In vitro evidence for the involvement of at least two forms of human liver UDP-glucuronosyltransferase in morphine 3-glucuronidation

Miners, John O.; Lillywhite, K. J.; Birkett, Donald

doi:10.1016/0006-2952(88)90048-2

Cited by 50 publications

(20 citation statements)

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“…The present study using morphine concentrations of 0.1-10 mM produced linear Eadie-Hofstee plots, consistent with the involvement of a single enzyme or multiple enzymes with similar affinity constants, and is in agreement with the majority of previous studies using human liver microsomes. In contrast, the only group to date to investigate morphine glucuronidation at morphine concentrations below 0.1 mM (2.5-5000 µM) observed nonlinear Eadie-Hofstee plots for the formation of M3G in human liver microsomes and concluded that two enzymes were involved [19]. The present study was limited to concentrations above 0.1 mM as morphine concentrations below this would …”

Section: Discussionmentioning

confidence: 64%

“…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 − 1 ) and lowaffinity, high-capacity enzyme (with mean K m and V max of 1203 µ M and 653 nmol mg − 1 protein h − 1 ) [19]. Methadone, a µ -opioid receptor agonist, is the most widely used substitution treatment for opioid dependence [20] and is becoming increasingly accepted as a treatment for cancer pain [21].…”

Section: Introductionmentioning

confidence: 99%

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

Morrish

Foster

Somogyi

2005

Brit J Clinical Pharma

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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...

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Section: Discussionmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

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

Morrish

Foster

Somogyi

2005

Brit J Clinical Pharma

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“…That is, the inhibitory actions of repeated heroin on morphine glucuronidation were not dependent on the presence of heroin in the microsomal preparations at the moment of testing, as entailed by a mechanism of action based on competitive inhibition. Indeed, the reported ability of codeine, flunitrazepam, chloramphenicol, probenecid, and amitriptyline to act as competitive inhibitors of morphine UGTs (Puig and Tephly, 1986;Miners et al, 1988;Thomassin and Tephly, 1990;Yue et al, 1990) requires in vitro incubation with these drugs. Also, the ability of repeated heroin to increase the synthesis of M6G is not easy to explain.…”

Section: Discussionmentioning

confidence: 99%

Repeated Exposures to Heroin and/or Cadmium Alter the Rate of Formation of Morphine Glucuronides in the Rat

Antonilli¹,

Suriano²,

Paolone³

et al. 2003

J Pharmacol Exp Ther

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After absorption, heroin is transformed into mono-acetyl-morphine and then into morphine. Morphine, in turn, is metabolized to morphine-3-glucuronide (M3G), an inactive compound, and morphine-6-glucuronide (M6G), a potent opioid agonist. Thus, changes in the rate of formation of M6G may alter the pharmacological consequences of a treatment with heroin or morphine. In this study, we investigate the effect of repeated exposures (10 daily i.p. injections) to heroin, morphine, cadmium (which has been previously shown to inhibit M3G formation in vitro), or heroin ϩ cadmium on morphine glucuronidation both in vivo and ex vivo (i.e., microsomal preparation obtained from rats treated in vivo). Repeated heroin (2.5, 5.0, and 10 mg/kg) increased plasma levels of M6G (which was undetectable in all other groups) and reduced those of M3G. Also, the microsomal preparations obtained from the liver of repeated heroin rats, when incubated with morphine, yielded significant amounts of M6G (which was undetectable in all other groups) and decreased levels of M3G relative to the control groups. These effects were reversible upon discontinuation of heroin administration. In contrast, repeated morphine (10, 20, and 40 mg/kg) only slightly reduced M3G formation at the dose of 40 mg/kg. Repeated cadmium (5, 15, and 45 g/kg) reduced the rate of M3G formation without inducing M6G synthesis. The effects of the repeated coadministration of heroin (10 mg/kg) and cadmium (15 g/kg) were virtually identical to those of repeated heroin alone. In summary, repeated exposure of rats to heroin can shift morphine glucuronidation toward the formation of the active metabolite M6G.

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“…Indeed, there is evidence indicating that other isoforms have the capacity to glucuronidate morphine (Green et al, 1998;Cheng et al, 1999), and the apparent biphasic M3G formation kinetics in human liver microsomes (Miners et al, 1988) further suggests that multiple isoforms may catalyze the formation of this metabolite. We describe studies that aimed to characterize the selectivity and kinetics of M3G and M6G formation by recombinant human UGTs.…”

Section: Introductionmentioning

confidence: 99%

Isoform Selectivity and Kinetics of Morphine 3- And 6-Glucuronidation by Human Udp-Glucuronosyltransferases: Evidence for Atypical Glucuronidation Kinetics by Ugt2b7

Stone¹,

Mackenzie²,

Galetin³

et al. 2003

Drug Metab Dispos

189

152

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Morphine elimination involves UDP-glucuronosyltransferase (UGT) catalyzed conjugation with glucuronic acid to form morphine 3-and 6-glucuronides (M3G and M6G, respectively). It has been proposed that UGT2B7 is the major enzyme involved in these reactions, but there is evidence to suggest that other isoforms also catalyze morphine glucuronidation in man. Thus, we have characterized the selectivity and kinetics of M3G and M6G formation by recombinant human UGTs. UGT 1A1, 1A3, 1A6, 1A8, 1A9, 1A10, and 2B7 all catalyzed M3G formation, but only UGT2B7 formed M6G. The kinetics of M3G formation by the UGT1A family isoforms was consistent with a single enzyme Michaelis-Menten model, with apparent K m values ranging from 2.6 to 37.4 mM. In contrast, M3G and M6G formation by UGT2B7 exhibited atypical kinetics. The atypical kinetics may be described by a model with high-and low-affinity K m values (0.42 and 8.3 mM for M3G, and 0.97 and 7.4 mM for M6G) from fitting to a biphasic Michaelis-Menten model. However, a multisite model with an interaction between two identical binding sites in a negative cooperative manner provides a more realistic approach to modeling these data. According to this model, the respective binding affinities (K s ) for M3G and M6G were 1.76 and 1.41 mM, respectively. These data suggest that M6G formation may be used as a selective probe for UGT2B7 activity, and morphine glucuronidation by UGT2B7 appears to involve the simultaneous binding of two substrate molecules, highlighting the need for careful analysis of morphine glucuronidation kinetics in vitro.Conjugation with glucuronic acid is an important metabolic pathway for the inactivation and elimination of a myriad of compounds, including drugs, dietary chemicals, environmental pollutants, and endobiotics. In particular, glucuronidation represents an important clearance mechanism for drugs from all therapeutic classes (Miners and Mackenzie, 1991). Glucuronidation reactions are catalyzed by enzymes of the UDP-glucuronosyltransferase (UGT 1 ) gene superfamily. The individual forms of UGT (isoforms) tend to exhibit distinct, but overlapping, substrate specificities. Twenty-eight human UGT genes have been identified to date, and these have been classified into families and subfamilies based on evolutionary divergence . However, only 14 of the known human UGTs appear to be catalytically active: UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B15, 2B17, and 2B28.Morphine is the preferred opioid for the relief of moderate to severe pain. Conversion to morphine 3-and 6-glucuronides (M3G and M6G, respectively) accounts for approximately two-thirds of the elimination of a parenteral dose of morphine in humans (Milne et al., 1996). Morphine 3-glucuronidation is the dominant pathway, and metabolic clearance to M3G is, on average, 5.4-fold higher than metabolic clearance to M6G. Although the liver appears to be the principal organ responsible for morphine glucuronidation in v...

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In vitro evidence for the involvement of at least two forms of human liver UDP-glucuronosyltransferase in morphine 3-glucuronidation

Cited by 50 publications

References 16 publications

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

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

Repeated Exposures to Heroin and/or Cadmium Alter the Rate of Formation of Morphine Glucuronides in the Rat

Isoform Selectivity and Kinetics of Morphine 3- And 6-Glucuronidation by Human Udp-Glucuronosyltransferases: Evidence for Atypical Glucuronidation Kinetics by Ugt2b7

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