Differential metabolism of midazolam in mouse liver and intestine microsomes: a comparison of cytochrome P450 activity and expression.
1. Although multiple cytochrome P450s (CYP) contribute to hepatic phase I metabolism, CYP3A is the principal subfamily present in human and mouse small intestine. 2. Differences in phase I metabolism were investigated using midazolam (MDZ) hydroxylation in mouse liver and intestinal microsomes. The net MDZ metabolite formation rate in intestinal microsomes was approximately 30% that of liver microsomes (at 250 micro M MDZ). 3. Quantitative Western blotting with anti-CYP3A1 antibody detected two bands of immunoreactive protein in both liver and intestinal samples, 2.24 +/- 0.27 (mean +/- SD, n = 3) and 0.64 +/- 0.08 pmol mg(-1) protein, respectively. Qualitative Western blotting with anti-CYP2C11 antibody detected a single band of immunoreactive protein in liver microsomes and no signal in intestinal samples (1 micro g sample). 4. Ketoconazole potently inhibited formation of both alpha- and 4-OH-MDZ metabolites in intestinal microsomes (IC(50)' of 0.126 +/- 0.010 and 0.0955 +/- 0.014 micro M, respectively) and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) of 0.041 +/- 0.003 micro M). However, ketoconazole (5 micro M) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Inhibition by ritonavir (5 micro M) produced similar results. 5. MDZ hydroxylation is predominately CYP3A dependent in mouse intestine (compared with mouse liver) since CYP2C is not expressed in the intestine. The importance of CYP3A in the mouse intestine appears to mirror that in humans.
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine and the Division of Clinical Pharmacology, Tufts New England Medical Center, Boston, MA 02111, USA.
M D
MD
Perloff
L L
LL
Von Moltke
D J
DJ
Greenblatt
D000465:Algorithms; D000818:Animals; D014151:Anti-Anxiety Agents / Q000378:metabolism*; D019138:Antibodies, Blocking / Q000494:pharmacology; D001711:Biotransformation; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D008861:Microsomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D008874:Midazolam / Q000378:metabolism*; D009928:Organ Specificity; D019438:Ritonavir / Q000494:pharmacology; D014217:Troleandomycin / Q000494:pharmacology
D014151:Anti-Anxiety Agents; D019138:Antibodies, Blocking; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D014217:Troleandomycin; D019438:Ritonavir; D008874:Midazolam; D007654:Ketoconazole