| Trantinterol,2-(4-amino-3-chloro-5-trifluoromethylphenyl)-2-t-butylaminoethanol, is a novel P2-adrenoceptor agonist for the treatment of asthma. 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. The main purposes of this study are to systematically investigate its metabolic mechanism, including identification of the main metabolic enzymes in human and the site of metabolism in rat, assess the potential for drug-drug interactions mediated by CYP450 enzymes, and investigate its transport mechanism as well as its effect on the transport ability of P-glycoprotein (P-gp).1. Study on the metabolic mechanism in vitroAn HPLC method was developed to determine trantinterol in rat S9. The study with S9 from rat liver, kidney, lung, and small intestine indicated that trantinterol could be metabolized in liver primarily and small intestine could also participate in the metabolism.The metabolite profile of trantinterol was investigated using liquid chromatography-mass spectrometric (LC-MS/MS) techniques. A total of 9 metabolites (M1-M9) were found in rat urine, and glucuronide of hydroxyl trantinterol (M6) was a new metabolite detected in the present study. The main metabolites of trantinterol in rat were carbonyl trantinterol (M1) and hydroxyl trantinterol (M2). Rat liver microsomes (RLM) were prepared by using calcium chloride condensed method. The in vitro metabolism of trantinterol was studied by incubation with RLM or HLM and the metabolites were investigated by LC-MS/MS. The metabolites generated in RLM were the same as those in HLM, and the metabolites were in accordance with the phase I metabolites in rat urine except for M4. The in vitro metabolic system established in the present study could be appropriate for metabolic mechanism study of trantinterol.2. Identification of the CYP450 isozymes involved in the formation of Ml and M2A rapid UPLC-MS/MS method had been developed to determine M1 in HLM and human recombinant CYP450 enzymes. The method was employed for the semi-quantitative determination of M2 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 M1 and M2. The result indicated that the formation of M1 was mainly catalyzed by CYP2E1, and the formation of M2 was mainly catalyzed by CYP2C19 and CYP3A4/5.3. Assessment of trantinterol for interference with rat and human liver CYP450 enzymes activitiesTrantinterol did not inhibit CYP1A, CYP2C, CYP2D, and CYP3A activities in RLM. The administration of trantinterol to rat for 7 days at doses of 0.3 mg/kg,1.0 mg/kg, and 3 mg/kg per day did not significantly induce CYP450 enzyme activities. No significant increases in liver weight, concentrations of liver microsomal protein, and CYP450 concentrations were observed after a 7-day administration.The posibility of trantinterol to inhibit human liver CYP450 activities was evaluated in vitro in HLM. Trantinterol did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 activities. To evaluate its CYP450 induction, human cryohepatocytes (n=3) were used and treated once daily for 3 days with trantinterol (0.01,0.1, and 1 ng/mL), the activities of CYP1A2 and CYP3A4/5 were then measured. No induction effect was observed on the activities of CYP1A2 and CYP3A4/5. These in vitro results indicated that, at pharmacological relevant concentrations, trantinterol would not produce clinically significant CYP450 inhibition or induction.4. The transportation of trantinterol across the Caco-2 cell monolayers and its effect on the P-gp transport ability in Caco-2 cellsA UPLC-MS/MS method had been developed to determine trantinterol in HBSS buffer, and the method was fully validated and applied to the transportation study of trantinterol in Caco-2 cell model. In Caco-2 cells, trantinterol showed a good absorption character with an apparent permeability coefficient (Papp) of more than 1×10-6 cm/s. The efflux ratio of trantinterol at 5,10, and 20μmol/L were less than 1.5, which indicated that the main transportation of trantinterol in small intestine was passive diffusion. The efflux ratio of trantinterol was not affected by P-gp inhibitor verapamil and MRP1/2 inhibitor MK-571. The results indicated that trantinterol was not a substrate for P-gp and MRP 1/2.Using digoxin as the substrate probe of P-gp, the effect of trantinterol on P-gp transport ability was studied. A UPLC-MS/MS method had been developed to determine digoxin in HBSS buffer. Trantinterol had significant inhibition effect on the P-gp-mediated transportation of digoxin across the Caco-2 cell monolayers. No induction effect was observed after the Caco-2 cells exposed to trantinterol for 72 h. |
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