Expression And Activity Comparison Of Recombinant Minipig And Human CYP3As

Objective:To obtain recombinant minipig CYP3A22, CYP3A29, CYP3A46and human CYP3A4, CYP3A5, and to compare the CYP3A activity between human and minipig by using the specific probe substrates.Methods:Bac-to-Bac baculovirus expression system was used to coexpress recombinant CYP3A with POR and Cyt b5. The enzymes’metabolic and inhibition activity was determined by HPLC, using testosterone, midazolam and nifedipine as probe substrates.Results:The recombinant CYP3A4, CYP3A5, CYP3A22, CYP3A29and CYP3A46were obtained. Their Km values for testosterone6β-hydroxylation were135.7±9.3,188.5±24.8,86.5±4.8,97.55±4.8and184.8±15.5μM, Vwere7.39±0.16,1.56±0.07,3.49±0.05,6.23±0.09and0.914±0.03nmol/min/nmol P45Q, CLint were54.5±2.8,8.3±0.8,40.4±1.8,63.8±2.4and4.95±0.29μL/min/nmol P450, respectively. The Km values for midazolam1-hydroxylation were3.27±0.16,6.14±0.21,5.58±0.12,16.95±0.87and25.57±1.63μM, Vwere1.11±0.01,2.88±0.03,0.297±0.002,0.505±0.011and0.416±0.010nmol/min/nmol P450, CLint were340.5±13.65,469.4±12.5,53.02±0.85,29.80±0.95and16.28±0.70μL/min/nmol P450, respectively. The Km values for nifedipine oxidization were13.59±0.90,33.26±1.63,24.21±1.22,40.33±2.02and51.88±2.25μM, V were1.50±0.03,2.07±0.03,0.992±0.002,1.85±0.03and1.11±0.02nmol/min/nmol P450, CLint were110.3±5.9,62.33±2.24, 40.96±1.59,45.81±1.63and21.41±0.63μL/min/nmol P450, respectively. The IC50of ketoconazole inhibition of testosterone6β-hydroxylation mediated by CYP3A4, CYP3A5, CYP3A22, CYP3A29and CYP3A46were0.1561±0.0022,1.281±0.043,0.5343±0.0087,0.7317±0.0109and0.7520±0.0510μM, respectively; The IC50of ketoconazole inhibition of midazolam1-hydroxylation were0.1496±0.0010,0.6772±0.0105,0.1224±0.0039，0.0791±0.0009and0.0546±0.0004μM, respectively; IC50of ketoconazole inhibition of nifedipine oxidization were0.2851±0.0081,2.122±0.026,0.1846±0.0031,0.2227±0.0025and0.6305±0.0117μM, respectively.Conclusion:The recombinant enzymes expressed can be used to study the similarity and differences between human and minipig CYP3As. Both human and minipig CYP3As have similar metabolic activity to their classic substrates, testosterone, midazolam and nifedipine, so minipig can be utilized as a model to study human CYP3As-mediated drug metabolism.