Animal Studies

Chronic pain can be challenging to treat and increases the risk of developing psychological disorders such as depression. Identifying novel treatment modalities that effectively alleviate pain is essential to improve clinical treatment and rehabilitation for patients with pain conditions. Ketamine is an effective analgesic for many types of pain. However, its widespread use is limited by its side effect profile and requirement for intranasal/intravenous administration under medical supervision. (2R,6R)-hydroxynorketamine (HNK) is a ketamine metabolite that lacks the psychotomimetic effects of its parent drug but retains ketamine's anti-stress effects. Therefore, it is natural to question whether (2R,6R)-HNK may also possess analgesic activity. Administration of (2R,6R)-HNK produced antinociception in healthy mice exposed to a noxious, painful stimulus 24 hours after injection. The dose response for the delayed-yet-persistent antinociception revealed an inverted U shape with significant antinociception at doses of 10-18 mg/kg for both sexes and 30 mg/kg in female mice. Mice pretreated with different receptor antagonists to examine the potential mechanism for (2R,6R)-HNK mediated antinociception revealed a mechanism dependent on AMPA receptors and not opioid receptors. In contrast, ketamine antinociception was not dependent on AMPA receptors and partially dependent on opioid receptors. These results demonstrate that both (2R,6R)-HNK and the parent drug ketamine produce antinociception but work via different neural mechanisms. In separate studies, (2R,6R)-HNK administration reversed mechanical allodynia associated with localized inflammatory pain induced in mice by injecting λ-carrageenan into the hind paw. The onset for this analgesia-like activity was less than 1 hour with a duration greater than 24 hours following a single administration. (2R,6R)-HNK was effective at reversing mechanical allodynia at doses of 10 & 30 mg/kg in both male and female animals. These results demonstrate (2R,6R)-HNK exhibits great promise for treating inflammatory pain in addition to other pain types. Overall, these data suggest that (2R,6R)-HNK may be a safe alternative therapy for pain that could be made widely available to patients and support the need for continued investigation and development of (2R,6R)-HNK as a novel non-opioid pain treatment.

Opioids have been increasingly prescribed to treat chronic pain since the 1980s, despite evidence that long-term use of opioids may lead to tolerance and pain sensitization called opioid-induced hyperalgesia (OIH). OIH has been demonstrated in both preclinical models and healthy human volunteers, but is understudied and there is need for novel analgesics capable of mitigating OIH. α /α -selective GABA receptor positive allosteric modulators (PAMs) act specifically at subunits of the GABA receptor found to mediate analgesia, and have demonstrated antinociceptive effects in models of chronic inflammatory and neuropathic pain. However, the efficacy of these compounds at relieving opioid-induced pain hypersensitivity have not yet been investigated. This study systematically examined the antinociceptive effects of α /α -selective GABA receptor PAMs alone and in combination with acetaminophen in an OIH rat model wherein repeated treatment with the opioid fentanyl induces mechanical hyperalgesia. The von Frey test was used to measure mechanical nociception. Duration of actions of α /α -selective GABA receptor PAMs (KRM-II-81, NS16085, HZ-166) alone were studied, and combinations of KRM-II-81 and acetaminophen were also studied at fixed ratios (1:1, 1:3, 3:1). Dose-addition analysis was used to assess the antinociceptive interactions between KRM-II-81 and acetaminophen. α /α -selective GABA receptor PAMs were able to fully reverse mechanical sensitivity caused by OIH. Furthermore, KRM-II-81/acetaminophen combinations produced additive to supra-additive interactions depending on the drug mixture ratios. These findings support the idea that α /α -selective GABA receptor PAMs could serve as novel analgesics for treating OIH, and may interact favorably with other non-opioid analgesics.

In most preclinical models of neuropathic pain, hypersensitivity to pain often resolves after a few weeks. However, spared nerve injury (SNI) produces a persistent pain state that lasts at least 18 months. The goal of the current study was to evaluate antihyperalgesic effects of mu-opioid receptor (MOR) agonists varying in potency and efficacy in the SNI model. We hypothesized that the MOR agonists, fentanyl, morphine, and nalbuphine, would produce antihyperalgesic effects in both male and female rats. Mechanical hypersensitivity was evaluated by measuring responses to increasing pressure (g) applied to each paw (paw pressure test or Randall-Selitto test) with a maximum cut-off of 300 g. Using a within-subjects design, responses were evaluated before and after SNI or sham surgery in male and female rats, and cumulative dose effects curves for fentanyl (0.01-0.1 mg/kg), morphine (0.3-10 mg/kg), and nalbuphine (0.3-10 mg/kg) were determined. Both male and female sham rats demonstrated an initial hypersensitivity on their ipsilateral paw following surgery that dissipated within 7 d. Following SNI surgery, paw pressure thresholds on the injured paw were lower as compared with the pre-surgical response and with the contralateral paw for at least 20 wks in male and female rats. In male rats, fentanyl, morphine, and nalbuphine dose-dependently alleviated SNI-induced hypersensitivity, with EC50 values of 0.03 (± 0.007), 3.9 (± 0.8), and 5.3 (± 0.8) mg/kg, respectively. In female rats, fentanyl, morphine, and nalbuphine also alleviated SNI-induced hypersensitivity in a dose-dependent manner, with EC50 values of 0.01 (± 0.002), 4.1 (± 0.8), and 5.5 (± 0.8) mg/kg, respectively. Naltrexone (0.3 mg/kg) produced rightward shifts in the fentanyl and morphine dose effect curves. Interestingly, fentanyl and morphine also increased paw pressure thresholds in the uninjured, contralateral paws and sham-treated ipsilateral paws in male and female rats. In contrast, gabapentin produced anti-hyperalgesic effects in both male and female rats at 180 mg/kg as demonstrated by a return to pre-surgical, paw pressure thresholds without altering thresholds in the non-injured paw. These results demonstrate MOR agonists produce dose-dependent increases in paw pressure thresholds following SNI surgery in both male and female rats. As expected, MOR agonists demonstrated a rank order in potency with fentanyl > morphine > nalbuphine in both sexes, and only fentanyl was slightly more potent in females. Fentanyl and morphine also increased paw pressure thresholds in non-injured paws, suggesting that large doses inhibited responses to mechanical stimulation perhaps due to sedation and/or behavioral suppression. Overall, these findings demonstrate that MOR agonists produce antihyperalgesic effects in male and female rats potentially with narrow therapeutic indices. Future studies will investigate other behavioral effects of MOR agonists, such as reinforcing and interoceptive effects, in this chronic neuropathic pain model.

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity or biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein-biased MOR agonists, SR-17018 and SR-14968 stabilize the mu opioid receptor in a wash-resistant, yet antagonist-reversible, G protein signaling state. Furthermore, we demonstrate that these structurally related biased agonists, are noncompetitive for radiolabeled MOR antagonist binding and while they stimulate G protein signaling in mouse brain, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR-17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.

Small extracellular vesicles (sEVs) are 30-150 nm membranous particles released by a variety of cells and serve as a mediator in intercellular communication between adjacent and distal cells. sEVs carry biomolecular cargo including miRNAs, mRNAs, proteins, and lipids, which are selectively sorted and packaged and mirror the physiological state of the donor cells. Disease states can alter sEV composition affecting the message carried and thereby, its functional impact. Microglia, as the tissue-resident macrophages and primary innate immune cells of the central nervous system, play an important role in neuropathic pain. Here, we investigated alterations in the composition of serum sEVs from a mouse model of neuropathic pain and assessed the functional consequences of sEV uptake by primary cortical microglia.

The worsening opioid epidemic has highlighted the need for the development of new, safe, and effective analgesic therapeutics. Opioid therapy currently is associated with the risk of conversion to addiction, diversion from patients for whom use is intended, and the development of analgesic tolerance, or the loss of efficacy with continued treatment. To this end, we have developed a line of non-opioid, agmatine-based compounds and assessed them for their efficacy in reversing behavioral expressions of pain in animal models, as well as evaluated their safety following chronic exposure. We have previously shown that agmatine, decarboxylated L-arginine, is an N-methyl-D-aspartate (NMDA) receptor antagonist that preferentially antagonizes receptors that express NR2B subunits. This preferential antagonism is desirable for NMDA-based therapeutics as it can lead to a widening of the therapeutic window and avoidance of the side effects commonly associated with NMDA antagonism including motor ataxia and psychoactive effects. However, agmatine has shown limited penetration through the blood brain barrier (BBB) and a short systemic half-life, limiting its clinical utility. We have designed strategically-substituted agmatine (SSA) compounds with the goal of improving its penetration through the BBB by increasing the lipophilicity of agmatine, potentially improving distribution across the BBB and increasing its half-life following systemic delivery. To this end, we have evaluated this series of SSAs for safety and efficacy in multiple animal models of pain. Mice (21-30 g, M/F) were assessed for their baseline mechanical sensitivity, and then one of several models of pain was induced: inflammatory pain (Complete Freund's Adjuvant, (CFA) injected into a hindpaw), neuropathic pain (spared nerve injury surgery), or post-surgical pain (hindpaw muscle incision). Mechanical sensitivity was again assessed, then an SSA compound, agmatine (the parent compound), or vehicle control was administered, and mechanical sensitivity was recorded for up to three hours following administration. The SSA compounds effectively reversed pain behaviors in mice following administration in the various pain models. Additionally, side effects characteristic of NMDA receptor antagonists were assessed and not found at the range of doses that produced analgesia, indicating a wide therapeutic window. These data indicate that the strategically-substituted agmatines are anti-hyperalgesic compounds with a wide therapeutic window, avoiding the motor impairment typical of drugs of this class.

Irritable Bowel Syndrome is a functional gastrointestinal (GI) disorder that leads to chronic abdominal pain. The perception of this visceral pain involves peripheral mechanisms, such as nociceptors with cell bodies in dorsal root ganglia (DRG) and axons in the gut wall, and central mechanisms, such as neuroplasticity within the spinal cord and brain. Lysophosphatidylcholine (LPC), which has recently been shown to bind to GPR55 in human prostate carcinoma cells, is released from cell membranes and is elevated in patients with IBS. Dorsal root ganglion (DRG) neurons express GPR55, and activation of GPR55 has been implicated in inflammatory pain. Therefore, we hypothesised that elevated LPC during IBS contributes to pain due to its activation of GPR55 on dorsal root ganglion (DRG) neurons, leading to the excitation of visceral pain pathways. Current clamp recordings revealed that application of LPC (10 µM) to murine DRG neurons depolarised the resting membrane potential (p = 0.0001) by approximately 8 mV and decreased the rheobase (p < 0.05) by approximately 20%. Using ratiometric Ca2+ imaging using FURA-2 AM, LPC (10 µM) doubled intracellular [Ca2+]i. This effect was significantly reduced by the selective GPR55 antagonist CID16020046 (10 µM) (p < 0.05), suggesting the response to LPC is at least partially mediated by GPR55. The source of the [Ca2+] elevation following LPC application was elucidated using cyclopiazonic acid (CPA; 10 µM), which depletes intracellular Ca2+ stores, and a 0-Ca2+ external solution to remove the contribution of Ca2+ influx from extracellular sources. While both significantly decreased the Ca2+ influx elicited by LPC, the 0-Ca2+ external solution almost abolished the effect (p < 0.0001) of LPC. Together, these data suggest that the increased [Ca2+] elicited by LPC activation of GPR55 is partially mediated through the release of Ca2+ from intracellular stores but is mostly due to influx of extracellular Ca2+.

Brain mechanisms linking chronic pain conditions and development of comorbid clinical depression are still largely unknown. Here, we used a genome-wide microarray analysis to examine the genetic profile of the hippocampus, a limbic region that regulates mood and stress responses, from male rats exposed to 21 days of inflammatory pain. Bioinformatic gene network/canonical pathways analyses have identified significantly dysregulated genes with known roles in either neuroinflammation or neurodegenerative processes. Akt (protein kinase B) was identified as the main network hub gene. Altered activity of Akt-related signaling pathways has been linked to both the development of depressive state and antidepressant treatment. Furthermore, lipocalin-2 (Lcn2) or NGAL was identified as one of the highest upregulated genes (~ 2-fold) within the hippocampus during chronic pain state. Lcn2/NGAL is an iron-related protein with roles in innate immune response and cell differentiation/maturation that was recently implicated in regulation of emotional behaviors and cognitive function through regulation of neuronal excitability and dendritic spine formation/maturation. Besides the hippocampus, robust increases in Lcn2/NGAL mRNA were also observed within the prefrontal cortex (PFC) and anterior cingulate cortex (ACC), as well as in the brains of female rats exposed to the same pain paradigm. Overall, the results of this study continue to strengthen the idea that dysregulation of genes involved in neuroinflammatory and neurodegenerative processes in the hippocampus and other limbic brain areas may be involved in the development of mood disorders during the chronic pain state.