Studies On M-Nisoldipine And Its Metabolism In Vitro And Vivo By HPLC-MS Technologies

m-Nisoldipine [chemname: 3-iosbutyl-5methyl1,4-dihydro-2, 6- dimethyl-4-(3-nitrophenyl)-pyridine-3,5dicarboxylate] as a new dihydropyridine calcium ion antagonist, was first developed by School of Pharmacy Hebei Medical University, currently available for the treatment of systematic hypertension and angina etc. m-Nisoldipine with the nisoldipine is the isomers, whose pharmacological effect is similar with the nisoldipine, and m-Nisoldipine displays significantly light stability. Regarding metabolism, prevenient studies indicated that m-nisoldipine was mainly excreted in the form of metabolites, and the excretion of original drug is very few. Therefore, it is necessary to study to the metabolism of m-nisoldipine, so as to provide an important reference for clinical research.This study developed the liquid chromatography–mass spectrometry (HPLC-MS/MS) methods for the m-nisoldipine and its metabolism in vitro and vivo, found and determine 14 metabolites of m-nisoldopine, according to the order of retention time named as M1-M14. We concluded m-nisoldipine and its metabolites fragmentation rules, and illustrates metabolic pathways transformation rules of m-nisoldipine in vitro and in vivo in rats. To determine the role of enzyme cytochrome P450 (CYP450) in the major metabolic pathways of m-nisoldipine, and to understanding metabolism mechanisms, prediction of this drug interaction, drug polymorphism and clinical reasonable treatment provide basis.Part one Studies on metabolism of m-nisoldipine in vitroObjective: To study m-nisoldipine and its metabolites by multiple means, such as incubation of rat liver microsome, anaerobic culture of intestinal tract bacteria, and prepare pure metabolite as far as possible.Methods: 1. Liver microsome experiment: To detected mass spectra for projection and confirm the structure of metabolites, and according to the structure of metabolites speculate the metabolic pathway of m-nisoldipine in rat liver microsomes in vitro. We accorded literature and improved, m-nisoldipine was added to the incubation liquid after some time, the incubation culture was treated and analyzed by HPLC-MS. 2. Culture solution of intestinal flora were produced from rat feces and then incubated with m-nisoldipine to study the metabolism of m-nisoldipine. The highest conversion yields of metabolites were prepared by preparative liquid chromatography. The chemical structures of the metabolites were identified by ¹H-NMR and ESI-MS₂, and inferred possible microbial transformation pathways of m-nisoldipine by rat intestinal flora.Results: 1. Liver microsome experiment: We found 10 metabolites in the liver microsome of rat, and conclude the possible metabolic pathway of m-nisoldipine in rat liver microsomes in vitro. 2. Intestinal flora incubated experimental: We found 2 metabolites of m-nisoldipine in rat intestinal flora, and infer the possible metabolic pathway of m-nisoldipine.Conclusion: Liver microsome experiment and intestinal flora incubated experimental all of are imitation metabolic of m-nisoldipine in vitro. And the two methods are a practical method for study drug metabolism.Part two Studies on metabolism of m-nisoldipine in vivoObjective: To detected m-nisoldipine and its metabolites in vivo, and study the transformation process in rat and illuminate the metabolic pathway of m-nisoldipine.Methods: We employed HPLC-MS/MS analysis technology of study the fragmentation and its retention time of m-nisoldipine, as the base of analysis and identification of metabolism. After taking orally 0.5% CMC-Na solution of m-nisoldipine 10 mg/kg, and then urine, feces, bile, blood, contents of intestine and liver tissue sample were pretreated and analyzed by HPLC-MS/MS for m-nisoldipine and its metabolism, and to detected the mass information of metabolites, speculation and confirm structure of m-nisoldipine. According to the speculate of metabolites speculation the possible metabolism pathway of m-nisoldipine in vivo.Results: We detected metabolites of m-nisoldipine for biological samples in vivo accorded to HPLC-MS/MS analysis. We detected 8 metabolites in rat urine, and no detected parent drug; detected 5 metabolites and parent drug in rat feces; detected 8 metabolites and parent drug in rat bile; detected 9 metabolites and parent drug in rat blood; detected 7 metabolites and parent drug in rat intestine sample; and detected 6 metabolites and parent drug in rat liver sample. Summary metabolism of m-nisoldipine in vivo, we detected 14 metabolites of m-nisoldipine in vivo.Conclusion: m-Nisoldipine was transformed extensively in rat body, and this HPLC-MS/MS method is very sensitive, specific, simple sample preparation, rapid and suitable for drug metabolism.Part three Characterization of m-nisoldipine metabolism by rat liver cytochrome P450 enzymes using liquid chromatography- mass spectrometryObjective: To study the metabolism of m-nisoldipine and the effects of selective cytochrome P-450(CYP450) inhibitors on the metabolism of cilnidipine in rat liver microsomes in vitro.Methods: Rat liver microsomes were used to perform metabolism studies. Various selective CYP450 inhibitors were used to investigate their effects on the metabolism of m-nisoldipine and the principal CYP450 isoform involved in dehydrogenation of dihydropyridine ring of m-nisoldipine in rat liver microsomes.Results: m-Nisoldipine was rapidly metabolized to three metabolites. They are dehydrogenated metabolites of dihydropyridine ring of m-nisoldipine(M1), hydroxylation metabolites of lateral chain of dihydropyridine ring in position 3 of m-nisoldipine(M2), and the dehydrogenated and hydroxylation metabolites of m-nisoldipine(M3). Ketoconazole (Ket) and Ticlopidine hydrochloride (Ticlo) competitively inhibited the dehydrogenation of dihydropyridine ring of m-nisoldipine and 3- hydroxylation of m-nisoldipine, respectively.Conclusion: m-Nisoldipine was rapidly metabolized in rat liver microsomes. CYP3A4 and CYP2C19 play an important role in metabolism of m-nisoldipine.