Studies On The Transport Of Trantinterol In Vitro And Its Pharmacokinetics In Human

Trantinterol, 2-(4-amino-3-chloro-5-trifluomethylphenyl)-2-t-butylaminoethanol, is a novel phenylmethylamineβ2-adrenoceptor agonist. It selectively excited through the airway smooth muscle of theβ2 receptor, so that the parts smooth muscle relaxation and play to their anti-asthma.In this paper we reported the first quantitative method to determine trantinterol in biological fluids by liquid chromatography-mass spectrometry (LC/MS/MS). The assay has been successfully applied to a pharmacokinetic study in healthy volunteers and provided some references and inspirations for the clinical studies. Meanwhile, MDCK-MDR1 cell monolayer and Caco-2 model were established to study the transport and absorption mechanisms.1. Pharmacokinetic study of trantinterol in humanA rapid and sensitive assay for trantinterol, a novelβ2-adrenoceptor agonist, in human plasma has been developed. The method was applicable to clinical pharmacokinetic study of trantinterol in Chinese healthy volunteers. Samples containing the analyte and internal standard, clenbuterol, were analyzed by LC/MS/MS.Chromatographic separation was performed on a Venusil MP-C18 column (50×4.6 mm, 5μm). The mobile phase consisted of methanol/1 % formic acid (50:50, v/v) at a flow rate of 0.8 mL/min. Mass spectrometric detection employed an API 4000 mass spectrometer equipped with an electrospray ionization (ESI) source operated in the positive ion mode and monitored by multiple reaction monitoring (MRM). Trantinterol and clenbuterol were monitored using the transitions of the protonated molecular ions at m/z 311.2 to 238.1 and m/z 277.2 to 203.1, respectively. The assay was validated for specificity, linearity, precision, accuracy, recovery, matrix effects and stability according to guidelines.After administrated a single dose of 25μg, 50μg and100μg of trantinterol, the results of pharmacokinetic parameters were as follows: 87.0±40.7 pg?h/mL, 125.1± 48.3 pg?h/mL and 222.9±64.8 pg?h/mL for AUC0-t; 2.2±4.2 pg/mL,20.0±4.3 pg/mL,48. 6±14.3 pg/mL for Cmax; Tmax was 1.1±0.5 h, 1.8±1.2 h and 1.3±0.4 h; t1/2 was 16.4±3.6 h, 15.4±4.7 h and 13.3±2. 9 h. The linear pharmacokinetic characteristics were shown in the doses range of 25-100μg because the pharmacokinetics parameters (AUC0-t and Cmax) were linearly increased with the increasing of doses. While the other pharmacokinetic parameters such as t1/2 were not varied with the increasing of doses. There was significant difference in AUC0-t between the food fed and fasted groups.2. The influence of trantinterol on CYP450 enzymes in rat liver microsomesThe aim of the present study was to investigate whether exposure to trantinterol leads to induction of cytochrome P450 (CYP450) enzymes. Wistar rats were given single daily oral doses of trantinterol (9.0μg/kg) for one week. Liver microsomes were prepared and the effect of treatment on the total CYP450 content and the activities of CYP1A2, CYP2D6, CYP3A4, CYP2C9 and CYP2C19 were determined. The results showed that trantinterol didn't influence the content of microsome proteins and CYP450 enzymes and the activity of the main CYP450 subfamilies.3. Effect of trantinterol on cytochrome P450 mRNA expression in HepG2 cellsTo study the effect of trantinterol on the cytochrome P450 enzymes (CYP450) mRNA expression in HepG2 cells and provide theoretical basis for clinical drug combination, the cell proliferation was analyzed by MTT assay in 2.4, 12, 60, 300, 1200 and 4800 pg/mL of trantinterol solution. The mRNA expression levels of CYP1A1, CYP2E1 and CYP3A5 were examined with real-time quantitative reverse-transcriptase polymerase chain reaction. The result showed that, different concentrations of trantinterol didn′t inhibit the proliferation of HepG2 cells (P>0.05). Compared with control group, the mRNA expressions of CYP2E1 and CYP3A5 showed the similar values in trantinterol groups, while the mRNA expression of CYP1A1 was inhibited (P<0.01). Exposure of HepG2 cells to trantinterol has no effect on the mRNA expressions of CYP2E1 and CYP3A5 but inhibit the mRNA expression of CYP1A1.4. Transport of trantinterol in MDCK-MDR1 cell monolayer modelTo establish and validate MDCK-MDR1 cell monolayer model for drug transport study, MDCK and MDCK-MDR1 cells were cultured on the Transwell plate. Transepithelial electrical resistance (TEER) values, apparent permeability coefficient (Papp) of Lucifer yellow and the Rho123 efflux ratio of MDCK-MDR1 were tested to validate the cell model. TEER values of MDCK and MDCK-MDR1 cells were 591.6±7.4 ?·cm2 and 140.4±6.6 ?·cm2, and Papp values were 2.35±0.23×10-6 cm/s and 4.51±0.28×10-6 cm/s. The Rho123 efflux ratio of MDCK-MDR1 is 5.16. MDCK-MDR1 cell is generated from MDCK cell, which can be stably transfected with the human mdr1 gene leading to the polarized overexpression of P-glycoprotein (P-gp). In conclusion, the MDCK-MDR1 cell monolayer model was successfully established and well validated with all the testing items.MDCK-MDR1 cell monolayer model was used to investigate the effect of trantinterol on the bi-directional transport of Rho123. The results shown that, efflux ratio of Rho123 were droped to 2.77 and 1.82 in different concentration of trantinterol. It suggesting that trantinterol is not a substrate for P-gp. The same result was confirmed by the study of bi-directional transport of trantinterol on MDCK and MDCK-MDR1 cells. Efflux rates of MDCK-MDR1 and MDCK were 1.20 and 1.05, and the net efflux ratio is 1.14.The effect of trantinterol on ATPase activity of P-gp was investigated. Trantinterol could inhibit the ATPase activity of P-gp, thus inhibit the transport.5. The cell transport of trantinterol on Caco-2 cell modelCaco-2 cell were cultured in Transwell inserts for 21 days. TEER value was higher than 250 ?·cm2 and Papp of Lucifer yellow was less than 0.5×10-6 cm/s. Bi-diretional transport studies of trantinterol were carried out in Caco-2 cell monolayer model. The efflux ratio of trantinterol was 1.078, while combined with verapamil was 1.11. The results indicated that trantinterol is not a substrate for P-gp, and inhibitor of P-gp could not change the efflux of trantinterol.