Study On The Metabolism And Excretion Of Trantinterol

Trantinterol,2-(3-chloro-4-amino-5-(triflummethyl)phenyl)-2-(tert-butylamino)-ehtanol,is a novel ?2-adrenoceptor agonist,and is currently under phase ? clinical trials in China.Preclinical trials have revealed that trantinterol is a potent and highly selective ?2-adrenergic receptor agonist with long duration of action and low cardiac side effects.The purposes of this study are to prepare the reference standards of trantinterol metabolites with the combination of different methods,and to confirm the structures;then,systematically investigate the metabolism pathway of trantinterol in rats,in microbial models as well as in rat and human microsomes;to study the excretion of trantinterol and its major metabolites in rats,and to identify the main CYP450 enzymes involved in the metabolism of trantinterol in human.Our study gives the usefull information for a comprehensive evaluation of the efficacy and safety of trantinterol,and provides guidance for its rational clinical use.1.Preparation of trantinterol derivativesStrain screening and optimization experiments were carried out,and the microbial model of Cunninghamella blakesleana AS 3.970 was established for the microbial transformation of trantinterol.Five metabolites were isolated and purified from the preparative scale microbial transformation samples and rat urine samples after oral multi-dose of trantinterol.One trantinterol derivative(4-hydroxylamine trantinterol)was synthesized by self-designed procedure,and was confirmed to be one of the major in vivo metabolites of trantinterol by comparison of their retention times and the MS/MS fragments.The structures of these six reference standards were characterized by NMR,ESI-MS/MS and TOF-MS as 2-(3-chloro-4-(hydroxyamino)-5-(trifluoromethyl)phenyl)-2-(tert-butylamino)-ethanol(4-hydroxylamine trantinterol,M2),2-(3-chloro-4-amino-5-(trifluoromethyl)phenyl)-2-(2-hydroxy-1,1-dimethyl)ethylamino-ethanol(tert-butyl hydroxylated trantinterol,M3),2-(3-chloro-4-amino-5-(trifluoromethyl)phenyl)-2-(tert-butylamino)-acetic acid(1-carbonyl trantinterol,M4),2-(3-methylsulfinyl-4-amino-5-(trifluoromethyl)phenyl)-2-(tert-butylamino)-ethanol(3-methyl sulfoxide-dechloro-trantinterol,one pair of epimers,M5 and M6),2-(3-methanesulfonyl-4-amino-5-(trifluoromethyl)phenyl)-2-(tert-butylamino)-ethanol(3-methyl sulfone-dechloro-trantinterol,M7).All the derivatives except M4 are all new compounds.In our study,the stability of M2 was also investigated,and we found that it is generally stable in acidic condition.The reference standards can be used in the further study such as the metabolism pathway,excretion study and the CYP450 enzyme identifification.2.Metabolism of trantinterol in rats and in microbial models.The metabolism of trantinterol in rats and in the microbial model was investigated using the HPLC-MS/MS method.A total of 20 metabolites were found with at least 14 metabolites reported for the first time.The structures of six metabolies were confirmed by comparison of their HPLC retention times and MS/MS fragments with the prepared reference standards,and the other metabolites were identified according to drug metabolism rule and the information obtained from LC-MS and LC-MS/MS analyses.Among these metabolites,4 phase I metabolites included:2-(or 6-)hydroxylated trantinterol(M1),4-hydroxylamine trantinterol(M2),tert-butyl hydroxylated trantinterol(M3)and 1-carbonyl trantinterol(M4);16 phase II metabolites included:N-or O-glucuronide conjugates of trantinterol(M8 and M9),glucuronide conjugates of monohydroxylated trantinterol(M10,M11,and M12),acetylated trantinterol(M13),N-or O-glucoside conjugates of trantinterol(M18 and M19),sulfate conjugate of trantinterol(M20),3-(glutathione-S-yl)-dechloro-trantinterol(M14)and its consecutive derivatives,including the 3-(cysteinylglycine-S-yl)-dechloro-trantinterol(M15),3-(cysteine-S-yl)-dechloro-trantinterol(M16),3-(N-acetylcysteine-S-yl)-dechloro-trantinterol(M17),3-methyl sulfoxide-dechloro-trantinterol(one pair of epimers,M5 and M6)and 3-methyl sulfone-dechloro-trantinterol(M7).The in vivo metabolism pathway of trantinterol in rats can be summarized as monohydroxylation,C-oxidation,acetylation and glucronide conjugation.A metabolite might go through more than one metabolism pathways.Another important pathway in rats is the glutathione conjugation and further catabolism and oxidation to form consecutive derivatives.In the microbial model,besides the monohydroxylation,C-oxidation and acetylation,two metabolism pathway including glucoside conjugation and sulfate conjugation were found for the first time.The major differences between the metabolism in rat and microbial models are on the phase II metabolism.The results demonstrat that microbial models could partly replace mammals in drug metabolism studies,but not completely.3.The excretion study of trantinterol and its major metabolites in ratsA rapid,selective,sensitive and accurate HPLC-MS/MS method for the simultaneous determination of trantinterol and its metabolites M2,M3,M4,M7 in rat urine and feces was developed,and was applied to the excretion study of trantinterol in rats.In order to determine the amount of glucuonide conjugates of trantinterol,M2 and M3,the urine sample was hydrolyzed with?-glucurondidase,and the dose percentage of these metabolites were calculated.After a single oral administration of trantinterol to rats,about 28%of administered trantinterol was recovered in rat urine and feces excreta in the form of trantinterol,M2,M3,M4,M7 and the glucuronide conjugates of trantinterol,M2 and M3 within 96 h post-dose.The analytes were excreted more through urine than feces,with the ratio of their cumulative excretion amounted about 7.6:1.M4 was excreted in largest dose percentage,then trantinterol and M2,their cumulative excretion amounts,expressed as dose percentage,were about 12.72%,4.40%and 4.36%.Trantinterol was excreted unchanged more through feces than urine,while M3 and M4 was the opposite case,other metabolites including M2,M7 and the glucuronide conjugates excreted only through urine.4.In vitro metabolism of trantinterol and the identification of the CYP450 isozymes involved in the formation of M2 and M3With the same analytical method used in the in vivo metabolites identification experiment,the in vitro metabolism of traninterol in rat liver microsomes(RLM)and humun liver microsomes(HLM)were also studied using NADPH as the coenzyme.In vitro incubation samples of RLM and HLM exhibited the same metabolites,including 3 phase I metabolites 4-hydroxylamine trantinterol(M2),tert-butyl hydroxylated trantinterol(M3)and 1-carbonyl trantinterol(M4),and 1 phase ?metabolite acetylated trantinterol.The results demonstrate no significant species difference exists in the in vitro phase I metabolism between rats and human.It is also suggested that cytochrome P450 enzyme plays an important role in the metabolism of trantinterol,and M2 and M3 are the most abundant in vitro metabolites.A rapid UPLC-MS/MS method was developed and validated for the simultaneous determination of metabolite M2 and M3 in HLM and human recombinant CYP450 enzymes.Chemical inhibitors of CYP450 and individual human recombinant CYP450 enzymes were used to characterize the CYP450 isozymes involved in the formation of M2 and M3,and six isozymes including CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP2E1 were investigated.The result indicates that CYP2C19,CYP2D6 and CYP3A4 can catalyze trantinterol to M2,and among the enzymes tested,CYP2C19 has the highest affinity,maximum velocity and intrinsic clearance.The formation of M3 is mainly catalyzed by CYP2C19 and CYP3A4,and CYP2C19 has the higher affinity to the substract,while CYP3A4 has the higher maximum velocity and intrinsic clearance.The results suggest that when trantinterol is taken with inhibitors or inducers of CYP2C19,CYP2D6 and CYP3A4,it may cause changes in the plasma concentration of trantinterol or its metabolites,resulting in a certain safety problems.