Effects Of Cytochrome P450 And P-glycoprotein Regulators On The Pharmacokinetics Of Levotetrahydropalmatine In Rats

Background:Levo-tetrahydropalmatine?l-THP?is a medicinally active ingredient found in several different plants,mainly in the genus Corydalis ambigua?Yan Hu Suo?and Stephania Rotunda Lour?Qian jin teng?.l-THP is marketed under the trade name Rotundine,which is generally used for analgesia and sedation with high efficacy and safety in China.Recently,a number of clinical studies demonstrated that l-THP is effective for inhibiting the drug addiction-producing,addiction-sustaining and preventing relapse,which makes it a promising clinical medication for the management of drug addiction.Pharmacokinetic studies showed that l-THP was extensively metabolized in rats and the major metabolites in plasma were demethylated metabolites and their glucuronidated conjugates.The metabolism of l-THP was mediated by multiple CYP isoforms,of which CYP3A4 and CYP2D6displayed predominant contribution.Since many anti-addiction drugs can interact with CYP enzymes or P-gp and l-THP is generally combinated with other anti-addiction drugs for rehabilitation.However,how the change of CYP enzymes or P-gp activities will influence the pharmacokinetic profile of l-THP and its metabolites still remains unclear.Thus,the aim of the study is to evaluate the effects of changes in enzymes?CYP3A and CYP2D6?and P-gp activities on the pharmacokinetic characteristics of l-THP and its metabolites.This study will provide scientific basis for rational use of l-THP and help improve its clinical safety and efficacy.Methods:1.Rat plasma,Agilent 6410 liquid-chromatography-triple four-stage mass spectrometry,Poroshell 120 EC-C18 column and positive ion mode were used as the basis to establish an LC-MS/MS method of the simultaneous quantification of l-THP and its demethylated metabolites?2-DM and 3-DM?in rat plasma.2.The single dose of solvent,ketoconazole(p.o.,30 mg?kg^-1,CYP3A inhibitor),quinidine(p.o.,50 mg?kg^-1,CYP2D6 inhibitor),1-aminobenzotriazole(p.o.,ABT,100 mg?kg^-1,non-selective CYP inhibitor),dexamethasone(p.o.,50 mg?kg^-1,CYP inducer)or tariquidar(i.v.,50 mg?kg^-1,P-gp inhibitor)was administered before l-THP(9 mg·kg^-1)was dosed into SD rats,respectively.Blood samples were collected at certain time points.The concentrations of l-THP,2-DM and 3-DM were determined by LC-MS/MS.The pharmacokinetic parameters of l-THP and its metabolites were calculated by the non-compartmental analysis using the DAS 3.2.4 and all data were processed using SPSS 22.0 statistical software.Results:1.A sensitive LC-MS/MS method for the simultaneous analysis of l-THP,2-DM and 3-DM was developed and fully validated for the sensitivity,specificity,linearity,precision,accuracy and recovery.The results showed that this method was suitable for the pharmacokinetics study of l-THP and its metabolites.2.Co-administration with CYP inhibitors.Comparing with the control,the AUC_0-48 and C_max of l-THP were significantly increased in the ketoconazole group,quinidine group and ABT group?P<0.05,P<0.01?.The T_max was significantly prolonged in the ABT group?P<0.01?.After pretreatment with ketoconazole or quinidine,the AUC_0-48 of 2-DM were significantly increased?P<0.05?and the C_max were increased by 3.45-and2.68-fold,respectively?P<0.05?.After pretreatment with ABT,the C_max of2-DM was significantly decreased?P<0.01?.The t_1/2 and T_max of 2-DM was significantly prolonged?P<0.05,P<0.01?.Anyone of the three inhibitors significantly decreased the formation of 3-DM.The above results confirmed CYP inhibitors,including ketoconazole,quinidine and ABT,inhibited the metabolism of l-THP in rats and the production of 2-DM might be compensated by the multiple CYP enzymes.3.Co-administration with the CYP inducer.Comparing with the control group,the AUC_0-48 of l-THP was significantly decreased?P<0.05?.The C_maxax and AUC_0-48 of 2-DM was significantly increased by 6.72-and 3.18-fold,respectively?P<0.05?.However,the pharmacokinetic parameters of 3-DM were not statistically changed in the dexamethasone group?P>0.05?.The results suggested that dexamethasone induced the metabolism of l-THP in rats.4.Co-administration with the P-gp inhibitor,comparing with the control group,the C_max and AUC_0-48 of l-THP were not statistically different?P>0.05?.which suggested that l-THP was not a P-gp substrate.Notably,the C_max and AUC_0-48 of 2-DM-Glu were significantly increased?P<0.05?.The results suggested that 2-DM-Glu may be a substrate of efflux transporter?P-gp?.Conclusions:A rapid and accurate LC-MS/MS method for the simultaneous analysis of l-THP,2-DM and 3-DM was developed.The metabolism of l-THP could be inhibited by ketoconazole,quinidine and ABT and induced by dexamethasone in rats.Meanwhile,the formation of 2-DM might be compensated by multiple CYP enzymes.2-DM-Glu may be a substrates of P-gp.Therefore,drug-drug interactions between l-THP and CYP regulators or P-gp inhibitors should of great concern,which ensures the efficiency and safety of l-THP.