Chronicling changes in the somatosensory neurons after peripheral nerve injury.

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.

Inhibition of experimental visceral pain in rodents by cebranopadol.

The aim of this study was to investigate the efficacy of cebranopadol in two rodent models of visceral pain. Cebranopadol is a first-in-class analgesic with agonist activity at the nociceptin/orphanin FQ opioid peptide receptor and classical µ-, δ- and κ-opioid peptide receptors. Colitis was induced in Naval Medical Research Institute mice by intra-rectal infusion of mustard oil. The effects of intravenous cebranopadol pretreatment on spontaneous pain behaviours and referred allodynia and hyperalgesia were assessed. Pancreatitis was induced in Sprague-Dawley rats by intravenous administration of dibutyltin dichloride. After 6 days, the effects of intravenous cebranopadol on withdrawal reactions to mechanical abdominal stimulation with von Frey filaments were assessed. In mice with experimental colitis, cebranopadol dose-dependently inhibited spontaneous pain behaviours and allodynic and hyperalgesic withdrawal reactions, with half-maximal effective dose values of 4.6 µg/kg [95% confidence interval (CI): 2.9-7.9] for inhibition of spontaneous pain behaviours, 2.2 µg/kg (95% CI: 1.3-3.4) for inhibition of referred allodynia and 2.4 µg/kg (95% CI: 1.4-3.6) for inhibition of referred hyperalgesia in mice with colitis. In rats with experimental pancreatitis, cebranopadol dose-dependently inhibited abdominal tactile allodynia (half-maximal effective dose, 0.13 µg/kg; 95% CI: 0.03-0.49). Behavioural manifestations of visceral pain were almost completely abolished at the highest doses tested in mice (17.2 µg/kg, intravenous) and rats (2.4 µg/kg, intravenous). We conclude that cebranopadol is a potent and effective antiallodynic and antihyperalgesic agent in rodent models of visceral pain.

Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine.

Objective The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey. Methods We used a comprehensive integrated multi-molecular immunofluorescence labeling strategy to extensively profile the rat DDI and to a lesser extent that of the Rhesus monkey. Results The DDI was distributed across a dense, pervasive capillary network and included free nerve endings of peptidergic CGRP-expressing C fibers that were closely intertwined with noradrenergic (NA) sympathetic fibers and thin-caliber nonpeptidergic "C/Aδ" fibers. These newly identified C/Aδ fibers were unmyelinated, like C fibers, but expressed NF200, usually indicative of Aδ fibers, and uniquely co-labeled for the CGRP co-receptor, RAMP1. Slightly-larger caliber NF200-positive fibers co-labeled for myelin basic protein (MBP) and terminated as unbranched corpuscular endings. The DDI peptidergic fibers co-labeled for the lectin IB4 and expressed presumably excitatory α1-adrenergic receptors, as well as inhibitory 5HT1D receptors and the delta opioid receptor (δOR), but rarely the mu opioid receptor (µOR). Labeling for P2X3, TRPV1, TRPA1, and parasympathetic markers was not observed in the DDI. Interpretation These results suggest potential functional interactions, wherein peptidergic DDI fibers may be activated by stress-related sympathetic activity, resulting in CGRP release that could be detected in the circulation. CGRP may also activate nonpeptidergic C/Aδ fibers that are likely mechanosensitive or polymodal, leading to activation of post-synaptic pain transmission circuits. The distribution of α1-adrenergic receptors, RAMP1, and the unique expression of the δOR on CGRP-expressing DDI fibers suggest strategies for functional modulation and application to therapy.

Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention.

Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.

Biocompatible PEDOT:Nafion composite electrode coatings for selective detection of neurotransmitters in vivo.

A Nafion and poly(3,4-ethylenedioxythiophene) (PEDOT) containing composite polymer has been electropolymerized on carbon-fiber microelectrodes with the goal of creating a mechanically stable, robust, and controllable electrode coating that increases the selectivity and sensitivity of in vivo electrochemical measurements. The coating is deposited on carbon-fiber microelectrodes by applying a triangle waveform from +1.5 V to -0.8 V and back in a dilute solution of ethylenedioxythiophene (EDOT) and Nafion in acetonitrile. Scanning electron microscopy demonstrated that the coating is uniform and ∼100 nm thick. Energy-dispersive X-ray spectroscopy demonstrated that both sulfur and fluorine are present in the coating, indicating the incorporation of PEDOT (poly(3,4-ethylenedioxythiophene) and Nafion. Two types of PEDOT:Nafion coated electrodes were then analyzed electrochemically. PEDOT:Nafion-coated electrodes made using 200 µM EDOT exhibit a 10-90 response time of 0.46 ± 0.09 s versus 0.45 ± 0.11 s for an uncoated fiber in response to a 1.0 µM bolus of dopamine. The electrodes coated using a higher EDOT concentration (400 µM) are slower with a 10-90 response time of 0.84 ± 0.19 s, but display increased sensitivity to dopamine, at 46 ± 13 nA/µM, compared to 26 ± 6 nA/µM for the electrodes coated in 200 µM EDOT and 13 ± 2 nA/µM for an uncoated fiber. PEDOT:Nafion-coated electrodes were lowered into the nucleus accumbens of a rat, and both spontaneous and electrically evoked dopamine release were measured. In addition to improvements in sensitivity and selectivity, the coating dramatically reduces acute in vivo biofouling.

Pain relief produces negative reinforcement through activation of mesolimbic reward-valuation circuitry.

Relief of pain is rewarding. Using a model of experimental postsurgical pain we show that blockade of afferent input from the injury with local anesthetic elicits conditioned place preference, activates ventral tegmental dopaminergic cells, and increases dopamine release in the nucleus accumbens. Importantly, place preference is associated with increased activity in midbrain dopaminergic neurons and blocked by dopamine antagonists injected into the nucleus accumbens. The data directly support the hypothesis that relief of pain produces negative reinforcement through activation of the mesolimbic reward-valuation circuitry.

CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells.

We previously reported that CAP1 (Cyclase-Associated Protein 1) regulates matrix adhesion in mammalian cells through FAK (Focal Adhesion Kinase). More recently, we discovered a phosphor-regulation mechanism for CAP1 through the Ser307/Ser309 tandem site that is of critical importance for all CAP1 functions. However, molecular mechanisms underlying the CAP1 function in adhesion and its regulation remain largely unknown. Here we report that Rap1 also facilitates the CAP1 function in adhesion, and more importantly, we identify a novel signaling pathway where CAP1 mediates the cAMP signals, through the cAMP effectors Epac (Exchange proteins directly activated by cAMP) and PKA (Protein Kinase A), to activate Rap1 in stimulating matrix adhesion in colon cancer cells. Knockdown of CAP1 led to opposite adhesion phenotypes in SW480 and HCT116 colon cancer cells, with reduced matrix adhesion and reduced FAK and Rap1 activities in SW480 cells while it stimulated matrix adhesion as well as FAK and Rap1 activities in HCT116 cells. Importantly, depletion of CAP1 abolished the stimulatory effects of the cAMP activators forskolin and isoproterenol, as well as that of Epac and PKA, on matrix adhesion in both cell types. Our results consistently support a required role for CAP1 in the cAMP activation of Rap1. Identification of the key role for CAP1 in linking the major second messenger cAMP to activation of Rap1 in stimulating adhesion, which may potentially also regulate proliferation in other cell types, not only vertically extends our knowledge on CAP biology, but also carries important translational potential for targeting CAP1 in cancer therapeutics.

Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors.

Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male Sprague-Dawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80% of these cells co-express both receptors. Agonist-directed cell lesion in the RVM with the cytotoxin, saporin, using either CCK-saporin to target CCK receptor expressing cells, or dermorphin-saporin to target MOR expressing cells, resulted in concomitant loss of CCK2 and MOR expressing cells, did not alter the basal sensory thresholds but abolished the hyperalgesia induced by microinjection of CCK into the RVM. The findings suggest that these CCK2-MOR co-expressing RVM neurons facilitate pain and can be directly activated by CCK input to the RVM. Furthermore, lesion of these RVM neurons did not affect the initial development of neuropathic pain in the hind paw upon injury to the sciatic nerve, but the abnormal pain states were short lived such that by about day 9 the sensory thresholds had reverted to pre-injury baselines despite the existing neuropathy. These data support our hypothesis and identify CCK2-MOR co-expressing neurons in the RVM as potential therapeutic targets for neuropathic pain.

Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion.

Cyclase-associated protein 1 (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated focal adhesion kinase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that cyclin-dependent kinase 5 (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.

Antihyperalgesic effects of Meteorin in the rat chronic constriction injury model: a replication study.

Data from preclinical research have been suggested to suffer from a lack of inherent reproducibility across laboratories. The goal of our study was to replicate findings from a previous report that demonstrated positive effects of Meteorin, a novel neurotrophic factor, in a rat model of neuropathic pain induced by chronic constriction injury (CCI). Notably, 5 to 6 intermittent subcutaneous (s.c.) injections of Meteorin had been reported to produce reversal of mechanical allodynia/thermal hyperalgesia after injury, wherein maximum efficacy of Meteorin was reached slowly and outlasted the elimination of the compound from the blood by several weeks. Here, we evaluated the efficacy of Meteorin in reversing hindpaw mechanical hyperalgesia and cold allodynia in male, Sprague-Dawley rats with CCI. Nociceptive behavior was monitored before and after CCI, and after drug treatment until day 42 after injury. Systemic administration of recombinant mouse Meteorin (0.5 and 1.8 mg/kg, s.c.) at days 10, 12, 14, 17, and 19 after CCI produced a prolonged reversal of neuropathic hypersensitivity with efficacy comparable with that obtained with gabapentin (100 mg/kg, orally). Despite some protocol deviations (eg, nociceptive endpoint, animal vendor, testing laboratory, investigator, etc.) being incurred, these did not affect study outcome. By paying careful attention to key facets of study design, using bioactive material, and confirming drug exposure, the current data have replicated the salient findings of the previous study, promoting confidence in further advancement of this novel molecule as a potential therapy for neuropathic pain.

Positive allosteric modulators of nonbenzodiazepine γ-aminobutyric acidA receptor subtypes for the treatment of chronic pain.

Chronic neuropathic pain may be caused, in part, by loss of inhibition in spinal pain processing pathways due to attenuation of local GABAergic tone. Nociception and nocifensive behaviors are reduced after enhancement of tonically activated extrasynaptic GABAAR-mediated currents by agonist ligands for δ subunit-containing GABAARs. However, typical ligands that target δ subunit-containing GABAARs are limited due to sedative effects at higher doses. We used the spinal nerve ligation (SNL) and gp120 models of experimental neuropathic pain to evaluate compound 2-261, a nonbenzodiazepine site positive allosteric modulator of α4ß3δ GABAARs optimized to be nonsedative by selective activation of ß2/3-subunit-containing GABAARs over receptor subtypes incorporating ß1 subunits. Similar levels of 2-261 were detected in the brain and plasma after intraperitoneal administration. Although systemic 2-261 did not alter sensory thresholds in sham-operated animals, it significantly reversed SNL-induced thermal and tactile hypersensitivity in a GABAAR-dependent fashion. Intrathecal 2-261 produced conditioned place preference and elevated dopamine levels in the nucleus accumbens of nerve-injured, but not sham-operated, rats. In addition, systemic pretreatment with 2-261 blocked conditioned place preference from spinal clonidine in SNL rats. Moreover, 2-261 reversed thermal hyperalgesia and partially reversed tactile allodynia in the gp120 model of HIV-related neuropathic pain. The effects of 2-261 likely required interaction with the α4ß3δ GABAAR because 2-301, a close structural analog of 2-261 with limited extrasynaptic receptor efficacy, was not active. Thus, 2-261 may produce pain relief with diminished side effects through selective modulation of ß2/3-subunit-containing extrasynaptic GABAARs.

Design and Synthesis of a Novel and Selective Kappa Opioid Receptor (KOR) Antagonist (BTRX-335140).

κ opioid receptor (KOR) antagonists are potential pharmacotherapies for the treatment of migraine and stress-related mood disorders including depression, anxiety, and drug abuse, thus the development of novel KOR antagonists with an improved potency/selectivity profile and medication-like duration of action has attracted the interest of the medicinal chemistry community. In this paper, we describe the discovery of 1-(6-ethyl-8-fluoro-4-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)quinolin-2-yl)- N-(tetrahydro-2 H-pyran-4-yl)piperidin-4 amine (CYM-53093, BTRX-335140) as a potent and selective KOR antagonist, endowed with favorable in vitro ADMET and in vivo pharmacokinetic profiles and medication-like duration of action in rat pharmacodynamic experiments. Orally administered CYM-53093 showed robust efficacy in antagonizing KOR agonist-induced prolactin secretion and in tail-flick analgesia in mice. CYM-53093 exhibited a broad selectivity over a panel of off-target proteins. This compound is in phase 1 clinical trials for the treatment of neuropsychiatric disorders wherein dynorphin is thought to contribute to the underlying pathophysiology.

Selective deficiencies in descending inhibitory modulation in neuropathic rats: implications for enhancing noradrenergic tone.

Pontine noradrenergic neurones form part of a descending inhibitory system that influences spinal nociceptive processing. Weak or absent descending inhibition is a common feature of chronic pain patients. We examined the extent to which the descending noradrenergic system is tonically active, how control of spinal neuronal excitability is integrated into thalamic relays within sensory-discriminative projection pathways, and how this inhibitory control is altered after nerve injury. In vivo electrophysiology was performed in anaesthetised spinal nerve-ligated (SNL) and sham-operated rats to record from wide dynamic range neurones in the ventral posterolateral thalamus (VPL). In sham rats, spinal block of α2-adrenoceptors with atipamezole resulted in enhanced stimulus-evoked and spontaneous firing in the VPL, and produced conditioned place avoidance. However, in SNL rats, these conditioned avoidance behaviours were absent. Furthermore, inhibitory control of evoked neuronal responses was lost, but spinal atipamezole markedly increased spontaneous firing. Augmenting spinal noradrenergic tone in neuropathic rats with reboxetine, a selective noradrenergic reuptake inhibitor, modestly reinstated inhibitory control of evoked responses in the VPL but had no effect on spontaneous firing. By contrast, clonidine, an α2 agonist, inhibited both evoked and spontaneous firing, and exhibited increased potency in SNL rats compared with sham controls. These data suggest descending noradrenergic inhibitory pathways are tonically active in sham rats. Moreover, in neuropathic states, descending inhibitory control is diminished, but not completely absent, and distinguishes between spontaneous and evoked neuronal activity. These observations may have implications for how analgesics targeting the noradrenergic system provide relief.

CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide.

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with (S)-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.

Cholecystokinin in the rostral ventromedial medulla mediates opioid-induced hyperalgesia and antinociceptive tolerance.

Opioid-induced hyperalgesia is characterized by hypersensitivity to innocuous or noxious stimuli during sustained opiate administration. Microinjection of lidocaine into the rostral ventromedial medulla (RVM), or dorsolateral funiculus (DLF) lesion, abolishes opioid-induced hyperalgesia, suggesting the importance of descending pain facilitation mechanisms. Here, we investigate the possibility that cholecystokinin (CCK), a pronociceptive peptide, may drive such descending facilitation from the RVM during continuous opioid administration. In opioid-naive rats, CCK in the RVM produced acute tactile and thermal hypersensitivity that was antagonized by the CCK2 receptor antagonist L365,260 or by DLF lesion. CCK in the RVM also acutely displaced the spinal morphine antinociceptive dose-response curve to the right. Continuous systemic morphine elicited sustained tactile and thermal hypersensitivity within 3 d. Such hypersensitivity was reversed in a time-dependent manner by L365,260 in the RVM, and blockade of CCK2 receptors in the RVM also blocked the rightward displacement of the spinal morphine antinociceptive dose-response curve. Microdialysis studies in rats receiving continuous morphine showed an approximately fivefold increase in the basal levels of CCK in the RVM when compared with controls. These data suggest that activation of CCK2 receptors in the RVM promotes mechanical and thermal hypersensitivity and antinociceptive tolerance to morphine. Enhanced, endogenous CCK activity in the RVM during sustained morphine exposure may diminish spinal morphine antinociceptive potency by activating descending pain facilitatory mechanisms to exacerbate spinal nociceptive sensitivity. Prevention of opioid-dose escalation in chronic pain states by CCK receptor antagonism represents a potentially important strategy to limit unintended enhanced clinical pain and analgesic tolerance

Positive emotions and brain reward circuits in chronic pain.

Chronic pain is an important public health problem that negatively impacts the quality of life of affected individuals and exacts enormous socioeconomic costs. Chronic pain is often accompanied by comorbid emotional disorders including anxiety, depression, and possibly anhedonia. The neural circuits underlying the intersection of pain and pleasure are not well understood. We summarize recent human and animal investigations and demonstrate that aversive aspects of pain are encoded in brain regions overlapping with areas processing reward and motivation. We highlight findings revealing anatomical and functional alterations of reward/motivation circuits in chronic pain. Finally, we review supporting evidence for the concept that pain relief is rewarding and activates brain reward/motivation circuits. Adaptations in brain reward circuits may be fundamental to the pathology of chronic pain. Knowledge of brain reward processing in the context of pain could lead to the development of new therapeutics for the treatment of emotional aspects of pain and comorbid conditions.

Efficacy of (S)-Lacosamide in preclinical models of cephalic pain.

Migraine is one of the world's most common neurological disorders. Current acute migraine treatments have sub-optimal efficacy and new therapeutic options are needed. Approaches targeting calcitonin gene related peptide (CGRP) signaling are clinically effective but small molecule antagonists have not been advanced due to toxicity. In this study, we explored the axonal growth/specification collapsin response mediator protein 2 (CRMP2) as a novel "druggable" target for inhibiting CGRP release and for potential relevance for treatment of migraine pain. CRMP2 has been demonstrated to regulate N-type voltage gated Ca2+ channel (CaV2.2) activity and Ca2+-dependent CGRP release in sensory neurons. The co-expression of CRMP2 with CaV2.2 and CGRP in trigeminal ganglia (TG) sensory neurons suggested the possibility of a novel approach to regulate CGRP release in the trigeminal system. Screening protocols surprisingly revealed that (S)-Lacosamide ((S)-LCM), an inactive analog of the clinically-approved small molecule anti-epileptic drug (R)-Lacosamide (Vimpat®), inhibited CRMP2 phosphorylation by cyclin dependent kinase 5 (Cdk5) in rat TG slices and decreased depolarization-evoked Ca2+ influx in TG cells in culture. (S)-LCM significantly blocked capsaicin-evoked CGRP release from dural nerve terminals in the rat an ex vivo cranial cup preparation. Additionally, cephalic and extracephalic cutaneous allodynia (CA) induced in rats by activation of dural nociceptors with a cocktail of inflammatory mediators, was inhibited by oral administration of (S)-LCM. The confirmation of CRMP2 as an upstream mediator of CGRP release together with the brain penetrance of this molecule suggest (S)-LCM as a potential therapy for acute migraine.

(S)-lacosamide inhibition of CRMP2 phosphorylation reduces postoperative and neuropathic pain behaviors through distinct classes of sensory neurons identified by constellation pharmacology.

Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2-CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration-approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.

Sustained relief of ongoing experimental neuropathic pain by a CRMP2 peptide aptamer with low abuse potential.

Uncoupling the protein-protein interaction between collapsin response mediator protein 2 (CRMP2) and N-type voltage-gated calcium channel (CaV2.2) with an allosteric CRMP2-derived peptide (CBD3) is antinociceptive in rodent models of inflammatory and neuropathic pain. We investigated the efficacy, duration of action, abuse potential, and neurobehavioral toxicity of an improved mutant CRMP2 peptide. A homopolyarginine (R9)-conjugated CBD3-A6K (R9-CBD3-A6K) peptide inhibited the CaV2.2-CRMP2 interaction in a concentration-dependent fashion and diminished surface expression of CaV2.2 and depolarization-evoked Ca influx in rat dorsal root ganglia neurons. In vitro studies demonstrated suppression of excitability of small-to-medium diameter dorsal root ganglion and inhibition of subtypes of voltage-gated Ca channels. Sprague-Dawley rats with tibial nerve injury had profound and long-lasting tactile allodynia and ongoing pain. Immediate administration of R9-CBD3-A6K produced enhanced dopamine release from the nucleus accumbens shell selectively in injured animals, consistent with relief of ongoing pain. R9-CBD3-A6K, when administered repeatedly into the central nervous system ventricles of naive rats, did not result in a positive conditioned place preference demonstrating a lack of abusive liability. Continuous subcutaneous infusion of R9-CBD3-A6K over a 24- to 72-hour period reversed tactile allodynia and ongoing pain, demonstrating a lack of tolerance over this time course. Importantly, continuous infusion of R9-CBD3-A6K did not affect motor activity, anxiety, depression, or memory and learning. Collectively, these results validate the potential therapeutic significance of targeting the CaV-CRMP2 axis for treatment of neuropathic pain.

Behavioral and neurochemical analysis of ongoing bone cancer pain in rats.

Cancer-induced bone pain is described as dull, aching ongoing pain. Ongoing bone cancer pain was characterized after intratibial injection of breast cancer cells in rats. Cancer produced time-dependent bone remodeling and tactile hypersensitivity but no spontaneous flinching. Conditioned place preference (CPP) and enhanced dopamine (DA) release in the nucleus accumbens (NAc) shell was observed after peripheral nerve block (PNB) selectively in tumor-bearing rats revealing nociceptive-driven ongoing pain. Oral diclofenac reversed tumor-induced tactile hypersensitivity but did not block PNB-induced CPP or NAc DA release. Tumor-induced tactile hypersensitivity, and PNB-induced CPP and NAc DA release, was blocked by prior subcutaneous implantation of a morphine pellet. In sham rats, morphine produced a modest but sustained increase in NAc DA release. In contrast, morphine produced a transient 5-fold higher NAc DA release in tumor bearing rats compared with sham morphine rats. The possibility that this increased NAc DA release reflected the reward of pain relief was tested by irreversible blockade of rostral anterior cingulate cortex (rACC) µ-opioid receptors (MORs). The rACC MOR blockade prevented the morphine-induced transient increased NAc DA release in tumor bearing rats but did not affect morphine-induced effects in sham-operated animals. Consistent with clinical experience, ongoing cancer pain was controlled by morphine but not by a dose of diclofenac that reversed evoked hypersensitivity. Additionally, the intrinsic reward of morphine can be dissociated from the reward of relief of cancer pain by blockade of rACC MOR. This approach allows mechanistic and therapeutic assessment of ongoing cancer pain with likely translation relevance.