Abstract
Human cytochrome P450 3A4 (CYP3A4) is the most abundant hepatic and intestinal phase I drug-metabolizing enzyme, and participates in the oxidative metabolism of approximately 50% of drugs on the market. In the present study, a transgenic-CYP3A4(Tg-CYP3A4) mouse model that expresses CYP3A4 in the intestine and is phenotypically normal was generated, which was genotyped by both polymerase chain reaction and Southern blotting. Intestinal microsomes prepared from Tg-CYP3A4 mice metabolized midazolam (MDZ) to 1′-hydroxymidazolam about 2 times, and to 4-hydroxymidazolam around 3 times faster than that from wild-type (WT) mice. These increased MDZ hydroxylation activities were completely inhibited by an anti-CYP3A4 monoclonal antibody. The time course of plasma MDZ and its metabolite concentrations was measured after intravenous (0.25 mg/kg) and oral (2.5 mg/kg) administration of MDZ, and pharmacokinetic parameters were estimated by fitting to a noncompartmental model. Pretreatment with ketoconazole increased orally dosed MDZ maximum plasma concentration (Cmax), time of the maximum concentration, area under the plasma concentration-time curve from zero to infinity (AUC0-∞), and elimination half-life (t1/2) to 3.2-, 1.7-, 7.7-, 2-fold, and decreased MDZ apparent oral clearance about 8-fold in Tg-CYP3A4 mice. The ratios of MDZCmax, AUC0-∞,t1/2 and bioavailability between Tg-CYP3A4 and WT mice after the oral dose of MDZ were 0.3, 0.6, 0.5, and 0.5, respectively. These results suggest that this Tg-CYP3A4 mouse would be an appropriate in vivo animal model for the evaluation of human intestine CYP3A4 metabolism of drug candidates and potential food-drug and drug-drug interactions in preclinical drug development.
Footnotes
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↵1 Current address: CombinatoRx Inc., 650 Albany St., Boston, MA 02118.
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↵2 Current address: Departamento De Farmacologia Y Toxicologia, Seccion De Toxicologia Ambiental, Cinvestav-IPN, AP 14-740, México D.F. 07000.
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↵3 Current address: Merck Research Laboratories, 126 East Lincoln Ave., Rahway, NJ 07065.
- Abbreviations used are::
- P450
- cytochrome P450
- MDZ
- midazolam
- 1′-OH-MDZ
- 1′-hydroxymidazolam
- 4-OH-MDZ
- 4-hydroxymidazolam
- PBS
- phosphate-buffered saline
- mAb
- monoclonal antibody
- Tg-CYP3A4
- transgenic-CYP3A4
- WT
- wild-type
- PCR
- polymerase chain reaction
- mEH
- microsomal epoxide hydrolase
- SSC
- standard saline citrate
- LC-MS/MS
- liquid chromatography tandem mass chromatography
- AUC
- area under the concentration-time curve
- MRT
- mean residence time
- Received December 3, 2002.
- Accepted January 27, 2003.
- U.S. Government
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