alexa Development of Caco-2 cells expressing high levels of cDNA-derived cytochrome P4503A4.
Pharmaceutical Sciences

Pharmaceutical Sciences

Journal of Bioequivalence & Bioavailability

Author(s): Crespi CL, Penman BW, Hu M

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Abstract PURPOSE: To develop Caco-2 cell derivatives expressing high levels of human cytochrome P450 drug metabolizing enzymes. METHODS: The cDNAs for two cytochrome P450 forms, CYP2A6 and CYP3A4, were introduced into an extrachromosomal vector under control of the cytomegalovirus early intermediate promoter. Vector-bearing cells were selected via resistance to hygromycin B. RESULTS: Transfected cells exhibited high levels of cDNA-derived protein as measured by Western blot, spectrophotometric P450 determination and/or cytochrome P450 form-selective enzyme assay. CYP3A4 and CYP2A6 catalytic activities were about 100 fold higher than in control cells. cDNA-expressing cells were found to form tight monolayers and were suitable for study of xenobiotic transport and metabolism. The permeabilities of cephalexin, phenylalanine, mannitol and propranolol across transfected monolayers were found to be similar to those across untransfected monolayers. The appropriate transfected monolayers metabolized the CYP2A6 substrate coumarin and the CYP3A4 substrates testosterone and nifedipine. CONCLUSIONS: A Caco-2 cell system to simultaneously study drug transport and metabolism has been developed.
This article was published in Pharm Res and referenced in Journal of Bioequivalence & Bioavailability

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