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

Abstract

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)-and (S)-methadone and structurally related opioids Department of Clinical Pharmacology, Royal Adelaide Hospital, Adelaide, Australia AimsTo 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. MethodsM3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 µ M ) and the mechanism of inhibition determined for (R)-and (S)-methadone (70-500 µ M ) using three microsomal samples. ResultsGlucuronide formation displayed single enzyme kinetics. The M3G V max (mean ± SD) was 4.8-fold greater than M6G V max (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 ). K m values for M3G and M6G formation were not significantly different (1.12 ± 0.37 vs. 1.11 ± 0.31 m M ; P = 0.89, 0.02; − 0.29, 0.32 m M ). 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 K i of 320 ± 42 µ M and 192 ± 12 µ M for M3G and M6G, respectively, and (S)-methadone K i of 226 ± 30 µ M and 152 ± 20 µ M for M3G and M6G, respectively. K i values for M3G inhibition were significantly greater than for M6G for (R)-methadone ( P = 0.017, 128; 55, 202 µ M ) and (S)-methadone ( P = 0.026, 75; 22, 128 µ M ). ConclusionsBoth 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 methadonemaintained patients and reduced relative formation of the opioid active M6G compared with M3G. IntroductionMorphine is the gold standard opioid for the treatment of moderate to severe acute and chronic pain. Morphine is primarily cleared via glucuronidation to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). M6G is a potent µ -opioid agonist [1] with clinical studies suggesting it contributes to analgesia after the administration of morphine [2,3]. M3G has no anal- [11,12,14]. Investigations with expressed recombinant human UDPglucuronosyltransferase (UGT) enzymes have shown UGT1A1, 1A3, 1A6, 1A8, 1A9, 1A10 and 2B7 all metabolize morphine to M3G [15][16][17]. However, only UGT2B7 has been shown to form M6G [17,18]. The relative contribution of these isoforms to the overall glucuronidation of morphine is still unknown. Only one investigation using human liver microsomes has shown the possible involvement of more than one UGT isoform in M3G formation, with the suggested involvement of a high-affinity, low-capacity enzyme (with a mean K m and V max of 5.3 µ M and 18 nmol mg − 1 protein h...

Methadone inhibits CYP2D6 and UGT2B7/2B4 in vivo: a study using codeine in methadone‐ and buprenorphine‐maintained subjects

“…The reason for this discrepancy between plasma and urine is uncertain. Nevertheless, the plasma metabolic ratio data are consistent with a previous study that found both the ( R )‐ and the ( S )‐methadone enantiomers noncompetitively inhibited UGT2B7 in vitro using liver microsomes, through reducing the formation of M3G and M6G from morphine [18].…”

“…The analgesic effect of codeine, therefore, depends on two metabolites, morphine and M6G. In the presence of methadone, however, the predominant enzyme responsible for the formation of both of these conjugated metabolites, UGT2B7 [16, 17], has been found to be noncompetitively inhibited in vitro , with the formation of M6G being more strongly inhibited than that of M3G [18]. Thus, giving morphine on its own, in the presence of methadone, may result in increased plasma morphine concentrations and reduced plasma M3G and M6G concentrations, the clinical consequences of which remain speculative.…”

In Vitro–In Vivo Extrapolation Predicts Drug–Drug Interactions Arising from Inhibition of Codeine Glucuronidation by Dextropropoxyphene, Fluconazole, Ketoconazole, and Methadone in Humans

“…Indeed, K i values obtained here for fluconazole (from incubations with and without BSA) of human liver microsomal COD glucuronidation were approximately 30% higher than the corresponding K i s we previously reported for inhibition of zidovudine glucuronidation (Uchaipichat et al, 2006b). K i values determined here for metha- Table 5 corrected for the predicted binding to incubation constituents (i.e., K i /fu inc ): dextropropoxyphene, 12 M; fluconazole, 220 M; ketoconazole, 7.5 M; and methadone, 0.6 M. Foster et al, 2004. done and ketoconazole inhibition of COD glucuronidation also differ from inhibition studies with the predominantly UGT2B7 substrate morphine, which reported less potent inhibition (Morrish et al, 2005;Takeda et al, 2006). While this may be caused in part by the differing contributions of UGT2B4 and UGT2B7 to COD and morphine (3-and 6-) glucuronidation and the differential inhibition of each enzyme by methadone, binding to HLM was not accounted for in the morphine inhibition studies and effects of BSA were not investigated.…”

“…In contrast, data from both in vitro and in vivo studies indicate that inhibition of UGT2B7 may potentially result in significant drug-drug interactions (DDIs), with reduced clearance via glucuronidation. For example, DDIs in vivo have been reported between fluconazole and zidovudine (Sahai et al, 1994) and methadone and zidovudine (McCance-Katz et al, 1998), whereas dextropropoxyphene, fluconazole, ketoconazole, methadone, and valproic acid have been shown to inhibit human liver microsomal morphine or zidovudine glucuronidation (Trapnell et al, 1998;Morrish et al, 2005;Takeda et al, 2006;Uchaipichat et al, 2006b).…”

From Pediatric Covariate Model to Semiphysiological Function for Maturation: Part II—Sensitivity to Physiological and Physicochemical Properties

“…The obtained drug‐specific parameters for each drug are presented in Table 2 . With respect to the Michaelis–Menten parameters, values for the formation of the two morphine glucuronides were obtained from the liver microsomes from five separate adults 25 and values for the glucuronidation of zidovudine were obtained from the liver microsomes from four adults 26 . To verify the obtained drug‐specific parameters, predictions of morphine and zidovudine clearances for 1,000 healthy adult volunteers were compared with reported clearance values in adults of 93 l/h for morphine 27 , 28 , 29 and 91.2 l/h for zidovudine 30 , 31 , 32 .…”