Methadone is one of the most widely used drugs to treat opiate dependency (Kreek et al., 1994). We have recently reported that there is discordance between genotype and in vivo CYP2D6 activity in patients undergoing methadone maintenance treatment (MMT) such that some extensive metabolisers are phenocopied to poor metabolisers (Shiran et al; 2003). This was consistent with the finding that methadone inhibits CYP2D6 activity in vitro (Wu et al; 1993). However, the latter study used concentrations that are much higher than those expected in the blood of MMT patients. Thus, we have investigated the mechanism-based inhibition of CYP2D6 by methadone as a possible explanation for the inconsistency in the above reports.

Microsomal preparations from yeast expressing CYP2D6 (n=3) and human liver (HLM, n=3) were co-incubated or pre-incubated for 5 and 15 min respectively with methadone (0, 2.5, 5, 10, 15, 32, 48, 100 µM) in the presence of an NADPH generating system. Samples were then assayed for remaining CYP2D6 activity using dextromethorphan (5, 10 and 20 µM) as the substrate (5 min for yeast and 10 min for HLM).

Methadone inhibited CYP2D6 activity in yeast (mean IC₅₀ = 12 and 37 µM at 5 and 20 µM dextromethorphan, respectively) and HLM (mean IC₅₀ =128 µM for 3 livers at 20 µM dextromethorphan). Pre-incubation of methadone did not appear to increase the degree of inhibition of CYP2D6 activity in either yeast or HLM (Figure 1).

Thus, our findings confirm that methadone is a weak to moderate competitive inhibitor, but not a mechanism based inhibitor of CYP2D6. Unbound concentrations of methadone in vivo could be as low as 0.1-0.25 µM assuming fu=0.127 (Eap et al; 1990). Therefore, the suppressed CYP2D6 activity observed in MMT patients (Shiran et al; 2003), is not consistent with the degree of inhibition observed in our study. Decreased CYP2D6 activity in MMT patients may be related to the effects of other substances taken by these patients (e.g. heroine, morphine), or the metabolites of methadone, and/or to the active uptake of the methadone into hepatocytes.

Figure 1. The effects of co-incubation and pre-incubation of methadone on the activity of CYP2D6 expressed in yeast.

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