Study On The Metabolism And Pharmacokinetics Of Trantinterol In Animals

Trantinterol,2-(4-amino-3-chloro-5-trifluomethyl)-phenyl-2-t-butyl-amino-ethanol, is a novel phenyl-methyl-amineβ2-adrenoceptor agonist currently undergoing clinical trials in China. Preclinical trials have revealed that trantinterol is a potent and highly selectiveβ2-adrenergic receptor agonist with long duration of action and low cardiac side effects. Until now, studies on the metabolism and pharmacokinetics of trantinterol in vivo are limited. The main purposes of this study are to systematically investigate the metabolism of trantinterol in rat and dog, the pharmacokinetics of trantinterol in rat, binding ratio of trantinterol to human plasma proteins and enantioseparation of trantinterol.1. Metabolism of trantinterol in rat and dog.Two metabolites of trantinterol were prepared by isolation from rat urine and feces after oral administration of trantinterol. Their structures were identified by MS and NMR. They were identified as:2-(3-chloro-4-amino-5-trifluomethyl)-phenyl-2-tert-butylamino acetic acid (M1) and 2-(3-chloro-4-amino-5-trifluomethyl)-phenyl-2-tert-butylamino ethyl acetate (M7).The reference standards of M1 and M7 were used for the metabolism studies of trantinterol in rat and beagle dog using LC-ESI-MS/MS. A total of 13 metabolites were found in rat and beagle dog. The structures of 2 metabolites in vivo were identified by using reference standards. According to drug metabolism rule and the information obtained from MS/MS analyses, the structures of other 11 metabolites were elucidated as followed:hydroxyl trantinterol (M2, M3 and M4), dihydroxyl-trantinterol (M5), dinitro-trantinterol (M6), acetyl trantinterol (M8), glucuronide of N-debutyl trantinterol (M9), glucuronide of trantinterol (M10), glucuronide of M3 or M4 (M11), glucuronide of M5 (M12) and glucuronide of trantinterol acetylate (M13).8 metabolites were detected in rat. Trantinterol was metabolismed extensively in beagle dog and 13 metabolites were detected.There were 6 identical metabolism pathways in rat and dog. Phase I metabolism pathways included:hydroxylation, C-oxidation, C-nitration and N-dealkylation; phase II metabolism pathways included:acetylation and glucuronide conjugation. A metabolite might go through more than one metabolism pathways.2. Pharmacokinetic study of trantinterol in rat.In order to study the systemic exposure of M1, a sensitive and rapid method was established using liquid chromatography-mass spectrometry techniques. The LC-MS/MS method was developed for the simultaneous determination of trantinterol and M1 in plasma, urine and feces samples after oral administration for the first time.After oral administration of trantinterol, the concentrations of trantinterol and its metabolite M1 in rat plasma were determined and mean plasma concentration-time curves were simulated and their main pharmacokinetic parameters were calculated.The absorption of trantinterol was rapid and tmax was 0.6 h. The tmax of M1 was later than that of trantinterol. The ratios of Cmax and AUC of trantinterol to M1 in male rat plasma were 1:3 and 1:4, respectively. The two parameters of M1 were obviously higher than those of parent drug. The results indicated some of trantinterol were quickly metabolized into M1 and the plasma concentration of M1 was higher than that of trantinterol, and M1 indicated a higher systemic exposure than trantinterol in male rat. While the ratios of Cmax and AUC of trantinterol to M1 in female rat plasma were 1:0.7 and 1:1, respectively. The results indicated the plasma concentrations of trantinterol and M1 were almost the same and there was not difference in systemic exposures of trantinterol and M1 in female rat plasma.3. Excretion study of trantinterol in ratAfter oral administration of trantinterol, the concentration of trantinterol and its metabolite M1 in rat urine and feces were determined to acquire to excretion situation of them.In 0～96 h after oral administration, the excreted amounts of trantinterol and M1 accounted for 14.9% of the total dose in male rat. Trantinterol was excreted in smaller dose percentage than M1 and the ratio of cumulative excretion of trantinterol to M1 in male rat was almost 1:10. In female rat, the excreted amounts of trantinterol and Ml accounted for 9.31% of the total dose and the ratio of cumulative excretion of trantinterol to M1 was 1:2. In male rat, the fecal excretion amounts of trantinterol and M1 accounted for 2.46% of the total dose and the ratio of fecal cumulative excretion of M1 to trantinterol was almost 1:8. In female rat, the fecal excretion amounts of trantinterol and M1 accounted for 2.53% of the total dose and the ratio of fecal cumulative excretion of M1 to trantinterol was almost 1:35. Trantinterol was excreted in larger dose percentage than M1 in rat feces.The urinary cumulative excretion amount of rantinterol in male rat was more than in female rat and the fecal cumulative excretion amounts of rantinterol in male and female rat were almost the same. While urinary and fecal cumulative excretion amount of M1 in female rat was more than in male rat.4. Binding ratio of trantinterol to human plasma proteinsThe protein binding interaction of trantinterol with human plasma was investigated using equilibrium dialysis method and LC-MS technique. After being equilibrated for 72 h, the average plasma protein binding ratio in the concentration of 2.00～50.0 ng/mL was 30.2±6.5%. The plasma protein binding ratio of trantinterol was at moderate level and showed concentration-independent.5. Chiral separation of trantinterol enantiomers by HPLC.Tow simple and efficient analytical HPLC methods were developed for enantioseparation of trantinterol using pre-column derivatization with (R)-(+)-4-(N, N-Dimethylaminosulfonyl)-7-(2-chloroformylpyrrilidin-1-yl)-2,1,3-benzoxadiazole (DBD-Pro-COC1) or using Vancomycin chiral stationary phase (CSP). The effects of concentration of derivative agent, time of derivative reaction and chromatographic conditions were investigated for the enantioseparation of trantinterol. The enantiomers of trantinterol were directly resolved on Chirobiotic V column, and the separation mode, mobile phase and column temperature were investigated. The chiral separation method is significant in studying of pharmacokinetic processes, the synthesis and quality control of the enantiomer of trantinterol.