Pharmacology/Drug Development

Voltage-dependent sodium and calcium channels in pain-initiating nociceptor neurons are attractive targets for new analgesics. We made a permanently charged cationic derivative of an N-type calcium channel-inhibitor. Unlike cationic derivatives of local anesthetic sodium channel blockers like QX-314, this cationic compound inhibited N-type calcium channels more effectively with extracellular than intracellular application. Surprisingly, the compound is also a highly effective sodium channel inhibitor when applied extracellularly, producing more potent inhibition than lidocaine or bupivacaine. The charged inhibitor produced potent and long-lasting analgesia in mouse models of incisional wound and inflammatory pain, inhibited release of the neuropeptide calcitonin gene-related peptide (CGRP) from dorsal root ganglion neurons, and reduced inflammation in a mouse model of allergic asthma, which has a strong neurogenic component. The results show that some cationic molecules applied extracellularly can powerfully inhibit both sodium channels and calcium channels, thereby blocking both nociceptor excitability and pro-inflammatory peptide release.

Type-1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 (H3K9) methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects in an animal model of neuropathic pain. We show that nerve injury profoundly reduces mRNA levels CB1Rs but increases the expression of CB2 receptors in the rat DRG. Chromatin-immunoprecipitation results indicated increased enrichment of H3K9me2, a G9a-catalyzed repressive histone mark, at the promoter regions of the CB1R genes. G9a inhibition in nerve-injured rats not only upregulates CB1R expression level in the DRG but also potentiated the analgesic effect of a CB1R agonist on nerve injury-induced pain hypersensitivity. Furthermore, in mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce CB1R expression in the DRG and to decrease the analgesic effect of the CB1R agonist. Moreover, nerve injury diminished the inhibitory effect of the CB1R agonist on synaptic glutamate release from primary afferent nerves to spinal cord dorsal horn neurons in wild-type mice, but not in mice lacking Ehmt2 in DRG neurons. Our findings reveal that nerve injury diminishes the analgesic effect of CB1R agonists through G9a-mediated CB1R downregulation in primary sensory neurons.

Developing effective analgesics with fewer unwanted side effects is a pressing concern. Due to a lack of effective nonopioid options currently available, an alternative approach termed opioid-sparing evaluates the ability of a coadministered drug to reduce the amount of opioid needed to produce an antinociceptive effect. Opioids and benzodiazepines are often coprescribed. Although this approach is theoretically rational given the prevalent comorbidity of chronic pain and anxiety, it also has inherent risks of respiratory depression, which is likely responsible for the substantial percentage of fatal opioid overdoses that have involved benzodiazepines. Moreover, there have been no clinical trials to support the effectiveness of this drug combination nor has there been corroborative preclinical evidence using traditional animal models of nociception. The present studies examined the prescription µ-opioid analgesic oxycodone (0.003-0.1 mg/kg) and the prototypical benzodiazepine anxiolytic diazepam (0.03-1.0 mg/kg), alone and in combination, using an animal model of pain that examines the restoration of conflict-related operant behavior as evidence of analgesia. Results documented significant dose-related increases in thermal threshold following oxycodone treatment. Diazepam treatment alone did not produce significant antinociception. In combination, diazepam pretreatment shifted oxycodone functions upward in a dose-dependent manner, but the additive effects were limited to a narrow dose range. In addition, combinations of diazepam and oxycodone at higher doses abolished responding. Taken together, though intriguing, these findings do not provide sufficient evidence that coadministration of an anxiolytic will result in clinically relevant opioid-sparing for pain management, especially when considering the inherent risks of this drug class combination.

We have developed an experimental fibromyalgia (FM)-like mouse model using intermittent cold stress (ICS), where chronic pain is generalized, female-predominant and abolished in type 1 lysophosphatidic acid receptor-KO (LPA1-/-) mice, but is not reversed by systemic or brain treatment with morphine. In the present study, we investigated two issues whether both chronic pain mechanisms and lack of brain morphine analgesia are associated to each other in the ICS model, and what mechanisms are involved in the lack of morphine analgesia. The hyperalgesia was not affected in μ-opioid receptor-KO (MOPr-/-) mice, while the lack of brain morphine analgesia remained unchanged in LPA1-/- mice, which completely abolish the hyperalgesia in the ICS model. On the other hand, the lack of morphine analgesia was abolished in NR2A-NMDA receptor-KO (NR2A-/-) mice, and blocked by intracerebroventricular (i.c,v,) injection of (R)-CPP, an NR2A antagonist or by microinjection of siRNA for NR2A into PAG region, while no change was observed with Ro 04-5595, an NR2B antagonist (i.c.v.). The lack of morphine analgesia was also reversed by the concomitant treatment with 1 mg/kg (i.p.) of dextromethorphan possessing NMDA receptor antagonist activity, which has no analgesic activity. Finally, the hyperalgesia was completely reversed by methadone possessing MOPr agonist and NMDA receptor antagonist activity. Indeed, the methadone analgesia was abolished in MOPr-/- mice. All these results suggest that chronic pain status and lack of morphine analgesia are independent to each other, and the lack of morphine analgesia is mediated by an activation of NR2A-NMDA receptor system. SIGNIFICANCE STATEMENT: This study describes that a type of stress-induced wide-spread pain has chronic pain independent of endogenous opioid-mediated pain inhibitory system and lack of morphine analgesia independent of chronic pain status. This study describes that the lack of morphine analgesia is possibly mediated by endogenous opioid-induced analgesic tolerance and thereby reversed by anti-opioid NMDA receptor system. The latter view is evidenced by the findings that NMDA receptor blockades recover the morphine analgesia.

Endometriosis is a chronic painful disease highly prevalent in women that is defined by growth of endometrial tissue outside the uterine cavity and lacks adequate treatment. Medical use of cannabis derivatives is a current hot topic and it is unknown whether phytocannabinoids may modify endometriosis symptoms and development. Here we evaluate the effects of repeated exposure to Δ9-tetrahydrocannabinol (THC) in a mouse model of surgically-induced endometriosis. In this model, female mice develop mechanical hypersensitivity in the caudal abdomen, mild anxiety-like behavior and substantial memory deficits associated with the presence of extrauterine endometrial cysts. Interestingly, daily treatments with THC (2 mg/kg) alleviate mechanical hypersensitivity and pain unpleasantness, modify uterine innervation and restore cognitive function without altering the anxiogenic phenotype. Strikingly, THC also inhibits the development of endometrial cysts. These data highlight the interest of scheduled clinical trials designed to investigate possible benefits of THC for women with endometriosis.

Neuropathy is major source of chronic pain that can be caused by mechanically or chemically induced nerve injury. Intraplantar formalin injection produces local necrosis over a two-week period and has been used to model neuropathy in rats. To determine whether neuropathy alters dopamine (DA) receptor responsiveness in mesolimbic brain regions, we examined dopamine D-like and D-like receptor (DR) signaling and expression in male rats 14 days after bilateral intraplantar formalin injections into both rear paws. DR-mediated G-protein activation and expression of the DR long, but not short, isoform were reduced in nucleus accumbens (NAc) core, but not in NAc shell, caudate-putamen or ventral tegmental area of formalin-compared to saline-treated rats. In addition, DR-stimulated adenylyl cyclase activity was also reduced in NAc core, but not in NAc shell or prefrontal cortex, of formalin-treated rats, whereas DR expression was unaffected. Other proteins involved in dopamine neurotransmission, including dopamine uptake transporter and tyrosine hydroxylase, were unaffected by formalin treatment. In behavioral tests, the potency of a DR agonist to suppress intracranial self-stimulation (ICSS) was decreased in formalin-treated rats, whereas DR agonist effects were not altered. The combination of reduced DR expression and signaling in NAc core with reduced suppression of ICSS responding by a DR agonist suggest a reduction in D autoreceptor function. Altogether, these results indicate that intraplantar formalin produces attenuation of highly specific DA receptor signaling processes in NAc core of male rats and suggest the development of a neuropathy-induced allostatic state in both pre- and post-synaptic DA receptor function.