Bioactivation of Morphine in Human Liver: Isolation and Identification of Morphinone, a Toxic Metabolite

Abstract

Morphine is a potent analgesic and is used worldwide in the clinical management of severe acute and chronic pain. It is known, however, that the repeated administration of morphine results in tolerance and physical dependence. It has been shown that the treatment of the rat and mouse with high doses of morphine decreases the hepatic glutathione (GSH) content and increases the levels of serum transaminase activity.1-3) These adverse reactions of morphine have long been thought to be due, at least in part, to the generation of reactive metabolite(s) that can bind to GSH and tissue macromolecules. We found morphine 6-dehydrogenase, which catalyzes the conversion of morphine to morphinone in the guinea pig livers and purified the enzyme.4) The product with the enzyme was identified as the 2-mercaptoethanol (ME) adduct of morphinone (MO-ME) which was formed by nonenzymatic binding of morphinone to ME in the incubation medium via a Michael addition, indicating that morphinone is a reactive electrophile. This finding led us to the identification of the morphinone-GSH adduct (MO-GSH) in the bile of guinea pigs given morphine. 5) Thus the metabolic pathway of morphine to morphinone and subsequently to the MO-GSH was first demonstrated in the guinea pig (Fig. 1). The latter reaction was considered to occur mainly through a nonenzymatic binding of morphinone to GSH and, in part, in a reaction catalyzed by the GSH S-transferase.6) In vivo studies in the guinea pig 6) and rat 7) showed that most morphinone formed from morphine is commonly excreted as MO-GSH into the bile and that the sum of morphinone and MO-GSH accounted for about 10% in the guinea pig and 8% in the rat of the administered dose. These observations indicate that the pathway of morphine to morphinone catalyzed by morphine 6-dehydrogenase is one of the main routes in morphine metabolism, at least in guinea pigs and rats.In the mouse, morphinone is nine-fold more toxic than morphine 2) and is a potent antagonist of morphine. 8) This metabolite was also found to block naloxone binding irreversibly in the mitochondrial-synaptosomal fractions of the mouse brain, 8) to bind covalently to tissue macromolecules through sulfhydryl groups in the mouse, 9) and to cause hepatotoxicity in the rat.10,11) These observations suggest that morphinone formed from morphine may affect the analgesic action of morphine and has a potential role in morphine-induced toxicity including the development of tolerance, although most morphinone would be trapped by intracellular GSH to form MO-GSH and excreted readily into the bile. It is therefore of toxicologic and pharmacologic interest to assess the metabolic pathway of morphine to morphinone in animals and humans.We previously reported that the livers of rabbit, mouse, hamster, and bovine as well as guinea pig and rat have the ability to produce morphinone from morphine. 7) However, the in vivo and in vitro formation of morphinone and MO- Morphinone, identified in the bile of guinea pigs and rats given morphine, is a reactive electrophi...

Glutationa e enzimas relacionadas: papel biológico e importância em processos patológicos

“…Um dos caminhos envolve a participação da morfina 6-desidrogenase (M6D) na etapa de oxidação, levando à formação da morfinona 40. 51 A adição conjugada da glutationa à morfinona 40 fornece o conjugado 41 que é então excretado do meio celular (Figura 9C). 52 O outro caminho envolve as enzimas citocromo P450: a atuação destas enzimas sobre a morfina 39 produz a quinona 42, que após adição da glutationa (GSH) fornece o conjugado 43, que é então excretado.…”

Covalent Protein Adduction by Drugs of Abuse

“…Previous studies involving morphine's covalent adduction to biological sulfhydryls using NMR spectrometry have described two distinct products, one involving thiol attachment at C-10 of morphine 88,90 and the other at C-8 80,82,83 (represented by Figure 52a and b, respectively; see Section 2.6). Correia et al determined that adduction at the C-10 position was CYP-mediated in rats, requiring NADPH as a cofactor to elicit metabolic activation of morphine, although no specific mechanistic explanation was proposed.…”

“…This reduction event is catalyzed by the enzyme morphine-6-dehydrogenase and is not mediated by the CYP or FMO enzyme superfamilies (making the event atypical in the realm of Phase I xenobiotic biotransformation). 82,83 In place of NADPH, morphine-6-dehydrogenase utilizes NAD(P) + as an activating cofactor (with bias toward NAD + activation over NADP + in many species, including humans).…”

“…132 Furthermore, previous reports have demonstrated the formation of the morphine reduction product morphinone in human liver tissues. 82 However, the intrinsic reactivity of compounds containing α,β-unsaturated carbonyls such as morphinone would likely result in binding to nucleophilic targets in cellular media in vivo (e.g., glutathione, free thiols within proteins). 133,134 While morphinone was not detected in free fraction by the methods used in this study, it may be that any morphinone produced would be protein bound, with the free fraction of morphinone remaining below the detection limits of this method.…”

“…[80][81][82][83][84] Specific structural examination by Ishida et al determined the formation of the distinct 8α-stereospecific product. 85 Further in vivo and in vitro studies with mice and rats by Nagamatsu and colleagues have demonstrated that macromolecular binding of morphinone directly correlated with hepatotoxicity.…”