The Pharmacodynamics And Mechanism Of W1 On The Potent Of Antiplatelet Effects

According to reports in the literature: patients more than 137 million people in China suffer from Coronary Heart Disease(CHD), 3 million were dead because of CHD in 2011.Standard drug therapy combined with Percutaneous coronary intervention(PCI) is the principal therapies for the treatment of coronary heart disease. Percutaneous coronary intervention surgery can cause vascular endothelial damage and plaque rupture which promote platelet activation and aggregation, thereby induce thrombus formation.Clopidogrel combined with Aspirin, is as the gold standard therapy, which has been widely used to prevent recurrent ischemic events after percutaneous coronary interventions(PCI).Clopidogrel is a thienopyridine, inhibiting adenosine diphosphate(ADP) induced platelet activation by blocking the P2Y12 receptor on the platelet surface, and it’s a prodrug that requires in vivo transformed to an active metabolite, approximately 85% of clopidogrel dose is metabolized to inactive metabolites by esterase(ACBC1), only 10-15% of the remaining metabolized by hepatic CYP P450 isoenzymes via two successive oxidative steps conversed to the active metabolite(SR26334), which inhibit adenosine diphosphate(ADP) stimulated platelet activation by irreversibly binding to platelet P2Y12 receptors. However, inter-individual variability in the response to clopidogrel is multifactorial, can be influenced by drug-drug interactions、genetic polymorphisms and inadequate clopidogrel dosage,which leading to "clopidogrel resistance".W1 is a novel ADP receptor inhibitor for antiplatelet aggregation drugs, consist of 2-o-clopidogrel the first step of clopidogrel metabolite and aspirin, which aims to reduce the clopidogrel resistance and improve the efficiency of antiplatelet aggregation. The present study was designed to evaluate the pharmacodynamics and initial mechanism of W1, including the following three parts:In the first part: the pharmacodynamics study of W1. Clopidogrel was served as positive control drug. Wistar rats were single oral administration and randomly divided into normal group(1 ml- MC), clopidogrel group(10 mg/kg), W1 high dose group(3 mg/kg), W1 dose(1 mg/kg), W1 low dose group(0.3 mg/kg) by measuring platelet aggregation ratio and the mass of thrombosis in rats, the bleeding(clotting) time, to evaluate W1 antithrombotic effect. ADP(50 μm) were used in this experiment as standard platelet aggregation activator and clopidogrel 10 mg/kg were used as positive control group. Platelet aggregation results indicated that platelet aggregation was reduced in a dose-dependent manner when treated with W1. And W1(3 mg/kg) has similar antiplatelet effects with clopidogrel(10mg/kg)(P > 0.05); thrombus formation in mouse arterio-venous shunt model results W1(3 mg/kg) and clopidogrel 10 mg/kg can significantly inhibit thrombosis, and their effect has no statistical difference(P > 0.05); The vivo efficacy of time study of antiplatelet aggregation of W1(3 mg/kg)were examined in comparison with clopidogrel(10 mg/kg). Both W1 and clopidogrel were showed their effectiveness in 0.5h after dosing.The inhibitions of platelet aggregation by W1 and clopidogrel reached a plateau within 3 h and 4 h post-dose, respectively. Effect of antiplatelet aggregation of W1 and clopidogrel were both of long duration, there were no differences between them and the control group until 96 h. Bleeding time and Clotting time was measured to determine anticoagulant effects of W1 in comparison to clopidogrel. All the drugs tested significantly prolong BT and CT, increased the amount of bleeding in a dose-related manner. And there is no difference between W1(3 mg/kg) group with clopidogrel(10 mg/kg) group(P>0.05).The second part: W1 superior effect study. Because of clopidogrel in the body metabolized by hepatic CYP P450 isoenzymes via two successive oxidative steps conversed to the active metabolite(CYP2C19 and CYP3A4), we choose metabolized by the same CYP P450 isoenzymes: proton pump inhibitors(omeprazole), statins(simvastatin), calcium channel blockers( amlodipine), The purpose of this study was to determine the superiorities antiplatelet effects of W1 compared with clopidogrel on thrombus formation in vivo and platelet aggregation ex vivo to evaluate if CYP2C19 and CYP3A4 have been inhibited or deficiency, whether W1 antiplatelet aggregation and thrombus formation maintain efficient. Rats were randomized to receive Omeprazole(20、40 mg/kg) for 7days, clopidogrel(10 mg/kg) or W1(3 mg/kg) were combined administration on the 7day respectively. Amlodipine Besylate(10 mg/kg、20 mg/kg) combined with clopidogrel(10 mg/kg) or W1(3 mg/kg) were single orally administration respectively.Platelet aggregation and the weight of thrombus were tested to evaluate the superiorities of the pharmacodynamic actions of W1 compared with clopidogrel. As shown below, no matter platelet aggregation or thrombus formation, clobidogrel effects were both significant inhibited while combined with Omeprazole and Amlodipine. However, the pharmacodynamic of W1 that antiplatelet aggregation and antithrombus marked curative effect.The third part, this study was designed to evaluate the initial mechanism of W1. It’s mechanism of antiplatelet activity was investigated by ELISA to test the level of Cyclic adenosine monophosphate(c AMP) and the platelet reactivity index(PRI) using flow cytometry. Blood were collected from the heart to prepare PRP and mixed with Buffer solution, PGE1 was added to increases c AMP levels, and ADP was to deceased the level of c AMP, Concentrations of c AMP in platelet were measured using an enzyme-linked immune sorbent assay(ELISA) kits. Cyclic AMP levels were significant increased by PGE1(10 μm) stimulation in 2 min. The elevated cyclic AMP levels were suppressed by ADP(50 μm) which added at 2 min after PGE1 stimulation and the levels of c AMP in control group was drop off rapidly,while W1 group was not that obviously.W1 has the potent to inhibit the action of the ADP activated platelets. It is well known that the increasing level of c AMP was followed by PKA activation which positively regulates VASP phosphorylation in platelets. Reasults shown that, the use of W1 was associated with a remarkable inhibition of platelet reactivity measured using flow cytometry compared with control group(66.22±10.67 % vs 38.88±11.27 %, P<0.01).In summary, W1 is a novel agent of antiplatelet aggregation, has two advantages compared with clopidogrel.As the stronger antithrombotic effect, but didn’t increase the risk of bleeding; less affected by cytochrome P450 indicating that in the case of CYP2C19 and CYP3A4 suppression,W1 still have a potent antithrombotic effect; when combination with drugs which metabolized by the same CYP P450 isoenzymes(CYP2C19 and CYP3A4), it’s also has a steady potent to inhibit thrombus formation and platelet aggregation, It implies that W1 has a potential to play antiplatelet effect than the present similar class drugs.