【摘要】：目的：表達(dá)小型豬細(xì)胞色素CYP3A22、CYP3A29、CYP3A46和人細(xì)胞色素CYP3A4、CYP3A5重組酶,通過比較人CYP3A4、CYP3A5和小型豬CYP3A22、 CYP3A29、CYP3A46對經(jīng)典底物睪酮、咪達(dá)唑侖和硝苯地平的代謝和抑制特性,考察小型豬與人CYP3A酶的代謝相似性和差異,為小型豬作為藥物代謝動物模型提供依據(jù)。 方法：采用桿狀病毒-昆蟲細(xì)胞表達(dá)系統(tǒng)構(gòu)建人和小型豬CYP3A重組桿狀病毒；將CYP3A4、CYP3A5、CYP3A22、CYP3A29和CYP3A46重組桿狀病毒與含細(xì)胞色素氧化還原酶(POR)和細(xì)胞色素b5(Cyt b5)的重組病毒共同感染Sf9細(xì)胞,得到上述CYP3A各亞型、POR和Cytb5共表達(dá)的重組蛋白；通過體外孵育實(shí)驗(yàn)考察人和小型豬CYP3A重組酶對睪酮、咪達(dá)唑侖和硝苯地平等經(jīng)典底物的代謝情況；考察經(jīng)典抑制劑酮康唑?qū)θ伺c小型豬CYP3A酶代謝經(jīng)典底物的抑制能力。采用HPLC或HPLC-MS/MS法檢測孵育樣品中底物及其代謝物的濃度。 結(jié)果：成功表達(dá)了人CYP3A4、CYP3A5和小型豬CYP3A22、CYP3A29、CYP3A46重組酶,這些重組酶對人CYP3A經(jīng)典底物的代謝能力以及受經(jīng)典抑制劑抑制的情況均較為相似。CYP3A4、CYP3A5、CYP3A22、CYP3A29和CYP3A46催化睪酮生成6p-羥基睪酮的米氏常數(shù)Km分別為135.7±9.3、188.5±24.8、86.5±4.8、97.6+4.8和184.8±15.5μM,最大反應(yīng)速率Vmax分別為7.39±0.16、1.56±0.07、3.49±0.05、6.23±0.09和0.914±0.03nmol/min/nmol P450,內(nèi)在清除率CLint分別為54.5±2.8、8.3±0.8、40.4±1.8、63.8±2.4和4.95±0.29μL/min/nmol P450;催化咪達(dá)唑侖生成1-羥基咪達(dá)唑侖的米氏常數(shù)Km分別為3.27±0.16、6.14±0.21、5.58±0.12、16.95±0.87和25.57±1.63μM,最大反應(yīng)速率Vmax分別為1.11±0.01、2.88±0.03、0.297±0.002、0.505±0.011和0.416±0.010nmol/min/nmol P450,內(nèi)在清除率CLint分別為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數(shù)Km分別為13.59±0.90、33.26±1.63、24.21±1.22、40.33±2.02和51.88±2.25μM,最大反應(yīng)速率Vmax分別為150±0.03、2.07±0.03、0.992±0.002、1.85±0.03和1.11±0.02nmol/min/nmol P450,內(nèi)在清除率CLint分別為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催化睪酮生成6β-羥基睪酮的的IC50分別為0.1561±0.0022,1.281±0.043,0.5343±0.0087,0.7317±0.0109和0.7520±0.0510μM；抑制催化咪達(dá)唑侖生成1-羥基咪達(dá)唑侖的的IC50分別為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分別為0.2851±0.0081,2.122±0.026,0.1846±0.0031,0.2227±0.0025和0.6305±0.0117μM。 結(jié)論：構(gòu)建的重組酶可以作為小型豬與人CYP3A酶代謝特性比較的高效、快速研究模型,用于CYP3As底物和抑制劑的篩選和代謝特性研究。小型豬與人CYP3A在藥代動力學(xué)行為、代謝產(chǎn)物情況和抑制特性上存在相似性,可以作為CYP3A介導(dǎo)藥物的代謝研究動物模型。
[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 鹵9.3188.5 鹵24.8, 86.5 鹵4.8, 97.68 and 184.8 鹵15.5 渭 M, respectively. The maximum reaction rates (Vmax) were 7.39 鹵0.16,1.56 鹵0.07,3.49 鹵0.05,6.23 鹵0.09 and 0.914 鹵0.03nmol/min/nmol P450, respectively. The intrinsic clearance rates (CLint) were 54.5 鹵2.8, 8.3 鹵0.8, 40.4 鹵1.8, 63.8 鹵2.4 and 4.95 鹵0.29 渭 L / min/nmol P450, respectively. The Km of catalytic midazolam to 1-hydroxymidazolam was 3.27 鹵0.16,6.14 鹵0.21,5.58 鹵0.12,16.95 鹵0.87 and 25.57 鹵1.63 渭 M, respectively, and that of midazolam was 3.27 鹵0.16,6.14 鹵0.21, 5.58 鹵0.12,16.95 鹵0.87,25.57 鹵1.63um respectively. The maximum reaction rates (Vmax) were 1.11 鹵0.01,2.88 鹵0.03,0.297 鹵0.002,0.505 鹵0.011 and 0.416 鹵0.010nmol/min/nmol P450, respectively. The intrinsic clearance rates (CLint) were 340.5 鹵13.65469.4 鹵12.5, 53.02 鹵0.85, 29.80 鹵0.95 and 16.28 鹵0.70 渭 L / min/nmolP450;, respectively. The Km of catalytic oxidation of nifedipine to nifedipine was 13.59 鹵0.90,33.26 鹵1.63, 24.21 鹵1.22,40.33 鹵2.02 and 51.88 鹵2.25 渭 M, respectively. The maximum reaction rates (Vmax) were 150 鹵0.03,2.07 鹵0.03,0.992 鹵0.002,1.85 鹵0.03,1.11 鹵0.02nmol/min/nmol P450, and the intrinsic clearance rates (CLint) were 110.3 鹵5.9, 62.33 鹵2.24,40.96 鹵1.59, 45.81 鹵1.63 and 21.41 鹵0.63 渭 L / min/nmol P450, respectively, and the internal clearance rates were 110.3 鹵5.9, 62.33 鹵2.24,40.96 鹵1.59, 45.81 鹵1.63 and 21.41 鹵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 鹵0.0022,1.281 鹵0.0433,0.5343 鹵0.0087, 0.7317 鹵0.0109 and 0.7520 鹵0.0510 渭 M, respectively. The IC50 of 1-hydroxymidazolam was 0.1496 鹵0.0010,0.6772 鹵0.0105, 0.1224 鹵0.0039, 0.0791 鹵0.0009 and 0.0546 鹵0.0004 渭 M, respectively. The IC50 of nifedipine was 0.2851 鹵0.0081, 2.122 鹵0.026, 0.1846 鹵0.0031, 0.2227 鹵0.0025 and 0.6305 鹵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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R969.1

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