The Study On Metabolism-based Mechanism For Anti-platelet Activity Of Clopidogrel In Type 2 Diabetic Rats

Clopidogrel?Clop?,a thienopyridine antiplatelet agent,is widely prescribed to prevent and treat cardiovascular events.Clop is characterized by a high inter-individual variability in PD effects,and a part of patients exhibit impaired responses,called“clopidogrel resistance”.Importantly,clopidogrel resistance is associated with an increased risk of recurrent ischemic events,including stent thrombosis.More and more clinical data indicate that diabetes is a high risk factor for inducing clopidogrel resistance,but its metabolic mechanism is still unknown.As a prodrug,Clop requires metabolic biotransformation to a pharmacologically active metabolite,Clop-AM,to prevent platelet aggregation.It is estimated that about85%of Clop is rapidly hydrolyzed to an inactive metabolite,Clop-acid,by carboxylesterase 1?CES1?.The remaining 15%is oxidized by cytochrome P450s?CYPs?to Clop-AM in two steps.Clop-AM contains a reactive thiol group that covalently modifies the cysteinyl residues of the platelet P2Y₁₂2 receptor,leading to prevention of adenosine diphosphate?ADP?-induced platelet aggregation.Here,we explored ZDF and T2DM rats for hepatic CES1-and CYP-mediated Clop metabolism,pharmacokinetics of Clop,Clop-AM and Clop-acid,and the anti-platelet efficacy of Clop.In contrast to clinical findings,Clop-treated ZDF rats displayed significantly less?50%?maximum platelet aggregation at 4 h than control rats;the enhanced efficacy was accompanied by higher formation of Clop-AM and lower formation of Clop-acid.In vitro studies showed that hepatic levels of CES1protein and activity and Ces1e mRNA were significantly lower in ZDF than in control rats,as were the mRNA levels of CYP2B1/2,CYP2C11 and CYP3A2,and levels of CYP2B6-,CYP2C19-and CYP3A4-related proteins and enzymatic activities in liver microsomes of ZDF rats.Interestingly,liver microsomes of ZDF rats produced higher levels of Clop-AM than that of control rats despite their lower CYP levels,though the addition of fluoride ion,an esterase inhibitor,enhanced Clop-AM formation in control rats more than in ZDF rats.These results suggest that the reduction in CES1-based Clop inactivation indirectly enhances Clop efficacy in ZDF rats by making more Clop available for CYP-mediated Clop-AM formation.Furthermore,We established a T2DM rat model by combining high-fat diet feeding and low-dose streptozotocin injection,which successfully mimicked the clinical observation of reduced Clop-AM generation,also accompanied with reduced levels of Clop and Clop-acid.Interestingly,in vitro liver microsomes incubated with Clop showed increased Clop-AM levels in diabetes,which is attributed to significant Changes in the expression and function of Clop-metabolizing enzymes including CES,CYP1A2,CYP2B,CYP3A,or CYP2C9/2C19-related proteins.The opposite results in vitro and in vivo suggest that metabolism is not responsible for the reduced Clop-AM level in diabetic rats.Further,different from oral administration,the comparable PK profiles of Clop and Clop-AM were observed in the two strains after intravenous injection.Meanwhile,in situ single-pass intestinal perfusion study also showed lower P_effff and K_a values in the diabetic rats,indicating the decreased Clop absorption in diabetes.This is explained by increased expressions or functions of P-glycoprotein?P-gp?in duodenum and jejunum of diabetic rats.In addition,the decreased Clop-AM and Clop levels were reversed by the coadministration of classic P-gp inhibitor,cyclosporin A.It suggests that P-gp might be the key factor causing the reduction of Clop absorption,subsequently making less Clop available for CYP-mediated Clop-AM formation.Our research provide new ideas and methods for clarifying the metabolic mechanism of Clop anti-platelet effect in diabetes state.