小型猪与人CYP3A重组酶的体外表达和代谢特性比较
发布时间：2019-03-18 21:29
【摘要】：目的：表达小型猪细胞色素CYP3A22、CYP3A29、CYP3A46和人细胞色素CYP3A4、CYP3A5重组酶,通过比较人CYP3A4、CYP3A5和小型猪CYP3A22、 CYP3A29、CYP3A46对经典底物睾酮、咪达唑仑和硝苯地平的代谢和抑制特性,考察小型猪与人CYP3A酶的代谢相似性和差异,为小型猪作为药物代谢动物模型提供依据。 方法：采用杆状病毒-昆虫细胞表达系统构建人和小型猪CYP3A重组杆状病毒；将CYP3A4、CYP3A5、CYP3A22、CYP3A29和CYP3A46重组杆状病毒与含细胞色素氧化还原酶(POR)和细胞色素b5(Cyt b5)的重组病毒共同感染Sf9细胞,得到上述CYP3A各亚型、POR和Cytb5共表达的重组蛋白；通过体外孵育实验考察人和小型猪CYP3A重组酶对睾酮、咪达唑仑和硝苯地平等经典底物的代谢情况；考察经典抑制剂酮康唑对人与小型猪CYP3A酶代谢经典底物的抑制能力。采用HPLC或HPLC-MS/MS法检测孵育样品中底物及其代谢物的浓度。 结果：成功表达了人CYP3A4、CYP3A5和小型猪CYP3A22、CYP3A29、CYP3A46重组酶,这些重组酶对人CYP3A经典底物的代谢能力以及受经典抑制剂抑制的情况均较为相似。CYP3A4、CYP3A5、CYP3A22、CYP3A29和CYP3A46催化睾酮生成6p-羟基睾酮的米氏常数Km分别为135.7±9.3、188.5±24.8、86.5±4.8、97.6+4.8和184.8±15.5μM,最大反应速率Vmax分别为7.39±0.16、1.56±0.07、3.49±0.05、6.23±0.09和0.914±0.03nmol/min/nmol P450,内在清除率CLint分别为54.5±2.8、8.3±0.8、40.4±1.8、63.8±2.4和4.95±0.29μL/min/nmol P450;催化咪达唑仑生成1-羟基咪达唑仑的米氏常数Km分别为3.27±0.16、6.14±0.21、5.58±0.12、16.95±0.87和25.57±1.63μM,最大反应速率Vmax分别为1.11±0.01、2.88±0.03、0.297±0.002、0.505±0.011和0.416±0.010nmol/min/nmol P450,内在清除率CLint分别为340.5±13.65、469.4±12.5、53.02±0.85、29.80±0.95和16.28±0.70μL/min/nmolP450；催化硝苯地平生成氧化硝苯地平的米氏常数Km分别为13.59±0.90、33.26±1.63、24.21±1.22、40.33±2.02和51.88±2.25μM,最大反应速率Vmax分别为150±0.03、2.07±0.03、0.992±0.002、1.85±0.03和1.11±0.02nmol/min/nmol P450,内在清除率CLint分别为110.3±5.9、62.33±2.24、40.96±1.59、45.81±1.63和21.41±0.63μL/min/nmol P450。酮康唑抑制CYP3A4、CYP3A5、CYP3A22、 CYP3A29和CYP3A46催化睾酮生成6β-羟基睾酮的的IC50分别为0.1561±0.0022,1.281±0.043,0.5343±0.0087,0.7317±0.0109和0.7520±0.0510μM；抑制催化咪达唑仑生成1-羟基咪达唑仑的的IC50分别为0.1496±0.0010、0.6772±0.0105、0.1224±0.0039、0.0791±0.0009和0.0546±0.0004μM；抑制催化硝苯地平生成氧化硝苯地平的IC50分别为0.2851±0.0081,2.122±0.026,0.1846±0.0031,0.2227±0.0025和0.6305±0.0117μM。 结论：构建的重组酶可以作为小型猪与人CYP3A酶代谢特性比较的高效、快速研究模型,用于CYP3As底物和抑制剂的筛选和代谢特性研究。小型猪与人CYP3A在药代动力学行为、代谢产物情况和抑制特性上存在相似性,可以作为CYP3A介导药物的代谢研究动物模型。
[Abstract]:Objective: to express cytochrome CYP3A22,CYP3A29,CYP3A46 and human cytochrome CYP3A4,CYP3A5 recombinant enzyme in minipigs, and to compare the metabolism and inhibition characteristics of human CYP3A4,CYP3A5 and CYP3A22,CYP3A29,CYP3A46 on the classical substrates testosterone, midazolam and nifedipine. The metabolic similarity and difference between minipigs and human CYP3A enzymes were investigated in order to provide evidence for miniature pigs as an animal model of drug metabolism. Methods: recombinant baculovirus CYP3A was constructed by baculovirus-insect cell expression system. The recombinant baculovirus CYP3A4,CYP3A5,CYP3A22,CYP3A29 and CYP3A46 were co-infected with recombinant viruses containing cytochrome redox enzyme (POR) and cytochrome b5 (Cytb5). The recombinant proteins co-expressed by CYP3A subtypes, POR and Cytb5 were obtained. The metabolism of testosterone, midazolam and nifedipine by CYP3A recombinant enzymes in human and miniature pigs was investigated in vitro, and the inhibition ability of ketoconazole on classical substrates in human and miniature pig CYP3A enzymes was investigated. The concentrations of substrates and their metabolites in incubation samples were determined by HPLC or HPLC-MS/MS. Results: the recombinant enzymes of human CYP3A4,CYP3A5 and miniature pig CYP3A22,CYP3A29,CYP3A46 were successfully expressed. The metabolic ability of these recombinant enzymes to the classical substrate of human CYP3A and inhibition by classical inhibitors were similar. CYP3A4, CYP3A5,CYP3A22, The Michaelis constants (Km) of testosterone production by CYP3A29 and CYP3A46 were 135.7 卤9.3188.5 卤24.8, 86.5 卤4.8, 97.68 and 184.8 卤15.5 渭 M, respectively. The maximum reaction rates (Vmax) were 7.39 卤0.16,1.56 卤0.07,3.49 卤0.05,6.23 卤0.09 and 0.914 卤0.03nmol/min/nmol P450, respectively. The intrinsic clearance rates (CLint) were 54.5 卤2.8, 8.3 卤0.8, 40.4 卤1.8, 63.8 卤2.4 and 4.95 卤0.29 渭 L / min/nmol P450, respectively. The Km of catalytic midazolam to 1-hydroxymidazolam was 3.27 卤0.16,6.14 卤0.21,5.58 卤0.12,16.95 卤0.87 and 25.57 卤1.63 渭 M, respectively, and that of midazolam was 3.27 卤0.16,6.14 卤0.21, 5.58 卤0.12,16.95 卤0.87,25.57 卤1.63um respectively. The maximum reaction rates (Vmax) were 1.11 卤0.01,2.88 卤0.03,0.297 卤0.002,0.505 卤0.011 and 0.416 卤0.010nmol/min/nmol P450, respectively. The intrinsic clearance rates (CLint) were 340.5 卤13.65469.4 卤12.5, 53.02 卤0.85, 29.80 卤0.95 and 16.28 卤0.70 渭 L / min/nmolP450;, respectively. The Km of catalytic oxidation of nifedipine to nifedipine was 13.59 卤0.90,33.26 卤1.63, 24.21 卤1.22,40.33 卤2.02 and 51.88 卤2.25 渭 M, respectively. The maximum reaction rates (Vmax) were 150 卤0.03,2.07 卤0.03,0.992 卤0.002,1.85 卤0.03,1.11 卤0.02nmol/min/nmol P450, and the intrinsic clearance rates (CLint) were 110.3 卤5.9, 62.33 卤2.24,40.96 卤1.59, 45.81 卤1.63 and 21.41 卤0.63 渭 L / min/nmol P450, respectively, and the internal clearance rates were 110.3 卤5.9, 62.33 卤2.24,40.96 卤1.59, 45.81 卤1.63 and 21.41 卤0.63 渭 L / min/nmol P450, respectively. The IC50 of ketoconazole inhibited testosterone production by CYP3A4,CYP3A5,CYP3A22, CYP3A29 and CYP3A46 was 0.1561 卤0.0022,1.281 卤0.0433,0.5343 卤0.0087, 0.7317 卤0.0109 and 0.7520 卤0.0510 渭 M, respectively. The IC50 of 1-hydroxymidazolam was 0.1496 卤0.0010,0.6772 卤0.0105, 0.1224 卤0.0039, 0.0791 卤0.0009 and 0.0546 卤0.0004 渭 M, respectively. The IC50 of nifedipine was 0.2851 卤0.0081, 2.122 卤0.026, 0.1846 卤0.0031, 0.2227 卤0.0025 and 0.6305 卤0.0117 渭 M, respectively. Conclusion: the recombinant enzyme can be used as an efficient and rapid model for comparing the metabolic characteristics of CYP3A enzyme between minipigs and human. The recombinant enzyme can be used to screen substrates and inhibitors of CYP3As and to study its metabolic characteristics. Small pigs and human CYP3A have similar pharmacokinetic behaviors, metabolic products and inhibitory properties. They can be used as animal models for CYP3A-mediated drug metabolism research.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969.1
本文编号：2443258
[Abstract]:Objective: to express cytochrome CYP3A22,CYP3A29,CYP3A46 and human cytochrome CYP3A4,CYP3A5 recombinant enzyme in minipigs, and to compare the metabolism and inhibition characteristics of human CYP3A4,CYP3A5 and CYP3A22,CYP3A29,CYP3A46 on the classical substrates testosterone, midazolam and nifedipine. The metabolic similarity and difference between minipigs and human CYP3A enzymes were investigated in order to provide evidence for miniature pigs as an animal model of drug metabolism. Methods: recombinant baculovirus CYP3A was constructed by baculovirus-insect cell expression system. The recombinant baculovirus CYP3A4,CYP3A5,CYP3A22,CYP3A29 and CYP3A46 were co-infected with recombinant viruses containing cytochrome redox enzyme (POR) and cytochrome b5 (Cytb5). The recombinant proteins co-expressed by CYP3A subtypes, POR and Cytb5 were obtained. The metabolism of testosterone, midazolam and nifedipine by CYP3A recombinant enzymes in human and miniature pigs was investigated in vitro, and the inhibition ability of ketoconazole on classical substrates in human and miniature pig CYP3A enzymes was investigated. The concentrations of substrates and their metabolites in incubation samples were determined by HPLC or HPLC-MS/MS. Results: the recombinant enzymes of human CYP3A4,CYP3A5 and miniature pig CYP3A22,CYP3A29,CYP3A46 were successfully expressed. The metabolic ability of these recombinant enzymes to the classical substrate of human CYP3A and inhibition by classical inhibitors were similar. CYP3A4, CYP3A5,CYP3A22, The Michaelis constants (Km) of testosterone production by CYP3A29 and CYP3A46 were 135.7 卤9.3188.5 卤24.8, 86.5 卤4.8, 97.68 and 184.8 卤15.5 渭 M, respectively. The maximum reaction rates (Vmax) were 7.39 卤0.16,1.56 卤0.07,3.49 卤0.05,6.23 卤0.09 and 0.914 卤0.03nmol/min/nmol P450, respectively. The intrinsic clearance rates (CLint) were 54.5 卤2.8, 8.3 卤0.8, 40.4 卤1.8, 63.8 卤2.4 and 4.95 卤0.29 渭 L / min/nmol P450, respectively. The Km of catalytic midazolam to 1-hydroxymidazolam was 3.27 卤0.16,6.14 卤0.21,5.58 卤0.12,16.95 卤0.87 and 25.57 卤1.63 渭 M, respectively, and that of midazolam was 3.27 卤0.16,6.14 卤0.21, 5.58 卤0.12,16.95 卤0.87,25.57 卤1.63um respectively. The maximum reaction rates (Vmax) were 1.11 卤0.01,2.88 卤0.03,0.297 卤0.002,0.505 卤0.011 and 0.416 卤0.010nmol/min/nmol P450, respectively. The intrinsic clearance rates (CLint) were 340.5 卤13.65469.4 卤12.5, 53.02 卤0.85, 29.80 卤0.95 and 16.28 卤0.70 渭 L / min/nmolP450;, respectively. The Km of catalytic oxidation of nifedipine to nifedipine was 13.59 卤0.90,33.26 卤1.63, 24.21 卤1.22,40.33 卤2.02 and 51.88 卤2.25 渭 M, respectively. The maximum reaction rates (Vmax) were 150 卤0.03,2.07 卤0.03,0.992 卤0.002,1.85 卤0.03,1.11 卤0.02nmol/min/nmol P450, and the intrinsic clearance rates (CLint) were 110.3 卤5.9, 62.33 卤2.24,40.96 卤1.59, 45.81 卤1.63 and 21.41 卤0.63 渭 L / min/nmol P450, respectively, and the internal clearance rates were 110.3 卤5.9, 62.33 卤2.24,40.96 卤1.59, 45.81 卤1.63 and 21.41 卤0.63 渭 L / min/nmol P450, respectively. The IC50 of ketoconazole inhibited testosterone production by CYP3A4,CYP3A5,CYP3A22, CYP3A29 and CYP3A46 was 0.1561 卤0.0022,1.281 卤0.0433,0.5343 卤0.0087, 0.7317 卤0.0109 and 0.7520 卤0.0510 渭 M, respectively. The IC50 of 1-hydroxymidazolam was 0.1496 卤0.0010,0.6772 卤0.0105, 0.1224 卤0.0039, 0.0791 卤0.0009 and 0.0546 卤0.0004 渭 M, respectively. The IC50 of nifedipine was 0.2851 卤0.0081, 2.122 卤0.026, 0.1846 卤0.0031, 0.2227 卤0.0025 and 0.6305 卤0.0117 渭 M, respectively. Conclusion: the recombinant enzyme can be used as an efficient and rapid model for comparing the metabolic characteristics of CYP3A enzyme between minipigs and human. The recombinant enzyme can be used to screen substrates and inhibitors of CYP3As and to study its metabolic characteristics. Small pigs and human CYP3A have similar pharmacokinetic behaviors, metabolic products and inhibitory properties. They can be used as animal models for CYP3A-mediated drug metabolism research.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969.1
【参考文献】
相关期刊论文 前1条
1 张德福,刘东;国内外小型猪实验动物化研究[J];生物学通报;2004年10期
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