Effects Of Antibiotic Cocktail On Clopidogrel Active Metabolite Systemic Exposure In Type 2 Diabetic Rats

Clopidogrel（Clop）in combination with aspirin has become the gold standard for patients with acute coronary syndrome（ACS）and undergoing percutaneous coronary interventions（PCI）for the treatment and prevention of atherothrombosis.However,ACS patients with diabetes mellitus have poor or even no response to Clop,referred to as Clop resistance,which markedly limits the clinical use and potential benefits of Clop in diabetic patients.Some studies have demonstrated that Clop resistance is strongly associated with absorption and metabolism of Clop.Clop is a substrate of P-gp,which affects the intestinal absorption.Once delivered to liver,Clop is largely（85%）hydrolyzed by carboxylesterase（CES）1 to an inactive metabolite,clopidogrel acid（Clop-acid）,while the remaining 15%is biotransformed to Clop-AM by two step oxidation processes using cytochrome P450（CYPs）enzymes.Clop-AM prevents platelet aggregation.Recent studies demonstrate that Clop resistance under diabetic condition is mainly attributed to the increased P-gp level,and subsequently the decreased Clop absorption and Clop-AM generation.Further,the underlying mechanism needs to be understood.Increasing evidence indicates that gut microbiota are strongly associated with the onset and development of type 2 diabetes mellitus（T2DM）.In addition,gut microbiota play an important role in pharmacokinetic process of drugs by influencing the levels of drug-metabolizing enzymes and transporter,consequently affecting individual response to drug treatment.Based on the double effects of gut microbiota on T2DM occurrence and drug disposition,it is worthwhile to explore whether gut microbiota affect Clop pharmacokinetics.In the study,we disrupted the gut microbiota with an antibiotic cocktail including ampicillin,neomycin,metronidazole,and vancomycin,then explored the influences of antibiotics on Clop absorption and metabolism in high fat diet and STZ-induced T2DM rats.It is helpful for revealing the relationship of diabetes,gut microbiota and drug metabolism,improving the safe and efficacy of anti-platelet drugs,and preventing the occurrence of thrombus in diabetic patients.First,we observed the changes of biochemical parameters in serum,liver and small intestine of T2DM rats pre and pro antibiotic treatment.The results showed that after 5days of antibiotic administration markedly alleviated T2DM rats’phenotype including hyperglycemia,hyperlipidemia,insulin resistance,liver dysfunction and inflammation.Meanwhile,antibiotic treatment also significantly increased the systemic exposure of Clop,Clop-AM and Clop-acid in T2DM rats.Next,we observed the effect of antibiotics on Clop-metabolizing enzyme levels in liver.Antibiotic treatment significantly increased the expression or activity of CES,CYP2B and CYP2C9-or 2C19-related protein.Meanwhile,liver microsome metabolism assay showed that antibiotic treatment only slightly inhibited the enhanced Clop-AM generation in liver microsomes of T2DM rats as compared to those of control rats with or without KF（P>0.05）,in contrast to the in vivo results,suggesting that Clop hepatic metabolism might not contribute to the altered plasma Clop-AM level in T2DM rats treated with antibiotic or not.Further,we explored the potential effect of absorption on the altered systemic exposure of Clop and its metabolites in antibiotics-treated T2DM rats via measuring P-gp and PXR levels in small intestine.The results showed that T2DM rats had higher m RNA and protein levels of P-gp and nuclear PXR than control group,while antibiotic treatment significantly reversed these changes.Because our previous studies have demonstrated that T2DM decreases systemic exposure of Clop-AM due to P-gp upregulation-caused the reduction of Clop absorption,suggesting that the decreased P-gp level in antibiotics-treated T2DM rats contributes to the enhanced plasma exposure of Clop,Clop-AM and Clop-acid.Furthermore,the effects of antibiotics on Clop-metabolizing enzymes and transporter were analyzed in vitro.The results showed that there were different patterns of change in the m RNA levels or catalytic activities of Clop-metabolizing enzymes and transporter between T2DM rats and LO₂/SW480 cells after antibiotic treatment.It suggests the change in CYPs and P-gp levels in antibiotics-treated T2DM rats are not due to the direct role of antibiotics.Finally,fecal 16S r RNA sequencing results showed that gut microbiota of T2DM rats had the decreased microbial diversity and the changed structure and composition,including a decrease in the relative abundance of Firmicutes and an increase in the relative abundance of Bacteroidetes or Proteobacteria.To our attention,5 days of antibiotic treatment sharply reduced the microbial amount（>95%）,and dramatically altered the microbial structure and composition with Proteobacteria being the dominant phylum（>90%）,suggesting a serious disruption of gut microbiota.In conclusion,our study demonstrated that antibiotics-induced disruption of gut microbiota increased systemic exposure of Clop-AM and alleviated diabetic phenotype in T2DM rats.Considering the effects of diabetes-related factors such as glucose,insulin,and cytokines on P-gp expression,the attenuation of T2DM phenotype may be responsible,at least partly for the reduced P-gp level,and subsequently increased Clop-AM systemic exposure due to the disruption of gut microbiota.The findings suggest that gut microbiota modulation is an effective therapeutic strategy to attenuate Clop resistance.