Preclinical Pain Models Research Articles

Grading facial expression is a sensitive means to detect grimace differences in orofacial pain in a rat model

Although pre-clinical models of pain are useful for defining relationships between biological mechanisms and pain, common methods testing peripheral sensitivity do not translate to the human pain experience. Facial grimace scales evaluate affective pain levels in rodent models by capturing and scoring spontaneous facial expression. But, the Rat Grimace Scale (RGS) has not assessed the common disorder of temporomandibular joint (TMJ) pain. A rat model of TMJ pain induced by jaw loading (1 hr/day for 7 days) was used to investigate the time course of RGS scores and compare them between different loading magnitudes with distinct peripheral sensitivity profiles (0N–no sensitivity, 2N–acute sensitivity, 3.5N–persistent sensitivity). In the 3.5N group, RGS is elevated over baseline during the loading period and one day after loading and is correlated with peripheral sensitivity (ρ = −0.48, p = 0.002). However, RGS is not elevated later when that group exhibits peripheral sensitivity and moderate TMJ condylar cartilage degeneration. Acutely, RGS is elevated in the 3.5N loading group over the other loading groups (p < 0.001). These findings suggest that RGS is an effective tool for detecting spontaneous TMJ pain and that spontaneous pain is detectable in rats that develop persistent TMJ sensitivity, but not in rats with acute resolving sensitivity.

GpTx-1 and [Ala5 , Phe6 , Leu26 , Arg28 ]GpTx-1, two peptide NaV 1.7 inhibitors: analgesic and tolerance properties at the spinal level.

The voltage-gated sodium channel NaV 1.7 is considered a therapeutic target for pain treatment based on human genetic evidence. GpTx-1 and its potent analogue [Ala5 , Phe6 , Leu26 , Arg28 ]GpTx-1 (GpTx-1-71) were recently characterized as NaV 1.7 inhibitors in vitro. Furthermore, the present work was conducted to investigate the analgesic properties of these two peptides in different pain models after spinal administration. The antinociceptive activities of both GpTx-1 and GpTx-1-71 were investigated in mouse models of acute, visceral, inflammatory and neuropathic pain. Furthermore, the side effects of GpTx-1 and GpTx-1-71 were evaluated in rotarod, antinociceptive tolerance, acute hyperlocomotion and gastrointestinal transit tests. The i.t. administration of both GpTx-1 and GpTx-1-71 dose-dependently produced powerful antinociception in the different pain models. This effect was attenuated by the opioid receptor antagonist naloxone, suggesting the involvement of the opioid system. In this study, repeated administration of these two_peptides produced spinal analgesia without a loss of potency over 8days in mouse models of acute, inflammatory and neuropathic pain. Moreover, spinal administration of GpTx-1 and GpTx-1-71 did not induce significant effects on motor coordination, evoke acute hyperlocomotion or increase gastrointestinal transit time. Our data indicate that the NaV 1.7 peptide inhibitors GpTx-1 and GpTx-1-71 produce powerful, nontolerance-forming analgesia in preclinical pain models, which might be dependent on the endogenous opioid system. In addition, at the spinal level, the limited side effects imply that these NaV 1.7 peptide inhibitors could be potentially developed as GpTx-1-based drugs for pain relief.

Systemic administration of the bifunctional opioid/neuropeptide FF receptors agonist BN-9 produced peripheral antinociception in preclinical mouse models of pain

We recently characterized a novel bifunctional agonist for opioid and neuropeptide FF receptors, named BN-9, which exhibited potent analgesia in the mouse tail-flick test when given centrally. To further evaluate its potential therapeutic efficacy for translational-medical development, the current work was performed to explore the antinociceptive activities of intraperitoneal (i.p.) administration of BN-9 in mouse models of tail-flick assay, formalin pain, visceral pain and post-operative pain. In the tail-flick test, BN-9 induced a dose-related antinociceptive effect, which was fully blocked by systemic pretreatment with the peripheral acting opioid receptor antagonist naloxone methiodide, but not supraspinal naloxone methiodide, implying the involvement of the peripheral opioid receptors. In addition, the systemic antinociception of BN-9 was antagonized by the selective antagonists of the μ- and κ-opioid receptors, independently of the δ-opioid and neuropeptide FF receptors. Similarly, dose-dependent analgesia was also produced by systemic BN-9 in different pain models via the peripheral opioid receptors, independently of the neuropeptide FF receptors. Furthermore, the side-effects of systemic BN-9 on motor performance, tolerance development and gastrointestinal transit inhibition were also evaluated. Repeated systemic injection of BN-9 produced non-tolerance analgesia over 8 days. Compared with morphine, intraperitoneal administration of BN-9 exerted less inhibition of gastrointestinal transit. These data show that BN-9 induced systemic analgesia with reduced side-effects on tolerance and constipation. This article suggests that systemic injection of BN-9 causes effective antinociception in different preclinical pain models via the peripheral opioid receptors, providing an attractive approach to develop peripherally acting opioid analgesics with multiple targeting properties.

Soluble Epoxide Hydrolase/Phosphodiesterase Dual Inhibitor is Efficacious Against Inflammatory Pain

The soluble epoxide hydrolase (sEH) is the principal regulator of epoxy fatty‐acids (EpFA) transforming them in to the less active diols. Inhibitors of sEH block this activity and maintain levels of EpFA in vivo which has demonstrated potent analgesia in several preclinical models of acute and chronic pain. However, sEH inhibitors have no observable action in the absence of a pain state [1]. It was therefore hypothesized that an additional factor was present in the painful state that was required for the antinociceptive action of the EpFA [2]. Investigation began with cyclic adenosine monophosphate (cAMP) and experiments demonstrated this was active but also that increasing cAMP with phosphodiesterase inhibitors increased EpFA as well. Phosphodiesterase enzymes hydrolyze the cyclic nucleotide into adenosine monophosphate altering its secondary messenger capacity. This led to the design and synthesis of several dual inhibitors of sEH and phosphodiesterase.Multi‐target ligands are designed to improve safety and efficacy in treatment of diseases. The use of drug combinations cannot be assumed to be safe given drug‐drug interactions. Additionally, the prediction of pharmacodynamic and pharmacokinetic relationships is less complicated with a single entity. The multi‐target ligands are selected to improve therapeutic efficacy by hitting multiple targets in physiological disorders. Thus, increasing the EpFA by halting their degradation and at the same time potentiating their activity with an increase in cAMP was the goal with synthesis of the dual inhibitor. We present the biochemical characterization as well as the in vivo efficacy in an inflammatory pain model for the selected dual inhibitor of sEH and PDE enzymes. The IC50 of RBH61 for sEH determined on the sEH human recombinant enzyme is 2.1 ± 0.5 nM. This is paired with a IC50 in the PDE assay of 8.1 ± 0.05 nM in human embryonic kidney (HEK293) cells. Evaluation of the dual inhibitor in a model of inflammatory pain in rats demonstrates its antinociceptive efficacy.Support or Funding InformationThis work was supported, in part, by National Institutes of Health grants: NIEHS R01 ES002710 and NIEHS/Superfund Research Program P42 ES004699, NINDS U54 NS079202 and Grants NIEHS T32ES007059, NIH 5T32DC008072‐05 and 4T32HL086350‐09.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain

A novel series of EP4 agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain, ultimately leading to the identification of the EP4 partial agonist GSK726701A.

VEGFR2 promotes central endothelial activation and the spread of pain in inflammatory arthritis

Chronic pain can develop in response to conditions such as inflammatory arthritis. The central mechanisms underlying the development and maintenance of chronic pain in humans are not well elucidated although there is evidence for a role of microglia and astrocytes. However in pre-clinical models of pain, including models of inflammatory arthritis, there is a wealth of evidence indicating roles for pathological glial reactivity within the CNS. In the spinal dorsal horn of rats with painful inflammatory arthritis we found both a significant increase in CD11b+ microglia-like cells and GFAP+ astrocytes associated with blood vessels, and the number of activated blood vessels expressing the adhesion molecule ICAM-1, indicating potential glio-vascular activation. Using pharmacological interventions targeting VEGFR2 in arthritic rats, to inhibit endothelial cell activation, the number of dorsal horn ICAM-1+ blood vessels, CD11b+ microglia and the development of secondary mechanical allodynia, an indicator of central sensitization, were all prevented. Targeting endothelial VEGFR2 by inducible Tie2-specific VEGFR2 knock-out also prevented secondary allodynia in mice and glio-vascular activation in the dorsal horn in response to inflammatory arthritis. Inhibition of VEGFR2 in vitro significantly blocked ICAM-1-dependent monocyte adhesion to brain microvascular endothelial cells, when stimulated with inflammatory mediators TNF-α and VEGF-A165a. Taken together our findings suggest that a novel VEGFR2-mediated spinal cord glio-vascular mechanism may promote peripheral CD11b+ circulating cell transmigration into the CNS parenchyma and contribute to the development of chronic pain in inflammatory arthritis. We hypothesise that preventing this glio-vascular activation and circulating cell translocation into the spinal cord could be a new therapeutic strategy for pain caused by rheumatoid arthritis.

Effects of ralfinamide in models of nerve injury and chemotherapy-induced neuropathic pain

Neuropathic pain is among the most common and difficult-to-treat types of chronic pain and is associated with sodium channel malfunction. The sodium channel blocker ralfinamide has exhibited potent analgesic effects in several preclinical pain models and in patients with mixed neuropathic pain syndromes (Phase II trials), but it failed to ameliorate neuropathic low back pain in Phase III trials. It is unclear whether ralfinamide is effective against neuropathic pain induced by specified etiologies. In the present study, the antinociceptive effects of ralfinamide in neuropathic pain models induced by spared nerve injury and chemotherapy were compared in a gabapentin-controlled manner. The effects of ralfinamide on physiological pain were evaluated in mechanical withdrawal, hot plate, and acetic acid writhing tests. We also investigated the effects of ralfinamide on cardiovascular function and locomotor activity. Oral ralfinamide dose-dependently alleviated spared nerve injury-induced allodynia in rats and mice. Ralfinamide increased mechanical withdrawal thresholds in oxaliplatin-induced and paclitaxel-induced neuropathic pain. Ralfinamide did not affect physiological pain, locomotion, or cardiovascular function. Together, ralfinamide attenuated mechanical allodynia in all the neuropathic pain models tested, with subtle differences in efficacy. The effect of ralfinamide is comparable to that of gabapentin, but with no interference in basal mechanical sensitivity. The present study supports the effectiveness of selective sodium channel blockade as an analgesic strategy, as well as the development of compounds similar to ralfinamide.

Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice.

Elevated N-methyl-D-aspartate receptor (NMDAR) activity is linked to central sensitization and chronic pain. However, NMDAR antagonists display limited therapeutic potential because of their adverse side effects. Novel approaches targeting the NR2B-PSD95-nNOS complex to disrupt signaling pathways downstream of NMDARs show efficacy in preclinical pain models. Here, we evaluated the involvement of interactions between neuronal nitric oxide synthase (nNOS) and the nitric oxide synthase 1 adaptor protein (NOS1AP) in pronociceptive signaling and neuropathic pain. TAT-GESV, a peptide inhibitor of the nNOS-NOS1AP complex, disrupted the in vitro binding between nNOS and its downstream protein partner NOS1AP but not its upstream protein partner postsynaptic density 95 kDa (PSD95). Putative inactive peptides (TAT-cp4GESV and TAT-GESVΔ1) failed to do so. Only the active peptide protected primary cortical neurons from glutamate/glycine-induced excitotoxicity. TAT-GESV, administered intrathecally (i.t.), suppressed mechanical and cold allodynia induced by either the chemotherapeutic agent paclitaxel or a traumatic nerve injury induced by partial sciatic nerve ligation. TAT-GESV also blocked the paclitaxel-induced phosphorylation at Ser15 of p53, a substrate of p38 MAPK. Finally, TAT-GESV (i.t.) did not induce NMDAR-mediated motor ataxia in the rotarod test and did not alter basal nociceptive thresholds in the radiant heat tail-flick test. These observations support the hypothesis that antiallodynic efficacy of an nNOS-NOS1AP disruptor may result, at least in part, from blockade of p38 MAPK-mediated downstream effects. Our studies demonstrate, for the first time, that disrupting nNOS-NOS1AP protein-protein interactions attenuates mechanistically distinct forms of neuropathic pain without unwanted motor ataxic effects of NMDAR antagonists.

CB1 cannabinoid receptor agonist mouse VD-hemopressin(α) produced supraspinal analgesic activity in the preclinical models of pain

Mouse VD-hemopressin(α) (VD-Hpα) is an undecapeptide that selectively activates CB1 cannabinoid receptor in in vitro functional tests, and exerts CB1-mediated central antinociception in the mouse tail-flick assay. The aim of the present study was to further investigate the analgesic properties of supraspinal mouse VD-Hpα in a range of preclinical pain models. Our results indicated that the classical cannabinoid agonist WIN 55,212-2 produced supraspinal analgesia in preclinical pain models, which was selectively antagonized by the CB1 antagonist/inverse agonist AM251, but not by the CB2 antagonist AM630. In contrast, in post-operative pain model and phase I of formalin test, intracerebroventricular administration of mouse VD-Hpα induced dose-related analgesia in mice, which were markedly reduced by pretreatment with the CB1 neutral antagonist AM4113, but not AM251, AM630 and the selective antagonists of opioid and Transient Receptor Potential Vanilloid Type 1 (TRPV1) receptors. Furthermore, in the acetic acid-induced visceral pain model, supraspinal administration of mouse VD-Hpα dose-dependently produced analgesic activities and the effects were significantly antagonized by both AM4113 and the TRPV1 receptor antagonist SB366791, but not AM251, AM630 and naloxone. In addition, central injection of mouse VD-Hpα did not have significant effect in phase II of formalin test. Taken together, the present work suggests that the CB1 receptor peptidic agonist mouse VD-Hpα produces supraspinal analgesia in preclinical pain models via a novel CB1 receptor-mediated mechanism, in a manner pharmacologically dissociable from WIN 55,212-2. In addition, TRPV1 receptor might also be involved in mouse VD-Hpα-induced analgesia in a visceral pain model.

DRG Voltage-Gated Sodium Channel 1.7 Is Upregulated in Paclitaxel-Induced Neuropathy in Rats and in Humans with Neuropathic Pain.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse effect experienced by cancer patients receiving treatment with paclitaxel. The voltage-gated sodium channel 1.7 (Nav1.7) plays an important role in multiple preclinical models of neuropathic pain and in inherited human pain phenotypes, and its gene expression is increased in dorsal root ganglia (DRGs) of paclitaxel-treated rats. Hence, the potential of change in the expression and function of Nav1.7 protein in DRGs from male rats with paclitaxel-related CIPN and from male and female humans with cancer-related neuropathic pain was tested here. Double immunofluorescence in CIPN rats showed that Nav1.7 was upregulated in small DRG neuron somata, especially those also expressing calcitonin gene-related peptide (CGRP), and in central processes of these cells in the superficial spinal dorsal horn. Whole-cell patch-clamp recordings in rat DRG neurons revealed that paclitaxel induced an enhancement of ProTx II (a selective Nav1.7 channel blocker)-sensitive sodium currents. Bath-applied ProTx II suppressed spontaneous action potentials in DRG neurons occurring in rats with CIPN, while intrathecal injection of ProTx II significantly attenuated behavioral signs of CIPN. Complementarily, DRG neurons isolated from segments where patients had a history of neuropathic pain also showed electrophysiological and immunofluorescence results indicating an increased expression of Nav1.7 associated with spontaneous activity. Nav1.7 was also colocalized in human cells expressing transient receptor potential vanilloid 1 and CGRP. Furthermore, ProTx II decreased firing frequency in human DRGs with spontaneous action potentials. This study suggests that Nav1.7 may provide a potential new target for the treatment of neuropathic pain, including chemotherapy (paclitaxel)-induced neuropathic pain.SIGNIFICANCE STATEMENT This work demonstrates that the expression and function of the voltage-gated sodium channel Nav1.7 are increased in a preclinical model of chemotherapy-induced peripheral neuropathy (CIPN), the most common treatment-limiting side effect of all the most common anticancer therapies. This is key as gain-of-function mutations in human Nav1.7 recapitulate both the distribution and pain percept as shown by CIPN patients. This work also shows that Nav1.7 is increased in human DRG neurons only in dermatomes where patients are experiencing acquired neuropathic pain symptoms. This work therefore has major translational impact, indicating an important novel therapeutic avenue for neuropathic pain as a class.

Analgesic effect of ADX71441, a positive allosteric modulator (PAM) of GABAB receptor in a rat model of bladder pain

Therapeutic use of GABAB receptor agonists for conditions like chronic abdominal pain, overactive bladder (OAB) and gastroesophageal reflux disease (GERD) is severely affected by poor blood-brain barrier permeability and potential side effects. ADX71441 is a novel positive allosteric modulator (PAM) of the GABAB receptor that has shown encouraging results in pre-clinical models of anxiety, pain, OAB and alcohol addiction. The present study investigates the analgesic effect of ADX71441 to noxious stimulation of the urinary bladder and colon in rats. In female Sprague-Dawley rats, systemic (i.p), but not intrathecal (i.t), administration of ADX71441 produced a dose-dependent decrease in viscero-motor response (VMR) to graded urinary bladder distension (UBD) and colorectal distension (CRD). Additionally, intra-cerebroventricular (i.c.v.) administration of ADX71441 significantly decreased the VMRs to noxious UBD. In electrophysiology experiments, the drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD. In contrast, ADX71441 significantly decreased the responses of UBD-responsive lumbosacral (LS) spinal neurons in spinal intact rats. However, ADX71441 did not attenuate these LS neurons in cervical (C1-C2) spinal transected rats. During cystometrogram (CMG) recordings, ADX71441 (i.p.) significantly decreased the VMR to slow infusion without affecting the number of voiding contraction. These results indicate that ADX71441 modulate bladder nociception via its effect at the supra-spinal sites without affecting the normal bladder motility and micturition reflex in naïve adult rats.

Breaking barriers to novel analgesic drug development.

Acute and chronic pain complaints, although common, are generally poorly served by existing therapies. This unmet clinical need reflects a failure to develop novel classes of analgesics with superior efficacy, diminished adverse effects and a lower abuse liability than those currently available. Reasons for this include the heterogeneity of clinical pain conditions, the complexity and diversity of underlying pathophysiological mechanisms, and the unreliability of some preclinical pain models. However, recent advances in our understanding of the neurobiology of pain are beginning to offer opportunities for developing novel therapeutic strategies and revisiting existing targets, including modulating ion channels, enzymes and G-protein-coupled receptors.

Cannabinoid CB2 Agonist GW405833 Suppresses Inflammatory and Neuropathic Pain through a CB1 Mechanism that is Independent of CB2 Receptors in Mice.

GW405833, widely accepted as a cannabinoid receptor 2 (CB2) agonist, suppresses pathologic pain in preclinical models without the unwanted central side effects of cannabinoid receptor 1 (CB1) agonists; however, recent in vitro studies have suggested that GW405833 may also behave as a noncompetitive CB1 antagonist, suggesting that its pharmacology is more complex than initially appreciated. Here, we further investigated the pharmacologic specificity of in vivo antinociceptive actions of GW405833 in models of neuropathic (i.e., partial sciatic nerve ligation model) and inflammatory (i.e., complete Freund's adjuvant model) pain using CB2 and CB1 knockout (KO) mice, their respective wild-type (WT) mice, and both CB2 and CB1 antagonists. GW405833 (3, 10, and 30 mg/kg i.p.) dose dependently reversed established mechanical allodynia in both pain models in WT mice; however, the antiallodynic effects of GW405833 were fully preserved in CB2KO mice and absent in CB1KO mice. Furthermore, the antiallodynic efficacy of GW405833 (30 mg/kg i.p.) was completely blocked by the CB1 antagonist rimonabant (10 mg/kg i.p.) but not by the CB2 antagonist SR144528 (10 mg/kg i.p.). Thus, the antinociceptive properties of GW405833 are dependent on CB1 receptors. GW405833 (30 mg/kg i.p.) was also inactive in a tetrad of tests measuring cardinal signs of CB1 activation. Additionally, unlike rimonabant (10 mg/kg i.p.), GW405833 (10 mg/kg, i.p.) did not act as a CB1 antagonist in vivo to precipitate withdrawal in mice treated chronically with Δ9-tetrahydrocannabinol. The present results suggest that the antiallodynic efficacy of GW405833 is CB1-dependent but does not seem to involve engagement of the CB1 receptor's orthosteric site.

Intra-spinal microstimulation may alleviate chronic pain after spinal cord injury

Chronic pain after spinal cord injury (SCI) is a form of central neuropathic pain that is debilitating and often refractory to current pharmacological treatments. Neurostimulation pain therapies, such as epidural spinal cord stimulation, have only moderate success in reducing SCI pain. The pathogenesis of SCI pain may involve a state of central neuronal hyperexcitability, especially in the spinal cord dorsal horn, that develops after injury. We hypothesize that the neuronal structures near the spinal cord injury site may be an important pain generator, and intraspinal microstimulation (ISMS) may normalize dorsal horn neuronal hyperexcitability and hence alleviate SCI pain. Specifically, ISMS may induce frequency-dependent conduction block on axons of afferent sensory neurons, in the spinothalamic tract and Lissauer’s tract. ISMS may also facilitate primary afferent depolarization that elicits presynaptic inhibition of incoming afferent inputs. Together, these actions will reduce abnormal afferent inputs and ascending pain signals before they can reach the brain. Furthermore, ISMS may directly induce inhibitory postsynaptic potentials in dorsal horn neurons, and trigger the release of endogenous inhibitory neurotransmitters, opioids and serotonin to inhibit postsynaptic neurons and restore the compromised segmental pain inhibition after SCI. Finally, ISMS may alter the frequency and pattern of discharge such that the rostrally conducted impulses no longer code pain or activate brain areas concerned with pain signaling. Based on recent progress in understanding spinal learning and plasticity, we also postulate that repetitive or long-term ISMS may help the dorsal horn “reset” neuronal excitability and regain normal pain processing for a prolonged period. By finely tuning the stimulation parameters (e.g., intensity, pulse width, frequency), position, and geometry of ISMS electrode, multiple spinal structures (e.g., dorsal horn, dorsal column, spinothalamic tract) may be modulated to induce synergistic pain inhibition. Our hypothesis can be readily tested in preclinical models of SCI pain by using a combination of in vivo electrophysiological (neuronal activity) and animal behavioral (pain response) approaches. Since ISMS electrodes stimulate the spinal structures directly, we expect that the effective stimulus intensity and energy consumption can be lower than that for epidural spinal cord stimulation. The proposed hypothesis may provide insights and rationales for developing a novel neurostimulation pain therapy by directly inhibiting the pain generators in the spinal cord, and ISMS may be an alternative strategy to treat SCI pain.

Benzoxazolinone aryl sulfonamides as potent, selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model

Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.

Modeling human lumbar spinal osteoarthritis and low back pain through preclinical studies

Low back pain (LBP) is a prevalent condition and the primary cause for global disability. This is likely a result, in part, of poor pain management that stems from a lack of understanding of the pathophysiological mechanisms that contribute to this complex condition. To better understand the underlying mechanisms of LBP, we use preclinical animal pain models to mimic the human condition and examine the neurobiological processes induced from spine‐related tissues that contribute to pain. We are currently investigating two common causes for LBP, lumbar facet joint degeneration and muscle pain. Facet joint degeneration that is consistent with spinal osteoarthritis (OA) has distinctive patterns of histopathological findings. We wanted to determine if a rat model of lumbar vertebral segmental hypomobility induces degeneration that replicates these characteristic findings to mimic spinal OA. We performed time‐dependent investigations to determine the location, severity, and progression of histopathological changes to the articular cartilage following the onset of hypomobility. Our findings suggest that lumbar spine hypomobility induces facet joint histopathological changes that mimic spinal OA histopathology, location (patterning), and progression. We have also identified the potential contribution of adjacent joint tissues including the synovium, capsule, and subchondral bone in the onset and progression of OA. The low back muscles are also a clinically relevant source of LBP. We are investigating a rat model to mimic chronic low back muscle pain (LBPm) using nerve growth factor (NGF), a critical regulator of pain signaling and essential to muscle pain including LBPm. Repeated lumbar paraspinal intramuscular NGF injections in rats induce prolonged pain behavioral responses (e.g., paraspinal mechanical hyperalgesia) and enhanced response profiles of dorsal horn nociceptive neurons. These findings are consistent with central (spinal) sensitization, a pathophysiological state implicated in chronic pain. Central sensitization is associated with a glial cell mediated inflammatory process called neuroimmune activation, which is defined experimentally as glial cell hyperactivation with the release of proinflammatory cytokines. We hypothesized that repeated NGF paraspinal muscle injections contribute to persistent mechanical hyperalgesia and central sensitization, in part, through spinal cord neuroimmune activation. Our time course studies revealed that the development of NGF induced LBPm is associated with lumbar spinal cord dorsal horn microglial hyperactivation; suggesting that neuroimmune activation contributes to LBPm. Collectively, these models are promising and may provide a better mechanistic understanding to develop therapeutic approaches that offer meaningful LBP relief.Support or Funding InformationSLU Center for Neuroscience and NCMIC

Broad spectrum efficacy with LY2969822, an oral prodrug of metabotropic glutamate 2/3 receptor agonist LY2934747, in rodent pain models.

A body of evidence suggests activation of metabotropic glutamate 2/3 (mGlu2/3 ) receptors would be an effective analgesic in chronic pain conditions. Thus, the analgesic properties of a novel mGlu2/3 receptor agonist prodrug were investigated. After oral absorption, the prodrug LY2969822 rapidly converts to the brain penetrant, potent and subtype-selective mGlu2/3 receptor agonist LY2934747. Behavioural assessments of allodynia, hyperalgesia and nocifensive behaviours were determined in preclinical pain models after administration of LY2969822 0.3-10mg·kg-1 . In addition, the ability of i.v. LY2934747 to modulate dorsal horn spinal cord wide dynamic range (WDR) neurons in spinal nerve ligated (SNL) rats was assessed. Following treatment with LY2934747, the spontaneous activity and electrically-evoked wind-up of WDR neurons in rats that had undergone spinal nerve ligation and developed mechanical allodynia were suppressed. In a model of sensitization, orally administered LY2969822 prevented the nociceptive behaviours induced by an intraplantar injection of formalin. The on-target nature of this effect was confirmed by blockade with an mGlu2/3 receptor antagonist. LY2969822 prevented capsaicin-induced tactile hypersensitivity, reversed the SNL-induced tactile hypersensitivity and reversed complete Freund's adjuvant - induced mechanical hyperalgesia. The mGlu2/3 receptor agonist prodrug demonstrated efficacy in visceral pain models, including a colorectal distension model and partially prevented the nocifensive behaviours in the mouse acetic acid writhing model. Following oral administration of the prodrug LY2969822, the mGlu2/3 receptor agonist LY2934747 was formed and this attenuated pain behaviours across a broad range of preclinical pain models.

Development of a pharmacodynamic biomarker to measure target engagement from inhibition of the NGF–TrkA pathway

BackgroundNGF signaling through TrkA triggers pathways involved in a wide range of biological effects. Clinical trials targeting either NGF or TrkA are ongoing to treat various diseases in the areas of oncology, neuroscience, and for pain, but there is no described measure of target engagement of TrkA in these studies. New methodWe have developed custom ELISA assays to measure NGF-induced phosphorylation of TrkA specific for rodent and human receptors. Optimized tissue processing methods allow for detection in both the brain and in skin. In addition, TrkB and TrkC assays have been in established to evaluate selectivity against other neurotrophin receptors. ResultsIn a preclinical NGF-induced pain model, we show that pre-dosing with a TrkA inhibitor prevents phosphorylation of TrkA in the skin at a dose that is efficacious in reversal of thermal hypersensitivity. In addition, we show data in non-human primate and human skin supporting the potential use of this approach to enable translational target engagement.Comparison with existing methods: Existing methods involve animal models expressing TrkA tumors or injection of over-expressing TrkA recombinant cells into animals. Our method can measure target engagement in both normal and disease tissues in preclinical animal models and human skin. ConclusionsWe have developed methods to assess target engagement for drug programs aimed at disrupting NGF-induced TrkA signaling. This includes preclinical determination of selectivity against other neurotrophin receptors and estimation of functional peripheral restriction. Preliminary data supports this method can be translated into a clinical pharmacodynamic readout using human skin biopsies.

Functional MRI of the Reserpine-Induced Putative Rat Model of Fibromyalgia Reveals Discriminatory Patterns of Functional Augmentation to Acute Nociceptive Stimuli

Functional neuroimaging, applied to pre-clinical models of chronic pain, offers unique advantages in the drive to discover new treatments for this prevalent and oppressive condition. The high spatial and temporal resolution of fMRI affords detailed mapping of regional pharmacodynamics that underlie mechanisms of pain suppression by new analgesics. Despite evidence supporting the translational relevance of this approach, relatively few studies have investigated fMRI abnormalities in rodent models of chronic pain. In this study, we used fMRI to map the BOLD response in a recently developed putative rat model of fibromyalgia to innocuous and acute nociceptive stimuli by applying a step-wise graded electrical forepaw stimulation paradigm, with comparison to healthy controls. We observed discriminatory functional signatures (p < 0.001) to 2 mA electrical forepaw stimulation, found to be innocuous in the control group. As such, this translational approach provides sensitive and quantitative neural correlates of the underlying chronic disease. The regional patterns of functional augmentation were found to be concordant with previous studies of nociception in the anaesthetised rat brain, supporting the specificity of this approach in the study of altered central pain processing in reserpine induced myalgia. The methodology introduced in this work represents a novel platform for emerging treatment evaluation in highly experimentally controlled conditions.

Nonpharmacological Interventions in Targeting Pain-Related Brain Plasticity.

Chronic pain is a highly prevalent and debilitating condition that is frequently associated with multiple comorbid psychiatric conditions and functional, biochemical, and anatomical alterations in various brain centers. Due to its widespread and diverse manifestations, chronic pain is often resistant to classical pharmacological treatment paradigms, prompting the search for alternative treatment approaches that are safe and efficacious. The current review will focus on the following themes: attentional and cognitive interventions, the role of global environmental factors, and the effects of exercise and physical rehabilitation in both chronic pain patients and preclinical pain models. The manuscript will discuss not only the analgesic efficacy of these therapies, but also their ability to reverse pain-related brain neuroplasticity. Finally, we will discuss the potential mechanisms of action for each of the interventions.