Inhibitory Effect Of Compound Chinese Medicine TA-05 On Human CYP450 Enzymes And Transporters And Its Pharmacokinetics Effects On Metformin

Objective:Diabetes mellitus(DM)is one of the most common diseases of chronic endocrine and metabolic disorders.It has become one of the most important diseases which affecting human health in the world.TA-05 which consists of rhubarb,coptis,white peony root,Bupleurum root,posseses functions of dealing with the blues and clearing stomach,nourishing Yin and removing fire,purgation and eliminating turbidity.Morever,it will be used in the treatment of type 2 diabetes.Preclinical pharmacodynamic studies have shown that the drug could significantly reduce blood glucose in both normal and diabetic animal models and also could effectively control HbAlc in patients with newly diagnosed and overweight type 2 diabetes and significantly lower fasting blood glucose and 2 h postprandial blood glucose.In view of TA-05 which may be combined with metformin or statins in clinic,we develope the study of the effect of TA-05 on the transporting activity of OATP1B1,OATP1B3 and OCT2 transporters of SLCO family and the inhibition of CYP450 enzymes.Meanwhile we also investigate the impact of TA-05 on pharmacokinetic characteristics in vivo of metformin in rats in order to provide datas for supporting the research of TA-05 in clinical Ⅱ trials.Methods:1.liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was established by using tolbutamide and propranolol as internal standard(IS)respectively to determine the substrate concentration.The inhibitory effects of TA-05 on the transporter activity of OATP1B1,OATP1B3 and OCT2 were investigated;2.Quantitative determination of the concentrations in human liver microsomes of acetaminophen,hydroxybupropion,N-deacetylglucosamine,4’-hydroxy diclofenac,4’-hydroxymembrane Doxorubicin,dextrorphan,dextrorphan,1’-hydroxy midazolam and 6 beta-hydroxytestosterone were measured by LC-MS/MS to assess the inhibitional effects of TA-05 on human liver microsomal cytochrome P450 isoenzymes(CYP1A2,CYP2B6,CYP2C8,CYP2C9,CYP2C19,CYP2D6 and CYP3A4);3.LC-MS/MS method with paracetamol as an internal standard was developed,the analytes and IS were separated on an inertsutain AQ-C18 column(2.1 μm × 75mm,3 μm)by using mobile phase of water with 2mM ammonium acetate and methanol as isocratic elution(70:30,V:V)at a flow rate of 0.2 mL/min,the column temperature was 35℃ and the injection volume was5μL;The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization(ESI)by multiple reactions monitoring(MRM)to investigate the pharmacokinetics of metformin in rats between the control group and TA-05 group.Results:1.HEK293-OATP1B1,OATP1B3 and OCT2 cells were co-incubated with transporter substrates in the presence of TA-05 or positive inhibitors.After protein precipitation by the cell lysate,the concentrations of substrates were quantified by LC-MS/MS and the values of IC50 were calculated by software.The corresponding the values of IC50 were 95.1 ± 17.6,36.9 ± 16.1 and 116 ± 28.9 μg/mL,respectively;2.After adding TA-05,nicotinamide adenine dinucleotide phosphate(NADPH)and cytochrome P450 isozymes specific substrates,the mixed human liver microsomal incubation system were incubated.The metabolites produced by specific substrates were determinated by liquid chromatography-tandem mass spectrometry.The experimental results of TA-05 showed CYP1A2(IC50=18.7μg/mL),CYP2B6(IC50=323μg/mL),CYP2C8(IC50=77.4μg/mL),CYP2C9(IC50=115μg/mL),CYP2C19(IC50=100μg/mL),CYP2D6(IC50=183μg/mL),CYP3A4(IC50=208μg/mL)and CYP3A4(IC50=194μg/mL);3.The LC-MS/MS method was developed for determination of acetaminophen and metformin hydrochloride(MET)at the same time.Its specificity,precision,accuracy,linearity,stability,extraction recovery of the established method were fully validated.The plasma concentrations of metformin hydrochloride in plasma of rats were detected by LC-MS/MS.The results showed that there was no significant difference in the main pharmacokinetic parameters between the TA-05-administered group and the control group(P>0.01).Conclusion:1.TA-05 had little certain inhibitory effects on the uptakes of OATP1B1,OATP1B3 and OCT2 transporters,and the IC50 was 95.1 ± 17.6,36.9 ± 16.1 and 116 ± 28.9μg/mL,respectively;2.The results showed that TA-05 had no apparent effects on CYP1A2(IC50=18.7μg/mL),CYP2B6(IC50=323μg/mL),CYP2C8(IC50=77.4 μg/mL),CYP2C9(IC50=115μg/mL),CYP2C19(IC50=100μg/mL),CYP3A4(Midazolam)(IC50=208μg/mL)and CYP3A4(Testosterone)(IC50=194 μg/mL);3.The established LC-MS/MS method was sensitive and stable,an the plasma concentrations of metformin hydrochloride in rat plasma were determined and it could be successfully applied in pharmacokinetic experiments.The measured datas were sorted by DAS 4.0 software and made a statistical analysis by SPSS software.The main pharmacokinetic parameters were not statistically different(P>0.01)so that the effect of TA-05 on metformin hydrochloride Pharmacokinetics in rats has no significant effect,there is no interaction between TA-05 and metformin hydrochloride.