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 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.
Footnotes
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Send reprint requests to: Dr. Kenneth E. Thummel, Ph.D., Department of Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98195-7610. E-mail: thummel{at}u.washington.edu
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↵1 This study was supported in part by Eli Lilly & Co. and National Institutes of Health Grant GM 32165.
- Abbreviations:
- CYP
- cytochrome P-450
- P-gp
- P-glycoprotein
- MDZ
- midazolam
- 1′-OH MDZ
- 1′-hydroxymidazolam
- 1α
- 25-(OH)2-D3, 1α,25-di-hydroxy vitamin-D3
- FBS
- fetal bovine serum
- DMEM
- Dulbelcco’s modified Eagle’s medium
- NEAA
- non-essential amino acids
- DM
- differentiation medium
- PET
- polyethylene terephthalate
- TEER
- transepithelial electrical resistance
- DMSO
- dimethylsulfoxide
- ER
- extraction ratio
- Received October 19, 1998.
- Accepted January 16, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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