Abstract
Purpose. To develop model polarized cell systems expressingcytochrome P4503A4, NADPH P450 reductase, and P-glycoprotein (Pgp).
Methods. LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp,the product of the multidrug resistance gene (MDR1), were transfectedstably using either a mammalian neomycin selectable expression vector(CYP3A4-Neo) or an episomal vector based on Epstein—Barr virus(CYP3A4-Hygro). These CYP3A4 expressing cells were compared withLLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase(AdRed) transduction. Cells were characterized for expression of CYP3A4protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays wereperformed to determine whether infection with recombinant Ad3A4 ±AdRed affected Pgp function.
Results. The rank order of optimal CYP3A4 expression and activitiesin LLC-PK1 and L-MDR1 cells from highest to lowest was cellsco-transduced with Ad3A4 plus AdRed >> Ad3A4 >>>CYP3A4-Hygro > CYP3A4-Neo. Similarly, coexpression of Ad3A4 plus AdRedled to enhanced CYP3A4 mediated metabolism in Caco-2 cells overcells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolitein the medium. In microsomes from Caco-2 and LLC-PK1 cells, eachco-transduced with Ad3A4/AdRed, Vmax values for testosterone6β-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg,respectively. For either Caco-2 or LLC-MDR1 cells, TEER values and therate of apical to basal and basal to apical transport of vinblastine ordigoxin 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. Theresults document the utility of these polarized model systems forsimultaneous drug transport/drug metabolism studies. Since the experimentalapproach can be adapted to study the interplay of multipleenzyme/transporting systems, it may find significant application as a screeningtool for the pharmaceutical industry and as a more basic research toolto study the kinetics of intestinal drug bioavailability.
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Brimer, C., Dalton, J.T., Zhu, Z. et al. Creation of Polarized Cells Coexpressing CYP3A4, NADPH Cytochrome P450 Reductase and MDR1/P-glycoprotein. Pharm Res 17, 803–810 (2000). https://doi.org/10.1023/A:1007599923694
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DOI: https://doi.org/10.1023/A:1007599923694