First-Pass Midazolam Metabolism Catalyzed by 1α,25-Dihydroxy Vitamin D3-Modified Caco-2 Cell Monolayers

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 289; no. 2; p. 1134
• Main Authors: Jeannine M. Fisher, Steven A. Wrighton, Paul B. Watkins, Phyllissa Schmiedlin-Ren, Justina C. Calamia, Danny D. Shen, Kent L. Kunze, Kenneth E. Thummel
• Format: Journal Article
• Language: English
• Published: American Society for Pharmacology and Experimental Therapeutics 01.05.1999
• ISSN: 0022-3565; 1521-0103

Cytochrome P-450 (CYP) 3A4 accounts for approximately 50% of all P-450s found in the small intestine ( Paine et al., 1997 ) and contributes to the extensive and variable first-pass extraction of drugs such as cyclosporine and saquinavir. We recently demonstrated that CYP3A4 expression in a differentiated Caco-2 subclone is increased when cell monolayers are treated with 1α,25-dihydroxy-vitamin-D 3 ( Schmiedlin-Ren et al., 1997 ). This improved metabolic capacity permits the in vitro modeling of first-pass intestinal metabolic kinetics. Midazolam (MDZ) 1′-hydroxylation was used as a specific probe for CYP3A-mediated metabolism in modified Caco-2 monolayers. Caco-2 cells were grown to confluence on laminin-coated culture inserts, and then for two additional weeks in the presence of 1α,25-dihydroxy vitamin-D 3 . Cell monolayers were subsequently exposed to MDZ for varying lengths of time and concentrations. The amount of MDZ in the monolayer increased rapidly after apical drug administration, reaching a pseudo steady state within 6 min. The cellular uptake rate was considerably slower after a basolateral dose. By either route of administration, the rate of 1′-hydroxymidazolam formation was stable and linear for 2 h. Under basolateral sink conditions and low apical MDZ dosing concentration (1–8 μM), the first-pass extraction ratio was found to be ∼15%. Higher dosing concentrations led to saturation of the hydroxylation reaction and reduction in the extraction ratio. The modified Caco-2 cell monolayer is an excellent model for studying drug absorption and first-pass intestinal metabolic kinetic processes. In this system, the selective CYP3A probe MDZ was rapidly absorbed, yet extensively metabolized, as is observed in vivo.