PT  - JOURNAL ARTICLE
AU  - Fisher, Jeannine M.
AU  - Wrighton, Steven A.
AU  - Watkins, Paul B.
AU  - Schmiedlin-Ren, Phyllissa
AU  - Calamia, Justina C.
AU  - Shen, Danny D.
AU  - Kunze, Kent L.
AU  - Thummel, Kenneth E.
TI  - First-Pass Midazolam Metabolism Catalyzed by  1α,25-Dihydroxy Vitamin D<sub>3</sub>-Modified Caco-2 Cell Monolayers
DP  - 1999 May 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 1134--1142
VI  - 289
IP  - 2
4099  - http://jpet.aspetjournals.org/content/289/2/1134.short
4100  - http://jpet.aspetjournals.org/content/289/2/1134.full
SO  - J Pharmacol Exp Ther1999 May 01; 289
AB  - 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-D3 (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-D3. 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. The American Society for Pharmacology and Experimental Therapeutics