Mirogabalin Decreases Pain-like Behaviours and Improves Opioid and Ketamine Antinociception in a Mouse Model of Neuropathic Pain

Neuropathic pain remains a difficult clinical challenge due to its diverse aetiology and complex pathomechanisms, which are yet to be fully understood. Despite the variety of available therapies, many patients suffer from ineffective pain relief; hence, the search for more efficacious treatments continues. The new gabapentinoid, mirogabalin has recently been approved for clinical use. Although its main mechanism of action occurs at the α2σ-1 and α2σ-2 subunits of calcium channels and is well documented, how the drug affects the disturbed neuropathic interactions at the spinal cord level has not been clarified, which is crucial information from a clinical perspective. The findings of our study suggest that several indirect mechanisms may be responsible for the beneficial analgesic effect of mirogabalin. This is the first study to report that mirogabalin enhances the mRNA expression of spinal antinociceptive factors, such as IL-10 and IL-18BP, and reduces the concentration of the pronociceptive substance P. Importantly, mirogabalin improves the morphine-, buprenorphine-, oxycodone-, and ketamine-induced antinociceptive effects in a neuropathic pain model. Our findings support the hypothesis that enhancing opioid and ketamine analgesia by combining these drugs with mirogabalin may represent a new strategy for the effective pharmacotherapy of neuropathic pain.

Long-term two-photon imaging of spinal cord in freely behaving mice

The spinal cord is critical to integrating peripheral information under sensory-guided motor behaviors in health and disease. However, the cellular activity underlie spinal cord function in freely behaving animals is not clear. Here, we developed a new method for imaging the spinal cord at cellular and subcellular resolution over weeks under naturalistic conditions. The method involves an improved surgery to reduce spinal movement, and the installation of a miniaturized two-photon microscope to obtain high-resolution imaging in moving mice. In vivo calcium imaging demonstrated that dorsal horn neurons show a sensorimotor program-dependent synchronization and heterogeneity under distinct cutaneous stimuli in behaving mice. The long-term imaging of sensory neurons revealed that in the spinal cord, healthy mice demonstrated stereotyped responses. However, in a neuropathic pain model, plasticity changes and neuronal sensitization were observed. We provide a practical method to study the function of spinal cord on sensory perception and disorders in freely behaving mice.

Bifunctional Opioid/Melanocortin Peptidomimetics for Use in Neuropathic Pain: Variation in the Type and Length of the Linker Connecting the Two Pharmacophores

Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.

Abnormal Ca2+ Signals in Reactive Astrocytes as a Common Cause of Brain Diseases

In pathological brain conditions, glial cells become reactive and show a variety of responses. We examined Ca2+ signals in pathological brains and found that reactive astrocytes share abnormal Ca2+ signals, even in different types of diseases. In a neuropathic pain model, astrocytes in the primary sensory cortex became reactive and showed frequent Ca2+ signals, resulting in the production of synaptogenic molecules, which led to misconnections of tactile and pain networks in the sensory cortex, thus causing neuropathic pain. In an epileptogenic model, hippocampal astrocytes also became reactive and showed frequent Ca2+ signals. In an Alexander disease (AxD) model, hGFAP-R239H knock-in mice showed accumulation of Rosenthal fibers, a typical pathological marker of AxD, and excessively large Ca2+ signals. Because the abnormal astrocytic Ca2+ signals observed in the above three disease models are dependent on type II inositol 1,4,5-trisphosphate receptors (IP3RII), we reanalyzed these pathological events using IP3RII-deficient mice and found that all abnormal Ca2+ signals and pathologies were markedly reduced. These findings indicate that abnormal Ca2+ signaling is not only a consequence but may also be greatly involved in the cause of these diseases. Abnormal Ca2+ signals in reactive astrocytes may represent an underlying pathology common to multiple diseases.

Investigating the anti-cocaine, anti-nociceptive properties and side effects of MP1104, a novel mixed opioid receptor agonist

<p>Rationale: Drug addiction is a chronic, relapsing disease with great socioeconomic and morbidity costs. There are limited treatments, with no Food and Drug Administration approved pharmacotherapies available for psychostimulant addiction. In addition, the use of prescription opioid medications has reached epidemic proportions in the world. More than 40,000 deaths from prescription opioid overdose was reported in USA alone in the year 2017. There is an urgent need for the development of effective, non-addictive pain medications and addiction treatments. The opioid receptors play an important role in the modulation of pain and addiction. Mu opioid receptor (MOPr) agonists are widely used to treat pain, however, can also induce respiratory depression, tolerance and addiction. In contrast, drugs activating the kappa opioid receptor (KOPr) attenuate the rewarding properties of drugs, hence are promising non-addictive analgesics. However, side effects like aversion, sedation, anxiety and depression limit their clinical utility. Delta opioid receptor (DOPr) agonists have rewarding, anti-nociceptive and anti-depressive properties, but can also cause seizures. We hypothesise that development of mixed opioid receptor ligands may have therapeutic properties with reduced side effects. Therefore, this thesis evaluated MP1104, a potent mixed opioid receptor agonist, with full efficacy at all three receptors and 3- and 13-fold higher binding affinity for KOPr compared to MOPr and DOPr, respectively. MP1104 was evaluated for the ability to modulate cocaine-induced behaviours, the anti-nociceptive effects and side effects. Methods: Male Sprague-Dawley rats were used to investigate the effects of acute MP1104 treatment on cocaine self-administration and drug seeking behaviour. To determine the mechanism, the modulatory effect of MP1104 on dopamine transporter (DAT) function was assessed using rotating disk electrode voltammetry to measure dopamine uptake in rat dorsal striatum (dStr) and nucleus accumbens (NAc) tissue. Evaluation of side effects included sedation (spontaneous locomotor activity), anxiety (elevated plus maze (EPM)), aversion (conditioned place aversion (CPA)) and depression (forced swim tests (FST)) in rats. The anti-nociceptive effects were measured in the warm-water tail withdrawal assay in rats and male C57BL/6 mice. Acute and chronic administration of MP1104 were evaluated in the paclitaxel-induced neuropathic pain model in mice. Results: In rats trained to self-administer cocaine, acute MP1104 (0.3 and 1 mg/kg, i.p.) administration reduced cocaine-primed reinstatement of drug seeking behaviour and caused a significant downward shift in the cocaine dose-response curve. The anti-cocaine effects exerted by MP1104 are in part due to increased dopamine uptake by DAT in the NAc, which was KOPr-mediated. In the warm-water tail withdrawal assay in rats, acute administration of MP1104 (0.3 and 0.6 mg/kg, i.p.) was 4 times longer acting (8 h) than morphine (2 h). These effects were both KOPr and DOPr dependent. In the dose-response tail withdrawal assay, MP1104 was found to be potent in both rats (ED₅₀ = 0.58 mg/kg, s.c.) and mice (ED₅₀ = 0.35 mg/kg, s.c.). In the paclitaxel-induced neuropathic pain model, mice treated with MP1104 showed potent reductions in both mechanical (ED₅₀ = 0.449 mg/kg, s.c.) and cold (ED₅₀ = 0.479 mg/kg, s.c.) allodynia compared to morphine. Following chronic daily administration of the ED₈₀ dose, MP1104 (1.2 mg/kg, i.p.) was more potent than morphine in reducing mechanical and cold allodynia. Surprisingly, MP1104 reversed responding back to baseline (non-disease) levels. The most remarkable finding was that MP1104, unlike morphine did not produce tolerance when administered chronically. When the side effects of MP1104 were evaluated in rats, no significant anxiogenic effects were seen in the EPM, nor pro-depressive effects in the FST, nor aversion in CPA tests in rats. Furthermore, pre-treatment with a DOPr antagonist, led to MP1104 producing aversive effects. This data suggests that the DOPr agonist actions of MP1104 attenuate the KOPr-mediated aversive effects of MP1104. However, at higher doses, MP1104 (1 mg/kg, i.p.) was found to be sedative. Conclusions: MP1104 exerts potent anti-cocaine properties in self-administration tests. The reduced cocaine reward is at least in part due to the ability of MP1104 to modulate DAT function by increasing dopamine uptake in the NAc. MP1104 is also a potent and long-lasting anti-nociceptive agent in rats. Significantly, when evaluated in a chronic neuropathic pain model, MP1104 was potent with no tolerance to the anti-nociceptive effects observed. Moreover, MP1104 showed fewer side effects with reduced sedative effects and no observed anxiety, aversive, nor pro-depressive effects, unlike pure KOPr agonists. This data supports the therapeutic development of mixed opioid receptor agonists, particularly mixed KOPr/DOPr agonists as non-addictive pain medications and anti-cocaine pharmacotherapies with fewer side effects.</p>

Investigating the anti-cocaine, anti-nociceptive properties and side effects of MP1104, a novel mixed opioid receptor agonist

<p>Rationale: Drug addiction is a chronic, relapsing disease with great socioeconomic and morbidity costs. There are limited treatments, with no Food and Drug Administration approved pharmacotherapies available for psychostimulant addiction. In addition, the use of prescription opioid medications has reached epidemic proportions in the world. More than 40,000 deaths from prescription opioid overdose was reported in USA alone in the year 2017. There is an urgent need for the development of effective, non-addictive pain medications and addiction treatments. The opioid receptors play an important role in the modulation of pain and addiction. Mu opioid receptor (MOPr) agonists are widely used to treat pain, however, can also induce respiratory depression, tolerance and addiction. In contrast, drugs activating the kappa opioid receptor (KOPr) attenuate the rewarding properties of drugs, hence are promising non-addictive analgesics. However, side effects like aversion, sedation, anxiety and depression limit their clinical utility. Delta opioid receptor (DOPr) agonists have rewarding, anti-nociceptive and anti-depressive properties, but can also cause seizures. We hypothesise that development of mixed opioid receptor ligands may have therapeutic properties with reduced side effects. Therefore, this thesis evaluated MP1104, a potent mixed opioid receptor agonist, with full efficacy at all three receptors and 3- and 13-fold higher binding affinity for KOPr compared to MOPr and DOPr, respectively. MP1104 was evaluated for the ability to modulate cocaine-induced behaviours, the anti-nociceptive effects and side effects. Methods: Male Sprague-Dawley rats were used to investigate the effects of acute MP1104 treatment on cocaine self-administration and drug seeking behaviour. To determine the mechanism, the modulatory effect of MP1104 on dopamine transporter (DAT) function was assessed using rotating disk electrode voltammetry to measure dopamine uptake in rat dorsal striatum (dStr) and nucleus accumbens (NAc) tissue. Evaluation of side effects included sedation (spontaneous locomotor activity), anxiety (elevated plus maze (EPM)), aversion (conditioned place aversion (CPA)) and depression (forced swim tests (FST)) in rats. The anti-nociceptive effects were measured in the warm-water tail withdrawal assay in rats and male C57BL/6 mice. Acute and chronic administration of MP1104 were evaluated in the paclitaxel-induced neuropathic pain model in mice. Results: In rats trained to self-administer cocaine, acute MP1104 (0.3 and 1 mg/kg, i.p.) administration reduced cocaine-primed reinstatement of drug seeking behaviour and caused a significant downward shift in the cocaine dose-response curve. The anti-cocaine effects exerted by MP1104 are in part due to increased dopamine uptake by DAT in the NAc, which was KOPr-mediated. In the warm-water tail withdrawal assay in rats, acute administration of MP1104 (0.3 and 0.6 mg/kg, i.p.) was 4 times longer acting (8 h) than morphine (2 h). These effects were both KOPr and DOPr dependent. In the dose-response tail withdrawal assay, MP1104 was found to be potent in both rats (ED₅₀ = 0.58 mg/kg, s.c.) and mice (ED₅₀ = 0.35 mg/kg, s.c.). In the paclitaxel-induced neuropathic pain model, mice treated with MP1104 showed potent reductions in both mechanical (ED₅₀ = 0.449 mg/kg, s.c.) and cold (ED₅₀ = 0.479 mg/kg, s.c.) allodynia compared to morphine. Following chronic daily administration of the ED₈₀ dose, MP1104 (1.2 mg/kg, i.p.) was more potent than morphine in reducing mechanical and cold allodynia. Surprisingly, MP1104 reversed responding back to baseline (non-disease) levels. The most remarkable finding was that MP1104, unlike morphine did not produce tolerance when administered chronically. When the side effects of MP1104 were evaluated in rats, no significant anxiogenic effects were seen in the EPM, nor pro-depressive effects in the FST, nor aversion in CPA tests in rats. Furthermore, pre-treatment with a DOPr antagonist, led to MP1104 producing aversive effects. This data suggests that the DOPr agonist actions of MP1104 attenuate the KOPr-mediated aversive effects of MP1104. However, at higher doses, MP1104 (1 mg/kg, i.p.) was found to be sedative. Conclusions: MP1104 exerts potent anti-cocaine properties in self-administration tests. The reduced cocaine reward is at least in part due to the ability of MP1104 to modulate DAT function by increasing dopamine uptake in the NAc. MP1104 is also a potent and long-lasting anti-nociceptive agent in rats. Significantly, when evaluated in a chronic neuropathic pain model, MP1104 was potent with no tolerance to the anti-nociceptive effects observed. Moreover, MP1104 showed fewer side effects with reduced sedative effects and no observed anxiety, aversive, nor pro-depressive effects, unlike pure KOPr agonists. This data supports the therapeutic development of mixed opioid receptor agonists, particularly mixed KOPr/DOPr agonists as non-addictive pain medications and anti-cocaine pharmacotherapies with fewer side effects.</p>

Fear Extinction-Based Inter-Individual and Sex Differences in Pain-Related Vocalizations and Anxiety-like Behaviors but Not Nocifensive Reflexes

Inter-individual and sex differences in pain responses are recognized but their mechanisms are not well understood. This study was intended to provide the behavioral framework for analyses of pain mechanisms using fear extinction learning as a predictor of phenotypic and sex differences in sensory (mechanical withdrawal thresholds) and emotional-affective aspects (open field tests for anxiety-like behaviors and audible and ultrasonic components of vocalizations) of acute and chronic pain. In acute arthritis and chronic neuropathic pain models, greater increases in vocalizations were found in females than males and in females with poor fear extinction abilities than females with strong fear extinction, particularly in the neuropathic pain model. Female rats showed higher anxiety-like behavior than males under baseline conditions but no inter-individual or sex differences were seen in the pain models. No inter-individual and sex differences in mechanosensitivity were observed. The data suggest that vocalizations are uniquely suited to detect inter-individual and sex differences in pain models, particularly in chronic neuropathic pain, whereas no such differences were found for mechanosensitivity, and baseline differences in anxiety-like behaviors disappeared in the pain models.

Investigation of the Possible Allostery of Koumine Extracted From Gelsemium elegans Benth. And Analgesic Mechanism Associated With Neurosteroids

Translocator protein 18 kDa (TSPO) is an evolutionarily conserved 5-transmembrane domain protein, and has been considered as an important therapeutic target for the treatment of pain. We have recently reported the in vitro and in vivo pharmacological characterization of koumine as a TSPO positive allosteric modulator (PAM), more precisely ago-PAM. However, the probe dependence in the allostery of koumine is an important question to resolve, and the possible analgesic mechanism of koumine remains to be clarified. Here, we report the in vivo evaluation of the allostery of koumine when orthosteric ligand PK11195 was used and preliminarily explore the possible analgesic mechanism of koumine associated with neurosteroids. We find that koumine is an ago-PAM of the PK11195-mediated analgesic effect at TSPO, and the analgesic mechanism of this TSPO ago-PAM may be associated with neurosteroids as the analgesic effects of koumine in the formalin-induced inflammatory pain model and chronic constriction injury-induced neuropathic pain model can be antagonized by neurosteroid synthesis inhibitor aminoglutethimide. Although our results cannot fully clarify the allosteric modulatory effect of koumine, it further prove the allostery in TSPO and provide a solid foundation for koumine to be used as a new clinical candidate drug to treat pain.