Creation of polarized cells coexpressing CYP3A4, NADPH cytochrome P450 reductase and MDR1/P-glycoprotein

Purpose. To develop model polarized cell systems expressing cytochrome P450 3A4, NADPH P450 reductase, and P-glycoprotein (Pgp). Methods. LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp, the pro duct of the multidrug resistance gene (MDR1), were transfected stably using either a mammalian neomycin selectable expression vector (CYP3A4-Neo) or a n episomal vector based on Epstein-Barr virus (CYP3A4-Hygro). These CYP3A4 expressing cells were compared with LLC-PK1, L-MDR1, or Caco-2 cells transd uced with Adenovirus-3A4, vector (Ad3A4) with or without simultaneous Adeno virus-P450 Reductase (AdRed) transduction. Cells were characterized for exp ression of CYP3A4 protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays were performed to determine whether infection with recombinant Ad3A4 +/- Ad Red affected Pgp function. Results. The rank order of optimal CYP3A4 expression and activities in LLC- PK1 and L-MDR1 cells from highest to lowest was cells co-transduced with Ad 3A4 plus AdRed much greater than Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo. Simil arly, coexpression of Ad3A4 plus AdRed led to enhanced CYP3A4 mediated meta bolism in Caco-2 cells over cells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detect able metabolite in the medium. In microsomes from Caco-2 and LLC-PK1 cells, each co-transduced with Ad3A4/AdRed, Vmax values for testosterone 6 beta-h ydroxylase activity ranged from 414 to 1350 pmoles/min/mg, respectively. Fo r either Caco-2 or LLC-MDR1 cells, TEER values and the rate of apical to ba sal and basal to apical transport of vinblastine or digoxin were similar in cells with and without Ad3A4/Red transduction. Conclusions. Polarized cellular systems coexpressing Ad3A4, AdRed, and the MDR1/Pgp transporter were developed and characterized. The results document the utility of these polarized model systems for simultaneous drug transpo rt/drug metabolism studies. Since the experimental approach can be adapted to study the interplay of multiple enzyme/transporting systems, it may find significant application as a screening tool for the pharmaceutical industr y and as a more basic research tool to study the kinetics of intestinal dru g bioavailability.