Study On The Pharmacokinetics,Antiplatelet Effect And Mechanism Of Deuterium Clopidogrel

Thrombosis can lead to heart,brain,and lung circulatory diseases,such as acute myocardial infarction,stroke,and pulmonary embolism.The incidence of thromboembolic disorders,mainly coronary heart disease and cerebral thrombosis,rises yearly.The trend is getting younger,posing a severe threat to human health and life.Among many anti-thrombotic drugs,clopidogrel has been widely used in clinical practice because of its advantages of economy,effectiveness,and low bleeding risk.However,the disadvantages of resistance,low bioavailability,and drug interaction are increasingly prominent and cannot be ignored.Given the above drawbacks of clopidogrel,our research group designed and synthesized deuterium clopidogrel early.The elimination rate of deuterium clopidogrel in rats shows that deuterium could significantly improve the stability of clopidogrel to esterase,reduce the hydrolysis rate of methyl ester,and delay the decomposition process of clopidogrel in vivo.In addition,the previous experimental study on the dose-response relationship revealed that:4 h after the treatment,deuterium clopidogrel has significant anti-platelet aggregation effects and anti-thrombotic effects,there is no tendency to prolong the risk of bleeding.Based on this work,we will simulate the clinical dosing regimen to study the pharmacokinetics,anti-platelet effect,and the mechanism of deuterium clopidogrel.The specific research contents are as follows:1.Molecular docking studies on the binding energy of deuterium clopidogrel and carboxylesterase 1 and P2Y₁₂receptorsResearch methods:Autodock software was used to conduct docking analysis of clopidogrel,deuterium clopidogrel,and their active metabolites with carboxylesterase 1 and P2Y₁₂receptors,respectively,to analyze the possible benefits of deuterium generation at a theoretical level.Results:Deuterium can reduce the binding ability of drugs to carboxylesterase 1 and enhance the necessary power of the P2Y₁₂receptor.This helps to minimize clopidogrel acid production and increase in vivo exposure to active metabolites,which has the potential to enhance the anti-platelet effect of clopidogrel.2.Pharmacokinetic studies on the metabolic characteristics of deuterium clopidogrelA pharmacokinetic study verified the above theoretical results.Methods:Rats were randomly divided into the clopidogrel and deuterium clopidogrel groups.The loading dose was 40mg/kg for 1 day,and the maintenance dose was 10mg/kg for 6 days.Anticoagulant whole blood samples were collected at different time points before and after the last administration.HPLC-MS/MS determined the levels of deuterium clopidogrel derivative active metabolites and clopidogrel acid to investigate the advantages of deuterium clopidogrel over clopidogrel metabolic pathway.In addition,rat liver microsomes were prepared.CYP450 activity and carboxylesterase 1 activity were measured by Cocktail method and PNPA substrate method,respectively,to explore the metabolic causes of deuterium clopidogrel based on enzymatic mechanism.Results:Deuterium clopidogrel can significantly increase the exposure of active metabolites,reduce the production of clopidogrel acid,therefore,improving the bioavailability of clopidogrel.The mechanism is related to deuterium clopidogrel can weaken the inhibition of CYP2C19,CYP3A4,and other major activating metabolic enzymes.3.Study on the anti-platelet effect and mechanism of deuterium clopidogrelSince compared with clopidogrel,deuterium clopidogrel has better pharmacokinetic properties,how about its anti-platelet effect?We will study the anti-platelet effect and mechanism of deuterium clopidogrel.In addition,omeprazole and clopidogrel are often used in combination to prevent and treat gastrointestinal adverse reactions,but FDA has issued a warning for the combination of omeprazole and clopidogrel due to the serious drug interactions.Therefore,we will further studied the effects of combined omeprazole on the pharmacokinetics and anti-platelet effects of deuterium clopidogrel.Methods:1)Platelet aggregation function and platelet granules release function were detected by the Born method,ELISA,and flow cytometry,so the anti-platelet effect of deuterium clopidogrel were evaluated.2)The platelet morphology and internal granules storage state were observed by a transmission electron microscope to evaluate the effect of deuterium clopidogrel on platelet activation;3)The anti-thrombotic effect of deuterium clopidogrel was evaluated by establishing a rat model of electrically stimulated thrombosis.4)4D Label-free proteomics was used to detect platelet proteomics and find differential proteins.Through GO analysis,KOG analysis,functional enrichment analysis,and KEGG signal pathway analysis of differential proteins,the anti-platelet effects of deuterium clopidogrel and its mechanism were explored.5)HPLC-MS/MS method was used to determine the production of derivative active metabolites and the activity of CYP450enzyme in deuterium clopidogrel combination group and to investigate the effect of omeprazole on deuterium clopidogrel metabolism;6)The change of platelet aggregation rate after combined deuterium clopidogrel with omeprazole was measured by Born method,the rat electrically stimulated thrombus model was established to measure the length of thrombus after combined,and the effect of combined deuterium clopidogrel with omeprazole on thrombosis process was investigated by thromboelastography,to comprehensively evaluate the effect of combination therapy on the anti-platelet and anti-thrombotic effects of deuterium clopidogrel.Results:1)Compared with clopidogrel,deuterium clopidogrel has a better inhibitory effect on platelet aggregation,granules release,and activation,so it has a more significant anti-thrombotic effect;2)Deuterium clopidogrel can affect the expression of many proteins related to platelet activation,skeleton contraction,and granules release function.Deuterium clopidogrel exerts anti-platelet effect by inhibiting the P2Y₁₂-c AMP-VASP/P-VASP-PAC-1signaling pathway in platelets and inhibiting the SNARE-SNAP-23-VAMP-8pathway related to granules release;3)Compared with clopidogrel,the pharmacokinetic parameters of deuterium clopidogrel were less affected by omeprazole,had weaker inhibition of CYP450 enzyme activity,and had more significant inhibition of platelet aggregation and anti-thrombosis,suggesting that deuterium clopidogrel may be used in combination with more drugs metabolized by CYP450 enzyme.Deuterium clopidogrel has the advantage of being less affected by drug-drug interactions.4.Study on the effect of deuterium clopidogrel on blood system and hepatic tissueWhile treating and preventing diseases,long-term use of anti-thrombotic drugs may make patients'coagulation function and other indicators appear abnormal.The liver mainly metabolizes P2Y₁₂receptor antagonists represented by clopidogrel.While the liver metabolizes the drug,the prototype,intermediate metabolites,or end products may cause different degrees of damage to the liver.Therefore,this chapter will detect the blood routine,coagulation function,liver function,hepatic tissue,hepatocyte apoptosis,and other indicators to investigate deuterium clopidogrel's influence on the blood system and hepatic tissue.Methods:1)The influence of deuterium clopidogrel on blood routine and coagulation indexes was detected by hemocyte analyzer and coagulation analyzer;2)The effects of deuterium clopidogrel on liver function,hepatic tissue,hepatocyte apoptosis and apoptosis-related proteins expression were detected by the biochemical analyzer,H&E staining,flow cytometry and immunohistochemistry,so the effects of deuterium clopidogrel on hepatic tissue were investigated.Results:Deuterium clopidogrel had no effect on the blood system,liver function,and hepatic tissue.Although deuterium clopidogrel can induce apoptosis in hepatocyte,compared with clopidogrel,deuterium clopidogrel induced weaker apoptosis in hepatocyte,which may be related to deuterium clopidogrel can down-regulate the expression of pro-apoptotic proteins Bax and caspase-3.