川丁特罗体外转运和人体药代动力学研究
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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。
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 human
A 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 microsomes
The 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 cells
To 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 model
To 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 model
Caco-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.
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。
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 human
A 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 microsomes
The 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 cells
To 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 model
To 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 model
Caco-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.
引文
[1]陈灏珠.实用内科学[M].北京:人民卫生出版社, 2000.
[2] National institute of health. Global Initiative for Asthma:global strategy for asthma management and prevention. 1995
[3] Yong RP. Confirmation of genetic linkage between atopic IgE response and chromosome 11q13 [J]. J.Med. Genetic. 1992, 29: 236-239.
[4] Gao P, Mao XQ, Baldini M. et al. Serum total IgE levels and CD14 on chromosome 5q31 [J]. Clin Genet. 1999, 56 (2): 164-165.
[5] Manian P. Genetics of asthma [J].Chest, 1997, 112: 1397-1408.
[6]谢敏强.药理学前沿-哮喘和COPD的新概念及新药[M].北京:科学出版社, 2003.
[7]钟南山.支气管哮喘防治研究进展[J].国外医学内科学分册, 1993, 20 (5): 194-197.
[8]高海鹏.气道高反应性的机理及影响因素[J].国外医学呼吸系统分册, 1990, 10(1): 1-4.
[9]胡亚美.诸福棠实用儿科学[M].北京:人民卫生出版社, 2002.
[10]钟南山,徐军.哮喘发病机制及诊断进展[J].中华结核和呼吸杂志, 1995, 18 (3): 136-139.
[11]李明华,董竞成.支气管哮喘的现代治疗[M].北京:北京医科大学中国协和医科大学联合出版社, 1996.
[12]魏尔清,卞如濂.平喘药研究现状[J].新药与临床, 1999, 14(1): 34-38.
[13]潘华杰.支气管哮喘药物的治疗研究进展[J].中外医疗,2009, 35: 164-165.
[14] Haahtela T, Jarvinen M, Kava T, et al. Effect of reducing or discontinuing inhaled budesonide in patients with mild asthma [J]. Nengl J Med, 1994, 331(11): 700-705.
[15] Agertft L, Pedersen S. Effects of long-term treatment with an inhaled corticosteroid on growth and pulomonary function in asthmatic children [J]. Respir Med, 1994, 88 (5): 373-381.
[16] O’Byrne PM. Pharmacologic interventions to reduce the risk of asthmaexacerbations [J]. Proc Am Thorac Soc, 2004, 1 (1): 105- 108.
[17]王长征.支气管哮喘治疗新药的研究进展[J].中华哮喘杂志, 2008, 2 (2): 137-140.
[18] Humbert M. Ciclesonide: a novel inhaled corticosteroid [J]. Expert Opin Investig Drugs, 2004, 13(10): 1349-1360.
[19]谢灿茂.吞咽疾病与哮喘及慢性阻塞性疾病[J].新医学, 1997, 28 (11): 616-617.
[20]王山泽,邓琨.组胺受体与支气管哮喘[J].国外医学呼吸系统分册, 1990, 10 (2): 113.
[21] Dunn CJ, Goa KL, Zafirlukast: an update of its pharmacology and therapeutic efficacy in asthma [J].. Drugs, 2001, 61 (2): 285-315.
[22] Gomez FP, Roca J, Barbera JA, et al. Effect of a platelet-activating factor (PAF) antagonist, SR 27417, on PAF-induced gas exchange abnormalities in mild asthma [J]. Eur Respir J, 1998, 11 (4): 835-839.
[23] Connor BJ, Tows LJ, Barnes PJ. Prolonged effect of no bromide on methacoline-induced brochoconstriction in asthma [J]. Am J Respir Crit Care Med, 1996, 154 (4 Pt 1): 876-880.
[24]宋勉,王亚梅.肺肠同治法在治疗支气管哮喘急性发作期的运用[J].光明中医, 2005, 20 (1): 5-6.
[25]李兰铮,杨志文,单丽囡.加味小青龙汤治疗支气管哮喘急性发作期30例[J].陕西中医, 2005, 26 (8): 740-742.
[26]李明星.参术汤治疗支气管哮喘缓解期45例临床研究[J]..吉林中医药, 2006, 26 (3): 19-21.
[27]朱永忠,郑开明,陈炼炼等.加减阳和汤治疗寒性哮喘30例[J].陕西中医, 2000, 21 (10): 449.
[28]杨宝峰.药理学[M].北京:人民卫生出版社, 2004.
[29] Ahlquist, Rl. A study of adrenotropic receptors [J].Am J physiol, 1984, 153 (2): 586-600.
[30] Lands AM, Arnold A, McAuliff JP, et al. Differentiation of receptor systems activated by sympathomimetic amines. Nature, 1967, 214: 597-598.
[31] Wallukat G. The beta-adrenergic receptors [J]. Herz, 2002, 27(7): 683-690.
[32] Johnson M. Beta2-adrenceptors: mechanisms of action of beta2-agonists [J].Paediatr Respir Rev. 2001, 2 (1): 57-62.
[33]郑虎.药物化学[M].北京:人民卫生出版社, 2004.
[34]余健民,熊永华,胡建新.β2受体激动剂的新进展和临床评价[J].药品评价. 2005, 2 (5): 327-334.
[35]林材元.β2受体激动剂进展与应用[J].实用医学杂志, 2001, 17 (6): 476-477.
[36]黄绍光.长效β肾上腺素受体激动剂的临床应用[J].国外医药, 2000, 21 (4): 204-206.
[37]赵海燕,潘莉,冀蕾,等.抗哮喘药物β2受体激动剂的研究进展[J].中国药物化学杂志, 2004, 14(3): 187-192.
[38]夏锡荣.班布特罗-新一代长效支气管扩张剂[J].国外医学:呼吸系统分册, 1996, 19 (3): 161-164.
[39]包雅琳.长效抗哮喘药沙美特罗和福莫特罗的研究进展[J].国外医学:药学分册, 1997, 24 (5): 282-286.
[40] Zrinka K, Anita B, Goran S, et al. Exploring the active sites of cholinesterases by inhibition with bambuterol and haloxon [J]. Croat Chem Acta, 2003, 76 (1): 63-67.
[41]张洪泉,孙云,刘晓梅.国产与进口班布特罗对豚鼠实验性哮喘的药效学比较[J].中国新药与临床杂志, 2002, 21 (2): 79-80.
[42] D'Alonzo GE, Smolensky MH, Feldman S, et al. Bambuterol in the treatment of asthma. A placebo-controlled comparison of once-daily morning vs evening administration [J]. Chest, 1995, 107 (2): 406-412.
[43]潘莉,程卯生,冀蕾,等.新型苯乙胺类β2-肾上腺素受体兴奋剂的研究.中国, 01128234.7 [P]. 2001-09-30
[44]甘乐凌.新型β2受体激动剂SPFF的抗哮喘作用及机理研究[D].沈阳:沈阳药科大学, 2003.
[45] Gan LN, Wang MW, Cheng MS, et al. Trachea relaxing effects andβ2-selectivity of SPFF, a newly developed bronchodilating agent, in guinea pigs and rabbits [J]. Bio Pharm Bull, 2003, 26 (3):323-328.
[46] Gan LN, Wang C, Wang MW, et al. Effect of 2-(4-amino-3-chloro-5-tri-fluomethyl- phenyl)-2-tert-butylaminoethanol hydrochloride on airway smooth muscle [J]. Chin J New Drugs, 2003, 12(12):1007-1011.
[47] Gan LN, Wang C, Wang MW, et al. Bronchodilation effect of novelβ2 agonist SPFF in guinea pigs [J]. J Shenyang Pharm Univ, 2003, 20(5):367-369.
[48]杨威,甘乐宁,王敏伟,等.盐酸川丁特罗遗传毒性研究[J].中国新药杂志, 2006, 15 (1): 22-25.
[49]薛梅妍,王东凯,纪标,等.盐酸川丁特罗片的制备及其处方优化[J].中国药学杂志, 2006, 41 (19): 1483-1485.
[50]刘伟,刘伶.高效液相色谱法测定硫酸沙丁胺醇胶囊的含量[J].中国药业, 2006, 15 (14): 26.
[51]周天舒,施国跃,吴芳,等.毛细管电泳安培法测定班布特罗及其代谢物[J].华东师范大学学报(自然科学版), 2006, 11(6): 47-52.
[52] Sirichai S, Khanatharana P. Rapid analysis of clenbuterol, salbutamol, procaterol, and fenoterol in pharmaceuticals and human urine by capillary electrophoresis [J]. Talanta, 2008, 15, 76 (5): 1194-1198.
[53]孙春华,刘蕾,殷琦. HPLC/MS研究国产盐酸班布特罗片及其代谢物特布他林人体生物等效性[J].药学学报, 2001, 36(5): 368-372.
[54] Lee MS, Kerns EH. LC/MS applications in drug development [J]. Mass spectrometry reviews, 1999, 18 (3-4): 187-279.
[55]陈笑艳,徐海燕,钟大放,等.液相色谱-电喷雾串联质谱法测定人血浆中班布特罗:在药代动力学研究中的应用[J].药学学报, 2001, 36 (10): 762-765.
[56]徐友宣,秦旸,刘欣,等.小鼠尿中沙美特罗及其代谢物的LC/MS研究[J].质谱学报, 2003, 24 (8): 417-420.
[57]冯飞飞,张典瑞.口服药物吸收模型的研究进展[J].中国生化药物杂志, 2009, 30 (5): 351-353.
[58]陈伟薇,李俊,宋珏.药物肠吸收的研究方法进展[J].安徽医药, 2009, 13 (4): 349-352.
[59] Poelma FG, Tukker JJ. Evaluation of a chronically isolated internal loop in the rat for the study of drug absorption kinetics [J]. J Pharm Sci. 1987, 76 (6): 433-436.
[60] Braun A, H?mmerle S, Suda K, et al. Cell cultures as tools in biopharmacy [J]. Eur J Pharm Sci. 2000, 11(2): S51-60.
[61] Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability [J]. Gastroenterology, 1989,96 (3): 736-749.
[62] Troutman MD, Thakker DR. Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium [J]. Pharm Res, 2003, 20(8): 1210-1224.
[63] Soldner A, Benet LZ, Mutschler E, et al. Active transport of the angiotensin-II antagonist losartan and its main metabolite EXP 3174 across MDCK2MDR1 and caco-2 cell monolayers [ J ]. Br J Pharmacol, 2000, 129(6): 1235-1243.
[64]钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题[J].药物分析杂志, 1996, 16 (5): 343-346.
[65]卫生部药典委员会.中华人民共和国药典[R].二部. 2005年版.北京:化学工业出版社, 2005.附录173.
[66] Loo JC, Beaulieu N, Jordan N, et al. A specific radio-immunoassay (RIA) for salbutamol (albuterol) in human plasma [J]. Res Commun Chem Pathol Pharmacol. 1987, 55 (2): 283-286.
[67] Shen S, Ouyang J, Baeyens WR, et al. Determination ofβ2-agonists by ion chromatography with direct conductivity detection [J]. J Pharm Biomed Anal. 2005, 38 (1): 166-172.
[68] Wood SG, Chasseaud LF. Determination of tulobuterol in human plasma by capillary gas chromatography [J]. J Chromatogr. 1987, 416 (1): 154-159.
[69] Thienpont LM, Verhaeghe PG, De Leenheer AP. Measurement of tulobuterol in human plasma by capillary gas chromatography and selected ion monitoring detection [J]. Biomed Environ Mass Spectrom. 1987, 14 (11): 613-616.
[70] Spisso BF, Lopes CC, Marques MA, et al. Determination ofβ2-agonists in bovine urine: comparison of two extraction/clean-up procedures for high-resolution gas chromatography-mass spectrometry analysis [J]. J Anal Toxicol, 2000, 24 (2) :146-152.
[71] De Wasch K, De Brabander H, Courtheyn D. LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver [J]. Analyst, 1998, 123 (12): 2701-2705.
[72] Xu F, Zhang Z, Tian Y, et al. High-performance liquid chromatography electrospray ionization mass spectrometry determination of tulobuterol in rabbit'splasma [J]. J Pharm Biomed Anal, 2005, 37 (1): 187-193.
[73] Yang J, Guan J, Pan L, et al. Enantioseparation and impurity determination of the enantiomers of novel phenylethanolamine derivatives by high performance liquid chromatography on amylose stationary phase [J]. Anal Chim Acta, 2008, 610 (2): 263-267.
[74] Zhang J, Xu Y, Di X, et al. Quantitation of salbutamol in human urine by liquid chromatography-electrospray ionization mass spectrometry [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2006, 831 (2): 328-332.
[75]王英武,顾景凯,王玲,等.电喷雾电离-四极杆飞行时间质谱法研究三种β2-受体激动剂的质谱裂解特征[J].质谱学报, 2004, 25 (2): 69-72.
[76] SFDA.化学药物临床药代动力学研究技术指导原则[R], 2005.
[77]崔一民,赵侠,孙培红,等.单次口服盐酸川丁特罗片在健康人体的耐受性和药代动力学[J].中国临床药理学杂志, 2008, 24 (1): 25-28.
[78]赵侠,孙培红,周颖,等.进食对健康受试者口服盐酸川丁特罗片剂药代动力学的影响[J].中国临床药理学杂志, 2008, 24 (3): 204-206.
[79] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent [J]. J Bio Chem. 1951, 193 (1): 265-275.
[80] Omura T, Sato R. The carbon Monoxide-binding pigment of liver microsomes. I. evidence for its hemoprotein nature [J]. J Bio Chem. 1964, 239: 2370-2378.
[81] Lepper ER, Baker SD, Permenter M, et al. Effect of common CYP3A4 and CYP3A5 variants on the pharmacokinetics of the cytochrome P450 3A phenotyping probe midazolam in cancer patients [J]. Clin Cancer Res. 2005, 11 (20): 7398-7404.
[82] Piver B, Fer M, Vitrac X, et al. Involvement of cytochrome P450 1A2 in the biotransformation of trans-resveratrol in human liver microsomes [J]. Biochem Pharmacol., 2004, 68 (4): 773-782.
[83] Dorado P, Berecz R, Caceres MC, et al. Determination of debrisoquine and 4-hydroxydebrisoquine by high-performance liquid chromatography: application to the evaluation of CYP2D6 genotype and debrisoquine metabolic ratio relationship [J]. Clin Chem Lab Med, 2005, 43 (3): 275-279.
[84] Bu HZ, Magis L, Knuth K, et al.. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. II. Validation of a directinjection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2D6 inhibition assessment [J]. J.Chromatogr. B. 2001, 753 (2): 321-326.
[85] Testino SA Jr, Patonay G. High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography- tandem mass spectrometry [J]. J Pharm Biomed Anal. 2003, 30 (5): 1459-1467.
[86] Bu HZ, Knuth K, Magis L, et al. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: III. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2C19 inhibition evaluation [J ]. J Pharm Biomed Anal. 2001, 25 (4): 437-442.
[87] Maliakal PP, Coville PF, Wanwimolruk S. Tea consumption modulates hepatic drug metabolizing enzymes in Wistar rats [J]. J Pharm Pharmacol, 2001, 53(4): 569-577.
[88]刘明远.去甲基文拉法辛前体药物和比索洛尔的药物动力学研究[D].长春:吉林大学, 2007.
[89] FDA. Guidance for Industry Bioanalytical Method Validation [R], 2001.
[90]冷欣夫.细胞色素P450酶系的结构、功能与应用前景[M].北京:科学出版社, 2001.
[91]朱大岭,韩维娜,张荣.细胞色素P450酶系在药物代谢中的作用[J].医药导报, 2004, 23 (7): 440-443.
[92]王广基.药物代谢动力学[M].北京:化学工业出版社. 2005.
[93] Danielson P B. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans [J]. Curr Drug Metab. 2002, 3 (6): 561-597.
[94] Zakia B. Role of cytochrome P450 in drug interactions [J]. Nutrition&Metabolism , 2008, 5: 27.
[95] Bock KW, Lipp HP, Bock HBS. Induction of drug- metabolizing enzymes by xenobiotics [J]. Xenobiotica, 1990, 20 (1): 1101-1111.
[96] Netter KJ. Mechanisms of monooxygenase induction and inhibition [J]. Pharmacol Ther, 1987, 33(1):1-9.
[97] Lin JH, Lu AY. Inhibition and induction of cytochrome P450 and the clinical implications [J]. Clin Pharmacokinet, 1998, 35(5):361-390.
[98]柳晓泉,王广基,钱之玉.细胞色素P450酶在药物代谢及开发研究中的应用[J].药学进展, 2000, 24 (6): 334-338.
[99] Miehalets EL.Update: clinically significant cytochrome P450 drug interactions [J].Pharmacotherapy, 1998, 18 (l):84-112.
[100]欧阳松应,杨冬,欧阳红生,等.实时荧光定量PCR技术及其应用[J].生命的化学, 2004, 24 (1): 74-76.
[101] Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression [J]. Methods. 2001 25 (4): 386-401.
[102] Arya M, Shergill IS, Williamson M, et al. Basic principles of real-time quantitative PCR [J]. Expert Rev Mol Diagn. 2005, 5 (2): 209-219.
[103] Hernandez M, Esteve T, Prat S, et al. Development of real-time PCR systems based on SYBR? Green I, Amplifluor? and TaqMan? technologies for specific quantitative detection of the transgenic maize event GA21 [J] J Cereal Sci, 2004, 39 (1): 99-107.
[104] Simpson DA, Feeney S, Boyle C, Stitt AW. Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR [J]. Mol Vis, 2000, 5-6: 178-183.
[105] Helps C, Lait P, Tasker S, et al. Melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription PCR [J]. J Virol Methods. 2002, 106 (2): 241-244.
[106] Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays [J]. J Mol Endocrinol. 2000, 25 (2): 169-193.
[107] Watzinger F, Lion T. Multiplex PCR for quality control of template RNA/cDNA in RT-PCR assays [J]. Leukemia, 1998, 12 (12): 1984-1986.
[108] UllmannováV, Haskovec C. The use of housekeeping genes (HKG) as an internal control for the detection of gene expression by quantitative real-time RT-PCR [J]. Folia Biol (Praha). 2003, 49 (6): 211-216.
[109] Wong ML, Medrano JF. Real-time PCR for mRNA quantitation [J]. Biotechniques., 2005, 39 (1): 75-85.
[110] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods. 2001, 25 (4): 402-408
[111] Rendic S, Di Garlo FJ. Human cytochrome P450 enzymes: A status reportsummarizing their reactions, substrates, inducers, and inhibitors [J]. Drug Metab Rev, 1997, 29 (1): 413-580.
[112] Kellermann G, Shaw CR, Luyten-Kellerman M. Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma [J]. N Engl J Med, 1973, 289(18):934-937.
[113] Kouri RE, McKinney CE, Slomiany DJ, et al. Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes [J]. Cancer Res, 1982, 42(12):5030-5037.
[114]伍忠銮,谢红光,周宏灏.细胞色素P450 2E1的研究进展[J].中国临床药理学杂志, 1997, 13 (1): 57-62.
[115]刘晨晖,乐江.细胞色素P450 CYP2E1酶构型特征及其表达调控机制的研究进展[J].中国药理学与毒理学杂志, 2010, 24 (2): 155-160.
[116]许钟,谢斌,吴小翎. CYP2E1在肝细胞癌中的作用研究进展[J].国际消化病杂志, 2009, 29(4):271-272.
[117]丁昭珩,赵劲秋,陈芳源.细胞色素P450酶系3A亚家族中CYP3A5的研究进展[J].诊断学理论与实践, 2007, 6 (4): 388-390.
[118]刘瑶,俞春娜,曾苏. P-糖蛋白高表达马丁达比狗肾上皮细胞系的建立.中国药学杂志, 2009, 44 (21): 1608-1613.
[119] Karyekar CS, Eddington ND, Garimella TS, et al. Evaluation of P-glycoprotein-mediated renal drug interactions in an MDR1-MDCK model [J]. Pharmocotherapy, 2003, 23 (4): 436-442.
[120] Agarwal S, Jain R, Pal D, et al. Functional characterization of peptide transporters in MDCKII-MDR1 cell line as a model for oral absorption studies [J]. Int J Pharm. 2007, 332 (1-2): 147-152.
[121]刘瑶,曾苏. MDCK-MDR1细胞模型及其在药物透过研究中的应用进展.药学学报, 2008, 43 (6): 559-564.
[122] Simons K, Ikonen E. Functional rafts in cell membranes [J]. Nature, 1997, 387 (6633): 569-572.
[123] Luker GD, Piea CM, Kumar AS, et al. Effects of cholesterol and enantiomeric cholesterol on P-glycoprotein localization and function in low density membrane domains [J]. Biochemistry, 2000, :39 (29): 8692.
[124] Tang F, Horie K, Borchardt RT, et al. Are MDCK cells transfected with the human MDRI gene a good model of the human intestinal mucosa [J]. Pharm Res. 2002, 19 (6):765-772.
[125] Artursson P, Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells [J]. Biochem Biophys Res Commun, 1991, 175 (3): 880-885.
[126]杨海涛,王广基. Caco-2单层细胞模型及其在药学中的应用[J].药学学报, 2000, 35 (10): 797-800.
[127] Artursson P, Borchardt RT. Intestinal drug absorption and metabolism in cell cultures: Caco-2 and beyond [J]. Pharm Res, 1997, 14(12):1 655-1658.
[128] Liang E, Chessic K, Yazdanian M. Evaluation of an accelerated Caco-2 cell permeability model [J]. J Pharm Sci, 2000, 89 (3): 336-345.
[129] Yee S. In vitro permeability across Caco-2 cells (clonic) can predict in vivo (small intestinal), absorption in man-fact or myth [J]. Pharm Res, 1997, 14 (6): 763-736.
[130]赵静,梁爱华. Caco-2细胞模型及其在中药吸收转运研究中的应用[J].中国实验方剂学杂志, 2009, 15 (5): 79-83.
[131]胡晓渝,姚彤炜,曾苏. Caco-2细胞系及其在药物吸收、代谢中的应用[J].中国现代应用药学杂志, 2002, 19 (2 ): 88-90.
[2] National institute of health. Global Initiative for Asthma:global strategy for asthma management and prevention. 1995
[3] Yong RP. Confirmation of genetic linkage between atopic IgE response and chromosome 11q13 [J]. J.Med. Genetic. 1992, 29: 236-239.
[4] Gao P, Mao XQ, Baldini M. et al. Serum total IgE levels and CD14 on chromosome 5q31 [J]. Clin Genet. 1999, 56 (2): 164-165.
[5] Manian P. Genetics of asthma [J].Chest, 1997, 112: 1397-1408.
[6]谢敏强.药理学前沿-哮喘和COPD的新概念及新药[M].北京:科学出版社, 2003.
[7]钟南山.支气管哮喘防治研究进展[J].国外医学内科学分册, 1993, 20 (5): 194-197.
[8]高海鹏.气道高反应性的机理及影响因素[J].国外医学呼吸系统分册, 1990, 10(1): 1-4.
[9]胡亚美.诸福棠实用儿科学[M].北京:人民卫生出版社, 2002.
[10]钟南山,徐军.哮喘发病机制及诊断进展[J].中华结核和呼吸杂志, 1995, 18 (3): 136-139.
[11]李明华,董竞成.支气管哮喘的现代治疗[M].北京:北京医科大学中国协和医科大学联合出版社, 1996.
[12]魏尔清,卞如濂.平喘药研究现状[J].新药与临床, 1999, 14(1): 34-38.
[13]潘华杰.支气管哮喘药物的治疗研究进展[J].中外医疗,2009, 35: 164-165.
[14] Haahtela T, Jarvinen M, Kava T, et al. Effect of reducing or discontinuing inhaled budesonide in patients with mild asthma [J]. Nengl J Med, 1994, 331(11): 700-705.
[15] Agertft L, Pedersen S. Effects of long-term treatment with an inhaled corticosteroid on growth and pulomonary function in asthmatic children [J]. Respir Med, 1994, 88 (5): 373-381.
[16] O’Byrne PM. Pharmacologic interventions to reduce the risk of asthmaexacerbations [J]. Proc Am Thorac Soc, 2004, 1 (1): 105- 108.
[17]王长征.支气管哮喘治疗新药的研究进展[J].中华哮喘杂志, 2008, 2 (2): 137-140.
[18] Humbert M. Ciclesonide: a novel inhaled corticosteroid [J]. Expert Opin Investig Drugs, 2004, 13(10): 1349-1360.
[19]谢灿茂.吞咽疾病与哮喘及慢性阻塞性疾病[J].新医学, 1997, 28 (11): 616-617.
[20]王山泽,邓琨.组胺受体与支气管哮喘[J].国外医学呼吸系统分册, 1990, 10 (2): 113.
[21] Dunn CJ, Goa KL, Zafirlukast: an update of its pharmacology and therapeutic efficacy in asthma [J].. Drugs, 2001, 61 (2): 285-315.
[22] Gomez FP, Roca J, Barbera JA, et al. Effect of a platelet-activating factor (PAF) antagonist, SR 27417, on PAF-induced gas exchange abnormalities in mild asthma [J]. Eur Respir J, 1998, 11 (4): 835-839.
[23] Connor BJ, Tows LJ, Barnes PJ. Prolonged effect of no bromide on methacoline-induced brochoconstriction in asthma [J]. Am J Respir Crit Care Med, 1996, 154 (4 Pt 1): 876-880.
[24]宋勉,王亚梅.肺肠同治法在治疗支气管哮喘急性发作期的运用[J].光明中医, 2005, 20 (1): 5-6.
[25]李兰铮,杨志文,单丽囡.加味小青龙汤治疗支气管哮喘急性发作期30例[J].陕西中医, 2005, 26 (8): 740-742.
[26]李明星.参术汤治疗支气管哮喘缓解期45例临床研究[J]..吉林中医药, 2006, 26 (3): 19-21.
[27]朱永忠,郑开明,陈炼炼等.加减阳和汤治疗寒性哮喘30例[J].陕西中医, 2000, 21 (10): 449.
[28]杨宝峰.药理学[M].北京:人民卫生出版社, 2004.
[29] Ahlquist, Rl. A study of adrenotropic receptors [J].Am J physiol, 1984, 153 (2): 586-600.
[30] Lands AM, Arnold A, McAuliff JP, et al. Differentiation of receptor systems activated by sympathomimetic amines. Nature, 1967, 214: 597-598.
[31] Wallukat G. The beta-adrenergic receptors [J]. Herz, 2002, 27(7): 683-690.
[32] Johnson M. Beta2-adrenceptors: mechanisms of action of beta2-agonists [J].Paediatr Respir Rev. 2001, 2 (1): 57-62.
[33]郑虎.药物化学[M].北京:人民卫生出版社, 2004.
[34]余健民,熊永华,胡建新.β2受体激动剂的新进展和临床评价[J].药品评价. 2005, 2 (5): 327-334.
[35]林材元.β2受体激动剂进展与应用[J].实用医学杂志, 2001, 17 (6): 476-477.
[36]黄绍光.长效β肾上腺素受体激动剂的临床应用[J].国外医药, 2000, 21 (4): 204-206.
[37]赵海燕,潘莉,冀蕾,等.抗哮喘药物β2受体激动剂的研究进展[J].中国药物化学杂志, 2004, 14(3): 187-192.
[38]夏锡荣.班布特罗-新一代长效支气管扩张剂[J].国外医学:呼吸系统分册, 1996, 19 (3): 161-164.
[39]包雅琳.长效抗哮喘药沙美特罗和福莫特罗的研究进展[J].国外医学:药学分册, 1997, 24 (5): 282-286.
[40] Zrinka K, Anita B, Goran S, et al. Exploring the active sites of cholinesterases by inhibition with bambuterol and haloxon [J]. Croat Chem Acta, 2003, 76 (1): 63-67.
[41]张洪泉,孙云,刘晓梅.国产与进口班布特罗对豚鼠实验性哮喘的药效学比较[J].中国新药与临床杂志, 2002, 21 (2): 79-80.
[42] D'Alonzo GE, Smolensky MH, Feldman S, et al. Bambuterol in the treatment of asthma. A placebo-controlled comparison of once-daily morning vs evening administration [J]. Chest, 1995, 107 (2): 406-412.
[43]潘莉,程卯生,冀蕾,等.新型苯乙胺类β2-肾上腺素受体兴奋剂的研究.中国, 01128234.7 [P]. 2001-09-30
[44]甘乐凌.新型β2受体激动剂SPFF的抗哮喘作用及机理研究[D].沈阳:沈阳药科大学, 2003.
[45] Gan LN, Wang MW, Cheng MS, et al. Trachea relaxing effects andβ2-selectivity of SPFF, a newly developed bronchodilating agent, in guinea pigs and rabbits [J]. Bio Pharm Bull, 2003, 26 (3):323-328.
[46] Gan LN, Wang C, Wang MW, et al. Effect of 2-(4-amino-3-chloro-5-tri-fluomethyl- phenyl)-2-tert-butylaminoethanol hydrochloride on airway smooth muscle [J]. Chin J New Drugs, 2003, 12(12):1007-1011.
[47] Gan LN, Wang C, Wang MW, et al. Bronchodilation effect of novelβ2 agonist SPFF in guinea pigs [J]. J Shenyang Pharm Univ, 2003, 20(5):367-369.
[48]杨威,甘乐宁,王敏伟,等.盐酸川丁特罗遗传毒性研究[J].中国新药杂志, 2006, 15 (1): 22-25.
[49]薛梅妍,王东凯,纪标,等.盐酸川丁特罗片的制备及其处方优化[J].中国药学杂志, 2006, 41 (19): 1483-1485.
[50]刘伟,刘伶.高效液相色谱法测定硫酸沙丁胺醇胶囊的含量[J].中国药业, 2006, 15 (14): 26.
[51]周天舒,施国跃,吴芳,等.毛细管电泳安培法测定班布特罗及其代谢物[J].华东师范大学学报(自然科学版), 2006, 11(6): 47-52.
[52] Sirichai S, Khanatharana P. Rapid analysis of clenbuterol, salbutamol, procaterol, and fenoterol in pharmaceuticals and human urine by capillary electrophoresis [J]. Talanta, 2008, 15, 76 (5): 1194-1198.
[53]孙春华,刘蕾,殷琦. HPLC/MS研究国产盐酸班布特罗片及其代谢物特布他林人体生物等效性[J].药学学报, 2001, 36(5): 368-372.
[54] Lee MS, Kerns EH. LC/MS applications in drug development [J]. Mass spectrometry reviews, 1999, 18 (3-4): 187-279.
[55]陈笑艳,徐海燕,钟大放,等.液相色谱-电喷雾串联质谱法测定人血浆中班布特罗:在药代动力学研究中的应用[J].药学学报, 2001, 36 (10): 762-765.
[56]徐友宣,秦旸,刘欣,等.小鼠尿中沙美特罗及其代谢物的LC/MS研究[J].质谱学报, 2003, 24 (8): 417-420.
[57]冯飞飞,张典瑞.口服药物吸收模型的研究进展[J].中国生化药物杂志, 2009, 30 (5): 351-353.
[58]陈伟薇,李俊,宋珏.药物肠吸收的研究方法进展[J].安徽医药, 2009, 13 (4): 349-352.
[59] Poelma FG, Tukker JJ. Evaluation of a chronically isolated internal loop in the rat for the study of drug absorption kinetics [J]. J Pharm Sci. 1987, 76 (6): 433-436.
[60] Braun A, H?mmerle S, Suda K, et al. Cell cultures as tools in biopharmacy [J]. Eur J Pharm Sci. 2000, 11(2): S51-60.
[61] Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelialpermeability [J]. Gastroenterology, 1989,96 (3): 736-749.
[62] Troutman MD, Thakker DR. Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium [J]. Pharm Res, 2003, 20(8): 1210-1224.
[63] Soldner A, Benet LZ, Mutschler E, et al. Active transport of the angiotensin-II antagonist losartan and its main metabolite EXP 3174 across MDCK2MDR1 and caco-2 cell monolayers [ J ]. Br J Pharmacol, 2000, 129(6): 1235-1243.
[64]钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题[J].药物分析杂志, 1996, 16 (5): 343-346.
[65]卫生部药典委员会.中华人民共和国药典[R].二部. 2005年版.北京:化学工业出版社, 2005.附录173.
[66] Loo JC, Beaulieu N, Jordan N, et al. A specific radio-immunoassay (RIA) for salbutamol (albuterol) in human plasma [J]. Res Commun Chem Pathol Pharmacol. 1987, 55 (2): 283-286.
[67] Shen S, Ouyang J, Baeyens WR, et al. Determination ofβ2-agonists by ion chromatography with direct conductivity detection [J]. J Pharm Biomed Anal. 2005, 38 (1): 166-172.
[68] Wood SG, Chasseaud LF. Determination of tulobuterol in human plasma by capillary gas chromatography [J]. J Chromatogr. 1987, 416 (1): 154-159.
[69] Thienpont LM, Verhaeghe PG, De Leenheer AP. Measurement of tulobuterol in human plasma by capillary gas chromatography and selected ion monitoring detection [J]. Biomed Environ Mass Spectrom. 1987, 14 (11): 613-616.
[70] Spisso BF, Lopes CC, Marques MA, et al. Determination ofβ2-agonists in bovine urine: comparison of two extraction/clean-up procedures for high-resolution gas chromatography-mass spectrometry analysis [J]. J Anal Toxicol, 2000, 24 (2) :146-152.
[71] De Wasch K, De Brabander H, Courtheyn D. LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver [J]. Analyst, 1998, 123 (12): 2701-2705.
[72] Xu F, Zhang Z, Tian Y, et al. High-performance liquid chromatography electrospray ionization mass spectrometry determination of tulobuterol in rabbit'splasma [J]. J Pharm Biomed Anal, 2005, 37 (1): 187-193.
[73] Yang J, Guan J, Pan L, et al. Enantioseparation and impurity determination of the enantiomers of novel phenylethanolamine derivatives by high performance liquid chromatography on amylose stationary phase [J]. Anal Chim Acta, 2008, 610 (2): 263-267.
[74] Zhang J, Xu Y, Di X, et al. Quantitation of salbutamol in human urine by liquid chromatography-electrospray ionization mass spectrometry [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2006, 831 (2): 328-332.
[75]王英武,顾景凯,王玲,等.电喷雾电离-四极杆飞行时间质谱法研究三种β2-受体激动剂的质谱裂解特征[J].质谱学报, 2004, 25 (2): 69-72.
[76] SFDA.化学药物临床药代动力学研究技术指导原则[R], 2005.
[77]崔一民,赵侠,孙培红,等.单次口服盐酸川丁特罗片在健康人体的耐受性和药代动力学[J].中国临床药理学杂志, 2008, 24 (1): 25-28.
[78]赵侠,孙培红,周颖,等.进食对健康受试者口服盐酸川丁特罗片剂药代动力学的影响[J].中国临床药理学杂志, 2008, 24 (3): 204-206.
[79] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent [J]. J Bio Chem. 1951, 193 (1): 265-275.
[80] Omura T, Sato R. The carbon Monoxide-binding pigment of liver microsomes. I. evidence for its hemoprotein nature [J]. J Bio Chem. 1964, 239: 2370-2378.
[81] Lepper ER, Baker SD, Permenter M, et al. Effect of common CYP3A4 and CYP3A5 variants on the pharmacokinetics of the cytochrome P450 3A phenotyping probe midazolam in cancer patients [J]. Clin Cancer Res. 2005, 11 (20): 7398-7404.
[82] Piver B, Fer M, Vitrac X, et al. Involvement of cytochrome P450 1A2 in the biotransformation of trans-resveratrol in human liver microsomes [J]. Biochem Pharmacol., 2004, 68 (4): 773-782.
[83] Dorado P, Berecz R, Caceres MC, et al. Determination of debrisoquine and 4-hydroxydebrisoquine by high-performance liquid chromatography: application to the evaluation of CYP2D6 genotype and debrisoquine metabolic ratio relationship [J]. Clin Chem Lab Med, 2005, 43 (3): 275-279.
[84] Bu HZ, Magis L, Knuth K, et al.. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. II. Validation of a directinjection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2D6 inhibition assessment [J]. J.Chromatogr. B. 2001, 753 (2): 321-326.
[85] Testino SA Jr, Patonay G. High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography- tandem mass spectrometry [J]. J Pharm Biomed Anal. 2003, 30 (5): 1459-1467.
[86] Bu HZ, Knuth K, Magis L, et al. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: III. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2C19 inhibition evaluation [J ]. J Pharm Biomed Anal. 2001, 25 (4): 437-442.
[87] Maliakal PP, Coville PF, Wanwimolruk S. Tea consumption modulates hepatic drug metabolizing enzymes in Wistar rats [J]. J Pharm Pharmacol, 2001, 53(4): 569-577.
[88]刘明远.去甲基文拉法辛前体药物和比索洛尔的药物动力学研究[D].长春:吉林大学, 2007.
[89] FDA. Guidance for Industry Bioanalytical Method Validation [R], 2001.
[90]冷欣夫.细胞色素P450酶系的结构、功能与应用前景[M].北京:科学出版社, 2001.
[91]朱大岭,韩维娜,张荣.细胞色素P450酶系在药物代谢中的作用[J].医药导报, 2004, 23 (7): 440-443.
[92]王广基.药物代谢动力学[M].北京:化学工业出版社. 2005.
[93] Danielson P B. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans [J]. Curr Drug Metab. 2002, 3 (6): 561-597.
[94] Zakia B. Role of cytochrome P450 in drug interactions [J]. Nutrition&Metabolism , 2008, 5: 27.
[95] Bock KW, Lipp HP, Bock HBS. Induction of drug- metabolizing enzymes by xenobiotics [J]. Xenobiotica, 1990, 20 (1): 1101-1111.
[96] Netter KJ. Mechanisms of monooxygenase induction and inhibition [J]. Pharmacol Ther, 1987, 33(1):1-9.
[97] Lin JH, Lu AY. Inhibition and induction of cytochrome P450 and the clinical implications [J]. Clin Pharmacokinet, 1998, 35(5):361-390.
[98]柳晓泉,王广基,钱之玉.细胞色素P450酶在药物代谢及开发研究中的应用[J].药学进展, 2000, 24 (6): 334-338.
[99] Miehalets EL.Update: clinically significant cytochrome P450 drug interactions [J].Pharmacotherapy, 1998, 18 (l):84-112.
[100]欧阳松应,杨冬,欧阳红生,等.实时荧光定量PCR技术及其应用[J].生命的化学, 2004, 24 (1): 74-76.
[101] Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression [J]. Methods. 2001 25 (4): 386-401.
[102] Arya M, Shergill IS, Williamson M, et al. Basic principles of real-time quantitative PCR [J]. Expert Rev Mol Diagn. 2005, 5 (2): 209-219.
[103] Hernandez M, Esteve T, Prat S, et al. Development of real-time PCR systems based on SYBR? Green I, Amplifluor? and TaqMan? technologies for specific quantitative detection of the transgenic maize event GA21 [J] J Cereal Sci, 2004, 39 (1): 99-107.
[104] Simpson DA, Feeney S, Boyle C, Stitt AW. Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR [J]. Mol Vis, 2000, 5-6: 178-183.
[105] Helps C, Lait P, Tasker S, et al. Melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription PCR [J]. J Virol Methods. 2002, 106 (2): 241-244.
[106] Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays [J]. J Mol Endocrinol. 2000, 25 (2): 169-193.
[107] Watzinger F, Lion T. Multiplex PCR for quality control of template RNA/cDNA in RT-PCR assays [J]. Leukemia, 1998, 12 (12): 1984-1986.
[108] UllmannováV, Haskovec C. The use of housekeeping genes (HKG) as an internal control for the detection of gene expression by quantitative real-time RT-PCR [J]. Folia Biol (Praha). 2003, 49 (6): 211-216.
[109] Wong ML, Medrano JF. Real-time PCR for mRNA quantitation [J]. Biotechniques., 2005, 39 (1): 75-85.
[110] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods. 2001, 25 (4): 402-408
[111] Rendic S, Di Garlo FJ. Human cytochrome P450 enzymes: A status reportsummarizing their reactions, substrates, inducers, and inhibitors [J]. Drug Metab Rev, 1997, 29 (1): 413-580.
[112] Kellermann G, Shaw CR, Luyten-Kellerman M. Aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma [J]. N Engl J Med, 1973, 289(18):934-937.
[113] Kouri RE, McKinney CE, Slomiany DJ, et al. Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes [J]. Cancer Res, 1982, 42(12):5030-5037.
[114]伍忠銮,谢红光,周宏灏.细胞色素P450 2E1的研究进展[J].中国临床药理学杂志, 1997, 13 (1): 57-62.
[115]刘晨晖,乐江.细胞色素P450 CYP2E1酶构型特征及其表达调控机制的研究进展[J].中国药理学与毒理学杂志, 2010, 24 (2): 155-160.
[116]许钟,谢斌,吴小翎. CYP2E1在肝细胞癌中的作用研究进展[J].国际消化病杂志, 2009, 29(4):271-272.
[117]丁昭珩,赵劲秋,陈芳源.细胞色素P450酶系3A亚家族中CYP3A5的研究进展[J].诊断学理论与实践, 2007, 6 (4): 388-390.
[118]刘瑶,俞春娜,曾苏. P-糖蛋白高表达马丁达比狗肾上皮细胞系的建立.中国药学杂志, 2009, 44 (21): 1608-1613.
[119] Karyekar CS, Eddington ND, Garimella TS, et al. Evaluation of P-glycoprotein-mediated renal drug interactions in an MDR1-MDCK model [J]. Pharmocotherapy, 2003, 23 (4): 436-442.
[120] Agarwal S, Jain R, Pal D, et al. Functional characterization of peptide transporters in MDCKII-MDR1 cell line as a model for oral absorption studies [J]. Int J Pharm. 2007, 332 (1-2): 147-152.
[121]刘瑶,曾苏. MDCK-MDR1细胞模型及其在药物透过研究中的应用进展.药学学报, 2008, 43 (6): 559-564.
[122] Simons K, Ikonen E. Functional rafts in cell membranes [J]. Nature, 1997, 387 (6633): 569-572.
[123] Luker GD, Piea CM, Kumar AS, et al. Effects of cholesterol and enantiomeric cholesterol on P-glycoprotein localization and function in low density membrane domains [J]. Biochemistry, 2000, :39 (29): 8692.
[124] Tang F, Horie K, Borchardt RT, et al. Are MDCK cells transfected with the human MDRI gene a good model of the human intestinal mucosa [J]. Pharm Res. 2002, 19 (6):765-772.
[125] Artursson P, Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells [J]. Biochem Biophys Res Commun, 1991, 175 (3): 880-885.
[126]杨海涛,王广基. Caco-2单层细胞模型及其在药学中的应用[J].药学学报, 2000, 35 (10): 797-800.
[127] Artursson P, Borchardt RT. Intestinal drug absorption and metabolism in cell cultures: Caco-2 and beyond [J]. Pharm Res, 1997, 14(12):1 655-1658.
[128] Liang E, Chessic K, Yazdanian M. Evaluation of an accelerated Caco-2 cell permeability model [J]. J Pharm Sci, 2000, 89 (3): 336-345.
[129] Yee S. In vitro permeability across Caco-2 cells (clonic) can predict in vivo (small intestinal), absorption in man-fact or myth [J]. Pharm Res, 1997, 14 (6): 763-736.
[130]赵静,梁爱华. Caco-2细胞模型及其在中药吸收转运研究中的应用[J].中国实验方剂学杂志, 2009, 15 (5): 79-83.
[131]胡晓渝,姚彤炜,曾苏. Caco-2细胞系及其在药物吸收、代谢中的应用[J].中国现代应用药学杂志, 2002, 19 (2 ): 88-90.