Metabolism Of Vindoline And Its Effect On Rat Cytochrome P450 Enzyme In Vitro And In Vivo

Vindoline（VDL）is an indole alkaloid,and highly presents in young leaves and twigs of Catharanthus roseus（Linn.）G.Don,which has been being used as a folk medicine for diabetes.Pharmacological studies indicated that VDL might have hypoglycemic effect.Therefore,VDL is expected to become a potential anti-diabetic drug.Liver is the major organ involved in the biotransformation of drugs,cytochrome P450 is one of the most important representatives of drug metabolism enzymes,which exists mainly in liver mocrosomes.Cytochrome P450 enzymes shows wide range,poor selectivity,large structure difference,inter-individual variation,overlapping substrate specificities and can be easily induced and inhibited by a number of chemical compounds.When multiple drugs are used to treat disease,drug-drug interactions may occur,affecting the metabolic characteristics of drugs.Therefore,for new drugs,it is necessary to study which enzymes can metabolize the drugs and how they affect CYP450 enzymes.But this research of VDL is still almost in blank.In the present study,based on the ultra high performance liquid chromatography coupled with triple quadrupole time of flight mass spectrometery（UHPLC-QTOF-MS/MS）,we systematically studied the metabolic characteristics of VDL,related metabolic CYP450 isoforms,and its effects on the activity of CYP450 enzymes,providing a scientific basis for its clinical application in a safe,rational and effective manner to avoid or reduce adverse reactions.Part one Analysis of vindoline’s metabolites and related metabolic CYP450 isoforms in rat liver microsomesObjective:To develop a UHPLC-QTOF-MS/MS method for identifying the metabolites of VDL in rat liver microsomes,and determining the related metabolic CYP450 isoforms of rat liver microsomes.Methods:（1）Based on UHPLC-QTOF-MS/MS analysis technology,we developed the fragmentation of VDL,which was used as the basis for the analysis and identification of the metabolites of VDL.（2）In order to examine the metabolites of VDL in vitro,the rat liver microsome incubation systems included the blank group,the control group and the sample group.The sample group added in VDL solution;the blank group replaced the VDL solution with equal amount of methanol;while the control group replaced the active liver microsomes with equal amounts of inactivated liver microsomes.The three groups were incubated with the same method.UHPLC-QTOF-MS/MS was used for analysis and data acquisition,the fragmentation rules of VDL combined with the collected mass spectral data to predict the structure of metabolites and determine their metabolic pathways.（3）α-naphthoflavone,diphenhydramine hydrochloride,sulfaphenazole,quinine,diethyldithiocarbamate and ketoconazole were applied as specific inhibitors for CYP1A2,CYP2B,CYP2C11,CYP2D1,CYP2E1 and CYP3A.By comparing the changes in the amount of metabolites generated before and after the addition of the chemical inhibitors,the isoforms of enzymes related to VDL metabolism were elucidated.Results:A total of 22 potential metabolites of VDL were detected in vitro,the main biotransformation reactions of VDL included deacetylation,oxidation,methylation and dealkylation.CYP3A might was the major isoform for all metabolites in rat liver microsomes,and CYP1A2,CYP2B,CYP2C11,CYP2D1 and CYP2E1 were also involved in VDL metabolism.Conclusion:In this study,we successfully detected and identified the metabolites of VDL in vitro by UHPLC-QTOF-MS/MS,and determined the isoforms of CYP enzyme involved in VDL metabolism.Moreover,this study will provide references for further research of VDL.Part two Identification of metabolites of vindoline in ratsObjective:To develop UHPLC-QTOF-MS/MS method for indentifying of VDL metabolites in rats plasma,urine,faeces and bile after oral administration,as well as to illustrate the pathways rule of VDL.Methods:The VDL solution was prepared using CMC-Na solution.Plasma,urine,faeces and bile samples at different time points were collected after single oral administration of VDL(20 mg·kg^-1)to rats in test group,and the rats in blank group were given the same volume of CMC-Na solution.Then the samples were pretreated by liquid-liquid extraction with ethyl acetate,and analyzed by UHPLC-QTOF-MS/MS.The chromatographic separation was performed on Poroshell 120 EC-C₁₈（2.1×100 mm,2.7μm）with a mobile phase consisted of 3 mM ammonium acetate buffer and acetonitrile.The mass spectral analysis was conducted in a positive electrospray ionization mode,an effective multiple mass defect filter（MMDF）and dynamic background subtraction（DBS）were used in the biological samples analysis to trace all the potential metabolites of VDL.Based on the obtained metabolites spectra information,the rules of VDL fragmentation and various data post-processing software,we inferred and confirmed the structure and type of metabolites,and then elucidated the metabolic pathway of VDL in rats.Results:A total of 25 metabolites（including 23 phase I and 2 phase II metabolites）were identified in rats after oral administration of VDL,of which 16 metabolites were found in the urine,14 metabolites were found in the bile,12 metabolites were found in the faeces and 6 metabolites were found in the plasma.These data suggested that the biotransformation of VDL was deacetylation,oxidation,deoxidization,methylation,dealkylation and sulfate conjugation.Conclusion:Based on UHPLC-QTOF-MS/MS method,the main metabolites of VDL in vivo are successfully detected and identified,and elucidated the metabolic regulation of VDL in vivo,which lays the foundation for further research.Part three Effect of vindoline on the activity of cytochrome P450 isoforms enzymes in rat liver microsomes in vitroObjective:To investigate the effect of VDL on the activities of cytochrome P450 isoforms enzymes in vitro by using“cocktail”probe substrate assay.Methods:VDL was incubated with five specific probes（tacrine,bupropion,diclofenac sodium,dextromethorphan hydrobromide and midazolam）belong to five cytochrome P450 enzymes（CYP1A2、CYP2B、CYP2C11、CYP2D1 and CYP3A）originated from rat liver microsome,respectively.Their five metabolites（1-hydroxylation tacrine,1-hydroxylation bupropion,4-hydroxylation diclofenac,o-demethylation dextromethorphan and 1-hydroxylation midazolam）were analyzed by UHPLC-QTOF-MS/MS assay,and calculated the IC₅₀ value.Results:VDL IC₅₀ values of CYP1A2、CYP2B、CYP2C11、CYP2D1 and CYP3A were 113.4μmol·L^-1、83.78μmol·L^-1、22.50μmol·L^-1、9.081μmol·L^-11 and 52.76μmol·L^-1,respectively.Conclusion:The inhibition effects of VDL on the activites of CYP2D1 were medium,and CYP2C11,CYP3A were weak,but no effect on the activites of CYP1A2、CYP2B.Part four Effect of vindoline on the activity of cytochrome P450 isoforms enzymes in rat liver microsomes in vivoObjective:To find out VDL whether influences the effect on rat CYP450 enzymes by using cocktail probe drugs in vivo.Methods:Using bupropion,diclofenac sodium,dextromethorphan hydrobromide and midazolam as the probe drugs of CYP2B、CYP2C11、CYP2D1 and CYP3A,we established a method for quantitative determination of probe substrates based on UHPLC-QTOF-MS/MS technology.The rats were randomly divided into two groups with seven rats per group,the test group was administrated VDL by oral gavage at a dose of 20 mg·kg^-1 once a day for fifteen days,and an equivalent CMC-Na solution with no VDL was given to the blank groups,on the sixteen day,all rats were administrated with single doses of the cocktail probe drugs,and the plasma samples were collected at different time points.The plasma concentrations of four mixed probes in rats plasma were analyzed by UHPLC-QTOF-MS/MS,and then pharmacokinetic parameters were calculated by DAS 3.0.Results:After 15 days of continuous administration of VDL to rats,the main pharmacokinetic parameters AUC_0-t,AUC_0-∞and C_max of bupropion,diclofenac sodium,dextromethorphan hydrobromide and midazolam in the test group were all higher than those in the blank group.However,there was no significant difference between the two groups in statistical analysis of pharmacokinetic parameters(AUC_0-t、AUC_0-∞、CL、C_max、T_max、T_1/2).Conclusion:VDL has no significant effect on the activity of CYP2B,CYP2C11,CYP2D1 and CYP3A in rats,so it is less likely to induce potential drug-drug interactions.