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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

CYP3A4-Transfected Caco-2 Cells as a Tool for Understanding Biochemical Absorption Barriers: Studies with Sirolimus and Midazolam

Carolyn L. Cummins, Wolfgang Jacobsen, Uwe Christians and Leslie Z. Benet
Journal of Pharmacology and Experimental Therapeutics January 2004, 308 (1) 143-155; DOI: https://doi.org/10.1124/jpet.103.058065
Carolyn L. Cummins
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Wolfgang Jacobsen
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Uwe Christians
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Leslie Z. Benet
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Abstract

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 μM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.

Footnotes

  • We gratefully acknowledge financial support provided by National Institutes of Health CA 72006 (to L.Z.B.) and Affymax Research Institute (to C.L.C.). L.Z.B. has a financial interest in and serves as Chairman of the Board of AvMax, Inc., a biotechnology company whose main interest is in increasing drug bioavailability by inhibiting intestinal CYP3A and P-glycoprotein. This work was presented in part as an oral presentation at the American Society for Pharmacology and Experimental Therapeutics Meeting in Orlando, FL, March 2001.

  • DOI: 10.1124/jpet.103.058065.

  • ABBREVIATIONS: P-gp, P-glycoprotein; TPA, 12-O-tetradecanoylphorbol 13-acetate; MDR, multidrug resistance; MDCK, Madin-Darby canine kidney; CsA, cyclosporine; GG918, GF120918: N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine; PET, polyethylene terephthalate; HPLC, high-performance liquid chromatography; FBS, fetal bovine serum; TEER, transepithelial electrical resistance; LC/MS, liquid chromatography/mass spectrometry; M2, dihydrosirolimus; A, apical; B, basolateral; ER, extraction ratio; K77, K11777: N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl.

  • ↵1 Current address: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.

  • ↵2 Current address: Biotechnology Centre of Oslo, University of Oslo, Oslo 0349, Norway.

  • ↵3 Current address: Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, CO 80262.

    • Received August 6, 2003.
    • Accepted October 8, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 308 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 308, Issue 1
1 Jan 2004
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

CYP3A4-Transfected Caco-2 Cells as a Tool for Understanding Biochemical Absorption Barriers: Studies with Sirolimus and Midazolam

Carolyn L. Cummins, Wolfgang Jacobsen, Uwe Christians and Leslie Z. Benet
Journal of Pharmacology and Experimental Therapeutics January 1, 2004, 308 (1) 143-155; DOI: https://doi.org/10.1124/jpet.103.058065

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

CYP3A4-Transfected Caco-2 Cells as a Tool for Understanding Biochemical Absorption Barriers: Studies with Sirolimus and Midazolam

Carolyn L. Cummins, Wolfgang Jacobsen, Uwe Christians and Leslie Z. Benet
Journal of Pharmacology and Experimental Therapeutics January 1, 2004, 308 (1) 143-155; DOI: https://doi.org/10.1124/jpet.103.058065
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