Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway.

Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating µ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.

Isobolographic analysis of the opioid-opioid interactions in a tonic and a phasic mouse model of induced nociceptive pain.

BACKGROUND: Opioids have been used for the management of pain and coadministration of two opioids may induce synergism. In a model of tonic pain, the acetic acid writhing test and in a phasic model, the hot plate, the antinociceptive interaction between fentanyl, methadone, morphine, and tramadol was evaluated. RESULTS: The potency of opioids in the writhing test compared to the hot plate assay was from 2.5 (fentanyl) to 15.5 (morphine) times, respectively. The ED50 was used in a fixed ratio for each of the six pairs of opioid combinations, which, resulted in a synergistic antinociception except for methadone/tramadol and fentanyl/tramadol which were additive, in the hot plate. The opioid antagonists naltrexone, naltrindole and nor-binaltorphimine, suggests that the synergism of morphine combinations are due to the activation of MOR subtypes with partially contribution of DOR and KOR, however fentanyl and methadone combinations are partially due to the activation of MOR and DOR subtypes and KOR lack of participation. The antinociceptive effects of tramadol combinations, are partially due to the activation of MOR, DOR and KOR opioid subtypes. CONCLUSION: These results suggets that effectiveness and magnitude of the interactions between opioids are dependent on pain stimulus intensity.

Inhibition of Gßγ-subunit signaling potentiates morphine-induced antinociception but not respiratory depression, constipation, locomotion, and reward.

Inhibition of Gßγ-subunit signaling to phospholipase C ß3 has been shown to potentiate morphine-mediated antinociception while attenuating the development of tolerance and dependence in mice. The objective of this study was to determine the effect of Gßγ-subunit inhibition on antinociception and other pharmacological effects, such as respiratory depression, constipation, and hyperlocomotion, mediated by the µ-opioid receptor. The Gßγ-subunit inhibitor, gallein, was administered to C57BL/6J mice by intraperitoneal injection before morphine, and data were compared with mice treated with vehicle, morphine, or gallein alone. Morphine-induced antinociception was measured using the 55°C warm-water tail-withdrawal test. Pretreatment with gallein produced a dose-dependent potentiation of morphine-mediated antinociception, producing up to a 10-fold leftward shift in the morphine dose-response curve and extending the duration of antinociception induced by a single dose of morphine. Gallein pretreatment also prevented acute antinociceptive tolerance induced by morphine. In contrast, the dose-dependent respiratory depression and hyperlocomotion induced by morphine were not potentiated by gallein pretreatment. Similarly, gallein pretreatment did not potentiate morphine-conditioned place preference responses or morphine-induced constipation, as measured as a reduction in excreta. These results suggest that selectively inhibiting Gßγ-mediated signaling may selectively increase µ-opioid receptor-mediated antinociception without matching increases in adverse physiological effects.

A novel knock-in mouse reveals mechanistically distinct forms of morphine tolerance.

The role of µ-opioid receptor (MOR) down-regulation in opioid tolerance remains controversial. In this study, we used a novel knock-in mouse to examine how changing the extent of MOR down-regulation alters the development of morphine tolerance. These mice express a mutant MOR, degrading MOR (DMOR), that differs from the wild-type (WT) MOR in two ways: 1) unlike the recycling WT MOR, the mutant DMOR is targeted for degradation after its internalization, thus facilitating down-regulation; and 2) unlike the WT MOR, DMOR is efficiently internalized in response to morphine activation. We found that both WT MOR and DMOR mice develop tolerance to morphine, but DMOR mice exhibit a more rapid onset of tolerance and show receptor down-regulation. WT MOR mice develop morphine tolerance more slowly but even once profoundly tolerant show no receptor down-regulation. Furthermore, WT mice show significantly more morphine dependence than DMOR mice after long-term treatment as indicated by withdrawal. Taken together these data indicate that tolerance mediated by receptor down-regulation manifests differently both at the behavioral and biochemical level than does the actual morphine tolerance that occurs in WT mice and that loss of receptor function is not a major contributor to morphine tolerance in WT MOR mice.

The roles of nerve growth factor and cholecystokinin in the enhancement of morphine analgesia in a rodent model of central nervous system inflammation.

Animal models of inflammatory pain are characterized by the release of inflammatory mediators such as cytokines and neurotrophic factors, and enhanced analgesic sensitivity to opioids. In this study, we examine the mechanisms underlying this effect, in particular the roles of cholecystokinin (CCK) and nerve growth factor (NGF), in an animal model of central nervous system (CNS) inflammation induced by spinal administration of lipopolysaccharide (LPS). Although spinal administration of LY-225910 (25 ng), a CCK-B antagonist, enhanced morphine analgesia in naïve rats, it was unable to do so in LPS-treated animals. Conversely, spinal CCK-8S administration (1 ng) decreased morphine analgesia in LPS-treated rats, but not in naïve animals. Further, spinal anti-NGF (3 microg) was able to reduce morphine analgesia in LPS-treated rats, but not in naïve animals, an effect that was reversed by spinal administration of LY-225910. While CCK-8S concentration was increased in spinal cord extracts of LPS animals as compared to controls, morphine-induced spinal CCK release in the extracellular space, as measured by in-vivo spinal cord microdialysis was inhibited in LPS animals as compared to controls, and this was reversed by anti-NGF pretreatment. Finally, chronic spinal administration of beta-NGF (7 microg/day) for 7 days enhanced spinal morphine analgesia, possibly by mimicking a CNS inflammatory state. We suggest that in intrathecally LPS-treated rats, spinal CCK release is altered resulting in enhanced morphine analgesia, and that this mechanism may be regulated to an important extent by NGF.

Effect of concomitant ingestion of alcohol on the in vivo pharmacokinetics of KADIAN (morphine sulfate extended-release) capsules.

UNLABELLED: The recent withdrawal of hydromorphone hydrochloride extended-release capsules (Palladone; Purdue Pharma L.P., Stamford, CT) from the market after pharmacokinetic data revealed a risk of alcohol-induced dose-dumping prompted a re-examination of the risk-benefit profiles of extended-release drugs. Although warnings on concomitant alcohol use are included on opioid product labels, further investigations of extended-release formulations to determine the risk of dose-dumping were recommended by the US Food and Drug Administration. The present study was undertaken to assess the single-dose relative bioavailability of polymer-coated, extended-release morphine sulfate capsules (KADIAN, 100 mg; Alpharma Pharmaceuticals LLC, Piscataway, NJ). This open-label, randomized, 3-way crossover study with an additional index arm, conducted among 32 healthy male volunteers, found no significant evidence of a formulation interaction between KADIAN and alcohol, in vivo. The pharmacokinetics of serum morphine did not differ significantly among subjects taking KADIAN with water (fasted) or with 240 mL 40% alcohol under fasted or fed conditions. Analysis of variance ratios of least-squares means for ln-transformed AUC(infinity) and C(max) satisfied the criteria (90% confidence intervals within 80%-125%) to declare no drug formulation interaction among the KADIAN regimens dosed with alcohol compared with KADIAN taken with water. There were no serious adverse events or deaths reported during the study. PERSPECTIVE: Because of the high rate of alcohol use in the United States, the potential for drug-alcohol interactions is an important clinical concern. Although it is recommended that alcohol not be used while the patient is taking opioids, results of this in vivo study indicate that the risk of alcohol-induced dose-dumping in connection with the use of KADIAN is negligible.

mGluR5 antagonists that block calcium mobilization in vitro also reverse (S)-3,5-DHPG-induced hyperalgesia and morphine antinociceptive tolerance in vivo.

The present study comparatively evaluated the potency of a series of new phenylethyl[1,2,4]methyltriazines which are analogues of the classical metabotropic glutamate (mGlu) receptor subtype 5 (mGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) in blocking hyperalgesia induced by the group I mGlu receptor agonist (S)-3,5-DHPG as well as in reversing morphine antinociceptive tolerance in mice. Hyperalgesia was assessed in mice using the tail immersion test. Intrathecal (i.t.) pre-treatment with the test compounds 5-methyl-3-phenylethynyl-[1,2,4]triazine (RTI-4229-707), 5-methyl-3-(4-phenoxy-phenylethynyl-[1,2,4]triazine (RTI-4229-766), and 3-(3-methylphenylethynyl)-5-methyl-[1,2,4]triazine (RTI-4229-787) resulted in a dose-dependent blockade of (S)-3,5-DHPG-induced hyperalgesia. The inhibitory dose-50 (ID(50)) values were 0.49, 0.72 and 0.44 nmol/mouse, for RTI-4229-707, RTI-4229-766 and RTI-4229-787, respectively, compared to 18.63 nmol/mouse for MPEP. The other two compounds tested 3-(2,5-dimethylphenylethynyl)-5-methyl[1,2,4]triazine (RTI-4229-785) and 3-(2-methylphenylethynyl)-5-methyl[1,2,4]triazine (RTI-4229-828) were totally inactive. Morphine tolerance was induced in mice by implanting a 75 mg morphine pellet and assessing morphine-induced antinociception 72-h later. The morphine-pelleted mice showed a 5.5-fold tolerance to the antinociceptive effect of acute morphine compared to placebo-pelleted mice in the tail immersion test. Intracerebroventricular (i.c.v.) administration of the three active mGluR5 antagonists dose-dependently reversed morphine antinociceptive tolerance. The ID(50) values were 57.7, 25.8 and 64.3 nmol/mouse, for RTI-4229-707, RTI-4229-766 and RTI-4229-787, respectively, compared to 1050 nmol/mouse for MPEP. Similar to the hyperalgesia study, test compounds RTI-4229-785 and RTI-4229-828 were totally inactive in reversing morphine tolerance. These results are in agreement with our previous study in which we demonstrated that the same active mGluR5 antagonists blocked glutamate-mediated mobilization of internal calcium in a selective mGluR5 in vitro efficacy assay.

Comparison of naltrexone, 6alpha-naltrexol, and 6beta-naltrexol in morphine-dependent and in nondependent rhesus monkeys.

RATIONALE: Some opioid receptor ligands that appear to be neutral antagonists can have inverse agonist activity under conditions of increased constitutive activity (e.g., agonist treatment). OBJECTIVES: This study compared the opioid receptor antagonist naltrexone and its metabolites 6alpha-naltrexol and 6beta-naltrexol in nondependent and morphine-dependent monkeys to see whether their potencies varied according to drug treatment and, presumably, to differences in constitutive activity of mu opioid receptors. RESULTS: In monkeys (n = 4) receiving 3.2 mg/kg per day of morphine and discriminating 0.0178 mg/kg naltrexone, naltrexone and each metabolite increased responding on the naltrexone lever in a dose-related manner with naltrexone being 8- and 71-fold more potent than 6alpha- and 6beta-naltrexol, respectively. After 27 h of no-morphine treatment, monkeys responded on the naltrexone lever, and this effect was reversed by morphine. Naltrexone and each metabolite prevented morphine reversal of naltrexone-lever responding, and their rank order potency was the same as their substitution for naltrexone; however, the potency between naltrexone and each metabolite was slightly greater in morphine-dependent as compared to morphine-deprived monkeys. In a separate group (n = 3) of nondependent monkeys discriminating 1.78 mg/kg of morphine, all three compounds antagonized morphine with the same potency as in the reversal study (morphine-dependent monkeys), with Schild analyses showing no difference in apparent affinities (pA (2)) between nondependent and morphine-dependent monkeys. CONCLUSION: Naltrexone and 6alpha- and 6beta-naltrexol have qualitatively similar effects, and their potencies do not vary markedly with opioid treatment, suggesting that under these conditions, they do not vary with regard to inverse agonism.

The prolactin-releasing peptide antagonizes the opioid system through its receptor GPR10.

Prolactin-releasing peptide (PrRP) and its receptor G protein-coupled receptor 10 (GPR10) are expressed in brain areas involved in the processing of nociceptive signals. We investigated the role of this new neuropeptidergic system in GPR10-knockout mice. These mice had higher nociceptive thresholds and stronger stress-induced analgesia than wild-type mice, differences that were suppressed by naloxone treatment. In addition, potentiation of morphine-induced antinociception and reduction of morphine tolerance were observed in mutants. Intracerebroventricular administration of PrRP in wild-type mice promoted hyperalgesia and reversed morphine-induced antinociception. PrRP administration had no effect on GPR10-mutant mice, showing that its effects are mediated by GPR10. Anti-opioid effects of neuropeptide FF were found to require a functional PrRP-GPR10 system. Finally, GPR10 deficiency enhanced the acquisition of morphine-induced conditioned place preference and decreased the severity of naloxone-precipitated morphine withdrawal syndrome. Altogether, our data identify the PrRP-GPR10 system as a new and potent negative modulator of the opioid system.

Pretreatment with antiserum against dynorphin, substance P, or cholecystokinin enhances the morphine-produced anti-allodynia in the sciatic nerve ligated mice.

It is generally accepted that neuropathic pain is resistant to amelioration by morphine in clinical studies and insensitivity to intrathecal (i.t.) administered morphine in experimental models of neuropathic pain has been demonstrated. This study is to determine if endogenous dynorphin, substance P or cholecystokinin is involved in the lack of anti-allodynia of morphine in a partial sciatic nerve ligation (PSL) model of CD-1 mice. Mice exhibited tactile allodynia in the ipsilateral hind paw 1 day after PSL, and reached its maximal allodynic effect at 2 days and remained allodynic for 7 days. Morphine (3.0 nmol) given i.t. did not alter the tactile allodynic threshold in ipsilateral paw of mice pretreated i.t. with normal rabbit serum 2 days after PSL. However, the same dose of morphine (3.0 nmol) given i.t. reduced markedly allodynia in mice pretreated for 2h with antiserum against dynorphin A(1-17) (200 microg); the morphine-produced anti-allodynia developed slowly, reached its peak effect at 30 min and returned to an allodynic state in 60 min. Similarly, i.t. injection of morphine reduced the allodynia in PSL mice pretreated with antiserum against substance P (10 microg) or cholecystokinin (200 microg) for 2h. Intrathecal pretreatment with antiserum against dynorphin A(1-17), substance P or cholecystokinin for 2h injected alone did not affect the baseline mechanical tactile threshold in ipsilateral paw 2 days after PSL. The results indicate that endogenous dynorphin A(1-17), substance P and cholecystokinin are involved in PSL-induced neuropathic allodynia to attenuate the anti-allodynic effect of morphine.

From morphine clinics to buprenorphine: regulating opioid agonist treatment of addiction in the United States.

The practice of prescribing opioid drugs for opioid dependent patients in the U.S. has been subjected to special government scrutiny for almost 100 years. From 1920 until 1964, doctors who used opioids to treat addicts risked federal and/or state criminal prosecution. Although that period ended when oral methadone maintenance was established as legitimate medical practice, public concern about methadone diversion and accidental overdose fatalities, combined with political pressure from both hostile bureaucracies and groups committed to drug-free treatments, led to the development of unprecedented and detailed Food and Drug Administration (FDA) regulations that specified the manner in which methadone (and later, levo-alpha-acetyl methadol, or levomethadyl acetate, (LAAM)) could be provided. In 1974, Congress gave the Drug Enforcement Administration (DEA) additional oversight of methadone treatment programs. Efforts to liberalize the FDA regulations over the past 30 years have been resisted by both the DEA and existing treatment providers. Additional flexibility for clinicians may evolve from the most recent effort to create an accreditation system to replace some of the FDA regulations. The development of buprenorphine, a partial opioid agonist, as an effective treatment for opioid addiction reopened the possibility for having a less burdensome oversight process, especially because of its reduced toxicity if ingested by non-tolerant individuals. New legislation, the Drug Addiction Treatment Act (DATA) of 2000, created an opportunity for clinicians with special training to be exempted from both federal methadone regulations and the requirement to obtain a special DEA license when using buprenorphine to treat addicts. Some details of how the DATA was developed, moved through Congress, and signed into law are described.

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments.

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.

Induction of G protein-coupled receptor kinases 2 and 3 contributes to the cross-talk between mu and ORL1 receptors following prolonged agonist exposure.

The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction.

Effects of adrenoceptor agonists and antagonists on morphine-induced Straub tail in mice.

Straub-tail behavior was induced by subcutaneous injection of different doses (10-60 mg/kg) of morphine to mice. The maximum response was obtained with 20-40 mg/kg of the drug. The response induced by morphine (40 mg/kg) was decreased by intraperitoneal administration of different doses of clonidine (0.05-0.1 mg/kg). Pretreatment of animals with yohimbine (1-4 mg/kg i.p.) reversed the inhibitory action of clonidine. Yohimbine did not elicit any response by itself. Administration of prazosin (0.25, 0.5, and 1 mg/kg) reduced the morphine response. The combination of prazosin with yohimbine (1 mg/kg), but not with clonidine (0.05 mg/kg), caused a potentiated inhibition of the morphine effect. Phenylephrine (2-6 mg/kg i.p.) did not elicit any effect by itself and also did not alter the response induced by morphine or morphine plus clonidine. Dobutamine (2.5-10 mg/kg i.p.), atenolol (2.5-10 mg/kg i.p.), salbutamol (2.5-10 mg/kg i.p.), and propranolol (2.5-10 mg/kg i.p.) did not alter morphine-induced Straub-tail behavior in mice. In conclusion, activation of alpha2-adrenergic pathways contributes to morphine-induced Straub tail, while alpha1- and beta2-adrenergic may not be involved in this phenomenon.

Opioid receptor stimulation acts as mediator of protection in ischaemic preconditioning.

Involvement of the opioid receptors in preconditioning-induced protection has recently been described. The aims of this study were to establish whether: (i) opioid receptor stimulation acts as a trigger ( during the preconditioning protocol) or as a mediator ( during sustained ischaemia) of cardioprotection using either morphine or [D-ala(2), D-leu(5)] enkephalin (DADLE), a synthetic delta-opioid receptor agonist; ( ii) the beneficial effects of DADLE are protein kinase C ( PKC) -mediated; and (iii) inhibitory 'cross-talk' occurs between the beta-adrenergic and phosphatidylinositol pathways activated by release of endogenous catecholamines and opioids respectively during sustained ischaemia. The isolated, perfused working rat heart, subjected to 25 minutes' global ischaemia and 30 minutes' reperfusion, was used as the experimental model. The results showed that delta-opioid receptor stimulation with DADLE (10(-8) M), when administered for 3 x 5 minutes, had no effect, while when given 10 minutes before sustained ischaemia the drug significantly improved functional recovery during reperfusion. This indicates that opioid receptor stimulation acts as a mediator rather than a trigger in the protection elicited. Morphine ( 3 x 10(-7)) when administered in the same manner was without effect. Opioid receptor stimulation caused a marked reduction in the beta -adrenergic response to isoproterenol, indicating inhibitory cross-talk between the phosphatidyl-inositol and beta-adrenergic signal transduction pathways. However, reduction of the beta-adrenergic response to ischaemia does not appear to be the mechanism of opioid-induced protection, as indicated by 3',5' -cyclic adenosine monophosphate (cAMP) levels at the end of 25 minutes' global ischaemia. Opioid receptor-mediated protection against ischaemic damage is PKC-dependent, since DADLE-induced protection could be abolished by the inhibitor chelerythrine.

Signal transduction within G-protein coupled receptors via an ion tunnel: a hypothesis.

Based on molecular modeling of the complexes between the mu-opioid receptor and its ligands, we present a hypothesis that accounts for several of the experimental data including the importance of conserved polar residues in rhodopsin-like G-protein-coupled receptors and the effect of Na+ on the binding of ligands to these receptors. We propose that agonists, but not antagonists, would displace Na+ from its initial binding site at the conserved D2.50 residue in the second transmembrane alpha-helical segment, H2. The displaced Na+ would pass through a "gate" of conserved hydrophobic residues and move along a tunnel-like interface (formed of H2, H3 and H7) enriched with several conserved hydrophilic residues including D3.49. Interaction of Na+ with D3.49 would result in the breaking of a salt-bridge between D3.49 and the conserved R3.50 residue thus exposing the latter for interaction with the G-protein.

Effect of mu-opioids morphine and buprenorphine on the development of adjuvant arthritis in rats.

OBJECTIVE AND DESIGN: On the basis that endogenous opioids play a role in the physiological response to inflammation, this study tests the anti-arthritic effects of a mu-opioid agonist, morphine and the partial mu-agonist, buprenorphine. MATERIAL: Male Lewis rats were used. TREATMENT: Rats were inoculated subcutaneously with 0.05 ml of Freund's complete adjuvant (5 mg/ml) into the right hind paw to produce adjuvant arthritis. Morphine (either 10 to 60 mg/kg/day s.c. bolus or 60 mg/kg/day s.c. infusion) and buprenorphine (0.65 +/- 0.06 mg/kg/day, orally), respectively, were administered for 3 days during the primary inflammatory phase of adjuvant arthritis. METHODS: The progression of adjuvant arthritis was monitored every three days by body weight change and hind limb oedema (ipsilateral and contralateral). On day 21 the animals were sacrificed and histology and radiography of the contralateral limb were performed. In rats receiving Freund's adjuvant and no drug treatment, the incidence of arthritis was 89%. Effect was expressed as the pooled severity index (PSI) derived from the arithmetic average of the volume, histology and radiography scores in the contralateral hind limb. RESULTS: Buprenorphine had no effect on experimental arthritis (PSI control vs treated: 242 +/- 28 vs 253 +/- 28%). In contrast, morphine by subcutaneous injection twice daily (10 to 60 mg/kg/day) but not by subcutaneous infusion (60 mg/kg/day) was found to attenuate the progression of adjuvant arthritis in a dose-dependent manner. This indicates that the anti-arthritic effects of morphine are opioid receptor mediated (ED50, 58 +/- 9 mg/kg) and suggests that the local concentration reached effective levels only after subcutaneous injection. It is also possible that the high doses of morphine were anti-inflammatory through effects at the kappa receptor. However, these high doses of morphine produced death in one third of the rats, the calculated lethal dose (LD50, 63 +/- 2 mg/kg) being close to the effective dose. CONCLUSION: Anti-arthritic effects of morphine are opioid receptor mediated but morphine use for this indication is restricted by its adverse effects.

The role of serotonergic receptors in the effects of mu opioids in squirrel monkeys responding under a titration procedure.

This experiment was conducted to determine whether drugs acting on brain serotonin modulate the effects of the mu opioid, morphine, as measured by the squirrel monkey shock titration procedure and, if so, whether serotonergic modulation is mediated via specific 5HT receptor subtypes. Under this procedure, electric shock was delivered to the monkey's tail and scheduled to increase once every 15 s from 0.01 to 2.0 mA in 30 steps. Five responses on a lever during the 15-s shock period terminated the shock for 15 s, after which the shock resumed at the next lower intensity. The intensity below which monkeys maintained shock 50% of the time (median shock level or MSL) and rate of responding (RR) in the presence of shock were determined under control conditions and after administration of morphine alone and in combination with various serotonergic compounds. Morphine increased median shock level and decreased rate of responding in a dose-dependent manner. These effects of morphine was attenuated by the 5HT1A receptor agonists, 8-OH-DPAT [(+)-8-hydroxy-2(di-n-propylamino tetralin HBr] and ipsapirone. The effects of morphine were not altered by the 5HT1A receptor antagonist, NAN-190 [1-(2-methoxyphenyl-4-[4-(2-phthalimido) butyl] piperazine HBr], and 5HT2 receptor antagonist, ketanserin, the 5HT3 receptor antagonist, MDL 72222 [3-tropanyl-3,5-dichlorobenzoate], the alpha 2 adrenergic antagonist, yohimbine, or the alpha2 adrenergic agonist, clonidine. These results suggest that 5HT1A receptors may be involved in the effects of morphine in the shock titration procedure, whereas 5HT2, 5HT3 and alpha 2 adrenergic receptors do not appear to play a role in morphine's effects in this procedure.

VV-hemorphin-7 and LVV-hemorphin-7 released during in vitro peptic hemoglobin hydrolysis are morphinomimetic peptides.

Two opioid peptides were generated by in vitro pepsin treatment of bovine hemoglobin. These peptides were identified using a GPI test and purified using HPLC chromatographic techniques. They correspond to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin-7) of the beta-chain of bovine hemoglobin. Binding experiments strongly confirm that VV-hemorphin-7 and LVV-hemorphin-7 are opioid peptides since they inhibited [3H]naloxone binding to rat brain membranes. Our results indicate that VV-hemorphin-7 and LVV-hemorphin-7 exhibit a lesser potency both in GPI and binding tests. Selectivity and affinity of these purified peptides and synthetic hemorphin-7 for opioid receptors is discussed.