Drug-drug Interaction And Metabolism Of M-nisoldipine Based On Cytochrome P450

m-Nisoldipine,1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedi carboxylate methyl-2-methyl propyl esters, is 1,4-dihydropyridine calcium ion antagonist, to be presented as a couple of enantiomers(R-(-)-, S-(+)-m-nisoldipine), which were firstly composed and separated in School of Pharmacy, Hebei Medical University. m-Nisoldipine enantiomers are relatively more stable than other 1,4-dihydropyridines under lighting condition. These new compounds have many advantages including an increased cardiac output and cardiac index when studied in rabbits and a less negative inotropic effect on myocardium. Regarding as 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.Liver is the major organ involved in the biotransformation of various endogenous compounds and drugs. The cytochrome P450 superfamily is a large and diverse group of enzymes that catalyze the oxidation of organic substances. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for most of different metabolic reactions. In recent years, a dramatic increase in the number of drug-drug interactions have been reported and have aroused attention with regard to clinic drug safety, pushing the need for the development of a high-through put screening approach for drugs.While the similar research of m-nisoldipine is still almost in blank.High performance liquid chromatography-tandem mass spectrometry(HPLC-MS) has become a powerful and reliable analytical approach for metabolite identification because of its high sensitivity, low consumption and high speed of analysis.Based on HPLC-MS and some metabolic models, we determined the role of CYP450 in the major metabolic pathways of m-nisoldipine, and understood in vivo and in vitro metabolism mechanisms of m-nisoldipine. Moreover, by use of HPLC-MS and real time PCR, the effects of fermentation on the CYP-mediated drug-drug interaction potential of m-nisoldipine enantiomers were determined by HepG2 cell line and human liver microsomes. Furthermore, a method for simultaneously assessing the effect potency of m-nisoldipine on five major in vivo CYP450 enzymes has found utilization by using a cocktail of probe substrates. This research can be applied to predict m-nisoldipine-other drug interaction and evaluate drug polymorphism and clinical reasonable treatment. Part one Studies on Metabolic enzyme phenotype of m-nisoldipine enantiomers in vitro by using HPLC-Q-TRAP/MSObjective: Relying on High performance liquid chromatography coupled to triple quadrupole linear ion trap mass spectrometry(HPLC-Q-TRAP/MS), the effects of selective CYP450 inhibitors were evaluated.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 enantiomers in rat liver microsomes.Results: It showed that the increased concentration of CYP 3A inhibitor ketoconazole resulted in significant decrease in metabolism of both m-nisoldipine enantiomers and the maximum inhibitory effect. However, the other inhibitors of CYP1A1/2, CYP2B1/2, 2D and 2C11 had no obvious inhibitory effects on whichever m-nisoldipine enantiomer metabolism.Conclusions: Result showed CYP3 A might primarily metabolize m-nisoldipine in rat liver microsomes. Part two Studies on metabolism of m-nisoldipine enantiomers in vitro by using UHPLC-Q-TOF-MS/MSObjective: Relying on Ultra high performance liquid chromatography coupled to triple quadrupole time of flight mass spectrometery(UHPLC-Q-TOF-MS/MS), the metabolites of m-nisoldipine enantiomers in rat liver microsomes in vitro were evaluated.Methods: 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 UHPLC-Q-TOF-MS/MS.Results: Ultimately, a total of 18 potential metabolites of(R)-m-nisoldipine and 16 potential metabolites of(S)-m-nisoldipine were detected in the pooled rat liver microsomes samples.Conclusions: According to identification and classification of metabolites, it was demonstrated that some biotransformation reactions, such as oxidation, ester hydrolysis and dehydrogenation, were major metabolic modes. Part three Cytochrome P450-mediated drug-drug interaction potential of m-nisoldipine enantiomersObjective: To evaluate the drug-drug interaction potential of m-nisoldipine enantiomers, mediated by CYP450 inhibition/induction.Methods: Real-time PCR was performed to determine the effects of aescin on the mRNA expression of CYP1A2, CYP2B6, CYP2D6 and CYP3A4 in HepG2 cell line. In addition, a cocktail probe assay was performed for determination of the selective inhibitory effect of m-nisoldipine enantiomers on CYP450 enzymes in human liver microsomes(HLM). Incubation of isoform-specific substrate probes with m-nisoldipine enantiomers in HLM.Results: The m-nisoldipine enantiomers can change the expression of obtained isoforms except CYP2D6.What’s more. The m-nisoldipine enantiomers can inhibit CYP2C8, CYP2C9, CYP2D6 and CYP3A4, respectively. However, both m-nisoldipine enantiomers had no obvious inhibitory effects on the other isoforms of CYP1A2, CYP2B6 and CYP2C19.Conclusions: m-Nisoldipine enantiomers could affect the expression of some isoforms of CYP enzyme, and showed a inhibition of some isoforms. Part four Studies on metabolism of m-nisoldipine in vivo by using UHPLC-Q-TOF-MS/MSObjective: Relying on Ultra high performance liquid chromatography coupled to triple quadrupole time of flight mass spectrometery(UHPLC-Q-TOF-MS/MS), the metabolites of m-nisoldipine enantiomers in rat liver microsomes in vitro were evaluated.Methods: We employed UHPLC-Q-TOF-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 and feces sample were pretreated and analyzed by UHPLC-Q-TOF-MS/MS for m-nisoldipine and its metabolism, and to detected the mass information of metabolites, speculation and confirm structure of m-nisoldipine, which indicated a possible metabolism pathway of m-nisoldipine in vivo.Results: Ultimately, a total of 25 potential metabolites of m-nisoldipine were detected in the biological samples in vivo.Conclusions: According to identification and classification of metabolites, it was demonstrated that some biotransformation reactions, such as oxidation, ester hydrolysis, nitro reduction, methylation and dehydrogenation, were major metabolic modes. Part five Effects of m-nisoldipine on cytochrome P450 enzymes in rats by using UHPLC-Q-TOF-MSObjective: To find out whether m-nisoldipine influences the effect on rat CYP450 enzymes by using cocktail probe drugs in vivo.Methods: A method for simultaneously assessing the effect potency of m-nisoldipine on five major in vivo CYP450 enzymes has found utilization by using a cocktail of probe substrates. A mixed cocktail solution at a dose of 5mL/kg, was given as oral administration to rats treated with a multiple doses of m-nisoldipine via i.g. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by UHPLC-Q-TOF-MS The corresponding pharmacokinetic parameters were calculated by the software of DAS 3.0.Results: Treatment with multiple doses of m-nisoldipine had inductive effects on rat CYP2 B, CYP2C11 and 3A1, while CYP1A2 and 2D1 enzyme activities were inhibited.Conclusions: m-Nisoldipine can either inhibit or induce activities of CYP1A2, CYP2 B, CYP2C11, CYP2D1 and CYP3A1. Therefore, caution is needed when m-nisoldipine is co-administration with these isoforms substrates in clinic, which may result in treatment failure and drug-drug interactions.