First-pass midazolam metabolism catalyzed by 1 alpha,25-dihydroxy vitamin D-3-modified Caco-2 cell monolayers

Cytochrome P-450 (CYP) 3A4 accounts for approximately 50% of all P-450s fou nd in the small intestine (Paine et al., 1997) and contributes to the exten sive and variable first-pass extraction of drugs such as cyclosporine and s aquinavir. We recently demonstrated that CYP3A4 expression in a differentia ted Caco-2 subclone is increased when cell monolayers are treated with 1 al pha,25-dihydroxy-vitamin-D-3 (Schmiedlin-Ren et al,, 1997). This improved m etabolic capacity permits the in vitro modeling of first-pass intestinal me tabolic kinetics. Midazolam (MDZ) 1'-hydroxylation was used as a specific p robe for CYP3A-mediated metabolism in modified Caco-2 monolayers, Caco-2 ce lls were grown to confluence on laminin-coated culture inserts, and then fo r two additional weeks in the presence of 1 alpha,25-dihydroxy vitamin-D-3. Cell monolayers were subsequently exposed to MDZ for varying lengths of ti me 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 d ose. By either route of administration, the rate of 1'-hydroxymidazolam for mation was stable and linear for 2 h. Under basolateral sink conditions and low apical MDZ dosing concentration (1-8 mu M), the first-pass extraction ratio was found to be similar to 15%. Higher dosing concentrations led to s aturation of the hydroxylation reaction and reduction in the extraction rat io. The modified Caco-2 cell monolayer is an excellent model for studying d rug absorption and first-pass intestinal metabolic kinetic processes. In th is system, the selective CYP3A probe MDZ was rapidly absorbed, yet extensiv ely metabolized, as is observed in vivo.