Inhibition Of Rat CYP450 And Liver Microsomal Metabolism By Kudiezi Injection And Its Sesquiterpene Lactones

Ischemic cardiovascular and cerebrovascular diseases always plagued clinicians,and Chinese medicine has extensive clinical experience in the treatment of cardiovascular and cerebrovascular injuries caused by ischemia.Kudiezi injection(KDZI)is one kind of Chinese patent medicine,which includes an effective active substance extracted from the whole plant of compositae,and mainly used for the treatment of cerebral thrombosis,coronary heart disease,angina pectoris and myocardial infarction.In clinical medicine,KDZI constantly combined with other drugs,such as Xueshuantong,for the treatment of cardiovascular and cerebrovascular diseases.Hepatocyte cytochrome P450 enzyme was involved in the metabolism of the vast majority of drugs and,the enzyme activities can be induced or inhibited by drugs.In recent years,adverse events caused by drug interaction of concomitant medications are gradually increasing.From the perspective of drug metabolism enzymes,drug interactions have already became the exploration focus of many scholars.However,it is unknown that concomitant medication of KDZI produces undesirable drug interactions,and related comprehensive research has not been reported.Owing to the great significance of the effect of drug on hepatic cytochrome P450 enzyme probed into the drug interactions,the aim of this article was to study the inhibitory effect of KDZI and its two major sesquiterpene lactones(IZ and DIZ)on rat hepatocyte cytochrome P450 enzyme by a method of "Cocktail"probe coupled with ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Meanwhile,metabolisms of KDZI and its two major sesquiterpene lactones by liver microsomes using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UPLC-LTQ-Orbitrap)in negative ion mode.Test results were conducive to provide support for drug-drug interaction,clinical safety and toxicology studies.Primary contents and results in this thesis are as follows:1.A quantitative method was established for the simultaneous determination of 5 specific metabolisms of probe substrates of rat hepatocyte cytochrome P450 enzymeThe rat liver microsomes were prepared by referring to "Pharmacological Experimental Methodology".According to the FDA guidelines,phenylephrine,tolbutamide,omeprazole,testosterone and chlorzoxazone were selected as probe substrates for CYP1A2,CYP2C9,CYP2C19,CYP3A and CYP2E1,respectively.And the specific metabolic products of the five probe substrates were acetaminophen,4-hydroxytoluene-butoxide,5-hydroxy omeprazole,6?-hydroxy testosterone and 6-hydroxychlorozamine.In this article,5 specific metabolisms of probe substrates were quantitatively determined based on the internal standard of donepezil by a method of "Cocktail" probe combined with UPLC-MS/MS in rat hepatocyte cytochrome P450 enzyme.Liver microsomal incubation samples were treated with 2 fold volume ice-cold acetonitrile protein precipitation.The method was performted on a Waters ACQUITY BEH C18 column(1.7 ?m,2.1×100 mm)with the mobile phase of methanol/acetonitrile(1:1,v/v)-0.1%(v/v)formic acid in water with elution gradient and a column temperature at 40?.The flow rate of the mobile phase was set as 0.4 mL·min-1.Multiple reaction monitoring was applied to the quantitative analysis by ESI positive mode.And the results of methodological study demonstrated that the method,which has good linearity,high specificity and sensitivity,met the requirements of the determination about the study.2.Inhibitory effect of KDZI and its two major sesquiterpene lactones on rat liver cytochrome P450Using the method of UPLC-MS/MS combined with "Cocktail",KDZI,IZ and DIZ were used as the research object.By optimizing the liver microsomes incubation system,inhibitory effect of KDZI,IZ and DIZ on the activities of CYP1A2,CYP2C9,CYP3A4 and CYP2E1 in rat hepatocyte cytochrome P450 enzyme of the liver microsomes were studied.And the incubation system was validated by positive inhibitors,which was recommended in the FDA guidelines of the CYP450 enzyme isoform.The results showed that the established incubation system met the requirements of the study,the IC50 value of IZ to CYP2C19 was 21.76?mol·L-1,to CYP3A4 was 145?Lmol·L-1,to CYP1A2 and CYP2C9 was over 200 ?mol·L-1.The IC50 value of DIZ to CYP2C9 was 9.46 ?mol·L-1,to CYP1A2 and CYP2C19 was over 100?mol·L-1.The IC50 value of KDZI to CYP3A4 was 9.18%,to CYP2C9/19 was over 8.00%,to CYP1A2 was over 10.00%.These consequences indicated a weak inhibitory effect on CYP2C19 for IZ,moderate inhibition inhibitory effect on CYP2C9 for DIZ,and no inhibitory effect on CYPs for KDZI.3.Metabolism of KDZI and its two major sesquiterpene lactones in vitro The metabolism of KDZI,IZ and DIZ in vitro were identified by UPLC-LTQ-Orbitrap MS.Total of 2 prototype compounds and 17 metabolites were selected out,and 16 metabolites were characterized based on the retention time,accurate mass measurements and mass fragmentation patterns,respectively.And they mainly experienced the oxidation,reduction,hydrolysis via phase I in rat liver microsomes,besides cysteine conjugation and glutathione(GSH)conjugation.This would help to clarify the metabolic regularity of KDZI,IZ and DIZ,and also can provide new train of thought for clinical safetyand toxicological effects.