Antinociceptive Effect Of Morphine Research Articles

The ventilatory depressant actions but not the antinociceptive effects of morphine are blunted in rats receiving intravenous infusion of L-cysteine ethyl ester

This study demonstrates that intravenous infusion of the cell-penetrant thiol ester, L-cysteine ethyl ester (L-CYSee), to adult male Sprague-Dawley rats elicited (a) minor alterations in frequency of breathing, expiratory time, tidal volume, minute ventilation, or expiratory drive but pronounced changes in inspiratory time, end-inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50, relaxation time, apneic pause, inspiratory drive and non-eupneic breathing index, (b) minimal changes in arterial blood-gas (ABG) chemistry (pH, pCO2, pO2, SO2) and Alveolar-arterial (A-a) gradient (index of alveolar gas exchange), and (c) minimal changes in antinociception (tail-flick latency). Subsequent injection of morphine (10 mg/kg, IV) elicited markedly smaller effects on the above parameters, ABG chemistry, and A-a gradient in rats receiving L-CYSee, whereas morphine antinociception was not impaired. Infusions of L-cysteine or L-serine ethyl ester (oxygen rather than sulfur moiety), did not affect morphine actions on ABG chemistry or A-a gradient. L-CYSee (250 μmol/kg, IV) injection elicited dramatic changes in ventilatory parameters given 15 min after injection of morphine in rats receiving L-CYSee. Our findings suggest that (a) L-CYSee acts in neurons that drive ventilation, (b) L-CYSee reversal of the adverse actions of morphine on ventilation, ABG chemistry and A-a gradient may be via modulation of intracellular signaling pathways activated by morphine rather than by direct antagonism of opioid receptors since morphine antinociception was not diminished by L-CYSee, and (c) the thiol moiety of L-CYSee is vital to efficacy, (d) intracellular conversion of L-CYSee to an S-nitrosylated form may be part of its mechanism of action.

S-nitroso-L-cysteine stereoselectively blunts the adverse effects of morphine on breathing and arterial blood gas chemistry while promoting analgesia

S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 μmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO2 and a decrease in pCO2, (d) a decrease in mean arterial blood pressure (MAP), and (e) an increase in tail-flick (TF) latency (antinociception). Infusion of S-nitroso-D-cysteine (SNO-D-CYS, 1 μmol/kg/min, IV), did not elicit similar responses, except for a sustained decrease in MAP equivalent to that elicited by SNO-L-CYS. A bolus injection of morphine (2 mg/kg, IV) in rats receiving an infusion of vehicle elicited (a) sustained decreases in frequency of breathing tidal volume, and therefore minute ventilation, (b) a sustained decrease in MAP, (c) sustained decreases in pH, pO2 and maximal sO2 with sustained increases in pCO2 and A-a gradient, and (d) a sustained increase in TF latency. In rats receiving SNO-L-CYS infusion, morphine elicited markedly smaller changes in minute ventilation, arterial blood gas chemistry, A-a gradient and MAP. In contrast, the antinociceptive effects of morphine were enhanced in rats receiving the infusion of SNO-L-CYS. The morphine-induced responses in rats receiving SNO-D-CYS infusion were similar to vehicle-infused rats. These data are the first to demonstrate that infusion of an S-nitrosothiol, such as SNO-L-CYS, can stereoselectively ameliorate the adverse effects of morphine on breathing and alveolar gas exchange while promoting antinociception.

Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Co-administration of morphine and levamisole increases death risk, produces neutropenia and modifies antinociception in mice.

Levamisole is a veterinary anthelmintic drug and a common adulterant of misused drugs. This study analyses the lethal, antinociceptive and haematological effects produced by acute or repeated levamisole administration by itself or combined with morphine. Independent groups of male Swiss Webster mice were i.p. injected with 100 mg/kg morphine, 31.6mg/kg levamisole (lethal doses at 10%, LD10 ) or the same doses combined. Naloxone pretreatment (10mg/kg, i.p.) prevented morphine-induced death, as did 2.5mg/kg, i.p. mecamylamine with levamisole. Co-administration of levamisole and morphine (Lvm + Mor) increased lethality from 10% to 80%. This augmented effect was prevented by 30 mg/kg, i.p. naloxone and reduced with 10mg/kg naloxone plus 2.5mg/kg, i.p. mecamylamine. In independent groups of mice, 17.7mg/kg, i.p. levamisole antagonized the acute morphine's antinociceptive effect evaluated in the tail-flick test. Repeated 17.7mg/kg levamisole administration (2×/day/3weeks) did not affect tolerance development to morphine (10mg/kg, 3×/day/1week). Blood samples obtained from mice repeatedly treated with levamisole showed leukopenia and neutropenia. Morphine also produced neutropenia, increased erythrocyte count and other related parameters (e.g. haemoglobin). Lvm + Mor had similar effects on leukocyte and neutrophil counts to those seen with levamisole only, but no erythrocyte-related alterations were evident. Blood chemistry analysis did not indicate liver damage but suggested some degree of electrolyte balance impairment. In conclusion, Lvm + Mor increased death risk, altered morphine-induced antinociceptive effects and produced haematologic abnormalities. The importance of studying combinations of drugs of abuse lies in the fact that drug users frequently combine drugs, which are commonly adulterated.

Central metabolism as a potential origin of sex differences in morphine antinociception but not induction of antinociceptive tolerance in mice.

In rodents, morphine antinociception is influenced by sex. However, conflicting results have been reported regarding the interaction between sex and morphine antinociceptive tolerance. Morphine is metabolised in the liver and brain into morphine-3-glucuronide (M3G). Sex differences in morphine metabolism and differential metabolic adaptations during tolerance development might contribute to behavioural discrepancies. This article investigates the differences in peripheral and central morphine metabolism after acute and chronic morphine treatment in male and female mice. Sex differences in morphine antinociception and tolerance were assessed using the tail-immersion test. After acute and chronic morphine treatment, morphine and M3G metabolic kinetics in the blood were evaluated using LC-MS/MS. They were also quantified in several CNS regions. Finally, the blood-brain barrier (BBB) permeability of M3G was assessed in male and female mice. This study demonstrated that female mice showed weaker morphine antinociception and faster induction of tolerance than males. Additionally, female mice showed higher levels of M3G in the blood and in several pain-related CNS regions than male mice, whereas lower levels of morphine were observed in these regions. M3G brain/blood ratios after injection of M3G indicated no sex differences in M3G BBB permeability, and these ratios were lower than those obtained after injection of morphine. These differences are attributable mainly to morphine central metabolism, which differed between males and females in pain-related CNS regions, consistent with weaker morphine antinociceptive effects in females. However, the role of morphine metabolism in antinociceptive tolerance seemed limited. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

In Vitro and In Vivo Pharmacological Comparison of Mu‐Opioid Receptor Activity of the Kratom (Mitragyna speciosa) Alkaloid Mitragynine and its Metabolite 7‐Hydroxymitragynine

AbstractBackgroundThe µ‐opioid receptor (MOR) pharmacology of mitragynine and its 7‐hydroxy metabolite have not been fully established.MethodFor both we assessed binding affinity and efficacy in vitro, as well as discriminative stimulus and antinociceptive effects in rats.ResultBoth mitragynine (MG) and 7‐hydroxymitragynine (7‐MG) had higher binding affinities at human MOR (Ki values 77.9 and 709 nM, respectively) than at the human k‐ (KOR) or d‐opioid receptors (DOR). MG was identified as a MOR antagonist while 7‐MG was identified as a MOR partial agonist (Emax=41%) using the [35S]GTPγS functional assay. In Sprague Dawley rats trained to discriminate 3.2 mg/kg morphine from vehicle (i.p.), MG produced a maximum of 72% morphine‐lever responding. Whereas morphine produced a maximum of 65% MG‐lever responding in a separate group of rats trained to discriminate 32 mg/kg MG from vehicle (i.p.), other MOR agonists (i.p.) produced high percentages of morphine‐lever and MG‐lever responding (respectively): 7‐MG (100% and 98%), fentanyl (100% and 80%), buprenorphine (100% and 79%), and nalbuphine (99% and 98%). MG (up to 56 mg/kg, i.p.) did not produce significant antinociception, whereas 7‐MG (17.8 mg/kg, i.p.) produced robust antinociception in the hotplate assay with the temperature set at 52 °C. Naltrexone (0.032 mg/kg, i.p.) antagonized the discriminative stimulus, rate‐decreasing, and antinociceptive effects of morphine and 7‐MG. Conversly, naltrexone (0.032 mg/kg, i.p.) antagonized the discriminative stimulus effects of MG, but not its rate‐decreasing effects. Pretreatment with MG (5.6 mg/kg, i.p.) potentiated the discriminative‐stimulus effects of morphine but antagonized the antinociceptive effects of morphine.ConclusionStriking differences in MOR efficacy in vitro are consistent with 7‐MG having greater MOR efficacy than MG in vivo. MG exerts both MOR antagonist and agonist activity depending on the MOR efficacy required.

Chemokine receptor antagonists enhance morphine's antinociceptive effect but not respiratory depression

AimsWe have shown that chemokines injected into the periaqueductal gray region of the brain blocks opioid-induced analgesia in the rat cold-water tail flick test (CWTF). The present experiments tested whether chemokine receptor antagonists (CRAs), in combination with sub-analgesic doses of morphine, would provide maximal analgesia in the CWTF test and the mouse formalin pain assay. The effect of CRAs on respiratory depression was also evaluated. Main methodsOne, two or four CRAs (AMD3100/CXCR4, maraviroc/CCR5, RS504393/CCR2 orAZD8797/CX3CR1) were used in combination with sub-analgesic doses of morphine, all given systemically. Pain was assessed using the rat CWTF test or formalin injection into the paw of mice scored by licking. Respiration and oxygen saturation were measured in rats using a MouseOX® Plus – pulse oximeter. Key findingsIn the CWTF test, a sub-maximal dose of morphine in combination with maraviroc alone, maraviroc plus AMD3100, or with the four chemokine receptor antagonists, produced synergistic increases in antinociception. In the formalin test, the combination of four CRAs plus a sub-maximal dose of morphine resulted in increased antinociception in both male and female mice. AMD3100 had an additive effect with morphine in both sexes. Coadministration of CRAs with morphine did not potentiate the opioid respiratory depressive effect. SignificanceThese results support the conclusion that combinations of CRAs can increase the potency of sub-analgesic doses of morphine analgesia without increasing respiratory depression. The results support an “opioid sparing” strategy for alleviation of pain using reduced doses of opioids in combination with CRAs to achieve maximal analgesia.

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine’s antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3–30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.

Effect of Umbelliprenin on Antinociceptive Activity of Morphine in a Rat Model of Neuropathic Pain Induced by Chronic Constriction Injury of the Sciatic Nerve

Objective: Although morphine is among of the first line medicines for treatment of neuropathic pain, evidence has shown that the morphine efficacy gradually decreases and a tolerance can occur. Rregarding the many reports concerning the antinociceptive and anti-inflammatory properties of umbelliprenin (UMB), this study aimed to investigate the effect of UMB on antinociceptive activity of morphine in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Methods: Twenty-four male Wistar rats were randomly divided into sham, CCI and CCI + UMB100 (100 μg UMB per rat) groups. UMB was intrathecally administered once daily for four consecutive days (from the day before surgery until day 2 after surgery). All the animals received a single dose of morphine (5 mg/kg, s.c.) on day 14. To evaluate the effect of UMB on antinociceptive activity of morphine, allodynia and hyperalgesia were measured using the von-Frey and hot plate tests, before and 30 min after morphine injection and the Percentage of Maximum Possible Effect (%MPE) was calculated. Besides, the expression and concentration of tumor necrosis factor-alpha (TNF-α), as a proinflammatory cytokine, was measured in the spinal cord using quantitative real-time PCR (RTPCR) and ELISA, respectively. Results: UMB significantly enhanced anti-allodynic and anti-hyperalgesic effects of morphine in the neuropathic animals. Moreover, UMB considerably downregulated TNF-α expression in the spinal cord of the animals. Conclusion: UMB can enhance the antinociceptive effects of morphine, and this action may be partially due to its anti-inflammatory property.

Persistent Exposure to Δ9-Tetrahydrocannabinol during Adolescence Does Not Affect Nociceptive Responding in Adult Mice.

Evidence suggests that Δ9-tetrahydrocannabinol (Δ9-THC), the intoxicating component of cannabis, causes enduring changes in the structure and function of adolescent brain circuits implicated in nociceptive responding. However, whether such changes might persistently disrupt nociceptive behaviors remains unknown. In the present study, we subjected C57BL6/J mice of both sexes to once-daily injections of Δ9-THC (5 mg-kg-1, i.p.) or vehicle throughout adolescence (PND 30-43) and, when the animals had reached adulthood (PND 70), assessed nociceptive behavior using the formalin and chronic constriction injury tests. We also investigated, using the tail immersion test, the antinociceptive effects of morphine and the development of tolerance to such effects. The results show that adolescent Δ9-THC exposure does not significantly impair nociceptive responding or morphine-related antinociception and tolerance. The findings suggest that frequent exposure to a moderate dose of Δ9-THC during adolescence does not permanently alter nociceptive circuits in male or female mice. SIGNIFICANCE STATEMENT: The endocannabinoid system serves critical functions in the central and peripheral nervous systems, including regulation of pain, and can be modified by prolonged exposure to the intoxicating constituent of cannabis, Δ9-tetrahydrocannabinol (Δ9-THC). This raises the possibility that regular use of Δ9-THC-containing cannabis during adolescence might cause changes in nociception that persist into adulthood. This study found that frequent early-life exposure to a moderate dose of Δ9-THC does not permanently alter nociceptive function in male or female mice.

Inhibition of Spinal Cord Hsp90 Enhances Src Kinase Signaling to Increase Opioid Anti‐Nociception

Chronic pain remains a huge social and economic burden on the United States and around the world. With few safe treatment options for these patients, many physicians turn to opioid analgesics such as morphine. These medications are not without serious side effects including constipation, respiratory depression, and addiction, which has led to an opioid addiction crisis affecting over 10 million Americans each year. This crisis emphasizes the need to find new ways to improve opioid therapy and decrease side effects like addiction. One such research area is the signal transduction cascades evoked by opioid receptor activation, which could be modulated to enhance their effects on pain management while decreasing negative side effects. Previously, our lab discovered that the molecular chaperone protein Heat shock protein 90 (Hsp90) plays a key role in mu opioid receptor (MOR) signal transduction. When inhibiting Hsp90 in the spinal cord through intrathecal administration of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) in mice we observed a significant increase in morphine anti-nociception, which could enable an opioid dose-reduction strategy. A preliminary proteomic evaluation in 17-AAG treated mouse spinal cord suggested that the inhibitory protein Src-kinase inhibitor 1 was downregulated by 17-AAG, thus suggesting that Src kinase signaling might be upregulated. To test this hypothesis, male and female CD-1 mice were treated with 0.5 nmol 17-AAG or vehicle intrathecally. After 24 hours, mice were treated with 10 nmol of the Src kinase inhibitor Src-I1 or vehicle intrathecally, then after 10 minutes, mice were administered 3.2 mg/kg morphine and a tail flick assay was performed. The enhanced anti-nociceptive effects seen with 17-AAG were completely abolished in groups that received the Src inhibitor, suggesting that Hsp90 inhibition enhances Src kinase signaling to lead to enhanced opioid pain relief. Interestingly, the group that did not receive 17-AAG but did receive Src-I1 also showed a decrease in the anti-nociceptive effects of morphine, thus suggesting a role for Src in the opioid receptor pathway at baseline without Hsp90 inhibitor treatment. Next, mice were treated with only Src-I1 or vehicle and then given morphine or vehicle, showing that Src-I1 administration had no effect on pain by itself. We also tested a post-surgical paw incision pain model to test if Src regulates opioid anti-nociception in other pain types. Similar results were seen with a complete loss of the enhanced anti-nociception of 17-AAG as well as a decrease in the anti-nociceptive effects of morphine with Src inhibitor treatment. Lastly, initial analysis by Western Blotting confirmed what we observed behaviorally. Groups treated with 17-AAG and the opioid agonist DAMGO had a significant increase in phosphorylated Src compared to groups treated with vehicle alone, thus supporting our hypothesis that Hsp90 inhibition in the spinal cord enhances opioid pain relief by upregulation of Src signaling. This has helped elucidate a key molecular mechanism by which Hsp90 regulates opioid signaling and leads to enhanced opioid antinociception, creating a potential target for future efforts to improve opioid treatment.

The Lofexidine‐Like Discriminative Effects of the Primary Kratom Alkaloid Mitragynine in Rats

The primary kratom alkaloid mitragynine has received attention due to its µ-opioid receptor (MOR) activity; however, the antinociceptive effects of mitragynine in mice were blocked by the adrenergic-α2 receptor (Aα2R) antagonist idazoxan. Here we examined the Aα2R pharmacology of mitragynine with radioligand receptor binding in human cell membranes and drug discrimination, the hot plate test (antinociception), and measurement of rectal temperature (hypothermia) in rats. The affinity of mitragynine at human MOR (Ki=706 nM) was 2- to 10-fold higher than its affinity at other opioid (k and d) and adrenergic (a2A and a2C) receptors. In rats discriminating mitragynine (32mg/kg, i.p.) from vehicle, MOR agonists (morphine, fentanyl, and methadone), Aα2R agonists (lofexidine and clonidine), and Aα2R antagonists (yohimbine and atipamezole) produced up to 84%, 74%, and 40% drug-lever responding, respectively. In rats discriminating lofexidine (0.032 mg/kg, i.p.) from vehicle, the Aα2R agonists (lofexidine and clonidine), mitragynine, Aα2R antagonists (yohimbine and atipamezole), and morphine produced up to 97%, 95%, 7.1%, and 7.3% drug-lever responding, respectively. In rats discriminating morphine (3.2mg/kg, i.p.) from vehicle, MOR agonists (morphine, fentanyl, and methadone) produced at least 98% drug-lever responding, and mitragynine produced up to 72% drug-lever responding. The Aα2R agonists (lofexidine and clonidine) and antagonists (yohimbine and atipamezole) produced a maximum of 30% drug-lever responding. The full substitution of mitragynine for lofexidine was pharmacologically specific. In rats discriminating the cannabinoid receptor agonist D9-THC (3.2mg/kg, i.p.) from vehicle, another cannabinoid receptor agonist CP55,940, mitragynine, and morphine produced up to 99%, 23%, and 18.4% drug-lever responding, respectively. In the hotplate (52°C) test, the MOR agonists (morphine, fentanyl, and methadone) produced at least 90 % maximum possible effects, whereas MG was ineffective. Aα2R agonists (lofexidine and clonidine) produced up to 63% maximum possible effects, and the Aα2R antagonists (yohimbine and atipamezole) produced up to 85% maximum possible effects. The cannabinoid receptor agonists (D9-THC and CP55,940) produced up to 67% maximum possible effects. Rectal temperature was decreased by 0.3°C for the MOR agonists, 1.0°C for Mitragynine, up to 4.8°C for the Aα2R agonists, up to 3.4°C for the Aα2R antagonists, and up to 3.8°C for the cannabinoid receptor agonists. Atipamezole antagonized the discriminative stimulus effects of mitragynine as well as lofexidine but not the discriminative or antinociceptive effects of morphine. Both lofexidine and clonidine potentiated mitragynine discrimination but not the discriminative or antinociceptive effects of morphine. These results suggest that mitragynine exerts its in vivo effects via dual agonism at µ-opioid and adrenergic-α2 receptors.

Supra‐additive effects of morphine and the α2/3 preferring GABA A receptor ligand MP‐III‐024 on mechanical hyperalgesia and thermal nociception

Opioid and GABAA receptors are both located in central nociceptive pathways and compounds that activate these receptors have pain-relieving properties. To date, the interactive effects of concurrent administration of these compounds in preclinical models of pain-like behaviors have not been assessed. The purpose of this study was to examine the interactive effects of the µ-opioid agonist morphine and the α2GABAA and α3GABAA receptor positive allosteric modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024) in preclinical models of mechanical hyperalgesia and thermal nociception. Initially, the antihyperalgesic and antinociceptive effects of morphine and MP-III-024 administered alone were assessed, followed by fixed-ratio mixtures of MP-III-024/morphine combinations. Drug interaction data were analyzed using isobolographic and dose-addition analyses. In the assay of mechanical hyperalgesia, each compound produced dose-dependent antihyperalgesic effects, whereas only morphine was effective on thermal nociception. Fixed-ratio mixtures of MP-III-024/morphine were also dose-dependently effective in both procedures. These drug combination studies revealed that morphine and MP-III-024 produced supra-additive (synergistic) effects in both assays, depending on their relative proportions. These results demonstrate an interaction between α2GABAA and α3GABAA receptor- and µ-opioid receptor-mediated signals, and suggest that combination therapy may be useful for the treatment of pain-related disorders.

Synergistic antihyperalgesic and antinociceptive effects of morphine and methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024): a positive allosteric modulator at α2GABAA and α3GABAA receptors.

Opioid and GABAA receptors are both located in central nociceptive pathways, and compounds that activate these receptors have pain-relieving properties. To date, the interactive effects of concurrent administration of these compounds in preclinical models of pain-like behaviors have not been assessed. The purpose of this study was to examine the interactive effects of the μ-opioid agonist morphine and the α2GABAA and α3GABAA receptor positive allosteric modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024) in preclinical models of mechanical hyperalgesia and thermal nociception. The antihyperalgesic and antinociceptive effects of morphine and MP-III-024 administered alone were assessed initially, followed by fixed-ratio mixtures of MP-III-024/morphine combinations. Drug interaction data were analyzed using isobolographic and dose-addition analyses. All studies were conducted in male CD-1 mice. In the assay of mechanical hyperalgesia, each compound produced dose-dependent antihyperalgesic effects, whereas only morphine was effective on thermal nociception. Fixed-ratio mixtures of MP-III-024/morphine were also dose-dependently effective in both procedures. These drug combination studies revealed that morphine and MP-III-024 produced supra-additive (synergistic) effects in both assays, depending on their relative proportions. These results demonstrate an interaction between α2GABAA and α3GABAA receptor- and μ-opioid receptor-mediated signals and suggest that combination therapy may be useful for the treatment of pain-related disorders.

A Comparison of the Antinociceptive Properties of SJP-005 and Morphine in Rats.

SJP-005 (a combination of ketotifen and ibuprofen) is being developed as a potential treatment for pain and for opioid use disorder. It is therefore important to investigate the potential antinociceptive properties of SJP-005. Two studies were conducted to evaluate the potential effects of SJP-005 in rats. Study 1 applied the von Frey test to examine the antinociceptive effect of morphine with and without SJP-005 in adjuvant-induced hypersensitivity to tactile stimulation. In a double-blind, between-groups design, groups of rats (n = 10 each) received morphine at 3, 10, or 30 mg/kg bodyweight (bw) (subcutaneous injection) with or without SJP-005 (oral). Mechanic allodynia and paw volume were assessed before and after treatment. Study 2 utilized the hot plate test. Using a crossover design, groups of rats (n = 10 each) received either morphine at 3, 10, or 30 mg/kg bw (subcutaneous injection) preceded by oral administration of placebo (Week 1) or SJP-005 (Week 2). In Study 1, in the von Frey up-and-down test, Δ paw withdrawal responses in Group 1 (3 mg/kg bw morphine) were significantly lower compared to those in Group 4 (3 mg/kg bw morphine plus SJP-005), whereas the differences in Δ paw withdrawal between Group 2 and Group 5 (10 mg/kg bw morphine with and without SJP-005) and between Group 3 and Group 6 (10 mg/kg bw morphine with and without SJP-005) did not reach statistical difference. Trendline analysis of the dose–response relationship for the morphine + placebo groups and morphine + SJP-005 groups revealed no significant differences in the intercepts and slopes. In Study 2, no significant differences were observed on hot plate performance between morphine and morphine in combination with SJP-005. In conclusion, the findings in the von Frey up-and-down test (Study 1) suggest that animals can withstand higher levels of painful stimuli when SJP-005 is co-administered. This may also suggest a possible opioid sparing effect. However, in the hot plate test (Study 2), animals did not respond more adaptively to stronger painful stimuli after co-administering SJP-005. These observations warrant further investigation of the antinociceptive properties of SJP-005.

Activation of trace amine-associated receptor 1 selectively attenuates the reinforcing effects of morphine.

Trace amine-associated TA1 receptors play critical roles in regulating dopamine transmission. Previous studies showed that pharmacologically or genetically manipulating the activity of TA1 receptors modulates addiction-like behaviours associated with psychostimulants. However, little is known about whether TA1 receptor modulation would regulate the behavioural effects of opioids. Effects of the selective TA1 receptor partial agonist RO5263397 on the addiction-related and antinociceptive effects of morphine were systematically assessed in male rats and mice. RO5263397 attenuated the expression of morphine-induced behavioural sensitization in wildtype but not TA1 receptor knockout mice. RO5263397 shifted the dose-effect curve of morphine self-administration downward and reduced the breakpoint in a progressive ratio schedule of reinforcement but did not affect food self-administration in rats. RO5263397 decreased the cue- and drug-induced reinstatement of morphine-seeking behaviour in rats. RO5263397 alone did not trigger reinstatement of morphine-seeking behaviour or change locomotor activity in rats with a history of morphine self-administration. However, RO5263397 did not affect the expression of morphine-induced conditioned place preference in mice or rats. RO5263397 did not affect naltrexone-precipitated jumping behaviour or naltrexone-induced conditioned place aversion in morphine-dependent mice. Furthermore, RO5263397 did not affect the analgesic effects of morphine in an acute nociception model in mice and a chronic pain model in rats. These results indicated that TA1 receptor activation selectively attenuated the reinforcing, but not withdrawal or antinociceptive effects of morphine, suggesting that selective TA1 receptor agonists might be useful to combat opioid addiction, while sparing the analgesic effects.

Restraint Stress Potentiated Morphine Sensitization: Involvement of Dopamine Receptors within the Nucleus Accumbens.

Sensitization to psychostimulant drugs, as well as morphine, subjected to cross-sensitization with stress. The development of morphine sensitization is associated with enhancements in dopamine overflow in the Nucleus accumbens (NAc). This study aimed to examine the role of accumbal D1/D2-like dopamine receptors in restraint stress (RS) induced sensitization to morphine antinociceptive effects. Adult male Wistar rats weighing 220-250 g underwent stereotaxic surgery. Two stainless steel guide cannulae were bilaterally implanted, 1 mm above the NAc injection site. Different solutions of SCH-23390, as a D1-like receptor antagonist or sulpiride, as a D2-like receptor antagonist, were microinjected into the NAc five min before exposure to RS. Restraint stress lasted for 3 h, 10 min after RS termination; animals received a subcutaneous injection of morphine (1 mg/kg) for 3 consecutive days. The procedure was followed by a 5-day drug and/or stress-free period. After that, on the 9th day, the nociceptive response was evaluated by the tail-flick test. The results revealed that intra-NAc administration of D1/D2-like dopamine receptor antagonists, SCH-23390 or sulpiride, respectively, blocked morphine sensitization-induced by RS and morphine co-administration in rats for three consecutive days. This work provides new insight into the determinant role of accumbal dopamine receptors in morphine sensitization produced by RS-morphine co-administration.

Persistent effects of the orexin-1 receptor antagonist SB-334867 on naloxone precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine dependent rats

Aim of the study In this study, we investigated the effect of long-term administration of orexin receptor 1 (OXR1) antagonist on naloxone-precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine-dependent rats. Materials and methods Wistar rats received subcutaneous (s.c.) injections of morphine (6, 16, 26, 36, 46, 56, and 66 mg/kg, 2 ml/kg) at an interval of 24 h for 7 days. In chronic groups, the OXR1 antagonist, SB-334867 (20 mg/kg, i.p.), or its vehicle, was injected repetitively from postnatal day 1 (PND1)-PND23 and then for the following seven days before each morphine injection. Meanwhile, in acute groups, SB-334867, or its vehicle, was administered before each morphine injection. In groups of rats that were designated for withdrawal experiments, naloxone (2.5 mg/kg, i.p.) was administered after the last injection of morphine. In the formalin-induced pain, the effect of OXR1 inhibition on the antinociceptive effects of morphine was measured by injecting formalin after the final morphine injection. Results Animals that received long-term SB-334867 administration before morphine injection demonstrated a significant reduction in chewing, defecation, diarrhea, grooming, teeth chattering, wet-dog shake, and writhing. Inhibiting OXR1 for a long time increased formalin-induced nociceptive behaviors in interphase and phase II of the formalin-induced pain. Conclusions Our results indicated that the inhibition of OXR1 significantly reduces the development of morphine dependence and behavioral signs elicited by the administration of naloxone in morphine-dependent rats. Furthermore, the prolonged blockade of OXR1 might be involved in formalin-induced nociceptive behaviors.

Agomelatine potentiates anti-nociceptive effects of morphine in a mice model for diabetic neuropathy: involvement of NMDA receptor subtype NR1 within the raphe nucleus and periaqueductal grey

ABSTRACT Objectives Opioid analgesics have been used for a long time in the treatment of acute and chronic pain. However, they have many side effects including tolerance development to a significant extent. Agomelatine, an atypical antidepressant, has been demonstrated to be effective in experimental studies on pain. However, the effect of agomelatine on morphine tolerance development and its mechanism of action are unknown. The antinociceptive effects of agomelatine, morphine and their combination were assessed in a mice model for painful diabetic neuropathy. The roles of glutamate ionotropic receptor N-methyl-D-aspartate (NMDA) type subunit-1 (GluN1) in raphe nucleus and periaqueductal gray (PAG) in the effect of agomelatine on neuropathic pain were also investigated in diabetic mice. Methods Agomelatine (10 mg/kg), morphine (10 mg/kg) and agomelatine + morphine were administered intraperitoneally for 15 consecutive days (twice per day), and the analgesic responses were assessed at days 1, 3, 6, 9, 12 and 15 in healthy and diabetic mice. Real time polymerase chain reaction (RT-PCR) was used to determine the changes in GluN1 expression. Results The tolerance development for morphine was evident, started at 6th day and remained thereafter, but not for agomelatine. GluN1 in raphe nucleus and PAG was upregulated in morphine treated but not in agomelatine-treated groups. Discussion The combination of agomelatine with morphine alone causes outlasting analgesic effects of repeated treatment, which can be interpreted as attenuated tolerance. Moreover, we also pointed out for the first time the modulatory effects of agomelatine on GluN1 expression in raphe nucleus and PAG after chronic morphine treatment. Abbreviations Ca2+: Calcium; D2DR: Dopamine D2 receptor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GluN1: Glutamate ionotropic receptor N-methyl-D-aspartate type subunit-1; 5-HT: 5-hydroxytryptamine; i.p.: intraperitoneal injection; MPE: Maximal possible effect; MT: Melatonin; NMDA: N-methyl-D-aspartate; NMDAR1: NMDA receptors-1; PAG: Periaqueductal grey; PKCγ: Protein kinase C gamma; RT-PCR: Real time polymerase chain reaction; STZ: Streptozotocin

Differences in antinociceptive signalling mechanisms following morphine and fentanyl microinjections into the rat periaqueductal gray.

Morphine and fentanyl are two of the most commonly used opioids to treat pain. Although both opioids produce antinociception by binding to mu-opioid receptors (MOR), they appear to act via distinct signalling pathways. This study will reveal whether differences in morphine and fentanyl antinociception are the result of selective activation of G-protein signalling and/or selective activation of pre- or postsynaptic MORs. The contribution of each mechanism to morphine and fentanyl antinociception was assessed by microinjecting drugs to alter G-protein signalling or block potassium channels linked to pre- and postsynaptic MORs in the ventrolateral periaqueductal gray (PAG) of male Sprague-Dawley rats. Both morphine and fentanyl produced a dose-dependent antinociception when microinjected into the PAG. Enhancement of intracellular G-protein signalling by microinjection of the Regulator of G-protein Signalling 4 antagonist CCG-63802 into the PAG enhanced the antinociceptive potency of morphine, but not fentanyl. Microinjection of α-dendrotoxin into the PAG to block MOR activation of presynaptic Kv + channels caused a significant rightward shift in the dose-response curve of both morphine and fentanyl. Microinjection of tertiapin-Q to block MOR activation of postsynaptic GIRK channels caused a larger shift in the dose-response curve for fentanyl than morphine antinociception. These findings reveal different PAG signalling mechanisms for morphine and fentanyl antinociception. In contrast with fentanyl, the antinociceptive effects of morphine are mediated by G-protein signalling primarily activated by presynaptic MORs. Microinjection of the opioids morphine and fentanyl into the periaqueductal gray (PAG) produce antinociception via mu-opioid receptor signalling. This study reveals differences in the signalling mechanisms underlying morphine and fentanyl antinociception in the PAG. In contrast with fentanyl, morphine antinociception is primarily mediated by presynaptic opioid receptors and is enhanced by blocking RGS proteins.