稳定表达人CYP3A4基因与Bama小型猪CYP3A基因的HepG_2细胞株的建立及探针药物代谢表征的比较研究
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摘要
一、研究背景
人CYP3A在肝肠中表达最为丰富,约占CYP450总量的30%-40%,底物谱广,并可分成CYP3A4、CYP33A5、CYP33A7和CYP3A43四种亚型,其中CYP3A4在成年个体的肝和肠中表达最多,占肝CYPs的30%和肠CYPs的70%,参与了60%现有临床药物的代谢。
新药临床前评价过程中需要其代谢相关数据。为获得可反映人体药物代谢概貌的最佳实验动物模型,通过对不同种属动物肝微粒体的比较研究发现,狗适合作为CYP2D代谢途径的动物模型;灵长类是最合适的CYP2CN家族动物模型,而小型猪的CYP1A、CYP2A、CYP3A与人类极为相似,且该动物体型较小、易于操作和较小的底物需求量,是人类CYP3A药物评价的最佳模型动物。
目前研究认为小型猪CYP3A亚家族蛋白共有4种亚型,即CYP3A22、CYP3A29、CYP3A39和CYP3A88,其中CYP3A29具有典型的硝苯地平和睾酮6β-羟基化活性(人CYP3A4和3A5的特异活性),被苯巴比妥、利福平和地塞米松诱导的活性,及对酮康唑和醋竹桃霉素抑制的敏感性。因此,除灵长类和狗以外,小型猪成为唯一被FDA推荐的临床前大动物实验动物,在药物研发、生产和监管等领域越来越多地用于狗的替代模型动物。
现有的临床前药物评价的小型猪主要有:Yucatan小型猪、德国的Gottingen小型猪、Hanford小型猪和Sinclair小型猪,特别是Gottingen在欧洲的得到了很好的推广运用;Bama小型猪产于我国的广西省,为香猪的进化品系,由于近交程度高,而且耐近交、遗传稳定、个体表型一致,在生物医药领域应用中已被证明是优秀的模型动物,并积累了丰富的解剖、生理生化、基础生物学特性及活体药物代谢背景资料,但有关其药物代谢表征的分子水平研究资料缺乏;由于重组酶成分单一,其药物代谢表征明确,被广泛用于酶表征的定性定量研究,是对酶进行特征研究的最佳工具。目前有关猪(小型猪)CYP3A亚家族同工酶的重组研究,在世界范围几乎是空白,猪(小型猪)CYP3A亚家族同工酶的药物代谢表征几乎全部都是通过人CYP3A探针药物的活体动物肝微粒体代谢特征获得,由于特异性探针药物对特定CYP450的相对性,以及猪肝微粒体成分的复杂性,其可靠性远低于重组酶。为了更好阐明Bama小型猪作为人CYP3A系统动物模型的有效性和合理性,以及各亚型在探针药物代谢中的作用特征,本研究通过对人CYP3A4和小型猪CYP3A基因克隆、HepG_2稳定表达细胞株的建立、以及在微粒体和全细胞两个水平对稳定表达细胞株的CYP3A特异性底物和抑制剂的鉴定,旨在分子水平阐明Bama小型猪的药物代谢特征,从而为Bama小型猪作为临床前药物代谢模型实验动物提供实验依据。
二、研究内容和结果
1.人CYP3A4基因和Bama小型猪CYP3A基因的克隆
用总RNA经逆转录PCR获得目的基因,并将目的基因亚克隆至pMD-19-T上,测序确认获得了人CYP3A4,以及小型猪的CYP3A22、CYP3A29、CYP3A39和CYP3A88共5个基因;其中人CYP3A4、小型猪CYP3A39和CYP3A88与GeneBank中公布的序列完全一致;CYP3A29基因与NCBI记录的CYP3A29基因序列(GENE ID:403324)有2个碱基不同,即G96A、T105A,但其编码相同的CYP3A29的氨基酸酸序列(NP 999588);但获得的CYP3A22基因与GeneBank中公开的基因序列(AB006010.1)有6个碱基不同,即G 36 C、A 169 G、C 546 T、A 612 C、C1329 T和A 1351G,将该序列推定的蛋白质氨基酸序列与CYP3A22(BAD06180.1)进行比对发现有3个氨基酸差异,即W 12 C、W 57 V、N 451 D,且对来自5头小型猪的基因经过PCR扩增后测序结果完全一致,故此序列可能为Bama小型猪特有。
2.稳定表达人CYP3A4基因和Bama小型猪CYP3A基因HepG_2细胞株的建立及探针药物代谢鉴定
将克隆载体pMD-19-T上人CYP3A4和小型猪CYP3A系列的基因,在其5′和端3′插入酶切位点,并在3′插入6个组氨酸核苷酸序列,克隆至表达载体pcDNA3.1上,分别测序确认;将表达载体经脂质体转染至HepG_2细胞,经10代抗性筛选及RT-PCR、West-blot检测表明本研究成功建立了所需CYP3A基因在HepG_2中的稳定表达体系;以人CYP3A的特异性底物硝苯地平和睾酮为探针鉴定重组细胞的药物代谢活性,结果表明所得人CYP3A4和小型猪CYP3A基因HepG_2稳定表达株具有硝苯地平氧化代谢和睾酮6β-羟基化活性。
3.稳定表达人CYP3A4基因和Bama小型猪CYP3A基因HepG_2细胞微粒体对探针药物的代谢表征分析
以硝苯地平和睾酮作为探针底物,酮康唑作为抑制剂进行体外药物代谢的动力学分析,结果如下。
(1)人CYP3A4与Bama小型猪CYP3A重组细胞中微粒体对硝苯地平的体外代谢符合米氏动力学特征,其中CYP3A4、CYP3A22、CYP3A29、CYP3A39和CYP3A88的Vmax分别为1.62、1.27、1.72、1.22和1.63nmol/min/mg,Km分别为12.12、18.98、12.83、18.42和12.46μM,内在清除率(Clint=Vm/Km)分别为133.66、66.91、134.06、66.23和130.29μL/min/mg。这些数据表明,CYP3A29和CYP3A88与CYP3A4的代谢特征极为相似,而CYP3A22和CYP3A39与CYP3A4的硝苯地平代谢差异较大。
(2)重组细胞微粒体代谢睾酮符合Hill动力学特征,CYP3A4、CYP3A22、CYP3A29、CYP3A39和CYP3A88的Hill系数分别为1.14、1.61、1.34、1.62和1.35,说明本实验获得的重组CYP3A酶对睾酮的代谢均属于正协同作用;Vmax分别为7.13、4.34、6.21、4.15和6.08nmol/min/mg,S_(50)分别为41.87、73.64、50.08、69.76和50.24μM mol/L,Clint(Vm/S50)分别为172.06、58.90、124.0、59.49和121.03μL/min/mg,表明在对睾酮的6β-羟基化代谢中CYP3A4、CYP3A29和CYP3A88的酶作用效能比较接近。
(3)本实验获得酮康唑对硝苯地平代谢反应抑制的IC_(50)值,CYP3A4、CYP3A22、CYP3A29、CYP3A39分别为0.132、0.233、0.090、0.262、0.104μM;对睾酮的代谢的IC_(50)值分别为0.032、0.0688、0.0562、0.077和0.065μM,这些数据表明酮康唑不论对硝苯地平或睾酮的代谢,其对CYP3A4、CYP3A29和CYP3A88抑制活性均较近。
4.稳定表达人CYP3A4基因和Bama小型猪CYP3A基因稳定表达HepG_2全细胞对探针药物的代谢表征分析
用基因重组全细胞体外孵育的方法,比较小型猪HepG_2-CYP3A与人HepG_2-CYP3A4的硝苯地平、睾酮和酮康唑代谢表征,其结果如下。
(1)重组全细胞的硝苯地平体外孵育代谢符合米氏动力学特征,HepG_2-CYP3A4、HepG_2-CYP3A22、HepG_2-CYP3A29、HepG_2-CYP3A39和HepG_2-CYP3A88的酶动力学参数,Vmax分别为2.21、1.52、2.51、1.40和2.46nmol/min/mg,Km分别为16.77、20.51、15.47、21.93和15.56μM,其内源性清除率Clint分别为131.78、74.11、162.25、63.84和157.97μL/min/mg,这些数据表明重组全细胞的硝苯地平代谢特征与微粒体实验类似,CYP3A29和CYP3A88对硝苯地平的代谢动力学特征与CYP3A4的代谢特征近似。
(2)重组全细胞的睾酮体外孵育代谢符合Hill动力学特征,HepG_2-CYP3A4、HepG_2-CYP3A22、HepG_2-CYP3A29、HepG_2-CYP3A39和HepG_2-CYP3A88的酶动力学参数,Vmax分别为2.55、1.83、2.37、1.72和2.39nmol/min/mg,Km分别为14.14、23.66、16.88、21.48和17.81μM,内源性清除率(Clint)180.40、77.59、140.36、93.20和134.40μL/min/mg,这些数据表明HepG_2-CYP3A4与HepG_2-CYP3A29和HepG_2-CYP3A88代谢特征较为相似,而与HepG_2-CYP3A22和HepG_2-CYP3A39的差别较大。
(3)重组全细胞酮康唑对硝苯地平代谢的IC_(50)值,HepG_2-CYP3A4、HepG_2-CYP3A22、HepG_2-CYP3A29、HepG_2-CYP3A39和HepG_2-CYP3A88,分别为0.539、0.8785、0.4985、0.8787和0.556μM;对睾酮的IC_(50)值分别为0.120、0.207、0.125、0.229和0.128μM,这些数据表明酮康唑对HepG_2-CYP3A4、HepG_2-CYP3A29和HepG_2-CYP3A88的睾酮代谢的抑制均较近,且显著高于对CYP3A22和CYP3A39的抑制活性。
5.结论
通过对人CYP3A4基因和Bama小型猪CYP3A22、CYP3A29、CYP3A39和CYP3A88基因重组细胞的微粒体和全细胞的CYP3A特异性探针底物的硝苯地平和睾酮代谢活性,以及CYP3A的特异性抑制剂的体外孵育实验表明,人CYP3A4与Bama小型猪CYP3A29和CYP3A88具有较近的底物代谢表征,证实了前人提出的CYP3A29是CYP3A4在小型猪体内的同源酶的假说;同时发现CYP3A88也具有同CYP3A4较近的药物代谢表征,提示有必要进行进一步的研究,探索其内在的生物学基础。CYP3A22和CYP3A39之间具有近似的体外探针药物代谢表征,二者与CYP3A4、CYP3A29和CYP3A88之间具有较大的体外探针药物代谢表征差异。
Research background
CYP3A enzymes,which are the most abundant CYPs expressed in the liver and intestine,and accounting for 30%-40%in the total CYPs contents expressed in liver and intestine,involve in the disposition of diverse exogenous chemical substances.There are four isoenzymes in CYP3A subfamily,namely,CYP3A4,CYP3A5,CYP3A7and CYP3A43.The CYP3A4,which is expressed most abundant in liver and intestine in the adults,accounting for 30%of liver CYPs contents and 70%of intestinal CYPs contents,is involved in the metabolism of 60%of clinical drugs.
Relevant data need for their metabolism in the process of pre-clinical drug evaluation In order to obtain the best experimental animals for human drug metabolism,comparative studies of the pooled microsomes from different species of animals found that dog is a suitable model animal for CYP2D subfamily;and primate is the most suitable model animal for the complex CYP2C subfamily,which has so many isoenzymes involving in so many complicated metabolic pathways;and pig(miniature pig),owing to its very similar characteristics of drug metabolism with human,along with its small size,easy operation and a smaller substrate demand comparing with dog,is the most suitable model animal for human CYP1A,CYP2A and CYP3A subfamily.
At present,there are four isoenzymes in CYP3A subfamily of pig (miniature pig),namely CYP3A22,CYP3A29,CYP3A39 and CYP3A88. CYP3A29,the most studied isoenzymes,has typical nifedipine oxidation activity and testosterone 6β-hydroxy-activity(of the human CYP3A4 and 3A5-specific activity),phenobarbital,rifampicin and dexamethasoneinduced activity,as well as triacetyloleandomycin and ketoconazole inhibitory activity.Therefor,the pig is the only big animal recommended by FDA for the pre-clinical drug in the big animal experiments in addition to primate and dog,and is increasingly used as alternative of dog in the pre-clinical drug evaluation by more and more drug reaserch and development,pharmaceutical industry and regulatory agencies.
At present,the famous strains of miniature pig used for the pre-clinical drug evaluation are mainly Yucatan,Gottingen,Hanford, Sinclair minpig.As the evolution of strains of pigs,Bama miniature pigs have a high extent of in breeding,and stable heredity,and a consistent phenotype,can bear inbreeding also.They have been proved to be excellent model animals,because there are much background information about anatomy,physiology,biochemistry and basic biological characteristics of Bama miniature pigs.
In order to elucidate the characteristics of drug metabolism at a more essential level,the genes of CYP 3A4,CYP3A22,CYP3A29,CYP3A39 andCYP3A88 were cloned,the cloned genes were transfected into HepG_2 cells,the transfected cells were screened for positive cells by G418,the stable expression cell lines were established after ten generation subculture,at last the characteristics of probe drugs were studied in the established HepG_2-CYP3A cell lines at the microsomes and whole-cell level,the present research aims to provide experimental evidences for the Bama miniature pig used as a preclinical experimental model animal of drug metabolism.
Research methods and results
1.Cloning human CYP3A4 and CYP3A gene of Bama miniature pig
The total RNA was extracted from human liver and miniature pig respectively,the target genes obtained by RT-PCR from total RNA were subcloned into T-vector pMD-19-T,the sequencing results and sequence analysis showed that five target genes of human CYP3A4,CYP3A22, CYP3A29,CYP3A39 and CYP3A88 of Bama miniature pig were obtained. The gene sequences of CYP3A4,CYP3A39 and CYP3A88 were completely consistent with the corresponding sequence published in GeneBank.There were two base differences in presently-cloned CYP3A29 compared with CYP3A29(403324) sequence in GeneBank,namely,G96A, T105A,there was no difference comparing the deduced amino acid sequence from the present CYP3A29 with amino acid sequence of CYP3A29((NP_999588)) from the NCBI protein data bank;there were six nucleotide differences in the CYP3A22 gene obtained in the present experiment comparing with CYP3A22 gene published in GeneBank, namely,G 36 C,A 169 G,C 546 T,A 612 C,C1329 T and A 1351G,and three amino acids differences were found in the present protein sequence of CYP3A22 compared with protein sequence of CYP3A22(BAD06180.1) published in NCBI Protein DataBank, namely,W12C,W57V,N451D,because the present CYP3A22,which out of the sequenced nine positive clones from five Bama miniature pigs, showed completely consistent results in the eight of the nine clones,It suggested that the present CYP3A22 gene cloned from Bama miniature pigs was the specific gene sequence for the pigs.
2.Establishment of HepG_2 cell lines stably expressing Human CYP3A4 and CYP3A gene of Bama minipig,and identification of the metabolic characteristics of probe drugs in the established cell lines.
The genes of human CYP3A4 and CYP3A of miniature pigs were cloned into expression vector pCDNA3.1(designated as pCDNA-CYP3A) from subcloning vector pMD-19-T by PCR,then the pCDNA-CYP3As were transfected into HepG_2 cells by Lipofectamine 2000 Reagent, RT-PCR and west-blot results showed the successful establishment of stably expression human CYP3A4 and CYP3A gene of miniature pigs in HepG_2 cell lines after ten generation selection of G418.The results from identification of metabolic activity by the specific CYP3A probe drugs, namely,nifedipine and testosterone,showed that the established HepG2-CYP3A cell lines had full oxidation of nifedipine activity and testosterone 6β-hydroxylation activity.
3.The metabolic analysis of probe drugs by microsomes in HepG2-CYP3A cell lines.
Kinetic parameters,including the Km,Vmax,are the key factors to determine the characteristics of enzymatic reaction curve.In order to compare the kinetic characteristics of human CYP3A4 and CYP3A of Bama miniature pigs,the microsomes extracted from HepG_2-CYP3A cell lines were incubated with nifedipine and testosterone as a probe substrates, and ketoconazole as inhibitors in regeneration system in vitro.The results showed as following.
(1) The metabolic kinetic characteristics of nifedipine were in line with the Michaelis-Menten kinetics.The Vmax for CYP3A4, CYP3A22,CYP3A29,CYP3A39 and CYP3A88 was 1.62,1.27,1.72,1.22 and 1.63 nmol/min/mg respectively;the Km for CYP3A4,CYP3A22, CYP3A29,CYP3A39 and CYP3A88 was 12.12,18.98,12.83,18.42 and12.46μM respectively,Intrinsic clearance(Clint) was 133.66,66.91, 134.06,66.23 and 130.29μL/min/mg respectively,These data indicated that CYP3A29 and CYP3A88 and CYP3A4 had the very similar characteristics in the metabolism of nifedipne,and were dissimilar to CYP3A22 and CYP3A39.
(2) Enzyme kinetic characteristics of testosterone metabolism were consistent with hill kinetics.For CYP3A4,CYP3A22,CYP3A29, CYP3A39 and CYP3A88,the n value was 1.14,1.61,1.34,1.62 and 1.35 respectively,all of them were more than 1,which indicated that the testosterone interacted with CYP3As in a synergy way;kinetic parameter of Vmax was 7.13,4.34,6.21,4.15 and 6.08 nmol/min/mg repectively, kinetic parameters of S_(50) was 1.87,73.64,50.08,69.76 and 50.24μM repectively,Clint was 172.06,58.90,124.0,59.49 and 121.03μL/min/mg repectively.
(3) For CYP3A4,CYP3A22,CYP3A29,CYP3A39 and CYP3A88, IC50 value obtained in the present experiment for the ketoconazole inhibition on the metabolism of nifedipine was 0.132,0.233,0.090,0.262, 0.104μM respectively,and IC50 value for the ketoconazole inhibition on the metabolism of testosterone was 0.032,0.0688,0.0562,0.077 and 0.065μM respectively for CYP3A4,CYP3A22,CYP3A29,CYP3A39 and CYP3A88,These data indicated that ketoconazole imposed stronger inhibition on the metabolism of testosterone than that of nifedipine,and the same did on CYP3A4,CYP3A29 and CYP3A88 than CYP3A22 and CYP3A39.
(4) The metabolic kinetic analysis of probe by whole-cell in HepG2-CYP3A cell lines.In order to compare the metabolic characteristics of human CYP3A4 and CYP3A of miniature pig,this study used recombinant cell microsomes and whole-cell in vitro incubation simultaneously.The analysis of the metabolic characteristics of nifedipine and testosterone in recombinant whole-cell of HepG2-CYP3A cell lines indicated that the metabolic kinetic characteristics of nifedipine were in line with the-Michaelis-Menten kinetics.For HepG_2-CYP3A4, HepG_2-CYP3A22,HepG_2-CYP3A29,HepG_2-CYP3A39 and HepG_2-CYP3A88,the kinetic parameter of Vmax was 2.21,1.52,2.51, 1.40 and 2.46 nmol/min/mg repectively,the kinetic parameter of Km was 16.77、20.51、15.47、21.93 and 15.56μM repectively,the Clint were 131.78, 74.11,162.25,63.84 and 157.97μL/min/mg respectively;
The data indicated that CYP3A29 and CYP3A88 and CYP3A4 shared the very similar characteristics in the metabolism of nifedipne in recombinant whole-cell,but were dissimilar to CYP3A22 and CYP3A39.The metabolic kinetic characteristics of testosterone were in line with the hill kinetics.For HepG_2-CYP3A4,HepG_2-CYP3A22, HepG_2-CYP3A29,HepG_2-CYP3A39 and HepG_2-CYP3A88,the kinetic parameter of Vmax was 2.55,1.83,2.37,1.72 and 2.39 nmol/min/mg respectively,the kinetic parameter of Km was 14.14,23.66,16.88,21.48 and 17.81μM respectively,intrinsic Clearance(Clint) was 180.40,77.59, 140.36,93.20 and134.40μL/min/mg respectively;The data indicated that CYP3A29 and CYP3A88 and CYP3A4 shared the very similar characteristics in the metabolism of testosterone,but were dissimilar to CYP3A22 and CYP3A39.
For HepG_2-CYP3A4,HepG_2-CYP3A22,HepG_2-CYP3A29, HepG_2-CYP3A39 and HepG_2-CYP3A88,IC_(50) value obtained in the present experiment for the ketoconazole inhibition on the metabolism of nifedipine was 0.639,0.8785,0.4985,0.8787 and 0.556μM respectively;- IC50 value for the ketoconazole inhibition on the metabolism of testosterone was 0.120, 0.207,0.125,0.229 and 0.128μM respectively.The data indicated that the ketococonazole imposed stronger inhibition on HepG_2-CYP3A4, HepG_2-CYP3A88 and HepG_2-CYP3A29 than that of CYP3A22 and CYP3A39.
Conclusion:This research confirmed the earlier hypothesis that CYP3A29 was homologous enzyme of human CYP3A4 in miniature pigs; that CYP3A88 shared similar pharmacokinetic characteristics and inhibitory activity with CYP3A4 and CYP3A29.It suggests that further research should be done to explore the biological basis for this phenomenon.
人CYP3A在肝肠中表达最为丰富,约占CYP450总量的30%-40%,底物谱广,并可分成CYP3A4、CYP33A5、CYP33A7和CYP3A43四种亚型,其中CYP3A4在成年个体的肝和肠中表达最多,占肝CYPs的30%和肠CYPs的70%,参与了60%现有临床药物的代谢。
新药临床前评价过程中需要其代谢相关数据。为获得可反映人体药物代谢概貌的最佳实验动物模型,通过对不同种属动物肝微粒体的比较研究发现,狗适合作为CYP2D代谢途径的动物模型;灵长类是最合适的CYP2CN家族动物模型,而小型猪的CYP1A、CYP2A、CYP3A与人类极为相似,且该动物体型较小、易于操作和较小的底物需求量,是人类CYP3A药物评价的最佳模型动物。
目前研究认为小型猪CYP3A亚家族蛋白共有4种亚型,即CYP3A22、CYP3A29、CYP3A39和CYP3A88,其中CYP3A29具有典型的硝苯地平和睾酮6β-羟基化活性(人CYP3A4和3A5的特异活性),被苯巴比妥、利福平和地塞米松诱导的活性,及对酮康唑和醋竹桃霉素抑制的敏感性。因此,除灵长类和狗以外,小型猪成为唯一被FDA推荐的临床前大动物实验动物,在药物研发、生产和监管等领域越来越多地用于狗的替代模型动物。
现有的临床前药物评价的小型猪主要有:Yucatan小型猪、德国的Gottingen小型猪、Hanford小型猪和Sinclair小型猪,特别是Gottingen在欧洲的得到了很好的推广运用;Bama小型猪产于我国的广西省,为香猪的进化品系,由于近交程度高,而且耐近交、遗传稳定、个体表型一致,在生物医药领域应用中已被证明是优秀的模型动物,并积累了丰富的解剖、生理生化、基础生物学特性及活体药物代谢背景资料,但有关其药物代谢表征的分子水平研究资料缺乏;由于重组酶成分单一,其药物代谢表征明确,被广泛用于酶表征的定性定量研究,是对酶进行特征研究的最佳工具。目前有关猪(小型猪)CYP3A亚家族同工酶的重组研究,在世界范围几乎是空白,猪(小型猪)CYP3A亚家族同工酶的药物代谢表征几乎全部都是通过人CYP3A探针药物的活体动物肝微粒体代谢特征获得,由于特异性探针药物对特定CYP450的相对性,以及猪肝微粒体成分的复杂性,其可靠性远低于重组酶。为了更好阐明Bama小型猪作为人CYP3A系统动物模型的有效性和合理性,以及各亚型在探针药物代谢中的作用特征,本研究通过对人CYP3A4和小型猪CYP3A基因克隆、HepG_2稳定表达细胞株的建立、以及在微粒体和全细胞两个水平对稳定表达细胞株的CYP3A特异性底物和抑制剂的鉴定,旨在分子水平阐明Bama小型猪的药物代谢特征,从而为Bama小型猪作为临床前药物代谢模型实验动物提供实验依据。
二、研究内容和结果
1.人CYP3A4基因和Bama小型猪CYP3A基因的克隆
用总RNA经逆转录PCR获得目的基因,并将目的基因亚克隆至pMD-19-T上,测序确认获得了人CYP3A4,以及小型猪的CYP3A22、CYP3A29、CYP3A39和CYP3A88共5个基因;其中人CYP3A4、小型猪CYP3A39和CYP3A88与GeneBank中公布的序列完全一致;CYP3A29基因与NCBI记录的CYP3A29基因序列(GENE ID:403324)有2个碱基不同,即G96A、T105A,但其编码相同的CYP3A29的氨基酸酸序列(NP 999588);但获得的CYP3A22基因与GeneBank中公开的基因序列(AB006010.1)有6个碱基不同,即G 36 C、A 169 G、C 546 T、A 612 C、C1329 T和A 1351G,将该序列推定的蛋白质氨基酸序列与CYP3A22(BAD06180.1)进行比对发现有3个氨基酸差异,即W 12 C、W 57 V、N 451 D,且对来自5头小型猪的基因经过PCR扩增后测序结果完全一致,故此序列可能为Bama小型猪特有。
2.稳定表达人CYP3A4基因和Bama小型猪CYP3A基因HepG_2细胞株的建立及探针药物代谢鉴定
将克隆载体pMD-19-T上人CYP3A4和小型猪CYP3A系列的基因,在其5′和端3′插入酶切位点,并在3′插入6个组氨酸核苷酸序列,克隆至表达载体pcDNA3.1上,分别测序确认;将表达载体经脂质体转染至HepG_2细胞,经10代抗性筛选及RT-PCR、West-blot检测表明本研究成功建立了所需CYP3A基因在HepG_2中的稳定表达体系;以人CYP3A的特异性底物硝苯地平和睾酮为探针鉴定重组细胞的药物代谢活性,结果表明所得人CYP3A4和小型猪CYP3A基因HepG_2稳定表达株具有硝苯地平氧化代谢和睾酮6β-羟基化活性。
3.稳定表达人CYP3A4基因和Bama小型猪CYP3A基因HepG_2细胞微粒体对探针药物的代谢表征分析
以硝苯地平和睾酮作为探针底物,酮康唑作为抑制剂进行体外药物代谢的动力学分析,结果如下。
(1)人CYP3A4与Bama小型猪CYP3A重组细胞中微粒体对硝苯地平的体外代谢符合米氏动力学特征,其中CYP3A4、CYP3A22、CYP3A29、CYP3A39和CYP3A88的Vmax分别为1.62、1.27、1.72、1.22和1.63nmol/min/mg,Km分别为12.12、18.98、12.83、18.42和12.46μM,内在清除率(Clint=Vm/Km)分别为133.66、66.91、134.06、66.23和130.29μL/min/mg。这些数据表明,CYP3A29和CYP3A88与CYP3A4的代谢特征极为相似,而CYP3A22和CYP3A39与CYP3A4的硝苯地平代谢差异较大。
(2)重组细胞微粒体代谢睾酮符合Hill动力学特征,CYP3A4、CYP3A22、CYP3A29、CYP3A39和CYP3A88的Hill系数分别为1.14、1.61、1.34、1.62和1.35,说明本实验获得的重组CYP3A酶对睾酮的代谢均属于正协同作用;Vmax分别为7.13、4.34、6.21、4.15和6.08nmol/min/mg,S_(50)分别为41.87、73.64、50.08、69.76和50.24μM mol/L,Clint(Vm/S50)分别为172.06、58.90、124.0、59.49和121.03μL/min/mg,表明在对睾酮的6β-羟基化代谢中CYP3A4、CYP3A29和CYP3A88的酶作用效能比较接近。
(3)本实验获得酮康唑对硝苯地平代谢反应抑制的IC_(50)值,CYP3A4、CYP3A22、CYP3A29、CYP3A39分别为0.132、0.233、0.090、0.262、0.104μM;对睾酮的代谢的IC_(50)值分别为0.032、0.0688、0.0562、0.077和0.065μM,这些数据表明酮康唑不论对硝苯地平或睾酮的代谢,其对CYP3A4、CYP3A29和CYP3A88抑制活性均较近。
4.稳定表达人CYP3A4基因和Bama小型猪CYP3A基因稳定表达HepG_2全细胞对探针药物的代谢表征分析
用基因重组全细胞体外孵育的方法,比较小型猪HepG_2-CYP3A与人HepG_2-CYP3A4的硝苯地平、睾酮和酮康唑代谢表征,其结果如下。
(1)重组全细胞的硝苯地平体外孵育代谢符合米氏动力学特征,HepG_2-CYP3A4、HepG_2-CYP3A22、HepG_2-CYP3A29、HepG_2-CYP3A39和HepG_2-CYP3A88的酶动力学参数,Vmax分别为2.21、1.52、2.51、1.40和2.46nmol/min/mg,Km分别为16.77、20.51、15.47、21.93和15.56μM,其内源性清除率Clint分别为131.78、74.11、162.25、63.84和157.97μL/min/mg,这些数据表明重组全细胞的硝苯地平代谢特征与微粒体实验类似,CYP3A29和CYP3A88对硝苯地平的代谢动力学特征与CYP3A4的代谢特征近似。
(2)重组全细胞的睾酮体外孵育代谢符合Hill动力学特征,HepG_2-CYP3A4、HepG_2-CYP3A22、HepG_2-CYP3A29、HepG_2-CYP3A39和HepG_2-CYP3A88的酶动力学参数,Vmax分别为2.55、1.83、2.37、1.72和2.39nmol/min/mg,Km分别为14.14、23.66、16.88、21.48和17.81μM,内源性清除率(Clint)180.40、77.59、140.36、93.20和134.40μL/min/mg,这些数据表明HepG_2-CYP3A4与HepG_2-CYP3A29和HepG_2-CYP3A88代谢特征较为相似,而与HepG_2-CYP3A22和HepG_2-CYP3A39的差别较大。
(3)重组全细胞酮康唑对硝苯地平代谢的IC_(50)值,HepG_2-CYP3A4、HepG_2-CYP3A22、HepG_2-CYP3A29、HepG_2-CYP3A39和HepG_2-CYP3A88,分别为0.539、0.8785、0.4985、0.8787和0.556μM;对睾酮的IC_(50)值分别为0.120、0.207、0.125、0.229和0.128μM,这些数据表明酮康唑对HepG_2-CYP3A4、HepG_2-CYP3A29和HepG_2-CYP3A88的睾酮代谢的抑制均较近,且显著高于对CYP3A22和CYP3A39的抑制活性。
5.结论
通过对人CYP3A4基因和Bama小型猪CYP3A22、CYP3A29、CYP3A39和CYP3A88基因重组细胞的微粒体和全细胞的CYP3A特异性探针底物的硝苯地平和睾酮代谢活性,以及CYP3A的特异性抑制剂的体外孵育实验表明,人CYP3A4与Bama小型猪CYP3A29和CYP3A88具有较近的底物代谢表征,证实了前人提出的CYP3A29是CYP3A4在小型猪体内的同源酶的假说;同时发现CYP3A88也具有同CYP3A4较近的药物代谢表征,提示有必要进行进一步的研究,探索其内在的生物学基础。CYP3A22和CYP3A39之间具有近似的体外探针药物代谢表征,二者与CYP3A4、CYP3A29和CYP3A88之间具有较大的体外探针药物代谢表征差异。
Research background
CYP3A enzymes,which are the most abundant CYPs expressed in the liver and intestine,and accounting for 30%-40%in the total CYPs contents expressed in liver and intestine,involve in the disposition of diverse exogenous chemical substances.There are four isoenzymes in CYP3A subfamily,namely,CYP3A4,CYP3A5,CYP3A7and CYP3A43.The CYP3A4,which is expressed most abundant in liver and intestine in the adults,accounting for 30%of liver CYPs contents and 70%of intestinal CYPs contents,is involved in the metabolism of 60%of clinical drugs.
Relevant data need for their metabolism in the process of pre-clinical drug evaluation In order to obtain the best experimental animals for human drug metabolism,comparative studies of the pooled microsomes from different species of animals found that dog is a suitable model animal for CYP2D subfamily;and primate is the most suitable model animal for the complex CYP2C subfamily,which has so many isoenzymes involving in so many complicated metabolic pathways;and pig(miniature pig),owing to its very similar characteristics of drug metabolism with human,along with its small size,easy operation and a smaller substrate demand comparing with dog,is the most suitable model animal for human CYP1A,CYP2A and CYP3A subfamily.
At present,there are four isoenzymes in CYP3A subfamily of pig (miniature pig),namely CYP3A22,CYP3A29,CYP3A39 and CYP3A88. CYP3A29,the most studied isoenzymes,has typical nifedipine oxidation activity and testosterone 6β-hydroxy-activity(of the human CYP3A4 and 3A5-specific activity),phenobarbital,rifampicin and dexamethasoneinduced activity,as well as triacetyloleandomycin and ketoconazole inhibitory activity.Therefor,the pig is the only big animal recommended by FDA for the pre-clinical drug in the big animal experiments in addition to primate and dog,and is increasingly used as alternative of dog in the pre-clinical drug evaluation by more and more drug reaserch and development,pharmaceutical industry and regulatory agencies.
At present,the famous strains of miniature pig used for the pre-clinical drug evaluation are mainly Yucatan,Gottingen,Hanford, Sinclair minpig.As the evolution of strains of pigs,Bama miniature pigs have a high extent of in breeding,and stable heredity,and a consistent phenotype,can bear inbreeding also.They have been proved to be excellent model animals,because there are much background information about anatomy,physiology,biochemistry and basic biological characteristics of Bama miniature pigs.
In order to elucidate the characteristics of drug metabolism at a more essential level,the genes of CYP 3A4,CYP3A22,CYP3A29,CYP3A39 andCYP3A88 were cloned,the cloned genes were transfected into HepG_2 cells,the transfected cells were screened for positive cells by G418,the stable expression cell lines were established after ten generation subculture,at last the characteristics of probe drugs were studied in the established HepG_2-CYP3A cell lines at the microsomes and whole-cell level,the present research aims to provide experimental evidences for the Bama miniature pig used as a preclinical experimental model animal of drug metabolism.
Research methods and results
1.Cloning human CYP3A4 and CYP3A gene of Bama miniature pig
The total RNA was extracted from human liver and miniature pig respectively,the target genes obtained by RT-PCR from total RNA were subcloned into T-vector pMD-19-T,the sequencing results and sequence analysis showed that five target genes of human CYP3A4,CYP3A22, CYP3A29,CYP3A39 and CYP3A88 of Bama miniature pig were obtained. The gene sequences of CYP3A4,CYP3A39 and CYP3A88 were completely consistent with the corresponding sequence published in GeneBank.There were two base differences in presently-cloned CYP3A29 compared with CYP3A29(403324) sequence in GeneBank,namely,G96A, T105A,there was no difference comparing the deduced amino acid sequence from the present CYP3A29 with amino acid sequence of CYP3A29((NP_999588)) from the NCBI protein data bank;there were six nucleotide differences in the CYP3A22 gene obtained in the present experiment comparing with CYP3A22 gene published in GeneBank, namely,G 36 C,A 169 G,C 546 T,A 612 C,C1329 T and A 1351G,and three amino acids differences were found in the present protein sequence of CYP3A22 compared with protein sequence of CYP3A22(BAD06180.1) published in NCBI Protein DataBank, namely,W12C,W57V,N451D,because the present CYP3A22,which out of the sequenced nine positive clones from five Bama miniature pigs, showed completely consistent results in the eight of the nine clones,It suggested that the present CYP3A22 gene cloned from Bama miniature pigs was the specific gene sequence for the pigs.
2.Establishment of HepG_2 cell lines stably expressing Human CYP3A4 and CYP3A gene of Bama minipig,and identification of the metabolic characteristics of probe drugs in the established cell lines.
The genes of human CYP3A4 and CYP3A of miniature pigs were cloned into expression vector pCDNA3.1(designated as pCDNA-CYP3A) from subcloning vector pMD-19-T by PCR,then the pCDNA-CYP3As were transfected into HepG_2 cells by Lipofectamine 2000 Reagent, RT-PCR and west-blot results showed the successful establishment of stably expression human CYP3A4 and CYP3A gene of miniature pigs in HepG_2 cell lines after ten generation selection of G418.The results from identification of metabolic activity by the specific CYP3A probe drugs, namely,nifedipine and testosterone,showed that the established HepG2-CYP3A cell lines had full oxidation of nifedipine activity and testosterone 6β-hydroxylation activity.
3.The metabolic analysis of probe drugs by microsomes in HepG2-CYP3A cell lines.
Kinetic parameters,including the Km,Vmax,are the key factors to determine the characteristics of enzymatic reaction curve.In order to compare the kinetic characteristics of human CYP3A4 and CYP3A of Bama miniature pigs,the microsomes extracted from HepG_2-CYP3A cell lines were incubated with nifedipine and testosterone as a probe substrates, and ketoconazole as inhibitors in regeneration system in vitro.The results showed as following.
(1) The metabolic kinetic characteristics of nifedipine were in line with the Michaelis-Menten kinetics.The Vmax for CYP3A4, CYP3A22,CYP3A29,CYP3A39 and CYP3A88 was 1.62,1.27,1.72,1.22 and 1.63 nmol/min/mg respectively;the Km for CYP3A4,CYP3A22, CYP3A29,CYP3A39 and CYP3A88 was 12.12,18.98,12.83,18.42 and12.46μM respectively,Intrinsic clearance(Clint) was 133.66,66.91, 134.06,66.23 and 130.29μL/min/mg respectively,These data indicated that CYP3A29 and CYP3A88 and CYP3A4 had the very similar characteristics in the metabolism of nifedipne,and were dissimilar to CYP3A22 and CYP3A39.
(2) Enzyme kinetic characteristics of testosterone metabolism were consistent with hill kinetics.For CYP3A4,CYP3A22,CYP3A29, CYP3A39 and CYP3A88,the n value was 1.14,1.61,1.34,1.62 and 1.35 respectively,all of them were more than 1,which indicated that the testosterone interacted with CYP3As in a synergy way;kinetic parameter of Vmax was 7.13,4.34,6.21,4.15 and 6.08 nmol/min/mg repectively, kinetic parameters of S_(50) was 1.87,73.64,50.08,69.76 and 50.24μM repectively,Clint was 172.06,58.90,124.0,59.49 and 121.03μL/min/mg repectively.
(3) For CYP3A4,CYP3A22,CYP3A29,CYP3A39 and CYP3A88, IC50 value obtained in the present experiment for the ketoconazole inhibition on the metabolism of nifedipine was 0.132,0.233,0.090,0.262, 0.104μM respectively,and IC50 value for the ketoconazole inhibition on the metabolism of testosterone was 0.032,0.0688,0.0562,0.077 and 0.065μM respectively for CYP3A4,CYP3A22,CYP3A29,CYP3A39 and CYP3A88,These data indicated that ketoconazole imposed stronger inhibition on the metabolism of testosterone than that of nifedipine,and the same did on CYP3A4,CYP3A29 and CYP3A88 than CYP3A22 and CYP3A39.
(4) The metabolic kinetic analysis of probe by whole-cell in HepG2-CYP3A cell lines.In order to compare the metabolic characteristics of human CYP3A4 and CYP3A of miniature pig,this study used recombinant cell microsomes and whole-cell in vitro incubation simultaneously.The analysis of the metabolic characteristics of nifedipine and testosterone in recombinant whole-cell of HepG2-CYP3A cell lines indicated that the metabolic kinetic characteristics of nifedipine were in line with the-Michaelis-Menten kinetics.For HepG_2-CYP3A4, HepG_2-CYP3A22,HepG_2-CYP3A29,HepG_2-CYP3A39 and HepG_2-CYP3A88,the kinetic parameter of Vmax was 2.21,1.52,2.51, 1.40 and 2.46 nmol/min/mg repectively,the kinetic parameter of Km was 16.77、20.51、15.47、21.93 and 15.56μM repectively,the Clint were 131.78, 74.11,162.25,63.84 and 157.97μL/min/mg respectively;
The data indicated that CYP3A29 and CYP3A88 and CYP3A4 shared the very similar characteristics in the metabolism of nifedipne in recombinant whole-cell,but were dissimilar to CYP3A22 and CYP3A39.The metabolic kinetic characteristics of testosterone were in line with the hill kinetics.For HepG_2-CYP3A4,HepG_2-CYP3A22, HepG_2-CYP3A29,HepG_2-CYP3A39 and HepG_2-CYP3A88,the kinetic parameter of Vmax was 2.55,1.83,2.37,1.72 and 2.39 nmol/min/mg respectively,the kinetic parameter of Km was 14.14,23.66,16.88,21.48 and 17.81μM respectively,intrinsic Clearance(Clint) was 180.40,77.59, 140.36,93.20 and134.40μL/min/mg respectively;The data indicated that CYP3A29 and CYP3A88 and CYP3A4 shared the very similar characteristics in the metabolism of testosterone,but were dissimilar to CYP3A22 and CYP3A39.
For HepG_2-CYP3A4,HepG_2-CYP3A22,HepG_2-CYP3A29, HepG_2-CYP3A39 and HepG_2-CYP3A88,IC_(50) value obtained in the present experiment for the ketoconazole inhibition on the metabolism of nifedipine was 0.639,0.8785,0.4985,0.8787 and 0.556μM respectively;- IC50 value for the ketoconazole inhibition on the metabolism of testosterone was 0.120, 0.207,0.125,0.229 and 0.128μM respectively.The data indicated that the ketococonazole imposed stronger inhibition on HepG_2-CYP3A4, HepG_2-CYP3A88 and HepG_2-CYP3A29 than that of CYP3A22 and CYP3A39.
Conclusion:This research confirmed the earlier hypothesis that CYP3A29 was homologous enzyme of human CYP3A4 in miniature pigs; that CYP3A88 shared similar pharmacokinetic characteristics and inhibitory activity with CYP3A4 and CYP3A29.It suggests that further research should be done to explore the biological basis for this phenomenon.
引文
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