Production of morphinone as a metabolite of morphine and its physiological role

Morphine is a potent analgesic and is widely used in the clinical management of severe acute and chronic pain; however, its clinical usefulness is limited due to the development of both tolerance and dependence after repeated morphine administration. The morphine metabolism has been studied in order to elucidate its pharmacological actions as well as its adverse effects. Thus far several metabolites have been identified and their analgesic potency and toxicity have been also investigated. In the toxicological viewpoint, the production of reactive metabolites that can bind cellular glutathione and protein has been postulated. We found morphine 6-dehydrogenase, which catalyzes the dehydrogenation of 6-hydroxy group of morphine to produce morphinone, in the guinea pig liver. It was also found that morphinone antagonizes the morphine analgesia and binds with glutathione and protein. We here demonstrate the presence of a metabolic pathway of morphine to morphinone and subsequently to morphinone-glutathione adduct, and compare the property including primary structure among the guinea pig, rabbit, mouse and hamster liver morphine 6-dehydrogenases. We also describe the toxicological significance of morphinone.     

The effects of opiate receptor agonists and antagonists on the stress-induced secretion of corticosterone in mice.

1 Intraperitoneal administration of normorphine, morphine or naloxone or exposure to ether vapour for 1 min, elevated plasma corticosteroid concentrations in mice. 2 Injection of saline or exposure to ether vapour rendered mice less sensitive to a subsequent exposure to ether vapour 15 min later. 3 Treatment with normorphine (50 mg/kg) potentiated the corticosteroid response to ether stress whilst pentazocine (20 mg/kg), naltrexone (10 mg/kg), morphine (24 mg/kg), levorphanol (20 mg/kg) and naloxone (50 mg/kg) prevented the stress-induced elevation of plasma corticosteroids. 4 Both naloxone and morphine inhibited the potentiation by normorphine of the response to ether, the dose of naloxone required being higher than that for inhibition of normorphine analgesia. 5 It is concluded that endogenous opioid peptides may be involved in the control of the response to ether stress in mice.     

Stimulation mechanism of guinea pig liver-mediated reduction of naloxone by morphine

The mechanism of the stimulatory effect of morphine on the reduction of naloxone has been elucidated using guinea pig liver naloxone reductase that is identical with morphine 6-dehydrogenase. The reaction products were quantitated by means of HPLC. When naloxone was incubated with the enzyme in the presence of NAD(P)H at pH 7.4 or pH optima, the production of 6 alpha-naloxol increased according to the added amount of morphine. The stimulation was predominant with NADH at pH 7.4. Under these conditions, the production of morphinone also increased in proportion to the amount of morphine. The enzymatic reduction of naloxone proceeded even if NAD(P)H was replaced by NAD(P)+ and morphine. At a fairly low concentration of NADH (0.01 mM), the enzyme produced 6 alpha-naloxol (0.3 mM), exceeding the stoichiometric amount in the presence of 16 mM morphine. Although the Vmax values for naloxone was increased by the addition of morphine, the Km values for naloxone remained unaltered. Besides other substrates for guinea pig liver morphine 6-dehydrogenase such as codeine, normorphine and ethylmorphine also enhanced the reduction of naloxone. From these results we concluded that the stimulation of guinea pig liver-mediated reduction of naloxone by morphine is caused by the acceleration of the redox of pyridine nucleotides conducted by the enzyme. These phenomena were further supported by the experiments with the liver cytosol. In addition, we confirmed that, in the guinea pig, the biliary excretion of the metabolites, naloxol and naloxol-3-glucuronide, increased after sc injection of naloxone with morphine.     

Acute opioid physical dependence in humans: effect of naloxone at 6 and 24 hours postmorphine

Previous studies in our laboratory have documented the occurrence of naloxone-precipitated opioid abstinence from 45 minutes to 6 hours after acute morphine administration in humans. This study extended the morphine-naloxone interval to 24 hours and examined the effect of repeated naloxone challenges on withdrawal responses. Six male nondependent opiate users participated in eight experimental sessions in which they received single IM injections of morphine (18 mg/70 kg) followed 6 and 24 hours later by challenge sessions with IM placebo or naloxone (10 mg/70 kg). Naloxone challenge at 6 hours postmorphine reversed morphine-induced miosis and subjective reports of opiate symptoms, drug high, good drug effects, and drug liking. At 24 hours postmorphine, naloxone had no effect on these measures, which had returned to premorphine levels. However, at 6 and 24 hours postmorphine, naloxone precipitated subjective symptoms and observer-rated signs of opioid abstinence. When naloxone challenge at 24 hours was preceded by naloxone at 6 hours postmorphine, the magnitude of abstinence symptoms and signs was attenuated. These data suggest that morphine-induced adaptational changes underlying the development of physical dependence persist beyond other measureable agonist effects, and that these changes are disrupted or reversed by repeated antagonist administration.     

Bioactivation of morphine in human liver: isolation and identification of morphinone, a toxic metabolite

Morphinone, identified in the bile of guinea pigs and rats given morphine, is a reactive electrophile and has the ability to bind to glutathione (GSH) and tissue macromolecules, leading to GSH depletion and cell damage. We previously demonstrated that the livers of various animal species are capable of forming morphinone from morphine. In this study, we examined whether the human liver can produce morphinone from morphine. HPLC analysis revealed that the incubation of morphine with the 9000xg supernatant of human liver in the presence of NAD(P) and 2-mercaptoethanol (ME) gave a peak corresponding to the synthetic morphinone-ME adduct (MO-ME), which is readily formed by a nonenzymatic reaction of morphinone with ME. The reaction product was isolated and was unambiguously identified as MO-ME using FAB-MS and NMR analyses in comparison with synthetic MO-ME. The conversion of morphine to morphinone required NAD(P), and NAD was a preferred cofactor over NADP. All the 9000xg supernatants from six human livers could produce morphinone at different rates, ranging from 30 to 120 nmol/g liver/30 min with NAD at pH 7.4. The enzyme activity responsible for the formation of morphinone from morphine was mainly localized in the microsomes. The microsomal enzyme activity was inhibited by steroids, lithocholic acid and indomethacin. Among these compounds, steroids with a 17beta-hydroxyl group almost completely depressed morphinone formation. In conclusion, the metabolic pathway of morphine to morphinone, a toxic metabolite, in human was shown for the first time in in vitro experiments.     

Naloxone, not proglumide or MK-801, alters effects of morphine preexposure on morphine-induced taste aversions

Both cholecystokinin (CCK) antagonists and N-methyl-D-aspartate (NMDA) antagonists block or reduce the development of morphine tolerance in several analgesic assays. The present experiments were performed to assess the ability of the CCK antagonist proglumide and the NMDA antagonist MK-801 to affect tolerance to the aversive properties of morphine as indexed by conditioned taste aversion (CTA) learning. Specifically, male Sprague-Dawley rats were exposed to either vehicle or morphine (5 mg/kg) in combination with either proglumide (5 mg/kg; Experiment 1), MK-801 (0.1 mg/kg; Experiment 2) or naloxone (1, 3.2 mg/kg; Experiment 3). Saccharin was then presented and was followed by an injection of either vehicle or morphine (10 mg/kg). Animals preexposed to and conditioned with morphine acquired an attenuated morphine-induced aversion to saccharin. While neither proglumide nor MK-801 had an effect on this attenuation, naloxone blocked the effects of morphine preexposure, suggesting that neither CCK nor NMDA may be involved in the aversive effects of morphine (or their modulation by drug exposure). That the attenuating effects of morphine preexposure on a morphine-induced CTA can be blocked suggests that the weakening of the aversive effects of morphine with chronic use can be prevented, an effect that may have implications for overall drug acceptability.     

Effects of morphine on myenteric plexus neurones

Intracellular recordings were made from neurones in the isolated myenteric plexus of the guinea-pig ileum. Approximately 50% of neurones responded to bath application of normorphine (10 nM?1 μM) with an immediate hyperpolarization. The hyperpolarization was usually associated with a fall in membrane resistance. The effect reversed on washing out the normorphine and was prevented by the addition of the opiate receptor antagonist naloxone (50–100 nM). It is suggested that the membrane hyperpolarization may be the basis for the inhibitory effects of morphine on neurone firing rates which have been reported both in the myenteric plexus and the central nervous system.     

Inhibition of neuroeffector transmission by morphine in the vas deferens of naive and morphine-treated mice.

1 The amplitude of excitatory junction potentials (e.j.ps) recorded intracellularly from smooth muscle cells of the mouse vas deferens varied with the strength of stimulation. Normorphine (0.4, 2 and 10 microM) shifted the stimulus-response curve to the right, without any change in slope. This shift of the curve was proportional to the concentration of the opiate in the bath. Naloxone (0.4 and 2 microM) antagonized this effect of normorphine. 2 The action of normorphine (2 and 10 microM) was studied in vasa deferentia prepared from control mice and mice that had been implanted with morphine pellets. Both groups of tissues were continuously exposed to a low concentration of normorphine (0.4 microM), to simulate the plasma concentration in the morphine-treated mice. Addition of 10 microM normorphine produced a parallel displacement of the curve in vasa deferentia from control animals, and a non-parallel displacement in tissues from morphine pellet-implanted mice. In the preparations from morphine-treated mice a pronounced degree of tolerance to normorphine was observed at a low stimulus strength. 3 Naloxone (0.4 and 2 microM) had a greater effect on vasa deferentia prepared from morphine-treated animals than on tissues from control mice, when both organs were continuously exposed to 0.4 microM normorphine. The difference in the effect of the antagonist in the two groups of preparations was absent when the incubating solution contained 2 microM normorphine. 4 It is concluded that a low intensity of stimulation the e.j.ps are more readily depressed by normorphine and also the degree of tolerance displayed is larger than at a high intensity of stimulation.     

Influence of morphine and naloxone on the release of noradrenaline from rat cerebellar cortex slices

Summary In slices of rat cerebellar cortex preincubated with (-)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow was not changed by 10^–5 M of either morphine or naloxone. On the other hand, morphine caused a concentration-dependent decrease of the overflow, of tritium evoked by electrical field stimulation. Naloxone did not change the stimulation-induced overflow, but prevented its inhibition by morphine. It is concluded that morphine, through an action on opiate receptors located on cerebellar noradrenergic neurones, inhibits the secretion of the transmitter in response to nerve impulses.     

The metabolism of N-acetyl-3,5-dimethyl-p-benzoquinone imine in isolated hepatocytes involves N-deacetylation

3,5-Dimethyl-N-acetyl-p-benzoquinone imine (3,5-dimethyl-NAPQI) was cytotoxic to isolated hepatocytes from Sprague Dawley rats at levels between 200 and 300 microM. It rapidly oxidized intracellular glutathione within 10 sec, with the formation of oxidized glutathione. The cytotoxicity of 3,5-dimethyl-NAPQI could be prevented over a 3.5-hr period with the carboxylesterase inhibitor bis(p-nitrophenyl) phosphate, indicating that cytotoxicity involved N-deacetylation. The N-deacetylated product could be trapped with glutathione as 3-(glutathion-S-yl)-4-amino-2,6-dimethylphenol in 3,5-dimethyl-NAPQI-treated hepatocytes but not in hepatocytes pretreated with bis(p-nitrophenyl) phosphate, indicating that N-deacetylation activity had been inhibited. 3,5-Dimethyl-NAPQI was readily N-deacetylated by rat liver microsomes, in contrast to 3,5-dimethylacetaminophen. The latter was also not cytotoxic to hepatocytes at up to 2 mM. The N-deacetylated product 4-amino-2,6-dimethylphenol rapidly underwent autoxidation to form 2,6-dimethylbenzoquinone imine and was highly cytotoxic to hepatocytes at 200-300 microM. The latter reacted with glutathione to give the above conjugate and no glutathione oxidation occurred. Dithioerythritol (2 mM) added at 10, 20, and 30 min after 3,5-dimethyl-NAPQI delayed but did not prevent cytotoxicity. Dithioerythritol also resulted in the partial restoration of GSH, presumably as a result of reduction of protein mixed disulphides. The mechanism of cytotoxicity of 3,5-dimethyl-NAPQI therefore appears to be a result of a combination of oxidative stress and deacetylation resulting in arylation.     

Interaction between histamine and morphine at the level of the hippocampus in the formalin-induced orofacial pain in rats

The present study explored the interaction between histaminergic and opioidergic systems at the level of the hippocampus in modulation of orofacial pain by intra-hippocampal microinjections of histamine, pyrilamine (an antagonist of histamine H(1) receptors), ranitidine (an antagonist of histamine H(2) receptors), morphine (an opioid receptor agonist) and naloxone (an opioid receptor antagonist) in separate and combined treatments. Orofacial pain was induced by subcutaneous (sc) injection of formalin (50 μl, 1%) in the upper lip region and the time spent face rubbing was recorded in 3 min blocks for 45 min. Formalin (sc) produced a marked biphasic (first phase: 0-3 min, second phase: 15-33 min) pain response. Histamine and morphine suppressed both phases of pain. Histamine increased morphine-induced antinociception. Pyrilamine and ranitidine had no effects when used alone, whereas pretreatments with pyrilamine and ranitidine prevented histamine- and morphine-induced antinociceptive effects. Naloxone alone non-significantly increased pain intensity and inhibited the antinociceptive effects of morphine and histamine. The results of the present study indicate that at the level of the hippocampus, histamine through its H(1) and H(2) receptors, mediates orofacial region pain. Moreover, morphine via a naloxone-reversible mechanism produces analgesia. In addition, both histamine H(1) and H(2) receptors, as well as opioid receptors may be involved in the interaction between histamine and morphine in producing analgesia.     

Model of opiate dependence in the guinea-pig isolated ileum

1 Segments of ileum, incubated for 2-24 h at 22°C with normorphine (0.01 - 1.0 μM), in the presence of hexamethonium, contracted when challenged with naloxone (0.03 μM). No response to this dose of naloxone was induced either by incubation in control solution without opiate for 2-24 h or by exposure of the preparation to opiate for 30 min at 37°C.

2 When segments were incubated for 24 h, the size of the response to naloxone was directly related both to the normorphine concentration in the incubation fluid (0.01 to 0.1 μM), and to the concentration of naloxone applied (0.03 to 0.1 μM).

3 A spontaneous withdrawal contracture was elicited in ilea that had been incubated with normorphine (1.0 μM), when the normorphine-containing bathing fluid was exchanged for one without opiate.

4 Normorphine restored to resting level the tension of the withdrawal contracture, whether it had been elicited spontaneously or by naloxone challenge.

5 Addition of naloxone (1.0 μM) to normorphine (1.0 μM) in the incubation fluid abolished the withdrawal contracture to subsequent challenge with naloxone.

6 Naloxone elicited a contracture from segments incubated for 24 h at 22°C with levorphanol (0.1 μM) but not from those incubated with dextrorphan.

7 Application of (+)-naloxone (0.03 μM) to segments previously incubated with normorphine (0.1 μM) did not elicit a contracture.

8 The contracture elicited by naloxone in preparations incubated with morphine (10 μM) was associated with a reduction in sensitivity to the acute inhibitory effect of morphine on the electrically-evoked response.

9 Addition of hyoscine (0.5 μM) immediately after challenge with naloxone restored the tension of the withdrawal contracture to resting level.

10 Tetrodotoxin (3.0 μM) given before challenge, prevented naloxone from eliciting a withdrawal contracture.

11 The inclusion of 5-hydroxytryptamine (10 μM) with morphine (10 μM) inhibited the induction of tolerance to morphine.

12 These experiments, together with those described earlier, indicate that incubation with opiate induces a dependence in the final cholinergic motor neurones of the myenteric plexus, manifested as a contracture of the longitudinal muscle on removal of opiate or administration of an antagonist. This dependence is associated with tolerance, expressed as a decrease in sensitivity to inhibition by morphine of the electrically-evoked contracture. Tolerance and dependence are induced and withdrawal precipitated through specific and stereospecific opiate receptors.

     

Covalent binding of morphine to isolated rat hepatocytes.

Incubation of [3H]morphine with isolated hepatocytes caused covalent binding of [3H]-morphine to hepatocellular proteins. Sulfhydryl compounds protected against morphine-induced toxicity and decreased covalent binding. Analysis of covalently bound proteins in the cytosol by electrophoresis indicated that covalently bound radiolabel was associated with macromolecules greater than 25 kDa and increased throughout the incubation. In contrast, covalent binding to the particulate fraction was highly selectively associated with three protein bands of 50-53 and 33 kDa. Covalent binding of morphine to particulate fraction proteins was observed in hepatocytes which exhibited cellular damage. We conclude that the covalent binding of morphine to protein is associated with morphine-induced cytotoxicity.     

Naloxone inhibits and morphine potentiates the adrenal steroidogenic response to ACTH

The administration of morphine to hypophysectomized rats potentiated the steroidogenic response of the adrenal cortex to exogenous adrenocorticotrophic hormone (ACTH) in a dose-dependent fashion. Conversely, the opiate antagonist naloxone inhibited the adrenal response to ACTH. Naloxone pretreatment also antagonized the potentiating effect of morphine on ACTH-induced steroidogenesis in a dose-dependent manner. Neither morphine nor naloxone, administered to hypophysectomized rats, had any direct effect on adrenal steroidogenesis. These adrenal actions were stereospecific since neither the (+)-stereoisomer of morphine, nor that or naloxone, had any effect on the adrenal response to ACTH. The administration of human beta-endorphin to hypophysectomized rats had no effect on the adrenal corticosterone concentration nor did it alter the response of the adrenal gland to ACTH. These results indicate that morphine can potentiate the action of ACTH on the adrenal by a direct, stereospecific, dose-dependent mechanism that is prevented by naloxone pretreatment and which may involve competition for ACTH receptors on the corticosterone-secreting cells of the adrenal cortex.     

Effects of d-amphetamine,morphine, naloxone,and drug combinations on visual discrimination in rats

The effects of d-amphetamine, morphine, and naloxone on visual discrimination were investigated using a two-choice discrete-trial procedure in which rats were trained to discriminate the position of a lightflash. Morphine (0.3–5.6 mg/kg) but not amphetamine (0.1–1.0 mg/kg) caused a significant dose-dependent disruption in discriminative performance. Both amphetamine and morphine increased response latencies. Naloxone (1.0 mg/kg) prevented the disruption of any aspect of performance by up to 100 mg/kg morphine. Performance after naloxone/amphetamine co-administration was not significantly different from that observed after amphetamine alone. Naloxone alone (0.3–10 mg/kg) had no effect on discrimination, spatial bias or response latencies. These results suggest that morphine and amphetamine affect different components of discrimination performance. Offprint requests to: S.G. Holzman     

Effect of morphine on circling behaviour in rats with 6-hydroxydopamine striatal lesions and electrolesions of the raphe nucleus

Morphine antagonized d-amphetamine circling in rats which had received unilateral 6-OHDA lesions of the striatum but failed to reduce the circling in rats with both a unilateral 6-OHDA striatal lesion and a raphe (5-HT) lesion. Naloxone precipitated withdrawal of morphine tolerant rats greatly enhanced d-amphetamine circling when the rats had a 6-OHDA lesion but not when both 6-OHDA and raphe lesions were present. It is concluded that 5-HT is necessary for the morphine-induced inhibition of the circling. The effect of morphine tolerance and naloxone precipitated withdrawal on brain 5-HT function was investigated using a putative 5-HT rotation model in which both dopamine and a 5-HT agonist were administered to rats with an asymmetrical medial raphe lesion. The findings suggest that chronic treatment with morphine increases striatal 5-HT function.     

Facilitation of morphine-induced tolerance and physical dependence by prolyl-leucyl-glycinamide

The pretreatment of mice with 30 mg/kg morphine s.c. did not alter the analgesic effect of morphine in mice pretreated with saline but decreased the analgesic effect of morphine in mice pretreated with prolyl-leucyl-glycinamide (PLG). Tolerance was evaluated by the effect of PLG on morphine-induced enhancement of naloxone potency which is a measure of the capacity of naloxone to antagonize morphine-induced analgesia and is postulated to be an indicator of tolerance development. The naloxone potency of PLG-treated mice was 2-fold greater than that of control mice. PLG did not alter the whole brain levels of morphine, nor did it alter the naloxone potency in mice which were not pretreated with 30 mg/kg morphine. In mice treated with 100 mg/kg morphine or implanted with 50 mg morphine pellets for 24 or 72 h, the amount of naloxone required to induce jumping was not altered by PLG. However, PLG treatment did increase the hypothermia and body weight loss seen after naloxone-induced withdrawal. Administration of PLG to morphine-dependent mice 1 h prior to naloxone did not modify the resultant hypothermia or body weight loss. These results indicate that PLG facilitated the development of morphine tolerance and dependence.     

In vivo evidence for the selectivity of ICI 154129 for the delta-opioid receptor

The in vivo selectivity of the novel delta opioid-receptor antagonist N,N-bisallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154129) was examined in several opioid-selective models. Antagonism at the delta receptor was demonstrated in the striatal head-turn model in the rat. Intrapallidal injection of the relatively selective delta-receptor agonist D-Ala2,D-Leu5-enkephalin (0.5 micrograms) slowed the head-turn time and this effect was completely prevented by prior subcutaneous administration of ICI 154129 (30 mg/kg). The role of delta receptors in two classical test situations was studied using the mixed opioid agonist etorphine and the antagonists naloxone and ICI 154129. The drug ICI 154129 (30 mg/kg, s.c.) failed to prevent the antinociceptive effects and stimulation of locomotor activity produced by etorphine, whereas the relatively selective mu-opioid receptor antagonist, naloxone was effective in both test situations. The possible involvement of delta receptors in morphine-induced dependence was studied by monitoring the abstinence behaviour precipitated in rats given pellets of morphine by either ICI 154129 or naloxone. Naloxone (0.5 mg/kg, i.p.) precipitated a characteristic withdrawal syndrome in conscious rats and, at a much smaller dose (0.02 mg/kg, i.p.), induced shaking behaviour in pentobarbitone-anaesthetised rats. No withdrawal signs were observed in either model after injection of ICI 154129 (30 mg/kg, s.c.), suggesting that the delta receptors are not involved in dependence on morphine.     

Plasma beta-endorphin and cortisol levels in morphine-tolerant rats and in naloxone-induced withdrawal

The relationship between morphine tolerance and pituitary-adrenocortical activity was examined. In rats made tolerant to morphine by implantation of morphine-containing pellets, there was a significant reduction in plasma levels of beta-endorphin-like immunoreactivity (beta-END-LI), whereas no significant changes in cortisol levels were seen. Naloxone treatment induced an increase in plasma beta-END-LI and cortisol levels in morphine-tolerant animals. Additionally, acute morphine administration induced an increase in plasma levels of beta-END-LI and cortisol, an effect which was prevented by naloxone. These results are consistent with an increased release of pro-opiomelanocortin-derived peptides after acute morphine and with a decreased release of these peptides in tolerant rats, and suggest that opioid peptides play an important role in the regulation of pituitary-adrenocortical function.     

Morphine inhibits the carrageenan-induced oedema and the chemoluminescence of leucocytes stimulated by zymosan

Morphine inhibited the oedema formation induced by carrageenan. The anti-inflammatory activity developed 120 min after carrageenan injection, suggesting that inhibition of the kinin phase might be partly responsible. This assumption is supported by the findings that morphine inhibited bradykinin oedema but did not influence oedema formation induced by histamine, 5-HT or PGE2. The anti-inflammatory activity of morphine was partially inhibited by naloxone (0.5-1 mg kg-1) in the carrageenan oedema test. Zymosan-stimulated chemoluminescence of neutrophils of the rat was inhibited both by morphine (0.1-10 microM) and naloxone (1-100 microM). When morphine and naloxone were administered simultaneously (10 microM) their inhibitory effects were additive. Naloxone also failed to antagonize the inhibitory action of morphine in lower dose (0.1 microM). These results suggest that the effect of morphine in inflammation might be mediated either by one of the opiate receptor subtypes insensitive to naloxone or a non-opiate mechanism might be involved.