Morphine Glucuronidation and Glucosidation Represent Complementary Metabolic Pathways That Are Both Catalyzed by UDP-Glucuronosyltransferase 2B7: Kinetic, Inhibition, and Molecular Modeling Studies

Abstract

Morphine 3-b-D-glucuronide (M3G) and morphine 6-b-D-glucuronide (M6G) are the major metabolites of morphine in humans. More recently, morphine-3-b-D-glucoside (M-3-glucoside) was identified in the urine of patients treated with morphine. Kinetic and inhibition studies using human liver microsomes (HLM) and recombinant UGTs as enzyme sources along with molecular modeling were used here to characterize the relationship between morphine glucuronidation and glucosidation. The M3G to M6G intrinsic clearance (CL int ) ratio (∼5.5) from HLM supplemented with UDP-glucuronic acid (UDP-GlcUA) alone was consistent with the relative formation of these metabolites in humans. The mean CL int values observed for M-3-glucoside by incubations of HLM with UDP-glucose (UDP-Glc) as cofactor were approximately twice those for M6G formation. However, although the M3G-to-M6G CL int ratio remained close to 5.5 when human liver microsomal kinetic studies were performed in the presence of a 1: 1 mixture of cofactors, the mean CL int value for M-3-glucoside formation was less than that of M6G. Studies with UGT enzymeselective inhibitors and recombinant UGT enzymes, along with effects of BSA on morphine glycosidation kinetics, were consistent with a major role of UGT2B7 in both morphine glucuronidation and glucosidation. Molecular modeling identified key amino acids involved in the binding of UDP-GlcUA and UDP-Glc to UGT2B7. Mutagenesis of these residues abolished morphine glucuronidation and glucosidation. Overall, the data indicate that morphine glucuronidation and glucosidation occur as complementary metabolic pathways catalyzed by a common enzyme (UGT2B7). Glucuronidation is the dominant metabolic pathway because the binding affinity of UDP-GlcUA to UGT2B7 is higher than that of UDP-Glc.

Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes

“…The specific enzyme‐mediating glucosidation of MPA (either phenol or acyl) has not yet been determined. Results so far implicate UGT2B7 as the main enzyme that glucosidates morphine (Chau et al ., ), ibuprofen (Buchheit et al ., ), an experimental endothelin antagonist (Tang et al ., ), and hyodeoxycholic acid (Mackenzie et al ., ), while UGT2B15 was found to glucosidate an experimental aldose reductase inhibitor (Toide et al ., ). Our results indicate that cats may express a UGT that has a higher catalytic efficiency for glucosidation of MPA than those expressed by humans and dogs.…”

“…This is despite the ready availability of UDPglucose as a cofactor in hepatocytes. Although there are relatively few examples of drugs that are glucosidated to any significant extent in mammals, some of them include: morphine, phenobarbital, amobarbital, ibuprofen, and varenicline (Kalow et al, 1979;Arima & Kato, 1990;Bernus et al, 1994;Nandi & Soine, 1997;Chau et al, 2014). In most instances, the same drugs are also metabolized by glucuronidation with higher efficiency, although a few exceptions have been identified, including acyl glucosidation of panoprofen in mice (Arima & Kato, 1990), and N-glucosidation of amobarbital and phenobarbital in humans (Kalow et al, 1979;Bernus et al, 1994;Nandi & Soine, 1997).…”

Posttranscriptional regulation of uridine diphosphate glucuronosyltransferases

“…There is also some evidence that negative cooperative kinetics can occur when different UDP-sugars are co-incubated with UGTs. For example, morphine metabolism by UGT2B7 in the presence of UDPGA and UDP-glucose resulted in a 70% lower Cl int for morphine-3-glucoside than without UDPGA [96]. These in vitro experiments produced metabolite concentrations more relevant to in vivo and suggest that co-incubations with UDP-sugars could be useful in examining inhibition and activation of UGTs [96].…”

Stable isotope‐labelled morphine to study in vivo central and peripheral morphine glucuronidation and brain transport in tolerant mice

“…Chronic administration of medication (Sweeney and Bromilow, 2006) such as anticancer drugs (Hu et al, 2015), pain killers [codeine (Antonilli et al, 2012)], antibiotics [rifampin (Lee et al, 2006)] or antiepileptic drugs [phenobarbital (Sakakibara et al, 2016)] can crucially affect metabolic enzymes and ultimately lead to physiological adaptations. Among painkillers, opiate metabolism in the liver has been extensively studied following acute morphine treatment (Smith, 2009;Chau et al, 2014). Although morphine's analgesic effect mainly involves binding to the μ opioid receptors in the CNS, its central metabolism is poorly characterized (Laux-Biehlmann et al, 2013).…”