Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type G(i)-linked P2T antagonist, CS-747

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.     

Prasugrel: a novel thienopyridine antiplatelet agent. A review of preclinical and clinical studies and the mechanistic basis for its distinct antiplatelet profile

Prasugrel (CS-747, LY640315) is a novel member of the thienopyridine class of oral antiplatelet agents that includes ticlopidine and clopidogrel. Like other thienopyridines, prasugrel is a prodrug that is inactive in vitro. Prasugrel's distinct chemical structure permits efficient conversion to its active metabolite with a less rigorous dependence on specific cytochrome P-450 enzymes. Prasugrel is rapidly converted in vivo to an active metabolite (R-138727) that binds specifically and irreversibly to the platelet P2Y 12 purinergic receptor inhibiting ADP-mediated platelet activation and aggregation. Preclinical studies indicated that prasugrel is approximately 10- and 100-fold more potent at inhibiting ex vivo platelet aggregation and in vivo thrombus formation than clopidogrel and ticlopidine, respectively. Early clinical data in healthy subjects confirmed the greater platelet inhibition and consistency with prasugrel compared to clopidogrel. While the active metabolites of prasugrel and clopidogrel resulted in similar levels of platelet inhibition in vitro, the amount of each active metabolite generated in vivo was quite different-prasugrel (60 mg) resulting in an approximately 12-fold greater exposure to its active metabolite compared with clopidogrel (300 mg). This observation provides a mechanistic basis for the faster, greater, and more consistent inhibition of platelet aggregation observed with prasugrel. Clinical studies in patients with cardiovascular disease confirmed the potent antiplatelet effect of prasugrel compared with clopidogrel. Collectively, these phase 1/1b studies and a phase 2 study (JUMBO-TIMI 26) aided in dose selection for the recently completed phase 3 trial (TRITON-TIMI 38) in patients with acute coronary syndrome undergoing percutaneous coronary intervention.     

Repeat oral dosing of prasugrel, a novel P2Y12 receptor inhibitor, results in cumulative and potent antiplatelet and antithrombotic activity in several animal species

Antiplatelet and antithrombotic activity of multiple oral dosing of prasugrel were evaluated in several animal species. Prasugrel's active metabolite concentration-relatedly inhibited in vitro ADP-induced aggregation of rat, rabbit, dog, monkey and human platelets. Oral administration of prasugrel to dogs (0.03-0.3 mg/kg/day) and monkeys (0.1 and 0.3 mg/kg/day) once a day for 14 days resulted in potent, dose-related and cumulative inhibition of ADP-induced platelet aggregation. The inhibitory effects reached a plateau on days 3 to 5 and thereafter were maintained during dosing. Inhibition decreased gradually after cessation of dosing with near full recovery by 7 days after last dose. Antiplatelet and antithrombotic activity of prasugrel and clopidogrel were further examined in rats. Multiple oral dosing of prasugrel (0.3-3 mg/kg/day) to rats resulted in more potent inhibition of platelet aggregation compared to clopidogrel (3-30 mg/kg/day) and ticlopidine (30-300 mg/kg/day). Separate experiments confirmed that platelet inhibition was associated with inhibition of [(3)H]-2-methylthio-ADP binding to rat platelets. In a rat model of electrically-induced arterial thrombosis, prasugrel (0.1-1 mg/kg/day, p.o.) significantly prolonged the time to arterial occlusion and increased the duration of arterial patency. The inhibition of platelet aggregation of prasugrel was about 10 and 300 times more potent than clopidogrel and ticlopidine, respectively. Overall these results show that in several species multiple oral administration of prasugrel results in more potent inhibition of platelet aggregation and thrombus formation than clopidogrel and ticlopidine, and that these effects are mediated by inhibition of platelet ADP receptors.     

Antithrombotic effects of KBT-3022, a novel antiplatelet agent,in an arterial thrombosis model in the guinea-pig

We examined the antithrombotic effect of KBT-3022, a novel antiplatelet agent, on photochemically induced arterial thrombosis in the guinea-pig saphenous artery, and compared its effect with that of aspirin and that of ticlopidine. Pretreatment with KBT-3022 [0.1 mg (0.2 μmole), 0.3 mg (0.7 μmole), 1 mg (2.2 μmole) and 3 mg (6.7 μmole)/kg, p.o.] 3 h prior to thrombosis induction prolonged the time required to achieve the thrombotic occlusion and inhibited the collagen (low concentration, 0.2 μg/ml; high concentration, 1 μg/ml)-induced platelet aggregation in whole blood ex vivo, each effect being concentration-dependent. The effects were tested of ticlopidine [30 mg (99.9 μmole), 100 mg (333.1 μmole) and 300 mg (999.2 μmole)/kg, p.o.] and aspirin [10 mg (55.5 μmole), 30 mg (166.5 μmole) and 100 mg (555.1 μmole)/kg, p.o.]. Both drugs prolonged the time to occlusion (but only at their highest concentration), and also inhibited the collagen (low concentration)- induced platelet aggregation in whole blood ex vivo. Aspirin [100 mg (555.1 μmole)/kg, p.o.], but not ticlopidine (at any concentration), inhibited the collagen (high concentration)-induced platelet aggregation. A good correlation was found between the antithrombotic and antiplatelet-aggregating effects of both KBT-3022 and aspirin, but not of ticlopidine. The antithrombotic potency of KBT-3022 was 300 times that of aspirin and 1000 times that of ticlopidine. These observations suggest that KBT-3022 may be clinically effective against platelet-rich thrombosis. Drug Dev. Res. 40:217–222, 1997. © 1997 Wiley-Liss, Inc.     

Antithrombotic effect of TRK-100, a novel, stable PGI2 analogue

TRK-100, a stable PGI2 analogue structurally different from carbacyclines, was compared with other antiplatelet drugs for its effect on platelet functions using animal models. TRK-100 (10-300 nM) inhibited rat platelet aggregation induced by ADP (3 microM), collagen (12.5 micrograms/ml) and A23187 (10 microM), and its potency was about 1/3-1/7 that of PGI2. TRK-100 (0.3-3 mg/kg, p.o.) dose-dependently inhibited rabbit platelet adhesion (ED50: 2.2 mg/kg), and its effect lasted over at least 5 hr. In contrast, aspirin and ticlopidine (both at 300 mg/kg, p.o.) showed only a slight inhibition (4-7%). In the thrombocytopenia induced by collagen injection in rats, TRK-100 (3-300 micrograms/kg, i.v.; 0.1-3 mg/kg, p.o.) dose-dependently inhibited a decrease in platelet number, and its ED50 was 0.48-0.62 mg/kg orally and 13.7-16.4 micrograms/kg intravenously, while the inhibition by aspirin and ticlopidine (both at 1000 mg/kg, p.o.) was 40 and 37%, respectively. In the experimental thread thrombosis in rats. TRK-100 (0.03-3 mg/kg, p.o.) dose-dependently inhibited thrombus formation, and its ED50 was 0.46 mg/kg, being 21 and 87 times as potent as aspirin and ticlopidine, respectively. These results reveal that TRK-100 has a potent antiplatelet activity and is orally and intravenously effective for a variety of thrombosis models, suggesting that it may have a therapeutic value as an antithrombotic drug.     

Antithrombotic activity of NSP-513, a novel selective phosphodiesterase 3 inhibitor, on femoral arterial thrombosis induced by physical stenosis and electrical current: comparison of antithrombotic and hemodynamic effects

NSP-513, a novel potent and selective phosphodiesterase 3 (PDE 3) inhibitor, and cilostazol, a previously developed PDE 3 inhibitor, were compared with respect to antiplatelet, antithrombotic, and hemodynamic effects. In the in vitro antiplatelet aggregation studies, NSP-513 and cilostazol inhibited collagen-induced canine platelet aggregation with median inhibitory concentration (IC50) values of 0.093 and 3.1 miccroM, respectively, and inhibited adenosine diphosphate (ADP)-induced canine platelet aggregation with IC50 values of 0.15 and 12 microM, respectively. For ADP-induced platelet aggregation, the presence of prostaglandin E1 (PGE1; 3 and 10 nM) further decreased the IC50 values for NSP-513 to 0.11 and 0.032 microM, respectively. In ex vivo antiplatelet aggregation studies, orally administered NSP-513 (0.03-1 mg/kg) and cilostazol (50 mg/kg) inhibited collagen-induced canine platelet aggregation. In an in vivo canine femoral arterial thrombosis model, intraduodenally administered NSP-513 (0.01-0.03 mg/ kg) dose-dependently prevented thrombus formation without any changes in blood pressure, heart rate, or bleeding time. In conscious dogs, NSP-513 at oral doses of > or =0.3 mg/kg produced hemodynamic changes such as decreased blood pressure and increased heart rate and LVdP/dt(max). Thus the minimal hemodynamically effective dose of NSP-513 was 0.3 mg/kg, and the hemodynamic effects of this dose were comparable to those of 50 mg/kg of cilostazol. In conclusion, these data suggest that NSP-513 has in vivo selectivity for antiplatelet and antithrombotic activities over hemodynamic activity, and that the selectivity of NSP-513 is higher than that of cilostazol in dogs.     

Interspecies comparison of the antiplatelet, antithrombotic, and hemorrhagic effects of SR 121566A, a novel nonpeptide GP IIb/IIIa antagonist.

We report the results from an interspecies comparison of the antiplatelet, antithrombotic, and hemorrhagic actions of SR 121566A, a novel nonpeptide antiplatelet agent with high affinity and specificity for the GP IIb/IIIa complex. SR 121566A exhibited in vitro antiplatelet activity against adenosine diphosphate (ADP)-induced aggregation with a rank order of potency [humans = baboons = dogs] > marmosets > guinea pigs > rabbits. These in vitro findings were predictive for the ex vivo antiplatelet potency after i.v. administration of SR 121566A to dogs, guinea pigs, and rabbits [median effective dose (ED50) values, 0.02, 0.05, and 0.15 mg/kg]. The antiplatelet actions of SR 121566A translated into an acute antithrombotic effect in an arteriovenous shunt model after i.v. administration in dogs, guinea pigs, rabbits, and marmosets (ED50, 0.08, 0.10, 0.50, and 0.007 mg/kg). Hemorrhagic effects of SR 121566A were observed in guinea pigs and rabbits at doses that represented 2-3 times the antithrombotic ED50, whereas in marmosets, no bleeding was observed at the antithrombotic ED90. These results demonstrate that SR 121566A exhibits favorable actions in terms of antithrombotic potency and hemostatic safety in different animal species, suggesting that, in humans, SR 121566A will be a good candidate as an antithrombotic compound.     

In-vitro and Ex-vivo Inhibition of Blood Platelet Aggregation by Naftazone

Because of the considerable interest in the role of platelets and antiplatelet therapy in cardiovascular disease, including the aggregation of platelets to each other during arterial thrombosis and atherogenesis, we have studied the effect of naftazone (Etioven), an original vasculotropic drug on platelet aggregation. Rat and human platelets were prepared and incubated in-vitro with different concentrations of naftazone. We found that naftazone inhibited both platelet secretion and aggregation in platelet-rich plasma (PRP) and washed platelets after stimulation with thrombin or ADP. Rats were also treated intraperitoneally for five days with various naftazone doses (0.125-10 mg kg^?1) and ex-vivo platelet aggregation compared, at various times after the last injection, with that of control animals. Inhibition by naftazone was dose-dependent in both PRP and isolated platelets. The inhibition was transient, a maximum value (～ 50%) being obtained about 3–6 h after the last injection, with a return to near-control values after 24 h. Naftazone also facilitated platelet deaggregation after in-vitro stimulation with thrombin or ADP. In another series of experiments, rats were treated intraperitoneally for five days with 10 mg kg^?1 of aspirin, ticlopidine, dipyridamole or naftazone. Platelets were prepared and tested for aggregation 90 min after the last injection. Thrombin-induced aggregation in PRP and washed platelets was significantly reduced after in-vivo treatment with ticlopidine and naftazone. Except for dipyridamole, all the drugs inhibited ex-vivo ADP-induced aggregation in PRP. In isolated platelet preparation, only naftazone induced a significant inhibition of ADP-or thrombin-stimulated aggregation. We conclude that naftazone inhibits platelet aggregation in-vitro and ex-vivo.     

T-686, a novel inhibitor of plasminogen activator inhibitor-1, inhibits thrombosis without impairment of hemostasis in rats

The aim of this study was to evaluate the antithrombotic potential of T-686 ((3E,4E)-3-benzylidene-4-(3,4,5-trimethoxy-benzylidene)-pyrrolidine-2,5-dione), a novel inhibitor of plasminogen activator inhibitor-1 (PAI-1), in rat thrombosis models. T-686 (0.1–100 mg/kg per day, p.o.) dose dependently decreased the weight of venous thrombi induced by a combination of stasis and hypercoagulability. The antithrombotic effect was enhanced by repeated administration of T-686. Warfarin (0.1 mg/kg per day for 3 days) also prevented thrombus formation. The antithrombotic action by warfarin was accompanied by prolongation of coagulation time, while no effect on coagulation time was observed in T-686-treated rats. T-686 lowered the activity of PAI-1 in plasma. In the arterio-venous shunt model, pretreatment with T-686 (10 mg/kg per day) or ticlopidine (100 mg/kg per day) for 8 days inhibited thrombus formation by 33% and 44%, respectively. T-686 had no effect on collagen-induced platelet aggregation ex vivo, while ticlopidine inhibited platelet aggregation. T-686 did not affect bleeding time at 10–100 times the antithrombotic dose, while warfarin dose dependently prolonged bleeding time at and around the antithrombotic dose. These results suggest that T-686 prevents thrombus formation in rats without impairment of hemostasis.     

Inhibition of ADP-induced platelet aggregation by clopidogrel is related to CYP2C19 genetic polymorphisms

1. Clopidogrel is one of the most important antithrombotic drugs but has different efficacies in different populations. The aim of the present study was to evaluate the contribution of CYP2C19 genetic polymorphisms to the inhibition of ADP-induced platelet aggregation by clopidogrel in healthy Chinese volunteers. 2. Eighteen healthy male volunteers (six CYP2C19*1/CYP2C19*1, six CYP2C19*1/CYP2C19*2and*3 and six CYP2C19*2/CYP2C19*2and*3) were enrolled in the study. Each subject took 300 mg clopidogrel on the first day and then 75 mg once daily for 2 consecutive days. Blood samples were taken to measure ADP-induced platelet aggregation at baseline and 4, 24 and 72 h after administration of the first dose of clopidogrel. 3. There were significant decrease in 2 and 5 micromol/L ADP-induced platelet aggregation at 4, 24 and 72 h after clopidogrel among the three CYP2C19 genotypes compared with baseline (P < 0.001). The change in 5 micromol/L ADP-induced platelet aggregation in subjects with the CYP2C19*1/CYP2C19*1 genotype was greater than that in subjects with the CYP2C19*2/CYP2C19*2and*3 genotype at 4 h (49.0 +/- 15.5 vs 29.7 +/- 17.4%, respectively; P = 0.029), 24 h (48.7 +/- 20.5 vs 25.0 +/- 17.6%, respectively; P = 0.035) and 72 h (45.5 +/- 15.2 vs 26.5 +/- 15.8%, respectively; P = 0.030) after clopidogrel administration. 4. In conclusion, CYP2C19*2 and CYP2C19*3 genetic polymorphisms reduced clopidogrel inhibition of ADP-induced platelet aggregation, with the degree of inhition dependent on the genetic polymorphism present.     

Effects of a new anti-inflammatory imidazole derivative, fenflumizole, on platelet aggregation in the rabbit

The antiplatelet effect of fenflumizole, compared with aspirin or ticlopidine, was examined in in vitro, ex vivo and in vivo situations of the rabbit. Unlike ticlopidine, fenflumizole and aspirin effectively inhibited in vitro the platelet aggregation elicited by arachidonate and collagen. The activity of fenflumizole was 350 times more potent than that of aspirin. Fenflumizole (0.3-3 mg/kg) given p.o. was 4.2 and 8.1 times more potent than aspirin in inhibiting arachidonate- and collagen-induced platelet aggregations, respectively. Ticlopidine (300 mg/kg, p.o.) resulted in only weak effects on the aggregations. Fenflumizole (3 mg/kg) as well as aspirin (10 mg/kg) given p.o., unlike ticlopidine (300 mg/kg), effectively prevented the arachidonate-induced sudden death.     

A multiple dose study of prasugrel (CS-747), a novel thienopyridine P2Y12 inhibitor, compared with clopidogrel in healthy humans

AIMS: This double-blind, placebo-controlled trial was designed to evaluate the pharmacodynamics, pharmacokinetics, safety, and tolerability of prasugrel (CS-747, LY640315), a novel thienopyridine P2Y(12) ADP receptor antagonist compared with clopidogrel, during multiple oral dosing in healthy subjects. METHODS: Thirty subjects received placebo, prasugrel 5 mg, 10 mg, or 20 mg, or clopidogrel 75 mg orally, daily for 10 days. Platelet aggregation, bleeding time, and prasugrel metabolites were measured and adverse events were recorded. RESULTS: Inhibition of ADP-induced platelet aggregation reached steady state by day 3 following prasugrel 10 and 20 mg compared with 5 days for clopidogrel 75 mg or prasugrel 5 mg. Compared with placebo, at 24 h after the last dose of study drug, inhibition of platelet aggregation using (20 microm) ADP was significantly higher in the prasugrel 10 mg group (58.2 +/- 4.9% vs. 9.2 +/- 4.0%, P < 0.001) with no difference in the clopidogrel group (15.7 +/- 6.8% vs. 9.2 +/- 4.0%, P = 0.78). With 5 microm ADP, inhibition of platelet aggregation with prasugrel 10 mg and clopidogrel 75 mg was significantly higher than with placebo (prasugrel 10 mg, 70.5 +/- 4.7%; clopidogrel 75 mg, 36.5 +/- 9.0%; vs. placebo, 11.3 +/- 5.1%; P < 0.0001 and P = 0.02). On day 10 at 4 h postdose, bleeding time was prolonged with prasugrel 10 mg (prasugrel 10 mg, 706 +/- 252 s vs. placebo, 221 +/- 38 s, P = 0.05) but not with clopidogrel (283 +/- 56 s, P = 0.98). There were no clinically significant bleeding events, serious adverse events, or discontinuations of the study drug. CONCLUSIONS: Compared with clopidogrel 75 mg, prasugrel 10 mg and 20 mg daily for 10 days resulted in more rapid, more consistent, and higher levels of platelet inhibition.     

Antithrombotic activity of AT-1015, a potent 5-HT(2A) receptor antagonist, in rat arterial thrombosis model and its effect on bleeding time.

The antithrombotic activity of N-[2-(4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidino)ethyl]-1-formyl-4-piperidinecarboxamide monohydrochloride monohydrate (AT-1015; a 5-HT(2A) receptor antagonist) was studied in a photochemically induced arterial thrombosis (PIT) model in the rat femoral artery, and in the tail transection bleeding time test. Ticlopidine (an antiplatelet agent) and sarpogrelate (a selective 5-HT(2A) receptor antagonist) were studied as reference compounds. Pretreatment with AT-1015 (1 mg/kg, p.o.) significantly prolonged the time required to occlusion of the artery with thrombus, and the effect (3 mg/kg, p.o.) persisted for 24 h with significant inhibition of 5-HT-induced vascular contraction. Ticlopidine and sarpogrelate also significantly prolonged the time to occlusion at 100 mg/kg, p.o. Sarpogrelate (300 mg/kg, p.o.) showed the similar antithrombotic efficacy to AT-1015 (3 mg/kg, p.o.), while the effect disappeared within 6 h. No significant bleeding time prolongation was observed at 10 mg/kg of AT-1015, which is 10 times higher than the antithrombotic effective dose; whereas ticlopidine significantly prolonged bleeding time at the same dose as the antithrombotic effective dose. These results suggested that AT-1015 is a potent and long-acting oral antithrombotic agent in this model, which may be elucidated by its potent and long-acting inhibition of vasoconstriction through 5-HT(2A) receptor.     

Effects of 2‐(1‐hydroxypentyl)‐benzoate on platelet aggregation and thrombus formation in rats

2‐(1‐hydroxypentyl)‐benzoate (dl‐PHPB), a derivate of 3‐n‐butylphthalide (NBP), is a novel therapeutic agent for treatment of cerebral ischemia. In the present study, the antiplatelet and antithrombotic activities of dl‐PHPB were evaluated in ex vivo platelet aggregation and in vivo arteriovenous (A‐V) shunt models. dl‐PHPB inhibited platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA), and collagen (COL) in a dose‐dependent manner when given orally (12.9–129.5 mg/kg). The inhibitory potency was similar to 3‐n‐butylphthalide (NBP) and aspirin (ASP). Inhibition on platelet aggregation was also observed after iv administration of dl‐PHPB (1.29–12.9 mg/kg). The time‐course of these effects showed the maximal inhibition on platelet aggregation at 1 h after oral administration and 30 min after iv injection. dl‐PHPB (12.9–129.5 mg/kg, p.o.) caused dose‐dependent inhibition of thrombus formation in the rat A‐V shunt thrombosis model. These results show that dl‐PHPB is an orally and iv antiplatelet and antithrombotic agent and may be useful for treatment of ischemia stroke. Drug Dev Res 63:174–180, (2004). © 2004 Wiley‐Liss, Inc.     

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.     

Overcoming clopidogrel resistance: discovery of vicagrel as a highly potent and orally bioavailable antiplatelet agent

A series of optically active 2-hydroxytetrahydrothienopyridine derivatives were designed and synthesized as prodrugs of clopidogrel thiolactone in order to overcome clopidogrel resistance. The final compounds were evaluated for their inhibitory effect on ADP-induced platelet aggregation in rats. Compound 9a was selected for further in vitro and in vivo metabolism studies, since its potency was comparable to that of prasugrel and was much higher than that of clopidogrel. Preliminary pharmacokinetic study results showed that the bioavailability of clopidogrel thiolactone generated from 9a was 6-fold higher than that generated from clopidogrel, implying a much lower clinically effective dose for 9a in comparison with clopidogrel. In summary, 9a (vicagrel) holds great promise as a more potent and a safer antiplatelet agent that might have the following advantages over clopidogrel: (1) no drug resistance for CYP2C19 poor metabolizers; (2) lower dose-related toxicity due to a much lower effective dose; (3) faster onset of action.     

Improved posthypoxic recovery in vitro on treatment with drugs used for secondary stroke prevention

Besides aspirin several new drugs for inhibition of platelet aggregation and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibition are used in secondary stroke prevention. Pharmacology and clinical effects, however, are not fully explained by current understanding of underlying mechanisms. Population spike amplitude (PSAP), an established marker of slice integrity, was measured during hypoxia and recovery thereof in hippocampal slices from control CD1 mice (25-35 g) and animals pretreated in vivo with a single i.p. injection of clopidogrel, ticlopidine, or atorvastatine at different time intervals and dosages. Posthypoxic recovery of PSAP was 20 +/- 35% in control CD1 mice. Upon pretreatment with clopidogrel (1-24 h, 0.5-2 mg/kg body weight) an increase up to 81 +/- 20% (p < 0.01 to control) was observed at 1h interval and 1mg/kg. Application of ticlopidine (1-24 h, 1-4 mg/kg body weight) resulted in an improvement of posthypoxic recovery to 61 +/- 41% (p < 0.05 to control) while administration of atorvastatine (1-24 h, 1-4 mg/kg body weight) caused an increase up to 87 +/- 31% (p < 0.01 to control) at 1h interval and 2 mg/kg. On application of these substances in vitro the NADH autofluorescence spectrum in hippocampal slices is blue-shifted suggesting an alteration of oxidative metabolism. The present data demonstrate a shared neuroprotective effect of agents known to inhibit platelets (acetylsalicylic acid, clopidogrel, and ticlopidine) and HMG-CoA reductase (atorvastatine). The time course of this neuroprotective action in the current experimental study (onset within an hour, duration of several hours in contrast to several days) resembles clinical practice in dosing these substances. We hypothesize that an increase of hypoxic tolerance resulting from mild mitochondrial inhibition by these substances is a principal constituent of the effectiveness of these drugs.     

Inhibitory effects of J78, a newly synthesized 1,4-naphthoquinone derivative, on experimental thrombosis and platelet aggregation

Several compounds with the backbone of 1,4-naphthoquinone chemical structure have been reported to display antiplatelet and antithrombotic activities, indicating that this congener compound may be a new source in the antithrombotic drug development. In the present study, the possible antiplatelet activity and antithrombotic efficacy of J78 (2-chloro-3-[2'-bromo, 4'-fluoro- phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, were examined. Orally administered J78 (50, 100 mg/kg) dose dependently protected mice against the collagen + epinephrine-induced thromboembolic death. Orally administered J78 also significantly inhibited the ADP- and collagen-induced rat platelet aggregation ex vivo, with inhibition values of 44 and 40%, respectively. J78 inhibited the collagen-, arachidonic acid- and thrombin-induced human platelet aggregation concentration dependently in vitro, with IC(50) values of 7.8 +/- 0.4, 10.1 +/- 0.4 and 18.4 +/- 2.0 micromol/l, respectively. It was also active in inhibiting Ca(2+) ionophore, A23187-induced platelet aggregation, suggesting that J78 may have an inhibitory effect on Ca(2+) mobilization. J78, however, did not alter coagulation parameters such as activated partial thromboplastin time and prothrombin time in human plasma. Taken together, these results suggest that J78 may be a promising antithrombotic agent, and its antithrombotic activity may be due to antiplatelet rather than anticoagulation activity.     

The platelet inhibitory effects and pharmacokinetics of prasugrel after administration of loading and maintenance doses in healthy subjects

Prasugrel (CS-747, LY640315), a novel thienopyridine, is a potent and orally active antiplatelet agent in vivo. The aims of this double-blind, double-dummy, placebo-controlled, randomized, parallel group phase 1 study were to investigate the antiplatelet effects of prasugrel after oral administration of a loading dose (LD) and subsequent 20 days of once-daily maintenance dosing (MD), to characterize the pharmacokinetics of prasugrel metabolites with an LD/MD regimen, and to assess the safety and tolerability of prasugrel in healthy subjects. Subjects were randomly assigned in a 1:1:1 ratio to prasugrel 40 mg LD/7.5 mg MD (n = 11), prasugrel 60 mg LD/15 mg MD (n = 10), or placebo LD/placebo MD (n = 11). Prasugrel 40 and 60 mg LDs provided rapid and consistent inhibition of 20 microM adenosine diphosphate (ADP)-stimulated platelet aggregation. Prasugrel 7.5 and 15 mg MDs maintained inhibition in a dose-dependent manner. The pharmacokinetic data indicate that exposure to prasugrel metabolites occurs rapidly after dosing and is consistent with dose proportionality. Within the limitations of this study, the safety and tolerability results suggest that prasugrel is well tolerated when dosed as an initial LD followed by a lower daily MD for 20 days. Prasugrel LDs and MDs provide rapid and sustained inhibition of ADP-mediated platelet aggregation.     

Comparison of in vitro and ex vivo antiplatelet effects of 1,2-benzisothiazolin-3-one and its 2-amino derivative

The in vitro and ex vivo antiplatelet effects of 2-amino-1,2-benzisothiazolin-3-one (1) are compared with those of its parent compound 1,2-benzisothiazolin-3-one (2) and with acetylsalicylic acid (ASA) against different agonists. 2-Amino-1,2-benzisothiazolin-3-one inhibits adenosine diphosphate (ADP)-, arachidonic acid (AA)- and collagen-induced human platelet aggregation in vitro, with IC50 values of 8.90 x 10(-5), 1.50 x 10(-6) and 5.11 x 10(-8) mol/l, respectively. The strong inhibitory activity is significant not only for collagen but also for AA-induced aggregation. The same compound inhibits ex vivo collagen- and particularly AA-induced rabbit platelet aggregation at the tested dose of 10 mg/kg i.m. In view of the potential use of 2-amino-1,2-benzisothiazolin-3-one as antithrombotic agent, the log P values for both 1,2-benzisothiazolin-3-one derivatives 1 and 2 are determined, to gain an understanding of the significance of the 2-amino group in the 1,2-benzisothiazolin-3-one moiety with respect to the biological activity under study.