Study On The Metabolism Of M-nisoldipine In Recombinant Cytochrome P450Enzymes And The Metabolites In Rat Tissues

m-Nisoldipine, as a new dihydropyridine calcium channel antagonists, isused for the prevention and treatment of hypertension and heart disease.Previous studies have shown that it is mainly excreted as metabolites. Liver isan important organ of drug metabolism where phase Ⅰ and phase Ⅱ reactionsare most likely to happen. Cytochrome P450enzymes are the major enzymesinvolved in drug metabolism and biotransformation. So understanding therelationship between drugs and the CYP450enzymes can provide us atheoretical basis of the pharmacological mechanism.In this study, incubations with human liver microsomes, CYP450enzymes and chemical inhibitors were conducted to identify the individualCYP450enzyme involved in the biotransformation of m-nisoldipine. Tissuesamples were analysed for metabolites of the rats after oral administration.Knowing the main metabolic pathway could help us to investigate thepharmacological mechanism. Samples of incubations with rat livermicrosomes, bile and urine of rats were analysed for glucuronidationmetabolites. Liquid chromatography-mass spectrometry was used toinvestigate the enzyme kinetics of m-nisoldipine enantiomers, optimize thereaction conditions and compare the parameters of the enantiomers.Part one Study on the metabolism of m-nisoldipine in recombinantcytochrome P450enzymesObjective: Incubations with human liver microsomes, cDNA expressedCYP enzymes and chemical inhibitors were analysed to identify the specificCYP450enzymes involved in the biotransformation of m-nisoldipine.Methods: The incubation samples were analysed in EMS-IDA-EPI,MRM-IDA-EPI and PREC-IDA-EPI modes for metabolites of m-nisoldipine.Incubation samples with human liver microsomes were analysed by usingHPLC-MS/MS to get structure information and MS2spectra for structure characterization.Specific CYP450inhibitors were incubated with human liver microsomesfor30min, after that m-nisoldipine was added. The production of metaboliteswas determined and compared with that in negative control sample in whichinhibitors were not added.Incubation samples with CYP450enzymes were detected for metabolites.And the production of metabolites in different CYP450enzymes wascompared.Results:8metabolites were found in the incubation with human livermicrosomes. Among them, the dehydrogenation product and hydroxylationproduct were generated much more than others.It was found in the inhibition experiment that ticlopidine hydrochloride(CYP2C19inhibitor) can significantly inhibit the formation of hydroxylationproduct in the side chain of position3, while ketoconazole (CYP3A4inhibitor)affected the dehydrogenation product obviously.The catalytic efficiency of CYP2C19and CYP3A4was found muchhigher than other CYP450enzymes. CYP2C19and CYP3A4mainly catalyzedthe hydroxylation and dehydrogenation reactions respectively.Conclusion: Both the inhibition experiment and CYP450enzymesincubation experiment showed that CYP2C19and CYP3A4were the majorenzymes involved in the biotransformation of m-nisoldipine in human livermicrosomes.Part two HPLC-MS/MS analysis of m-nisoldipine and its metabolites inrat tissuesObjective:6kinds of rat tissue samples were analysed for metabolites tosummarize the metabolic rules of m-nisoldipine and clarify itspharmacological mechanism.Methods:6kinds of rat tissues were obtained from SD rats after oraladministration of m-nisoldipine for1h. The tissues were cut into piece andmixed with the normal saline (NS) to get tissue homogenate. Samples wereextracted with diethyl ether and then analysed by HPLC-MS/MS. Based on our previous studies on the metabolism of m-nisoldipine, the structures ofdetected metabolites were characterized by comparing the retention times andthe MS2spectra.Results: Metabolites of m-nisoldipine were detected in all the6kinds oftissue samples. In liver,9metabolites were found with the production higherthan in other tissues.5metabolites were detected in spleen and kidney, while4metabolites were found in heart and lungs. Only2metabolites were found inbrain and the concentrations were really low.Conclusion: m-Nisoldipine could easily metabolize in vivo and convertinto many metabolites. Liver may be the most important organ for metabolism.And the main metabolic pathways of m-nisoldipine were dehydrogenation ofthe dihydropyridine core and hydroxylation and hydrolysis of ester bonds inside chain.Part three Primary study on glucuronidation phase Ⅱ metabolism ofm-nisoldipineObjective: Incubations with rat liver microsomes, bile and urine sampleswere analysed by HPLC-MS/MS to investigate the glucuronidation phase Ⅱmetabolism of m-nisoldipine.Methods: Incubations with rat liver microsomes were conducted in thesystem containing UDPGA, alamethicin and MgCl2. Rat bile and urinesamples were collected for36h after oral administration of m-nisoldipine. Thesamples were monitored in EMS-IDA-EPI, MRM-IDA-EPI andPREC-IDA-EPI modes for detecting the glucuronidation metabolites.Results: No glucuronidation metabolites were found in the samples aftercomparing with the blank sample and only phase Ⅰmetabolites were detectedin rat bile and urine.Conclusion: For m-nisoldipine, only the active H in dihydropyridine ringcan be conjugated with UDPGA. However, the active H was easily moved byoxidization. The conditions of the experiment and HPLC-MS/MS may alsohave effects. So we should do our best to optimize the experimental conditionsand further investigate the glucuronidation phase Ⅱ metabolism of m-nisoldipine.Part four Enzyme kinetics of m-nisoldipine enantiomers in human livermicrosomesObjective: To study the enzyme kinetics of m-nisoldipine enantiomers inhuman liver microsomes and get the parameters. The parameters ofm-nisoldipine enantiomers were compared.Methods: A HPLC-MS/MS method was established for quantitativeanalysis of m-nisoldipine enantiomers with nimodipine as internal standard.Separation on a Sapphire C18column (150mm×4.6mm,5μm) was achievedby acetonitrile and0.1%aqueous formic acid, isocratic elution. The massspectrometer was operated in the negtive mode in MRM mode.Linewrave-Burk graphic method was used to calculate the enzyme kineticsparameters Vmaxand Km.Results: The optimized reaction condition is that25μmol·L-1substrateincubated with1mg·mL-1protein for30mintutes. The enzyme kineticsparameters were as follow: R-m-nisoldipine Km=79.20μmol·L-1, Vmax=2.727μmol·(min·mg protein)-1; S-m-nisoldipine K-1m=83.80μmol·L, Vmax=2.892μmol·(min·mg protein)-1.Conclusion: The method was simple and reliable for the in vitrometabolism research of m-nisoldipine. There was no significant differencebetween R-m-nisoldipine and S-m-nisoldipine in enzyme kinetics.