ACCELERATED COMMUNICATION Mdr1 Limits CYP3A Metabolism in Vivo

We determined whether the drug efflux protein P-glycoprotein (Pgp) could influence the extent of CYP3A-mediated metabolism of erythromycin, a widely used model substrate for CYP3A. We compared CYP3A metabolism of erythromycin (a Pgp substrate) using the erythromycin breath test in mice proficient and deficient of mdr1 drug transporters. We first injected mdr1(1/1) mice with [C]N-methyl erythromycin and measured the rate of appearance of CO2 in the breath as a measure of hepatic CYP3A activity. Animals treated with CYP3A inducers or inhibitor showed accelerated or diminished CO2 in the breath, respectively. The erythromycin breath test was next administered to mdr1a(2/2) and mdr1a/1b(1/1) and (2/2) mice. These animals had equivalent levels of immunoreactive CYP3A and CYP3A activity as measured by erythromycin N-demethylase activity in liver microsomes. Nevertheless, the rate of CO2 appearance in the breath showed no relationship with these measurements of CYP3A, but changed proportionally to expression of mdr1. The average breath test CO2 area under the curves were 1.9and 1.5-fold greater in mdr1a/ 1b(2/2) and mdr1a(2/2) mice, respectively, compared with (1/1) mice, and CERmax was 2-fold greater in mdr1a/1b(2/2) compared with (1/1) mice. We conclude that Pgp, by limiting intracellular substrate availability can be an important determinant of CYP3A metabolism of numerous medications that are substrates for CYP3A and Pgp. Factors that contribute to interindividual variation in drug disposition influence drug toxicity, drug efficacy, and hence, therapeutic outcome. There is at least a 10-fold human variation in the systemic clearance of erythromycin (Watkins et al., 1985, 1990) and cyclosporin A (CsA). CYP3A is the exclusive P-450 involved in the N-demethylation of erythromycin, and the major CYP catalyzing the formation of CsA metabolites. Moreover, CYP3A has been estimated to metabolize 60% of all drugs (Wrighton et al., 1996). Because CYP3A expression varies 10to 40-fold between humans (Watkins et al., 1985; Shimada and Guengerich, 1989), it has been proposed that differences in the clearance of drugs such as erythromycin and CsA can be explained by variation in hepatic CYP3A. The “erythromycin breath test” (ERMBT) (Watkins et al., 1989; Watkins, 1991) was developed to phenotype the differences in hepatic CYP3A4 activity between humans. The ERMBT relies on the assumption that following i.v. administration of [C]N-methyl erythromycin, CYP3A4 in the liver limits the rate of erythromycin N-demethylation. CYP3A4 demethylates [C]N-methyl erythromycin, and at least half the radiolabeled carbon appears almost instantly in the breath as CO2 (Watkins, 1991, 1994). Thus, the rate of production of breath CO2 following i.v. [ C]N-methyl erythromycin has been proposed as a standard assay for measuring hepatic CYP3A4 activity (Watkins et al., 1989; Watkins, 1994). The ERMBT also relies on the assumption that other process, such as cellular efflux of substrate, are not