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 longerterm 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 mM, respectively, for up to 72 hours. At 30 mM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CL int was inhibited 58% by 3 mM of ERY, 75% by 15 mM of ERY, 89% by 30 mM of ERY, and 94% by 60 mM of ERY. ERY (30 mM) did not markedly affect CL int 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 mM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 mM), 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 mM 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 STATEMENTThis 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.

Effects of Cytochrome P450 3A4 Induction and Inhibition on the Pharmacokinetics of BI 425809, a Novel Glycine Transporter 1 Inhibitor

“…Micropatterned co-cultures of human hepatocytes (from a male donor, 31 years of age) and mouse fibroblasts (HepatoPac®, BioIVT Inc.) were used to assess the metabolism of BI 425809 by CYP3A [ 12 ]. The co-cultures were received in a 96-well plate in maintenance media containing 10% foetal bovine serum.…”

“…Next, 32 μl probing medium was added to each well of the 96-well plate, which was then placed into an incubator for 15 min in 10% carbon dioxide at 37 °C. [12]. Semi-quantitative data using the ratio of the peak area response of the analyte and internal standard (nevirapine for alprazolam and 13 C6-BI 425809) were used.…”

Latest impact of engineered human liver platforms on drug development

“… 59 The same company also showed that erythromycin, an inhibitor of CYP3A4, significantly inhibited in MPCCs the clearance of midazolam and alprazolam, two CYP3A4 substrates. 60 Finally, Vertex showed that drug-mediated induction of CYP2C enzymes was higher in MPCCs than in PHH monocultures; 61 furthermore, EC50 values (drug dose that causes 50% of maximal CYP induction) in MPCCs for rifampin-mediated CYP3A4 and CYP2C9 induction were more clinically relevant than monocultures, which was subsequently found to be due to higher uptake transporter activities in MPCCs that leads to more intracellular accumulation of drugs like rifampin than the monocultures, which were shown to have reduced transporter activities. 62 …”

Addressing Today’s Absorption, Distribution, Metabolism, and Excretion (ADME) Challenges in the Translation of In Vitro ADME Characteristics to Humans: A Case Study of theSMN2mRNA Splicing Modifier Risdiplam

“…This demonstrated that FMO and CYP3A enzymes are important contributors to risdiplam metabolism and provided potential f m ranges for planning of a definitive in vivo DDI study. Since the time of performing these studies the first demonstrations of chemical inhibitor effectiveness and application to f m estimation using long-term hepatocyte cultures have started to emerge (Chan et al, 2020). These approaches will give additional opportunities for f m estimation, based upon total drug depletion, when such validation is available for a panel of selective inhibitors.…”