Morphine-induced Antinociception Research Articles

Biasing Gβγ Downstream Signaling with Gallein Inhibits Development of Morphine Tolerance and Potentiates Morphine-Induced Nociception in a Tolerant State.

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gβγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gβγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gβγ and inhibit interactions of Gβγ with phospholipase-Cβ3 (PLCβ3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCβ3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gβγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gβγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cβ is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

Experimental Study: Interleukin-31 Augments Morphine-Induced Antinociceptive Activity and Suppress Tolerance Development in Mice.

Morphine-induced antinociception is partially reduced in interleukin-31 (IL-31) receptor A (IL-31RA)-deficient mice, indicating that IL-31RA is crucial for morphine-induced peripheral antinociception. Herein, we examined the combined effects of IL-31 and morphine on the antinociceptive activity and itch-associated scratching behavior (LLS) in mice and elucidated the regulatory mechanisms. A hot-plate test was used to assess antinociception. LLS was automatically detected and recorded via a computer. IL-31RA mRNA expression was assessed using real-time polymerase chain reaction. Repeated pre-treatment with IL-31 resulted in significant antinociceptive activity. Repeated administration of morphine decreased the morphine-induced antinociceptive activity, LLS counts, and regular dose and inhibited IL-31-induced LLS. These results suggested that the repeated administration of morphine depleted inter-neuronal IL-31RA levels, preventing morphine-induced antinociception. Therefore, IL-31 may be helpful as an adjunct analgesic to morphine. To explore the benefits of IL-31, its influence on morphine-induced antinociceptive tolerance in mice was examined. An IL-31 and morphine combination increased the analgesic action, which increased the expression of DRG neuronal IL-31RA, elucidating the site of peripheral antinociception of morphine. This site may induce exocytosis of IL-31RA in the sensory nervous system. Collectively, the suppressive effect of IL-31 on morphine-induced antinociceptive tolerance may result from IL-31RA supplementation in sensory nerves.

Analyzing the Antinociceptive Effect of Interleukin-31 in Mice.

The theory that an itch inhibits pain has been refuted; however, previous research did not investigate this theory for an interleukin-31 (IL-31)-induced itch. Previously, we have found that morphine-induced antinociception was partially reduced in IL-31 receptor A (IL-31RA)-deficient (IL-31RAKI) mice, indicating that IL-31RA may play an important role in morphine-induced peripheral antinociception. In the present study, we evaluated the effect of IL-31-induced analgesia on a 2,4,6-trinitrochlorobenzene (TNCB)-sensitized mice using a hot-plate test. This test evaluated the antinociceptive activity of morphine and non-steroidal anti-inflammatory drugs (NSAIDs). Repeated pretreatment with IL-31 showed significant antinociceptive action. Furthermore, its combination with morphine, but not with NSAIDs, increased the analgesic action. In contrast, treatment with TNCB and capsaicin decreased antinociception. Moreover, TNCB increased IL-31RA expression in the dorsal root ganglia at 24 h, whereas capsaicin inhibited it. The comparative action of several analgesics on TNCB or capsaicin was evaluated using a hot-plate test, which revealed that the antinociceptive activity was decreased or disappeared in response to capsaicin-induced pain in IL-31RAKI mice. These results indicate that the analgesic action of IL-31 involves the peripheral nervous system, which affects sensory nerves. These results provide a basis for developing novel analgesics using this mechanism.

Cannabidivarin alleviates neuroinflammation by targeting TLR4 co-receptor MD2 and improves morphine-mediated analgesia.

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor (PRR) that regulates the activation of immune cells, which is a target for treating inflammation. In this study, Cannabidivarin (CBDV), an active component of Cannabis, was identified as an antagonist of TLR4. In vitro, intrinsic protein fluorescence titrations revealed that CBDV directly bound to TLR4 co-receptor myeloid differentiation protein 2 (MD2). Cellular thermal shift assay (CETSA) showed that CBDV binding decreased MD2 stability, which is consistent with in silico simulations that CBDV binding increased the flexibility of the internal loop of MD2. Moreover, CBDV was found to restrain LPS-induced activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced pro-inflammatory factors NO, IL-1β, IL-6 and TNF-α. Hot plate test showed that CBDV potentiated morphine-induced antinociception. Furthermore, CBDV attenuated morphine analgesic tolerance as measured by the formalin test by specifically inhibiting chronic morphine-induced glial activation and pro-inflammatory factors expression in the nucleus accumbent. This study confirms that MD2 is a direct binding target of CBDV for the anti-neuroinflammatory effect and implies that CBDV has great translational potential in pain management.

Low-dose morphine reduces pain perception and blood pressure, but not muscle sympathetic outflow, responses during the cold pressor test.

Our knowledge about how low-dose (analgesic) morphine affects autonomic cardiovascular regulation is primarily limited to animal experiments. Notably, it is unknown if low-dose morphine affects human autonomic cardiovascular responses during painful stimuli in conscious humans. Therefore, we tested the hypothesis that low-dose morphine reduces perceived pain and subsequent sympathetic and cardiovascular responses in humans during an experimental noxious stimulus. Twenty-nine participants (14 females/15 males; 29 ± 6 yr; 26 ± 4 kg·m-2, means ± SD) completed this randomized, crossover, placebo-controlled trial during two laboratory visits. During each visit, participants completed a cold pressor test (CPT; hand in ∼0.4°C ice bath for 2 min) before and ∼35 min after drug/placebo administration (5 mg iv morphine or saline). We compared pain perception (100 mm visual analog scale), muscle sympathetic nerve activity (MSNA; microneurography; 14 paired recordings), and beat-to-beat blood pressure (BP; photoplethysmography) between trials (at both pre- and postdrug/placebo time points) using paired, two-tailed t tests. Before drug/placebo infusion, perceived pain (P = 0.92), ΔMSNA burst frequency (n = 14, P = 0.21), and Δmean BP (P = 0.39) during the CPT were not different between trials. After the drug/placebo infusion, morphine versus placebo attenuated perceived pain (morphine: 43 ± 20 vs. placebo: 57 ± 24 mm, P < 0.001) and Δmean BP (morphine: 10 ± 7 vs. placebo: 13 ± 8 mmHg, P = 0.003), but not ΔMSNA burst frequency (morphine: 10 ± 11 vs. placebo: 13 ± 11 bursts·min-1, P = 0.12), during the CPT. Reductions in pain perception and Δmean BP were only weakly related (r = 0.34, P = 0.07; postmorphine CPT minus postplacebo CPT). These data provide valuable information regarding how low-dose morphine affects autonomic cardiovascular responses during an experimental painful stimulus.NEW & NOTEWORTHY In this randomized, crossover, placebo-controlled trial, we found that low-dose morphine administration reduced pain perception and blood pressure responses during the cold pressor test via attenuated increases in heart rate and cardiac output. We also determined that muscle sympathetic outflow responses during the cold pressor test seem to be unaffected by low-dose morphine administration. Finally, our exploratory analysis suggests that biological sex does not influence morphine-induced antinociception in healthy adults.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Effects of prenatal opioid exposure and early life adversity on opioid‐induced antinociception

Diagnoses of maternal opioid use disorder (MOUD) at delivery increased more than 500% between 1999 and 2017 in the United States. Today, cases of in utero opioid exposure due to MOUD exceed cases of the most common birth defects, including cleft lip, cleft palate, clubfoot, and Down syndrome, combined. Children exposed to opioids in utero are at increased risk of adverse childhood experiences (ACEs) due to parental care being compromised by SUDs. These ACEs include abuse, neglect, loss of a parent from death or incarceration, parental divorce, and household domestic violence. Altered sensitivity to opioid analgesia later in life is one potential outcome shared by in utero opioid exposure and ACEs that may contribute to poorly controlled pain and increased risk of opioid addiction. Studies using rat models of prenatal opioid exposure (POE) or early life adversity (ELA) have shown that each decreases opioid‐induced antinociception long after exposure has ceased. However, the contributions of POE and ELA, alone and in combination, to opioid analgesic response in humans remains unknown. In this study, we used a rodent model of combined POE and ELA to disambiguate their long‐term effects more quickly and with greater experimental control than human studies. We hypothesized that combined POE and ELA will decrease morphine‐induced antinociception in adolescence relative to POE or ELA alone. We used a two‐by‐two between‐subjects factorial design in which timed‐pregnant Long‐Evans rats were exposed to morphine (15 mg/kg/day; “POE” group) or saline (“vehicle” group) via subcutaneous osmotic minipump from gestation day 9 until delivery. Litters were fostered to untreated dams then randomly assigned to normal housing or ELA conditions using a limited bedding and nesting procedure from postnatal days 3‐11. Morphine‐induced antinociception was measured between postnatal days 30 and 54 using a warm‐water tail withdrawal (WWTW) assay. Cumulative dose‐response of morphine‐induced antinociception was determined on test day 0 (1.0‐30 mg/kg morphine, s.c.) and test day 13 (3.0‐100 mg/kg, s.c.). On test days 1‐12, rats were dosed with 18 mg/kg, s.c., every 12 hours, and WWTW was conducted on odd test days. Interesting, preliminary results of this ongoing study indicate that morphine‐induced antinociception was enhanced on test day 0 in POE + normal‐housed males (n = 2). These rats exhibited 100% maximum possible effect (MPE) and significantly higher potency (mean ED50=5.96 mg/kg; 95% CI = ‐12.12, 24.05), while mean MPE for the other groups (n=2‐3) was less than 70% and mean ED50&gt;17 mg/kg. Males exhibited partial tolerance throughout the 12‐day chronic morphine treatment period. In the POE + normal‐housed males, MPE decreased from 100% on test day 1 to 58% on test day 11. For the remaining groups, MPE decreased from 44‐57% on test day 1 to 12‐23% on test day 11. Morphine was more potent in the POE + normal‐housed males on test day 13 (mean ED50=66.21 mg/kg; 95% CI = ‐281.6, 414) than in other males (mean ED50&gt;104.4 mg/kg). No clear group effect was observed in females. These unexpected results suggest a complex sex‐dependent interaction between POE and ELA on opioid antinociception.

Toll-like receptors change morphine-induced antinociception, tolerance and dependence: Studies using male and female TLR and signalling gene KO mice

Toll-like receptors (TLR) have been proposed as a site of action that alters opioid pharmacodynamics. However, a comprehensive assessment of acute opioid antinociception, tolerance and withdrawal behaviours in genetic null mutant strains with altered innate immune signalling has not been performed. Nor has the impact of genetic deletion of TLR2/4 on high-affinity opioid receptor binding. Here we show that diminished TLR signalling potentiates acute morphine antinociception equally in male and female mice. However, only male TIR8 null mutant mice showed reduced morphine analgesia. Analgesic tolerance was prevented in TLR2 and TLR4 null mutants, but not MyD88 animals. Withdrawal behaviours were only protected in TLR2−/− mice. In silico docking simulations revealed opioid ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. There was no binding of [3H](-)-naloxone or [3H]diprenorphine to TLR4 in the concentrations explored. These data confirm that opioids have high efficacy activity at innate immune pattern recognition binding sites but do not bind to TLR4 and identify critical pathway and sex-specific effects of the complex innate immune signalling contributions to opioid pharmacodynamics. These data further support the behavioural importance of the TLR-opioid interaction but fail to demonstrate direct evidence for high-affinity binding of the TLR4 signalling complex to ligands.

Different Roles of Beclin1 in the Interaction Between Glia and Neurons after Exposure to Morphine and the HIV- Trans-Activator of Transcription (Tat) Protein.

Previously we showed that Beclin1 has a regulatory role in the secretion of inflammatory molecules in glia after exposure to morphine and Tat (an HIV protein). Here we show increased secretion of neuronal growth factors and increased neuronal survival in Beclin1-deficient glia. However, without glia co-culture, neurons deficient in Beclin1 showed greater death and enhanced dendritic beading when compared to wild-type neurons, suggesting that glial-secreted growth factors compensate for the damage reduced autophagy causes neurons. To assess if our ex vivo results correlated with in vivo studies, we used a wild-type (Becn1+/+) and Beclin1-deficient (Becn1+/+) mouse model and intracranially infused the mice with Tat and subcutaneously administered morphine pellets. After morphine implantation, significantly impaired locomotor activities were detected in both Becn1+/+ and Becn1+/-mice, irrespective of Tat infusion. After induction of pain, morphine-induced antinociception was detected. Interestingly, co-exposure to morphine and Tat increased sensitivity to pain in Becn1+/+ mice, but not in similarly treated Becn1+/- mice. Brain homogenates from Becn1+/+ mice exposed to Tat, alone and in combination with morphine, showed increased secretion of pro-inflammatory cytokines and reduced expression of growth factors when compared to similarly treated Becn1+/- mice. Likewise, increased neuronal loss was detected when both Tat and morphine were administered to Becn1+/+ mice, but not in similarly treated Becn1+/- mice. Overall, our findings show that there is a Beclin1-driven interaction between Tat and morphine in glia and neurons. Moreover, reduced glial-Beclin1 may provide a layer of protection to neurons under stressful conditions, such as when exposed to morphine and Tat.

Evaluation of Kratom Opioid Derivatives as Potential Treatment Option for Alcohol Use Disorder.

Background and Purpose: Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder. Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice. Key Results: Paynantheine (10 mg∙kg−1, i.p.) produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mg∙kg−1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mg∙kg−1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures. Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window.

Mice with high FGF21 serum levels had a reduced preference for morphine and an attenuated development of acute antinociceptive tolerance and physical dependence

Because of increased opioid misuse, there is a need to identify new targets for minimizing opioid tolerance, and physical and psychological dependence. Previous studies showed that fibroblast growth factor 21 (FGF21) decreased alcohol and sweet preference in mice. In this study, FGF21-transgenic (FGF21-Tg) mice, expressing high FGF21 serum levels, and wildtype (WT) C57BL/6J littermates were treated with morphine and saline to determine if differences exist in their physiological and behavioral responses to opioids. FGF21-Tg mice displayed reduced preference for morphine in the conditioned place preference assay compared to WT littermates. Similarly, FGF21-Tg mice had an attenuation of the magnitude and rate of acute morphine antinociceptive tolerance development, and acute and chronic morphine physical dependence, but exhibited no change in chronic morphine antinociceptive tolerance. The ED50 values for morphine-induced antinociception in the 55 °C hot plate and the 55 °C warm-water tail withdrawal assays were similar in both strains of mice. Likewise, FGF21-Tg and WT littermates had comparable responses to morphine-induced respiratory depression. Overall, FGF21-Tg mice had a decrease in the development of acute analgesic tolerance, and the development of physical dependence, and morphine preference. FGF21 and its receptor have therapeutic potential for reducing opioid withdrawal symptoms and craving, and augmenting opioid therapeutics for acute pain patients to minimize tolerance development.

Involvement of the nociceptin opioid peptide receptor in morphine-induced antinociception, tolerance and physical dependence in female mice.

Nociceptin opioid peptide (NOP) receptor modulates pain transmission and is considered a prospective target for pain management. Under acute pain conditions in rodents, however, no definitive conclusions about effects of systemically intervening NOP receptors on nociception, classical opioid-induced antinociception, tolerance and physical dependence have been drawn. Given that opioid analgesia has sex differences, and females experience greater pain and consume more opioids, clarifying these issues in females will help develop novel analgesics. To clarify the role of NOP receptors on the pharmacological profiles of µ-opioid receptor agonists, in this study, a selective agonist (SCH221510) and antagonist (SB612111) of the NOP receptor were subcutaneously administered in female mice in multiple animal models. In hot-plate test, neither SCH221510 (3 and 10mg/kg, sc) nor SB612111 (10mg/kg, sc) produced significant antinociception. SCH221510 (3mg/kg, sc) attenuated but SB612111 (10mg/kg, sc) enhanced morphine-induced antinociception, with rightward and leftward shift of morphine dose-response curves, respectively. SCH221510 (3mg/kg, sc) combined with morphine (10mg/kg, sc) accelerated the development of morphine antinociceptive tolerance. Conversely, SB612111 (10mg/kg, sc) delayed morphine tolerance development. Neither SCH221510 (3mg/kg, sc) nor SB612111 (10mg/kg, sc) statistically significantly altered the development of morphine-induced physical dependence. Therefore, systemic activation of NOP receptors attenuated morphine antinociception to acute thermal stimuli, facilitated morphine-induced antinociceptive tolerance but did not robustly alter physical dependence in female mice. Systemic blockade of NOP receptors produced opposite actions. These findings demonstrate that N/OFQ-NOP receptor system plays diverse roles in modulating pharmacological profiles of µ-opioid receptor agonists.

Effects of the selective dopamine D3 receptor antagonist PG01037 on morphine-induced hyperactivity and antinociception in mice

Recent preclinical studies have reported that pretreatment with the novel and highly-selective dopamine D3 receptor (D3R) antagonists R-VK4-40 or VK4-116 attenuates the abuse-related behavioral effects of oxycodone while enhancing its analgesic properties. However, whether these observed effects are generalizable to the broad class of D3R antagonists and/or extend to opioids other than oxycodone has not been extensively explored. The present study sought to assess the impact of pretreatment with another selective D3R antagonist, PG01037, on several behavioral effects of morphine in mice. C57Bl/6 J mice were pretreated with PG01037 (0–10 mg/kg) and tested for 1) hyperlocomotion induced by acute morphine (5.6–56 mg/kg), 2) locomotor sensitization following repeated morphine (56 mg/kg), 3) antinociception following acute morphine (18 mg/kg), and 4) catalepsy following administration of PG01037 alone or in combination with morphine (56 mg/kg). PG01037 dose-dependently attenuated morphine-induced hyperlocomotion and morphine-induced antinociception at doses that did not alter basal locomotion or nociception alone, but did not prevent the induction of locomotor sensitization following repeated morphine administration. Moreover, PG01037 did not induce catalepsy either alone or in combination with morphine. These results suggest that attenuation of acute opioid-induced hyperactivity may be a behavioral effect shared among D3R-selective antagonists, thus supporting continued investigations into their use as potential treatments for opioid use disorder. However, PG01037 is unlike newer, highly-selective D3R antagonists in its capacity to reduce opioid-induced antinociception, indicating that modulation of opioid analgesia may vary across different D3R antagonists.

Loss of SUR1 subtype KATP channels alters antinociception and locomotor activity after opioid administration

Opioid signaling can occur through several downstream mediators and influence analgesia as well as reward mechanisms in the nervous system. KATP channels are downstream targets of the μ opioid receptor and contribute to morphine-induced antinociception. The aim of the present work was to assess the role of SUR1-subtype KATP channels in antinociception and hyperlocomotion of synthetic and semi-synthetic opioids. Adult male and female mice wild-type (WT) and SUR1 deficient (KO) mice were assessed for mechanical and thermal antinociception after administration of either buprenorphine, fentanyl, or DAMGO. Potassium flux was assessed in the dorsal root ganglia and superficial dorsal horn cells in WT and KO mice. Hyperlocomotion was also assessed in WT and KO animals after buprenorphine, fentanyl, or DAMGO administration. SUR1 KO mice had attenuated mechanical antinociception after systemic administration of buprenorphine, fentanyl, and DAMGO. Potassium flux was also attenuated in the dorsal root ganglia and spinal cord dorsal horn cells after acute administration of buprenorphine and fentanyl. Hyperlocomotion after administration of morphine and buprenorphine was potentiated in SUR1 KO mice, but was not seen after administration of fentanyl or DAMGO. These results suggest SUR1-subtype KATP channels mediate the antinociceptive response of several classes of opioids (alkaloid and synthetic/semi-synthetic), but may not contribute to the “drug-seeking” behaviors of all classes of opioids.

D-Cystine di(m)ethyl ester reverses the deleterious effects of morphine on ventilation and arterial blood gas chemistry while promoting antinociception

We have identified thiolesters that reverse the negative effects of opioids on breathing without compromising antinociception. Here we report the effects of d-cystine diethyl ester (d-cystine diEE) or d-cystine dimethyl ester (d-cystine diME) on morphine-induced changes in ventilation, arterial-blood gas chemistry, A-a gradient (index of gas-exchange in the lungs) and antinociception in freely moving rats. Injection of morphine (10 mg/kg, IV) elicited negative effects on breathing (e.g., depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive). Subsequent injection of d-cystine diEE (500 μmol/kg, IV) elicited an immediate and sustained reversal of these effects of morphine. Injection of morphine (10 mg/kg, IV) also elicited pronounced decreases in arterial blood pH, pO2 and sO2 accompanied by pronounced increases in pCO2 (all indicative of a decrease in ventilatory drive) and A-a gradient (mismatch in ventilation-perfusion in the lungs). These effects of morphine were reversed in an immediate and sustained fashion by d-cystine diME (500 μmol/kg, IV). Finally, the duration of morphine (5 and 10 mg/kg, IV) antinociception was augmented by d-cystine diEE. d-cystine diEE and d-cystine diME may be clinically useful agents that can effectively reverse the negative effects of morphine on breathing and gas-exchange in the lungs while promoting antinociception. Our study suggests that the d-cystine thiolesters are able to differentially modulate the intracellular signaling cascades that mediate morphine-induced ventilatory depression as opposed to those that mediate morphine-induced antinociception and sedation.

Inhibition of Heat shock protein 90 in the Spinal Cord Enhances Opioid Anti-Nociception by Disabling an Inhibitory GABA Circuit

One strategy to enhance the clinical treatment of chronic and acute pain is the development of dose-reductive compounds that, when co-administered with palliatives, can reduce the dose of opioid required for treatment while retaining analgesic efficacy and decreasing side-effects. We previously showed that intrathecal (IT) administration of the heat shock protein 90 (Hsp90) inhibitor 17-AAG potentiated the efficacy of morphine induced antinociception while reducing or not changing opioid side effects. Proteomic analysis of the translational alterations induced by IT 17-AAG administration revealed an upregulation of GABA transporter 2 (GAT-2). To elucidate the role of GAT-2 in mediating the enhancement of morphine antinociception, 17-AAG treated mice were administered a GAT inhibitor, Nipecotic Acid (NA). NA effectively blocked the 17-AAG enhancement of antinociception compared to controls, while not changing responses in controls, suggesting that GAT-2 mediates 17-AAG's antinociceptive enhancement. We hypothesized that an inhibitory GABA circuit is present in the spinal cord which reduces opioid antinociception and is disabled by Hsp90. To test this, mice were treated with IT GABA-A or GABA-B agonists prior to systemic morphine administration. The GABA-A agonist reversed 17-AAG enhanced antinociception much like NA. Intriguingly, the GABA-B agonist reversed 17-AAG antinociception in males only, suggesting a GABAergic sex-specific difference. To further test the inhibitory GABA circuit hypothesis, naive mice (no 17-AAG) were injected with IT GABA-A or GABA-B antagonists prior to morphine and tail flick. Males showed enhanced morphine antinociception with either antagonist. However, neither drug had an impact on female mice, again suggesting mechanistic sex differences. These results taken together suggest that IT 17-AAG potentiates morphine-induced antinociception via upregulation of GAT-2, relieving an inhibitory GABAergic brake on opioid antinociception. This study provides mechanistic insight into how Hsp90 inhibitors can be used to boost opioid antinociception and may have identified a novel inhibitory circuit of opioid antinociception. This study was funded by an Arizona Biomedical Research Commission New Investigator Award #ADHS18-198875 and institutional funds from the University of Arizona.

Enhancement of morphine-induced antinociception after electroconvulsive shock in mice.

Electroconvulsive therapy (ECT) has been applied for chronic pain for decades. The amounts of opioids to treat pain are sometimes reduced after a series of ECT. The effect of ECT on morphine-induced analgesia and its mechanism underlying the reduction of morphine requirement has yet to be clarified. Therefore, we administered electroconvulsive shocks (ECS) to mice and investigated the antinociceptive effect of morphine in a hot plate test. We examined the expression level of µ-opioid receptor in the thalami of mice 25 h after administration of ECS compared to the thalami of mice without ECS administration using western blotting. ECS disturbed the development of a decrease in the percentage of maximal possible effect (%MPE), which was observed 24 h after a morphine injection, when ECS was applied 25, 23, 21, and 12 h before the second administration of morphine. We also examined the effect of ECS on the dose-response curve of %MPE to morphine-antinociception. Twenty-five hours after ECS, the dose-response curve was shifted to the left, and the EC50 of morphine given to ECS-pretreated mice decreased by 30.1% compared to the mice that were not pretreated with ECS. We also found that the expression level of µ-opioid receptors was significantly increased after ECS administration. These results confirm previous clinical reports showing that ECT decreased the required dose of opioids in neuropathic pain patients and suggest the hypothesis that this effect of ECT works through the thalamus.

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition.

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.

Harmaline potentiates morphine-induced antinociception via affecting the ventral hippocampal GABA-A receptors in mice

Morphine is one of the most effective medications for treatment of pain, but its side effects limit its use. Therefore, identification of new strategies that can enhance morphine-induced antinociception and/or reduce its side effects will help to develop therapeutic approaches for pain relief. Considering antinociceptive efficacy of harmaline and also highlighted the important role of GABA-A receptors in the pain perception, this research aimed to determine whether the ventral hippocampal (vHip) GABA-A receptors are involved in the possible harmaline-induced enhancement of morphine antinociception. To achieve this, vHip regions of adult male mice were bilaterally cannulated and pain sensitivity was measured in a tail-flick apparatus. Intraperitoneally administration of morphine (0, 2, 4 and 6 mg/kg) or harmaline (0, 1.25, 5 and 10 mg/kg) increased the percentage of maximal possible effect (%MPE) and induced antinociception. Interestingly, co-administration of sub-effective doses of harmaline (5 mg/kg) and morphine (2 mg/kg) induced antinociception. Intra-vHip microinjection of muscimol (0, 200 and 300 ng/mice), a GABA-A receptor agonist, enhanced the anti-nociceptive effects of harmaline (2.5 mg/kg)+morphine (2 mg/kg) combination. Microinjection of the same doses of muscimol into the vHip by itself did not alter tail-flick latency. Intra-vHip microinjection of bicuculline (100 ng/mouse), a GABA-A receptor antagonist, did not cause a significant change in MPE%. Bicuculline (60 and 100 ng/mouse, intra-vHip) was administered with the harmaline (5 mg/kg)+morphine (2 mg/kg), and inhibited the potentiating effect of harmaline on morphine response. These findings favor the notion that GABAergic mechanisms in the vHip facilitate harmaline-induced potentiation of morphine response in the tail-flick test in part through GABA-A receptors. These findings shall provide insights and strategies into the development of pain suppressing drugs.

Effect of the morphine/heroin vaccine on opioid and non-opioid drug-induced antinociception in mice

Pain is a common symptom in patients with opioid use disorder (OUD), which increases synthetic and illicit synthetic opioid abuse and even fatalities due to opioid overdose. Many FDA-approved drugs are available for the treatment of OUD, however, the use of these medications is limited, mainly due to the development of various side effects. Active vaccination is a new therapeutic approach but the resulting antibodies may compromise the use and efficiency of opioid and non-opioid drugs.In this study, we evaluated whether the antibodies produced by the morphine/heroin vaccine (M-TT) would alter the antinociceptive effects of opioid and non-opioid drugs.Female Balb-c mice were immunized with the M-TT vaccine. A solid-phase antibody-capture ELISA was used for monitoring antibody titer responses after each booster dose in vaccinated animals, followed by tail-flick testing. This study found that the M-TT vaccine did not affect the antinociception induced by different doses of morphine or the ability of non-opioid and synthetic opioid drugs to decrease thermal pain. Moreover, the combination of vaccination and naloxone increased the time-course of morphine antagonism relative to either vaccination or naloxone alone. These results suggest that the antibody titers generated by the M-TT vaccine 1) are capable of reducing morphine-induced antinociception and 2) are selective enough not to alter antinociception induced by non-opioid or synthetic drugs. These characteristics support its potential as a treatment agent for patients with symptoms of pain comorbid to OUD.