic effects than sarpogrelate in a rat photochemically-induced thrombosis model.

Serotonin [5-hydroxytryptamine (5-HT)] has been implicated in platelet activation and vasoconstriction, two processes that contribute to arterial thrombosis in atherosclerotic diseases. In the present study, Japanese White rabbits fed 1% cholesterol for 5 weeks were used to investigate the response of hypercholesterolemic vascular arteries and platelets to 5-HT. Contractions of the thoracic aorta induced by 5-HT were comparable between the cholesterol-fed group and the age-matched control group. However, acetylcholine-induced vasodilation in arteries preconstricted with 5-HT was moderately but significantly attenuated in the cholesterol-fed rabbits. Platelet aggregation responses to 5-HT (0.1-3 micromol/l) in combination with epinephrine (5 micromol/l), adenosine diphosphate (ADP) (0.3-10 micromol/l), 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prostaglandin F2alpha (U-46619) (1-30 micromol/l) or collagen (3 microg/ml) were significantly enhanced in cholesterol-fed rabbits. In contrast, platelet 5-HT content determined with a high-performance liquid chromatography-electrochemical detector (HPLC-ECD) was significantly decreased in cholesterol-fed rabbits. These results suggest a possible association among the endothelial dysfunction, platelet aggregation and platelet 5-HT content in rabbits with dietary hypercholesterolemia.

1. 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. 2. Orally administered CS-747 (0.3 - 10 mg kg(-1)) partially but significantly decreased [(3)H]-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 E(1), suggesting that metabolites of CS-747 interfere with G(i)-linked P2T receptor. 3. 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 (ED(50) at 4 h=1.2 mg kg(-1)). 4. R-99224 (IC(50)=45 microM) inhibited in vitro PRP aggregation in a concentration-related manner. 5. CS-747 prevented thrombus formation in a dose-related manner with an ED(50) 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)). 6. 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. 7. 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.

A series of [2-(omega-phenylalkyl)phenoxy]alkylamines was synthesized and their receptor binding affinity was examined in vitro. These compounds showed an affinity for serotonin-2 (5-HT2) and dopamine-2 (D2) receptors. [2-(2-phenylethyl)phenoxy]alkylamine derivatives with a pyrrolidine or piperidine moiety in the structure showed higher affinity for 5-HT2 receptors but lower affinity for D2 receptors. Among these compounds, (S)-2-[2- [2-[2-(3-methoxyphenyl)ethyl]phenoxy]ethyl]-1-methylpyrrolidine, (S)-27, exhibited the most potent and selective affinity for 5-HT2 receptors. Furthermore, (S)-27 was effective in inhibiting 5-HT-induced vasoconstriction in vitro and platelet aggregation both in vitro and ex vivo.

The present study was undertaken to examine the involvement of platelet-derived serotonin (5-hydroxytryptamine; 5-HT) in thromboxane A2 (TXA2)-induced platelet aggregation. Pharmacological experiments with 5-HT2 and TXA2 inhibitors were conducted on platelet aggregation in platelet-rich plasma from cats. Exogenously added 5-HT, U-46619 (a stable TXA2 analogue) and collagen caused platelet aggregation in a concentration-dependent manner. The combination of low concentrations of 5-HT and U-46619 caused full platelet aggregation, whereas each agent alone, at these concentrations, caused a transient aggregation. 5-HT-induced aggregation was inhibited by ketanserin (0.01-0.3 micromol/l), a 5-HT2 receptor antagonist, in a concentration-dependent manner. Collagen-induced platelet aggregation was also inhibited by ketanserin, whereas the inhibition by indomethacin was modest even at the highest concentration tested (300 micromol/l). U-46619 triggered platelet aggregation in a biphasic manner. Ketanserin inhibited only the second phase of the aggregation. The inhibition of U-46619-induced aggregation by ketanserin occurred at a concentration range similar to that for 5-HT-induced platelet aggregation. Likewise, platelet aggregation induced by the combination of low concentrations of 5-HT and U-46619 was fully inhibited by ketanserin. These data suggest a major involvement of platelet-derived 5-HT in TXA2-dependent aggregation in cat platelets.