【作者】 孙严彤；

【导师】 顾景凯；

【作者基本信息】 吉林大学 ， 药理学， 2010， 博士

【摘要】 川丁特罗(2-(4-氨基-3-氯-5-三氟甲基苯基)-2-叔丁氨基乙醇,trantinterol),是沈阳药科大学程卯生教授采用Me-too策略,独立研发具有自主自主知识产权的一种新型β2受体激动剂。药理实验表明,川丁特罗通过选择性激动气道平滑肌上的β2受体,使平滑肌松弛而发挥其抗哮喘的作用,有效性和安全性良好。本试验首次建立了LC/MS/MS法用于测定生物样品中川丁特罗的浓度,对川丁特罗进行临床药代动力学研究,这为本品临床的安全、合理用药奠定了基础。同时,本试验建立了体外MDCK-MDR1和Caco-2细胞模型,并探索了川丁特罗在人体内的转运和吸收机制。一、川丁特罗人体药代动力学的研究首次建立了一种LC/MS/MS分析方法,用于测定人血浆中川丁特罗的浓度。采用Venusil MP C18色谱柱(50×4.6 mm, I.D., 5μm),以甲醇:水(含1%甲酸)(50:50,v/v)为流动相,流速为0.8 mL/min,LC/MS/MS配有电喷雾离子化源,采用正离子化方式,在选择反应监测(MRM)模式下,选择m/z 311.2→m/z 238.1和m/z 277.2→m/z 203.1为待测物川丁特罗和内标克伦特罗的定量离子对。方法确证表明,该方法的精密度和准确度、稳定性、提取回收率和基质效应等均符合SFDA规定和要求。应用该方法对人口服给予川丁特罗25μg、50μg和100μg后的吸收情况进行系统研究。结果表明:3个剂量组的血浆中Cmax分别为12.2±4.2 pg/mL,20.0±4.3 pg/mL和48.6±14.3 pg/mL;AUC0-t分别为87.0±40.7 pg?h/mL、125.1±48.3 pg?h/mL和222.9±64.8 pg?h/mL。采用SPSS 14.0软件,对参数进行相关性分析,人体内的低、中、高三个剂量下的主要药动学参数AUC0-t及Cmax均与给药剂量呈线性相关。表明在25～100μg内,川丁特罗的体内过程呈线性药代动力学特征,而其他参数如t1/2则不随剂量变化而变化。考察进食对药物吸收的影响结果表明,进食与空腹给药后,药物的Cmax无显著性差异,AUC0-t有差异,进食对药物的吸收有轻度影响。二、川丁特罗对大鼠肝微粒体细胞色素P450酶的影响为了研究川丁特罗对主要代谢酶的影响,本文考察了健康Wistar大鼠连续7天灌胃给予川丁特罗(9.0μg/kg)后,大鼠肝微粒体的蛋白含量和CYP450酶总含量的变化以及主要亚型(CYP1A2、CYP2D6、CYP3A4、CYP2C9和CYP2C19)的活性变化。结果表明:Wistar大鼠给药后,肝微粒体中蛋白含量和CYP450总含量与空白对照组相比没有显著性差异,含量未见变化。几种主要的亚型的活性也未受影响。由于CYP1A2、CYP2D6、CYP3A4、CYP2C9和CYP2C19这5种酶的含量约占肝微粒体CYP450酶的90%,本试验结果可初步推断,川丁特罗不会抑制或诱导CYP450酶的产生和活力,即不会改变合并给药时其他药物的代谢速率,进而影响其药效的发挥。三、川丁特罗HepG2细胞细胞色素P450 mRNA表达的影响建立人肝癌(HepG2)细胞体外模型,首次考察了川丁特罗对人CYP450酶mRNA表达的影响,为临床合联合用药提供理论依据。人肝癌细胞(HepG2)中分别加入浓度为2.4、12、60、300、1200和4800 pg/mL的川丁特罗溶液,MTT法检测药物作用下的细胞增殖。采用RT-PCR法检测HepG2细胞中CYP1A1,CYP2E1和CYP3A5亚型的mRNA表达量。结果显示:在2.4~4800 pg/mL的范围内,川丁特罗未抑制HepG2细胞的增殖。与对照组相比,川丁特罗给药组CYP3A5和CYP2E1 mRNA表达无明显变化,而CYP1A1 mRNA表达水平明显降低(P<0.01)。表明川丁特罗能明显抑制HepG2细胞中CYP1A1 mRNA表达,而对CYP3A5和CYP2E1表达则无抑制和诱导作用。四、川丁特罗在MDCK-MDR1细胞模型中的转运研究建立MDCK-MDR1单层细胞体外药物转运模型,对模型进行评价。将MDCK和MDCK-MDR1细胞在Transwell多聚碳酸酯膜上培养5天后,考察了跨膜电阻、荧光黄的表观渗透率和Rho123在细胞上的转运。MDCK和MDCK-MDR1细胞的跨膜电阻分别为591.6±7.4 ?·cm2和140.4±6.6 ?·cm2,荧光黄的表观渗透率为(Papp)分别为(2.35±0.23)×10-6和(4.51±0.28)×10-6 cm/s。在Rho123的转运试验中,MDCK-MDR1的外排比率为5.16,明显高于MDCK细胞,表明MDCK细胞在稳定转染人mdr1基因后,人源性P-gp产生高表达,外排功能增加。研究表明,成功建立MDCK-MDR1细胞模型,各项考察指标符合规定。川丁特罗对P-gp介导的Rho123在模型中外排的影响,结果显示:10μmol/L和100μmol/L川丁特罗不同程度上均降低了Rho123的外排比率。Rho123的外排率从3.11降到了2.27和1.82。统计分析发现,加入川丁特罗后,Rho123在MDCK-MDR1细胞的Papp值与对照组相比有显著性差异(P<0.05)。这表明川丁特罗抑制了P-gp介导的Rho123外排,可能为P-gp的抑制剂。在川丁特罗MDCK-MDR1细胞双向转运试验中,采用LC/MS/MS法测定川丁特罗的含量。结果显示:川丁特罗在MDCK-MDR1和MDCK细胞上外排率分别为1.2和1.05,净外排率为1.14,表明川丁特罗不是P-gp的底物。在川丁特罗对P-gp ATP酶的影响实验研究中发现,川丁特罗能够抑制ATP酶的活性,从而抑制P-gp的外排。五、川丁特罗在Caco-2细胞模型中的转运研究建立Caco-2单层细胞模型,观察Caco-2细胞间连接紧密,TEER>250 ?·cm2,荧光黄的Papp<0.5×10-6 cm/s。结果表明,本实验室所建立的Caco-2细胞模型指标符合要求。采用建立的Caco-2细胞模型考察川丁特罗的双向转运,将P-gp抑制剂维拉帕米作为对照组。结果显示,实验组川丁特罗的外排率为1.078;维拉帕米对照组的川丁特罗外排率为1.11。统计结果表明两者无显著性差异(P>0.05),P-gp的抑制不能显著改变川丁特罗的外排率,因此川丁特罗不是P-gp的底物。更多还原

【Abstract】 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.更多还原