Metabolism Of Arteannuin A/B And Their Induction Of CYP450 Enzymes

Patients in malaria-endemic areas often use the traditional Chinese medicine Artemisia annua L.(A.annua)to treat malaria when antimalarial drugs are not available.It was found that the dried leaves of A.annua could reverse the resistance of Plasmodium falciparum to artemisinin(QHS),the main active component in A.annua,probably via increasing the absorption of QHS when the dried leaves were administered.At the same time,clinical studies have shown that the cure rate is low and the recurrence rate is high when drinking A.annua tea continuously.To reduce the prognostic recurrence rate and slow down the rapid development of drug resistance,it is necessary to clarify the changes of matrix components in A.annua on the metabolic clearance of QHS.Previous in vitro reporter gene experiments showed that arteannuin A(Art A)and arteannuin B(Art B)can induce the expression and activity of metabolic enzymes CYP3A4 and CYP2B6 by activating nuclear receptors PXR and CAR.These two CYP enzymes mediate the metabolism of QHS,and it is proposed that Art A and Art B can induce the metabolic elimination of QHS,resulting in a high prognosis recurrence rate.Meanwhile,in vitro enzyme inhibition experiments showed that Art B significantly inhibited CYP3A4 activity.Based on the previous in vitro experiments,the in vivo induction effects of the two components on related metabolic enzymes were investigated,and the possible effects on the metabolism and elimination of QHS were also studied.In vitro and in vivo metabolism characteristics of the components themselves were evaluated in this thesis.The results of this paper can provide useful guidance for guiding the rational use of A.annua.1.Study on the effect of arteannuin A/B on CYP3A4 and CYP2B6 enzyme activitiesPrevious in vitro experiments showed both induction and inhibition of CYP enzymes by Art A/B in A.annua,and in vitro experiments could only provide a partial answer to the complexity of the in vivo situation.The actual effect of Art A/B on enzymatic activity in vivo at therapeutic concentrations was unknown.Based on the results of in vitro experiments,the cocktail in vivo probe method was used to detect the effects of Art A and Art B on the enzymatic activities of CYP3A and CYP2B in mice in this chapter,whether Art A and Art B played an inductive role in vivo was also evaluated.A quantitative bioanalytical method of probe drugs and their metabolites in mouse plasma was established.The enzyme activities of CYP3A4 and CYP2B6 were characterized by the biotransformation of the probe drug midazolam(MDZ)to hydroxymidazolam(OH-MDZ),and the hydroxy lation of bupropion(BUP)to hydroxy bupropion(OH-BUP).The drug metabolic capacities were expressed as the ratio of AUCOH-MDZ/AUCMDZ and AUCOH-BUP/AUCBUP.The effects of Art A and Art B on CYP3 A and CYP2B enzyme activities at two doses were studied in mice,with QHS as a positive control drug.A quantitative LC-MS/MS method for simultaneous determination of MDZ,OH-MDZ,BUP and OH-BUP in mouse plasma was established.The results showed that MDZ and OH-MDZ have good linearity in the range of 1.0-100.0 ng/mL;BUP and OH-BUP have good linearity in the range of 1.0-400.0 ng/mL.The accuracy,precision and extraction recovery of the four analytes met the requirements of bioanalysis,indicating the method could be used for the quantitative analysis of MDZ,OH-MDZ,BUP and OH-BUP in mouse plasma.The activity of the CYP3A enzyme was induced by oral administrations of QHS for 3 consecutive days with no dose correlation.Low-dose QHS(10 mg/kg)increased the activity of CYP3A by 1.6-fold,while 20 mg/kg QHS tended to induce CYP enzyme activity but with no significance(1.2-fold,p=0.35).QHS showed a dose-related induction effect on CYP2B enzyme activity.The enzyme activity of CYP2B6 was significantly increased by 1.4-fold(10 mg/kg)and 2.4-fold(20 mg/kg).The results showed that the positive control drug QHS could significantly induce CYP activity in mice.The induction effect of Art A on CYP3A enzyme activity was not dose-related.The CYP3A activity of mice was significantly increased by 1.6-fold(10 mg/kg)and 1.3-fold(20 mg/kg)after intragastric administrations of Art A for 3 consecutive days.The induction effect of Art A on CYP2B was dose-related.Low-dose Art A(10 mg/kg)increased CYP2B activity by 1.4-fold,while high-dose Art A(20 mg/kg)did not lead to a significant change in CYP2B6 activity(1.1-fold,p=0.92).No significant changes were observed in the enzyme activities of CYP3A or CYP2B in mice after being orally given 10 mg/kg or 20 mg/kg Art B for 3 consecutive days.The above results showed that Art A could induce the activity of CYP3 A in mice,and the induction effect was comparable to that of QHS;the effect on CYP2B enzyme activity was related to the dose.The experimental doses of Art B did not affect the activities of CYP3A4 and CYP2B6 in mice.2.In vitro metabolism study of arteannuin A/BDue to the complex environment in vivo,the effects of Art A and Art B on the enzyme activities in mice may be the confounding result of multiple factors.To explore the possible metabolic interactions between these two compounds and QHS,in vitro metabolic experiments were used to investigate the metabolic differences,main metabolites and metabolic enzyme phenotypes of Art A/B in liver microsomes of different species.The biotransformation of Art A/B in vitro was analyzed,and a reasonable animal model was provided for in vivo pharmacokinetic experiments.LC-MS analytical methods for the quantitative determination of Art A or Art B in the liver microsomal matrix were established.Art A and Art B had good linearity in the concentration range of 0.1-1.0 μM.The intra-day and inter-day precision were both less than 15.0%,and the accuracy was within±2.5%.The method could be used for the relative quantitative analysis of Art A and Art B in the liver microsomal matrix.Using the t1/2 elimination method,the intrinsic clearance(CLint)of Art A in human,mouse and rat liver microsomes was determined as 325.0 ± 46.6,754.4 ± 100.8 and 203.9±5.2 μL/min/mg,respectively.The CLint of Art B in human,mouse and rat liver microsomes was determined as 133.0±17.7,48.7 ± 2.3 和 62.1±5.7 μL/min/mg,respectively.The metabolic processes of Art A and Art B in human liver microsomes were more similar to that in rat liver microsomes.The main metabolites of Art A and Art B in liver microsomes in vitro were formed by hydroxylation.The recombinant enzyme incubation experiments showed that the metabolism of Art A and Art B was mainly mediated by CYP2B6 and CYP3A4,with a minor role of CYP2C19.These results showed that the metabolism of Art A and Art B in the liver was very rapid and had a short half-life.Rats are suitable animal models to study the metabolism of Art A and Art B in vivo.Art A and Art B mainly undergo phase I hydroxylation metabolism,which increases their polarity.Phenotypes of the main metabolic enzymes of Art A and Art B overlapped with QHS metabolic enzymes,suggesting that they may compete with QHS for metabolic enzymes and thus lead to metabolic interactions.3.Pharmacokinetic study of arteannuin B in ratsThe above studies indicated that there was a complex metabolic interaction process between Art A/B and QHS,and it was necessary to study the metabolic elimination of Art A/B in vivo.Art B was selected as the representative drug,and the pharmacokinetic characteristics of Art B in oral administration of Art B monomer and A.annua extracts were studied by using a healthy rat model.The metabolism of Art B in rats and the effect of the extracts’ matrix on the metabolism of Art B were investigated,and the difference between low-dose and high-dose,single-dose and multiple-doses were investigated.A quantitative analysis method of Art B in rat plasma was established.In this method,QHS was used as the internal standard,and protein precipitation was used for sample pretreatment.The results showed that Art B has good linearity in the concentration range of 0.5-50.0 ng/mL,the accuracy was within ± 4.0%,the intra-day and inter-day precisions were less than 15.0%,the extraction recovery rate was high,and the matrix interference could be ignored.The method could be used for the pharmacokinetic study of Art B in rat plasma.Compared with a single dose of pure Art B,after a single administration of total methanol extract(E-1),85%methanol extract(E-2)and ethyl acetate extract(E-3)of A.annua,the plasma exposures(AUC0-t and AUC0-∞)of Art B were significantly increased(p<0.05),while the CL/F were all significantly decreased(p<0.05).After repeated administrations of pure Art B,except for the significant increase in the mean residence time(MRT),there were no significant changes in other pharmacokinetic parameters(p>0.05).The plasma exposures(AUC0-t,AUC0-∞ and Cmax)and MRT of Art B in rats given multiple doses of A.annua extracts(E-1～E-3),compared with multiple administrations of pure Art B,were significantly increased(p<0.05),while CL/F was significantly decreased(p<0.05).There was no significant difference(p>0.05)between most of the pharmacokinetic parameters of Art B in rat plasma after a single dose of A.annua extracts(E-1～E-3)and no correlation with dose.After multiple administrations of A.annua extracts,most of the pharmacokinetic parameters of Art B in the three extracts were not significantly changed(p>0.05).However,compared with a single dose of the extracts,the plasma exposure of Art B was significantly increased(p<0.05),the CL/F was significantly decreased(p<0.05),and most other pharmacokinetic parameters had no significant changes(p>0.05).The above results showed that when pure Art B was administered,the plasma exposure was low,and the clearance was fast with a short half-life;while the extracts’ matrix will change the pharmacokinetic behavior of Art B in vivo,which significantly increased the plasma exposure with a decreased clearance rate.There were no significant changes in the in vivo pharmacokinetic behavior of Art B in the three extracts.The results of this study showed that Art A in A.annua was a new inducer of CYP3A activity in vivo,with a potency equivalent to QHS.Art A also showed induction of CYP2B activity in vivo,but not in a dose-dependent way,which may be caused by the confounding effect of inhibition.However,Art B could not induce CYP3A or CYP2B activity in mice.These findings indicated that there are potent inducers and inhibitors of CYP enzymes,which may lead to the alternation of the pharmacokinetics of QHS and then result in the high recrudescence and possible rapid development of drug resistance.