title: First-pass midazolam metabolism catalyzed by 1alpha,25-dihydroxy vitamin D3-modified Caco-2 cell monolayers.
abstract: 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 1alpha,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 1alpha,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 microM), the first-pass extraction ratio was found to be approximately 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.
authors.affiliation: Department of Drug Disposition, Eli Lilly & Co., Indianapolis, Indiana, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Fisher
authors.forename: S A
authors.initials: SA
authors.lastname: Wrighton
authors.forename: P B
authors.initials: PB
authors.lastname: Watkins
authors.forename: P
authors.initials: P
authors.lastname: Schmiedlin-Ren
authors.forename: J C
authors.initials: JC
authors.lastname: Calamia
authors.forename: D D
authors.initials: DD
authors.lastname: Shen
authors.forename: K L
authors.initials: KL
authors.lastname: Kunze
authors.forename: K E
authors.initials: KE
authors.lastname: Thummel
mesh_terms: D001711:Biotransformation; D001798:Blood Proteins / Q000378:metabolism; D015153:Blotting, Western; D018938:Caco-2 Cells; D002117:Calcitriol / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004553:Electric Conductivity; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008874:Midazolam / Q000031:analogs & derivatives / Q000097:blood / Q000378:metabolism* / Q000493:pharmacokinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010539:Permeability; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins; D003577:Cytochrome P-450 Enzyme System; C040081:1-hydroxymethylmidazolam; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D002117:Calcitriol; D008874:Midazolam