Evaluation of Erythromycin as a Tool to Assess CYP3A Contribution of Low Clearance Compounds in a Long-Term Hepatocyte Culture.

ABSTRACT

Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 µM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CLint was inhibited 58% by 3 µM of ERY, 75% by 15 µM of ERY, 89% by 30 µM of ERY, and 94% by 60 µM of ERY. ERY (30 µM) did not markedly affect CLint of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.