Kent L. Kunze

OBJECTIVE: To determine in humans the relative roles of intestinal and hepatic metabolism in the oral first-pass elimination of a CYP3A substrate using midazolam as a model compound. METHODS: Midazolam was administered intravenously (1 mg) or orally (2 mg) to 20 healthy young subjects (10 men and 10 women) in a random fashion, and the disposition of the drug and its 1'-hydroxy metabolite were determined. In separate in vitro studies, the CYP3A-mediated formation of 1'-hydroxymidazolam by human hepatic and intestinal microsomes was investigated. RESULTS: No gender-related differences were noted in either the systemic (370 +/- 114 ml/min [mean +/- SD]) or oral (1413 +/- 807 ml/min) clearance values of midazolam. Despite complete oral absorption, measured oral bioavailability was on average about 50% less than that predicted on the assumption that only the liver contributed to first-pass metabolism. Pharmacokinetic estimation of the intestinal component indicated an extraction ratio (0.43 +/- 0.24) that was similar to that of the liver (0.44 +/- 0.14). 1'-Hydroxymidazolam was extensively but variably formed in vitro by both hepatic and intestinal microsomes and, although the intrinsic clearance (V(max)/Km) was higher in the liver preparations (540 +/- 747 versus 135 +/- 92 microliters/min/mg protein), this difference was not statistically significant. CONCLUSIONS: These results show that the small intestine can be a major site for presystemic, CYP3A-mediated metabolism after oral administration. Moreover, it appears that this represents a true first-pass effect. In addition, intestinal and hepatic metabolism may be important factors in interindividual variability in disposition after oral administration of midazolam and similar CYP3A substrates. Finally, intestinal localization of CYP3A may be significant in metabolism-based drug-drug interactions.

Previous kinetic studies have identified a high-affinity (S)-warfarin 7-hydroxylase present in human liver microsomes which appears to be responsible for the termination of warfarin's biological activity. Inhibition of the formation of (S)-7-hydroxywarfarin, the inactive, major metabolite of racemic warfarin in humans, is known to be the cause of several of the drug interactions experienced clinically upon coadministration of warfarin with other therapeutic agents. In order to identify the specific form(s) of human liver cytochrome P-450 involved in this particular toxicity, we have determined the metabolic profiles of 11 human cytochrome P-450 forms expressed in HepG2 cells toward both (R)- and (S)-warfarin. Of the 11 forms examined only 2C9 displayed the regioselectivity and stereoselectivity appropriate for the high-affinity human liver microsomal (S)-7-hydroxylase. We further compared Michaelis-Menten and sulfaphenazole inhibition constants for (S)-warfarin 7-hydroxylation catalyzed by cDNA-expressed 2C9 and by human liver microsomes. Similar kinetic constants were obtained for each enzyme source. It is concluded that 2C9 is likely to be a principal form of human liver P-450 which modulates the in vivo anticoagulant activity of the drug. It is further concluded that those drug interactions with warfarin that arise as a result of decreased clearance of the biologically more potent S-enantiomer may have as their common basis the inhibition of P-450 2C9.

The CYP3A subfamily of cytochromes P450 metabolize many medications and environmental contaminants. CYP3A4 and, in 25% of patients, CYP3A5 seem to be the major CYP3A genes expressed in adult liver. Hepatic levels of CYP3A4 can be estimated by the erythromycin breath test and vary at least 10-fold among patients. CYP3A4 has also been shown to be present in small bowel where it is responsible for significant "first-pass" metabolism of orally administered substrates. However, it is not known whether there is significant interindividual variability in the intestinal expression of CYP3A4, or whether the liver and intestinal catalytic activities of CYP3A4 correlate within an individual. It is also not known whether CYP3A5 is expressed in the small intestine. To address these questions, we administered the erythromycin breath test to 20 patients and obtained biopsies from their small bowel. There was a 6-fold variation in CYP3A catalytic activity (midazolam hydroxylation), an 11-fold variation in CYP3A4 protein content, and an 8-fold variation in CYP3A4 mRNA content in intestinal biopsies. There was an excellent correlation between intestinal CYP3A4 protein level and catalytic activity (r = 0.86; p = 0.0001); however, neither parameter significantly correlated with hepatic CYP3A4 activity as measured by the erythromycin breath test result (r = 0.27; p = 0.24 and r = 0.33; p = 0.15, respectively). We also found that CYP3A5 protein was readily detectable in biopsies from 14 (70%) of the patients, indicating that CYP3A5 is commonly expressed in human small intestine.