Studies Of Metabolites In Intestinal Flora And Transcriptional Regulation Effects Of M-nisoldipine Enantiomers

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-m-nisoldipine,S-m-nisoldipine),which were firstly composed and separated in School of Pharmacy,Hebei Medical University.m-Nisoldipine with the nisoldipine is the isomers,whose pharmacological effect is similar with the nisoldipine,and m-Nisoldipine displays significantly light stability,and it is used for the prevention and treatment of hypertension and heart disease.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 enantiomers in the rat intestinal flora transformation,so as to provide an important reference for clinical research.Liver is an important organ of drug metabolism where metabolism reactions are most likely to happen.CYP450 enzymes are the major enzymes involved in drug metabolism and biotransformation which can be induced or inhibited.So understanding the relationship between drugs and the CYP450 enzymes can provide us a theoretical basis of the pharmacological mechanism.Research has proved CYP450 enzyme can be induced by the nuclear receptor PXR,but the study of m-nisoldipine in this aspect is still blank,so it is necessary to study this aspect research of m-nisoldipine.This study developed the UPLC-Q-TOF-MS methods for the m-nisoldipine enantiomer and its metabolism in rat intestinal flora transformation,identify the chemical structures of the metabolites and propose the possible metabolic pathways of m-nisoldipine enantiomer.Moreover,by use of RT-q PCR,the effects of m-nisoldipine on five major CYP450 enzymeswere determined by rat model.Furthermore,a method for vitro system to screen drug inducers of CYP450 enzymes has established,and to study whether h PXR is involved in the induction of CYP450 enzyme by m-nisoldipine enantiomers.This research can be applied to predict m-nisoldipine-other drug interaction and evaluate drug polymorphism and clinical reasonable treatment.Part one Studies of metabolites of m-nisoldipine enantiomers in vitro byrat intestinal flora by UPLC-Q-TOF-MSObjective: To establish the rat intestinal flora transformation method of m-nisoldipine enantiomers,identify the chemical structures of the metabolites and propose the possible metabolic pathways of m-nisoldipine.Methods: R,S-m-nisoldipine were anaerobically incubated with rat intestinal flora in vitro 2 h respectively,extracted by hexane-ether(1:1).The supernatant liquid after centrifugation was dried by nitrogen and analyzed by the UPLC-Q-TOF-MS.Analytes were separated on the Poroshell 120 EC-C18column(2.1 mm×100 mm,2.7 μm)with gradient elution the mobile phase consisting of 0.5% aqueous formic acid and acetonitrile,the flow rate was 400μL·min-1,and the column temperature was 40 ℃.Detection and quantitation was performed by electrospray ionization(ESI)in the negative ion mode.The metabolites were discovered by comparing the data of samples with the corresponding blanks.Results: The three metabolites were 1,4-dihydro-2,6-dimethyl-4-(3-am inophenyl)-3,5-pyridinedicarboxylic acid isobutyl methyl ester,1,4-dihydro-6-methyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid isobutyl methyl e ster and 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester.The drug and three metabolites were found in the incubation solution of enantiomers and inferred possible microbial transformation pathways of MNS by rat intestinal flora.Conclusions: R,S-m-nisoldipine can metabolize in rat intestinal flora into the same metabolites.The main metabolic pathways of enantiomers were reduction and oxidation.Part two Effects of m-nisoldipine on cytochrome P450 enzymes in ratsby using RT-q PCRObjective: To investigate the effects of m-nisoldipine on the m RNA expressions of CYP450 in CYP1A2,CYP2 B,CYP2C11,CYP2 D,CYP3A1.Methods: Male Sprague-Dawley rats were randomly divided into normal group,m-nisoldipine low dose,middle dose and high dose(2.5,5.0,12.5mg·kg-1)groups.The rats were killed after medicines administration once a day for consecutive 15 days by oral gavage.RT-q PCR was employed to examine the m RNA expressions of CYP1A2,CYP2 B,CYP2C11,CYP2 D,CYP3A1 enzymes in liver tissues of rats.Results: The results of quantitative RT-q PCR showed that,as compared with the normal group,CYP1A2,CYP2 B,CYP2D m RNA expression was decreased by m-nisoldipine,and CYP2C11,CYP3A1 m RNA expressions were increased m-nisoldipine.The m RNA expressiond of m-nisoldipine treatment group had statistical difference from normal group,and the effects of m-nisoldipine for CYP450 enzyme can be influence by dose of m-nisoldipine.Conclusions: m-Nisoldipine can regulation m RNA expressiond of CYP450 enzymes,indicating that m-nisoldipine can affect drug metabolism by CYP450 enzymes and in turn may result in some side-effect.Part three Transcriptional regulation effects of m-nisoldipineenantiomers for CYP450 enzyme mediated by h PXRObjective: To establish an in vitro system to screen drug inducers of CYP450 enzymes,and to investigate whether h PXR is involved in the induction of rat hepatic cytochrome P450 by m-nisoldipine enantiomers.Methods: Clonging the promoters of CYP2B6,CYP2C9 and CYP3A4 which contain the elements that h PXR,a kind of nuclear receptor,can recognize and bind to.The promoter elements were inserted to the upstream of firefly luciferasere porter gene in p GL3 vector.The reporter vectors and h PXR expression vector were co-transfected to Hep G2 cell line,and dual-luciferase activity was analyzed after the cell treated with different concentrationm-nisoldipine enantiomers.Results: The activation value of m-nisoldipine enantiomers treatment group had statistical difference from DMSO group(P<0.05).R-m-nisoldipine can induce CYP2B6,CYP2C9 and CYP3A4.S-m-nisoldipine can induce CYP2B6 and CYP2C9,but there was no statistical significant on CYP3A4.Conclusions: We successfully established an in vitro system which can be used for screen the drug agonist for h PXR and increase the expression of CYP450 enzyme.The inductive effects of m-nisoldipine enantiomers for CYP450 enzyme can be mediated by h PXR.