Our prior work indicated possessed higher degrees of estradiol and lower degrees of progesterone during past due puberty

Meanwhile, the potent anti-platelet activity of K-134 also make it a potential alternative agent for preventing secondary cerebral infarction because antiplatelet therapy for secondary stroke prevention has been proved to be beneficial in clinical trials. Moreover, a double-blind, randomized trial of cilostazol and aspirin demonstrated that cilostazol is non-inferior, and might be superior to aspirin for prevention of stroke after an ischemic stroke. For these reasons, we used cilostazol as a comparative drug to evaluate the efficacy of K-134 on the photothrombotic stroke model in this study. In the stroke model, 30 mg/kg of K-134 inhibited photothrombotic MCA occlusion and reduced cerebral infarct size more potently than 300 mg/kg of cilostazol. On the other hand, the lower dose of K-134 significantly prolonged MCA occlusion time and tended to decrease infarct volume, but this decrease was not statistically significant. This difference of effects might be due to a short half-life of K-134 when administered to rats at a dose of 10 mg/kg, because MCA occlusion time and infarct volume were evaluated 2 and 24 hours after administration of K-134, respectively. Thus, 10 mg/kg of K-134 may not have been able to inhibit platelet thrombus formation induced by Norgestimate endothelial injury during the later hours of treatment. The photothrombotic MCA occlusion model has numerous advantages for the study of antiplatelet inhibitors in vivo because this model permits observation of not only time to occlusion by thrombus formation but also effects on cerebral infarction at a certain period of time after endothelial injury by less-invasive approach without injuring dura mater, thereby enables taking account of effects of rate of drug metabolism and long-term pharmacological effects of drugs. We previously reported that K- 134 blocks stable platelet accumulation but not initial platelet adhesion onto Von Willebrand factor -coated surface under high shear conditions in vitro, but we could not determine whether K-134 can inhibit initial platelet adhesion to a damaged blood vessel in this in vivo model. Hence, intravital videomicroscopy analysis is needed to reveal the detailed mechanisms of antiplatelet action of K-134 in vivo. The photothrombotic stroke model is a suitable model for evaluating the effects of antiplatelet inhibitors because an arterial platelet aggregation is induced through endothelial damage by a photochemical reaction. However, we could not test the possibility that other effects of K-134 such as cerebral blood flow increase via its vasodilatory activity contribute to decrease of infarct volume in this model. Besides, true stroke has many causes other than platelet activation. Hence, care should be taken in interpreting the results obtained in the photothrombotic stroke model in the present study in terms of stroke prevention in humans, and additional studies using other stroke models, such as Stroke-Prone Spontaneously Hypertensive Rats, are necessary to obtain further insight into the therapeutic benefits of PDE3 inhibitors. Moreover, comparative studies of the effects of K-134 and other antiplatelet agents such as aspirin and P2Y12 inhibitors on stroke models are required to further extend our findings. In the photothrombotic cerebral infarction model, photoactivation of rose bengal by illumination with green light results in reactive oxygen intermediates. In our electron spin resonance experiments, K- 134, cilostazol, and OPC-13015 did not show scavenging activities against both singlet oxygen and superoxide anion radical. Therefore, we concluded that the observed inhibitory effects of K-134 on photothrombosis in this model are relevant to its antiplatelet activity but not radical scavenging activity. In the case of oral administration of cilostazol in in vivo experiments, antiplatelet activities of its active metabolites, OPC- 13015 and OPC-13213, could also contribute to inhibition of platelet thrombus formation. OPC-13015 has 3 times more potent antiplatelet activity than cilostazol, whereas OPC-13213 has 3 times less potent activity than cilostazol. Cmax of cilostazol, OPC-13015 and OPC-13213 were 2.4, 1.4 and 9.1 mM, respectively, and AUC0-24 h were 30.1, 18.3, and 127.3 mM?h, respectively, after a single oral administration of cilostazol at a dose of 300 mg/kg in non-fasting male SD rats. On the other hand, the results of the healthy male single dose study showed that in human after administration of 100 mg of cilostazol, Cmax of cilostazol, OPC-13015 and OPC-13213 were about 625, 122 and 64 mg/L, respectively, and AUC0-72 h were about 8087, 2423 and 617 mg/L?h, respectively. On the basis of these results, we concluded that the dosage of cilostazol used in the rat photothrombotic stroke model in our study could give sufficient plasma concentrations of cilostazol and its metabolites compared with their therapeutic plasma concentrations in human.