Differential in vitro inhibition of M3G and M6G formation from morphine by (R)- and (S)-methadone and structurally related opioids

Abstract

Aims: To determine the in vitro kinetics of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation and the inhibition potential by methadone enantiomers and structurally related opioids.

Methods: M3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 microm) and the mechanism of inhibition determined for (R)- and (S)-methadone (70-500 microm) using three microsomal samples.

Results: Glucuronide formation displayed single enzyme kinetics. The M3G Vmax (mean+/-SD) was 4.8-fold greater than M6G Vmax (555+/-110 vs. 115+/-19 nmol mg-1 protein h-1; P=0.006, mean of difference 439; 95% confidence interval 313, 565 nmol mg-1 protein h-1). Km values for M3G and M6G formation were not significantly different (1.12+/-0.37 vs. 1.11+/-0.31 mm; P=0.89, 0.02; -0.29, 0.32 mm). M3G and M6G formation was inhibited (P<0.01) with a significant increase in the M3G/M6G ratio (P<0.01) for all compounds tested. Detailed analysis with (R)- and (S)-methadone revealed noncompetitive inhibition with (R)-methadone Ki of 320+/-42 microm and 192+/-12 microm for M3G and M6G, respectively, and (S)-methadone Ki of 226+/-30 microm and 152+/-20 microm for M3G and M6G, respectively. Ki values for M3G inhibition were significantly greater than for M6G for (R)-methadone (P=0.017, 128; 55, 202 microm) and (S)-methadone (P=0.026, 75; 22, 128 microm).

Conclusions: Both methadone enantiomers noncompetitively inhibited the formation of morphine's primary metabolites, with greater inhibition of M6G formation compared with M3G. These findings indicate a mechanism for reduced morphine clearance in methadone-maintained patients and reduced relative formation of the opioid active M6G compared with M3G.

Figure 1

The effect of various concentrations of alamethicin on the formation of M3G (▪) and M6G (▴) from 10 mm(A) and 1 mm (B) morphine in human liver microsomes (0,4,8 µg alamethicin, n = 4 replicates; 2,6,12,16,20 µg alamethicin, n = 2 replicates). Data are expressed as mean ± SD

Figure 2

Eadie–Hosftee plot (A) and Michaelis–Menten plot (V vs. [S] mm) (B) of M3G (▪) formation from morphine. Eadie–Hosftee plot (C) and Michaelis–Menten plot (V vs. [S] mm) (D) of M6G (▴) formation from morphine. All plots using HLM no. 18. Solid line represents the single enzyme Michaelis–Menten equation fitted to the data

Figure 3

Percentage inhibition of M3G (□) and M6G (▪) formation (compared with control formation) by the individual methadone enantiomers and structurally related opioids (n = 3 human liver microsomes). Data are expressed as mean ± SD. *P < 0.01 compared with formation when no inhibitor was present (control). #P < 0.03 compared with inhibition of M3G formation

Figure 4

Michaelis–Menten noncompetitive inhibition plot of M3G (A) and M6G (B) formation from morphine in the presence of 0 (▪), 70 (▴), 100 (▾), 200 (♦), and 500 (•) µM of inhibitor ((R)-methadone). All plots using HLM no. 36. Solid lines represent the noncompetitive inhibition Michaelis–Menten equation fitted to the data