Abstract
Long-term hepatocyte culture systems such as HepatoPac® are well-suited to evaluate the metabolic turnover of low clearance (CL) drugs due to 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% for 3 and 10 μM, respectively, for up to 72 h. At 30 µM, ERY inhibited MDZ hydroxylation by >85% for the entire 144 h duration of the incubation. APZ CLint was inhibited 58% by 3 μM ERY, 75% by 15 μM ERY, 89% by 30 μM ERY and 94% by 60 μM ERY. ERY (30 μM) did not markedly affect CLint of substrates for several other major CYP isoforms evaluated and did not markedly inhibit 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 RIF induction) at 120 µM indicating that at effective concentrations for inhibition of CYP3A activity (30-60 uM), AhR (arylhydrocarbon receptor), CAR (constitutive androstane receptor) and PXR (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 hepatocytes 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 CYP3A inhibitor in a long-term hepatocyte model. Erythromycin is an ideal model in these systems as its inhibitory potency is long-lived and it does not markedly induce or inhibit other major drug-metabolizing enzymes.
- cytochrome P450
- drug development/discovery
- drug-drug interactions
- enzyme induction
- enzyme inhibitors
- hepatocytes
- in vitro-in vivo prediction (IVIVE)
- liver/hepatic
- nuclear receptors
- The American Society for Pharmacology and Experimental Therapeutics