Comparison Of Metabolic Characteristics Of The Probe Drugs In The HepG₂ Cell Lines Stably Expressing Genes Of Human CYP3A4 And CYP3A Subfamily Of Miniature Pig

Research backgroundCYP3A 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 HepG₂ 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 HepG₂-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 results1.Cloning human CYP3A4 and CYP3A gene of Bama miniature pigThe 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((NP₉99588)) 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 HepG₂ 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 HepG₂ 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 HepG₂ 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 HepG₂-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 S₅₀ 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 HepG₂-CYP3A4, HepG₂-CYP3A22,HepG₂-CYP3A29,HepG₂-CYP3A39 and HepG₂-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 HepG₂-CYP3A4,HepG₂-CYP3A22, HepG₂-CYP3A29,HepG₂-CYP3A39 and HepG₂-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 HepG₂-CYP3A4,HepG₂-CYP3A22,HepG₂-CYP3A29, HepG₂-CYP3A39 and HepG₂-CYP3A88,IC₅₀ 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 HepG₂-CYP3A4, HepG₂-CYP3A88 and HepG₂-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.