Characterization and pharmacological modulation of noci-responsive deep dorsal horn neurons across diverse rat models of pathological pain.

This overview compares the activity of wide dynamic range (WDR) and nociceptive specific (NS) neurons located in the deep dorsal horn across different rat models of pathological pain and following modulation by diverse pharmacology. The data were collected by our group under the same experimental conditions over numerous studies to facilitate comparison. Spontaneous firing of WDR neurons was significantly elevated (>3.7 Hz) in models of neuropathic, inflammation, and osteoarthritic pain compared with naive animals (1.9 Hz) but was very low (<0.5 Hz) and remained unchanged in NS neurons. WDR responses to low-intensity mechanical stimulation were elevated in neuropathic and inflammation models. WDR responses to high-intensity stimuli were enhanced in inflammatory (heat) and osteoarthritis (mechanical) models. NS responses to high-intensity stimulation did not change relative to control in any model examined. Several therapeutic agents reduced both evoked and spontaneous firing of WDR neurons (e.g., TRPV1, TRPV3, Nav1.7, Nav1.8, P2X7, P2X3, H3), other targets affected neither evoked nor spontaneous firing of WDR neurons (e.g., H4, TRPM8, KCNQ2/3), and some only modulated evoked (e.g, ASIC1a, Cav3.2) whereas others decreased evoked but affected spontaneous activity only in specific models (e.g., TRPA1, CB2). Spontaneous firing of WDR neurons was not altered by any peripherally restricted compound or by direct administration of compounds to peripheral sites, although the same compounds decreased evoked activity. Compounds acting centrally were effective against this endpoint. The diversity of incoming/modulating inputs to the deep dorsal horn positions this group of neurons as an important intersection within the pain system to validate novel therapeutics. NEW & NOTEWORTHY Data from multiple individual experiments were combined to show firing properties of wide dynamic range and nociceptive specific spinal dorsal horn neurons across varied pathological pain models. This high-powered analysis describes the sensitization following different forms of injury. Effects of diverse pharmacology on these neurons is also summarized from published and unpublished data all recorded under the same conditions to facilitate comparison. This comprehensive overview describes the function and utility of these neurons.

Efficacy and safety results of depatuxizumab mafodotin (ABT-414) in patients with advanced solid tumors likely to overexpress epidermal growth factor receptor.

BACKGROUND: Epidermal growth factor receptor (EGFR) alterations are associated with multiple cancers. Current EGFR-directed therapies have led to increased efficacy but are associated with specific side effects. The antibody-drug conjugate depatuxizumab mafodotin (depatux-m) targets EGFR with a monoclonal antibody linked to a cytotoxin, and is highly tumor-specific. METHODS: This phase 1/2 study evaluated the safety, pharmacokinetics, and efficacy of depatux-m in patients who had advanced solid tumors with known wild-type EGFR overexpression, amplification, or mutated EGFR variant III. A 3 + 3 dose escalation was used, and 2 dosing schedules were evaluated. Depatux-m also was manufactured under an alternate process to reduce the drug load and improve the safety profile, and it was tested at the maximum tolerated dose (MTD). In another cohort, prolonged infusion time of depatux-m was evaluated; and a cohort with confirmed EGFR amplification also was evaluated at the MTD. RESULTS: Fifty-six patients were treated. The MTD and the recommended phase 2 dose for depatux-m was 3.0 mg/kg. Common adverse events (AEs) were blurred vision (48%) and fatigue (41%). A majority of patients (66%) experienced 1 or more ocular AEs. Grade 3 or 4 AEs were observed in 43% of patients. One patient with EGFR-amplified, triple-negative breast cancer had a partial response. Stable disease was observed in 23% of patients. Pharmacokinetics revealed that depatux-m exposures were approximately dose-proportional. CONCLUSIONS: Depatux-m resulted in infrequent nonocular AEs but increased ocular AEs. Patient follow-up confirmed that ocular AEs were reversible. Lowering the drug-antibody ratio did not decrease the number of ocular AEs. A partial response in 1 patient with EGFR-amplified disease provides the opportunity to study depatux-m in diseases with a high incidence of EGFR amplification. Cancer 2018;124:2174-83. © 2018 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

TRPV3 modulates nociceptive signaling through peripheral and supraspinal sites in rats.

TRPV3 is a nonselective cation channel activated by temperatures above 33°C and is reported to be localized in keratinocytes and nervous tissue. To investigate a role for TRPV3 in pain modulation, we conducted a series of in vivo electrophysiological studies on spinal and brain nociceptive neurons. Structurally diverse TRPV3 receptor antagonists reduced responses of spinal wide dynamic range (WDR) neurons to low-intensity mechanical stimulation in neuropathic rats, but only CNS-penetrant antagonists decreased elevated spontaneous firing. Injections of an antagonist into the neuronal receptive field, into the L5 dorsal root ganglion, or intracerebroventricularly (ICV) attenuated the evoked firing, but only ICV injections reduced spontaneous activity. Intraspinal injections did not affect either. Spinal transection blocked the effect on spontaneous but not evoked firing after systemic delivery of a TRPV3 antagonist. Systemic administration of an antagonist to neuropathic rats also impacted the firing of On- and Off-cells in the rostral ventromedial medulla in a manner consistent with dampening nociceptive signaling. An assessment of nonevoked "pain," an EEG-measured pain-induced sleep disturbance induced by hind paw injections of CFA, was also improved with CNS-penetrant TRPV3 antagonists but not by an antagonist with poor CNS penetration. Antagonism of TRPV3 receptors modulates activity of key classes of neurons in the pain pathway in a manner consistent with limiting pathological nociceptive signaling and was mediated by receptors in the periphery and brain. Blockade of TRPV3 receptors is likely an effective means to alleviate mechanical allodynia and nonevoked pain. However, the latter will only be obtained by blocking supraspinal TRPV3 receptors.NEW & NOTEWORTHY Recent studies have linked TRPV3 to pain modulation, and much of this work has focused on its role in the skin-primary afferent interface. In this electrophysiological study, we demonstrate that receptor antagonists modulate evoked signals through peripheral mechanisms but blockade of supraspinal TRPV3 receptors contributes to dampening both evoked and nonevoked "pain" through descending modulation. Thus, the full therapeutic potential of TRPV3 antagonists may only be realized with the ability to access receptors in the brain.

A mixed Ca2+ channel blocker, A-1264087, utilizes peripheral and spinal mechanisms to inhibit spinal nociceptive transmission in a rat model of neuropathic pain.

N-, T- and P/Q-type voltage-gated Ca(2+) channels are critical for regulating neurotransmitter release and cellular excitability and have been implicated in mediating pathological nociception. A-1264087 is a novel state-dependent blocker of N-, T- and P/Q-type channels. In the present studies, A-1264087 blocked (IC50 = 1.6 µM) rat dorsal root ganglia N-type Ca(2+) in a state-dependent fashion. A-1264087 (1, 3 and 10 mg/kg po) dose-dependently reduced mechanical allodynia in rats with a spinal nerve ligation (SNL) injury. A-1264087 (4 mg/kg iv) inhibited both spontaneous and mechanically evoked activity of spinal wide dynamic range (WDR) neurons in SNL rats but had no effect in uninjured rats. The inhibitory effect on WDR neurons remained in spinally transected SNL rats. Injection of A-1264087 (10 nmol/0.5 µl) into the spinal cord reduced both spontaneous and evoked WDR activity in SNL rats. Application of A-1264087 (300 nmol/20 µl) into the receptive field on the hindpaw attenuated evoked but not spontaneous firing of WDR neurons. Using electrical stimulation, A-1264087 (4 mg/kg iv) inhibited Aδ- and C-fiber evoked responses and after-discharge of WDR neurons in SNL rats. These effects by A-1264087 were not present in uninjured rats. A-1264087 moderately attenuated WDR neuron windup in both uninjured and SNL rats. In summary, these results indicate that A-1264087 selectively inhibited spinal nociceptive transmission in sensitized states through both peripheral and central mechanisms.

Spontaneous firing and evoked responses of spinal nociceptive neurons are attenuated by blockade of P2X3 and P2X2/3 receptors in inflamed rats.

P2X3 and P2X2/3 receptors are selectively expressed on primary afferent nociceptors and have been implicated in modulating nociception in different models of pathological pain, including inflammatory pain. In an effort to delineate further the role of P2X3 receptors (homomeric and heteromeric) in the modulation of nociceptive transmission after a chronic inflammation injury, A-317491, a potent and selective P2X3-P2X2/3 antagonist, was administered to CFA-inflamed rats in order to examine its effects on responses of spinal dorsal horn neurons to mechanical and thermal stimulation. Systemic injection of A-317491 (30 µmol/kg, i.v.) reduced the responses of wide-dynamic-range (WDR) and nociceptive specific (NS) neurons to both high-intensity mechanical (pinch) and heat (49°C) stimulation. A-317491 also decreased low-intensity (10 g von Frey hair) mechanically evoked activity of WDR neurons but did not alter WDR neuronal responses to cold stimulation (5°C). Spontaneous firing of WDR neurons in CFA-inflamed rats was also significantly attenuated by A-317491 injection. By using immunohistochemistry, P2X3 receptors were demonstrated to be enhanced in lamina II of the spinal dorsal horn after inflammation. In summary, blockade of P2X3 and P2X2/3 receptors dampens mechanical- and heat-related signaling, as well as nonevoked activity of key classes of spinal nociceptive neurons in inflamed animals. These data suggest that P2X3 and/or P2X2/3 receptors have a broad contribution to somatosensory/nociceptive transmission in rats with a chronic inflammatory injury and are consistent with previous behavioral data demonstrating antiallodynic and antihyperalgesic effects of receptor antagonists.

Antagonism of supraspinal histamine H3 receptors modulates spinal neuronal activity in neuropathic rats.

There is growing evidence supporting a role for histamine H(3) receptors in the modulation of pathological pain. To further our understanding of this modulation, we examined the effects of a selective H(3) receptor antagonist, 6-((3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy)-N-methyl-3-pyridinecarboxamide (GSK189254), on spinal neuronal activity in neuropathic (L5 and L6 ligations) and sham rats. Systemic administration of GSK189254 (0.03-1 mg/kg i.v.) dose-dependently decreased both evoked (10-g von Frey hair for 15 s) and spontaneous firing of wide dynamic range (WDR) neurons in neuropathic, but not sham-operated, animals. The effects on spontaneous firing suggest that H(3) receptors may have a role in central sensitization and/or modulating non-evoked pain. Transection of the spinal cord (T9-T10) completely eliminated the effects (both evoked and spontaneous) of systemic GSK189254 (1 mg/kg, i.v.) on WDR neuronal firing in neuropathic rats, indicating that the descending modulatory system has an important role in the H(3)-related dampening of spinal neuronal activity. Subsequently, lesions of the locus coeruleus, or direct GSK189254 (3 and 10 nmol/0.5 µl) injections into this site, demonstrate that the locus coeruleus is a key component of the H(3) descending modulatory pathway. In summary, blockade of H(3) receptors reduces spontaneous firing as well as the responses of spinal nociceptive neurons to mechanical stimulation. This effect is in large part mediated via supraspinal sites, including the locus coeruleus, that send descending projections to modulate spinal neuronal activity.

TRPV1 antagonist, A-889425, inhibits mechanotransmission in a subclass of rat primary afferent neurons following peripheral inflammation.

TRPV1 (transient receptor potential vanilloid family type 1) is a nonselective cation channel that is activated and/or sensitized by noxious heat, protons, and other endogenous molecules released following tissue injury. In addition, a role for TRPV1 in mechanotransmission is emerging. We have recently reported that a selective TRPV1 receptor antagonist, A-889425, reduces mechanical allodynia and spinal neuron responses to mechanical stimulation of complete Freund's adjuvant (CFA)-inflamed rat hind paws. The population of peripheral nerve fibers through which TRPV1 antagonists mediate their effect on mechanotransmission have not yet been described. The objective of this study was to characterize TRPV1-mediated modulation of mechanically evoked activity in sensory axons innervating rat hind paws. We used an in vitro skin-nerve preparation to record neural activity from single axons isolated from rat tibial nerve. Single fibers were classified by conduction velocity, mechanical threshold, and stimulus-response relationships. We used A-889425 to investigate uninjured and inflamed skin afferent neuron populations to evoked mechanical stimulation. Application of A-889425 had no effect on the mechanical responsiveness of Aδ and C-fiber units innervating uninjured skin. In contrast, A-889425 inhibited responses of slowly conducting Aδ fiber units to noxious mechanical stimulation in a population of axons innervating CFA-inflamed hind paws. These data support a role for TRPV1 in mechanotransmission following peripheral inflammation, and highlight the importance of a distinct subclass of primary afferent neurons in mediating this effect.

TRPA1 modulation of spontaneous and mechanically evoked firing of spinal neurons in uninjured, osteoarthritic, and inflamed rats.

BACKGROUND: There is growing evidence supporting a role for TRPA1 receptors in the neurotransmission of peripheral mechanical stimulation. In order to enhance understanding of TRPA1 contributions to mechanotransmission, we examined the effects a selective TRPA1 receptor antagonist, A-967079, on spinal neuronal activity following peripheral mechanical stimulation in uninjured, CFA-inflamed, and osteoarthritc (OA) rats. RESULTS: Systemic injection of A-967079 (30 micromol/kg, i.v.) decreased the responses of wide dynamic range (WDR), and nociceptive specific (NS) neurons following noxious pinch stimulation of the ipsilateral hind paw in uninjured and CFA-inflamed rats. Similarly, A-967079 reduced the responses of WDR neurons to high-intensity mechanical stimulation (300 g von Frey hair) of the knee joint in both OA and OA-sham rats. WDR neuronal responses to low-intensity mechanical stimulation (10 g von Frey hair) were also reduced by A-967079 administration to CFA-inflamed rats, but no effect was observed in uninjured rats. Additionally, the spontaneous activity of WDR neurons was decreased after A-967079 injection in CFA-inflamed rats but was unaltered in uninjured, OA, and OA-sham animals. CONCLUSIONS: Blockade of TRPA1 receptors disrupts transmission of high-intensity mechanical stimulation to the spinal cord in both uninjured and injured rats indicating that TRPA1 receptors have an important role in noxious mechanosensation in both normal and pathological conditions. TRPA1 receptors also contribute to the transmission of low-intensity mechanical stimulation, and to the modulation of spontaneous WDR firing, but only after an inflammatory injury.

A selective Nav1.8 sodium channel blocker, A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide], attenuates spinal neuronal activity in neuropathic rats.

We have recently reported that systemic delivery of A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide], a selective Na(v)1.8 sodium channel blocker, reduces behavioral measures of chronic pain. In the current study, the effects of A-803467 on evoked and spontaneous firing of wide dynamic range (WDR) neurons were measured in uninjured and rats with spinal nerve ligations (SNLs). Administration of A-803467 (10-30 mg/kg i.v.) reduced mechanically evoked (10-g von Frey hair) and spontaneous WDR neuronal activity in SNL rats. In uninjured rats, A-803467 (20 mg/kg i.v.) transiently reduced evoked but not spontaneous firing of WDR neurons. The systemic effects of A-803467 in SNL rats were not altered by spinal transection or by systemic pretreatment with the transient receptor potential vanilloid type 1 (TRPV1) receptor agonist, resiniferatoxin, at doses that impair the function of TRPV1-expressing fibers. To determine sites of action, A-803467 was administered into spinal tissue, into the uninjured L4 dorsal root ganglion (DRG), or into the neuronal receptive field. Injections of A-803467 into the L4 DRG (30-100 nmol/1 mul) or into the hindpaw receptive field (300 nmol/50 mul) reduced evoked but not spontaneous WDR firing. In contrast, intraspinal (50-150 nmol/0.5 mul) injection of A-803467 decreased both evoked and spontaneous discharges of WDR neurons. Thus, Na(v)1.8 sodium channels on the cell bodies/axons within the L4 DRG as well as on peripheral and central terminals of primary afferent neurons regulate the inflow of low-intensity mechanical signals to spinal WDR neurons. However, Na(v)1.8 sodium channels on central terminals seem to be key to the modulation of spontaneous firing in SNL rats.

(R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102) blocks polymodal activation of transient receptor potential vanilloid 1 receptors in vitro and heat-evoked firing of spinal dorsal horn neurons in vivo.

The transient receptor potential vanilloid (TRPV) 1 receptor, a nonselective cation channel expressed on peripheral sensory neurons and in the central nervous system, plays a key role in pain. TRPV1 receptor antagonism is a promising approach for pain management. In this report, we describe the pharmacological and functional characteristics of a structurally novel TRPV1 antagonist, (R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102), which has entered clinical trials. At the recombinant human TRPV1 receptor ABT-102 potently (IC(50) = 5-7 nM) inhibits agonist (capsaicin, N-arachidonyl dopamine, anandamide, and proton)-evoked increases in intracellular Ca(2+) levels. ABT-102 also potently (IC(50) = 1-16 nM) inhibits capsaicin-evoked currents in rat dorsal root ganglion (DRG) neurons and currents evoked through activation of recombinant rat TRPV1 currents by capsaicin, protons, or heat. ABT-102 is a competitive antagonist (pA(2) = 8.344) of capsaicin-evoked increased intracellular Ca(2+) and shows high selectivity for blocking TRPV1 receptors over other TRP receptors and a range of other receptors, ion channels, and transporters. In functional studies, ABT-102 blocks capsaicin-evoked calcitonin gene-related peptide release from rat DRG neurons. Intraplantar administration of ABT-102 blocks heat-evoked firing of wide dynamic range and nociceptive-specific neurons in the spinal cord dorsal horn of the rat. This effect is enhanced in a rat model of inflammatory pain induced by administration of complete Freund's adjuvant. Therefore, ABT-102 potently blocks multiple modes of TRPV1 receptor activation and effectively attenuates downstream consequences of receptor activity. ABT-102 is a novel and selective TRPV1 antagonist with pharmacological and functional properties that support its advancement into clinical studies.

Characterization and comparison of rat monosodium iodoacetate and medial meniscal tear models of osteoarthritic pain.

Osteoarthritis (OA) is a degenerative form of arthritis that can result in loss of joint function and chronic pain. The pathological pain state that develops with OA disease involves plastic changes in the peripheral and central nervous systems, however, the cellular mechanisms underlying OA are not fully understood. We characterized the medial meniscal tear (MMT) surgical model and the intra-articular injection of monosodium iodoacetate (MIA) chemical model of OA in rats. Both models produced histological changes in the knee joint and associated bones consistent with OA pathology. Both models also increased p38 activation in the L3, but not L4 dorsal root ganglia (DRG), increased tyrosine hydroxylase immunostaining in the L3 DRG indicating sympathetic sprouting, and increased phosphorylated (p)CREB in thalamic neurons. In MIA-OA, but not MMT-OA rats, p38 and pERK were increased in the spinal cord, and pCREB was enhanced in the prefrontal cortex. Using in vivo electrophysiology, elevated spontaneous activity and increased responsiveness of wide dynamic range neurons to stimulation of the knee was found in both models. However, a more widespread sensitization was observed in the MIA-OA rats as neurons with paw receptive fields spontaneously fired at a greater rate in MIA-OA than MMT-OA rats. Taken together, the MIA and MMT models of OA share several common features associated with histopathology and sensitization of primary somatosensory pathways, but, observed differences between the models highlights unique consequences of the related specific injuries, and these differences should be considered when choosing an OA model and when interpreting data outcomes. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Effects of A-317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration.

We have recently reported that systemic delivery of A-317491, the first non-nucleotide antagonist that has high affinity and selectivity for blocking P2X3 homomeric and P2X2/3 heteromeric channels, is antinociceptive in rat models of chronic inflammatory and neuropathic pain. In an effort to further evaluate the role of P2X3/P2X2/3 receptors in nociceptive transmission, A-317491 was administered either intrathecally or into the hindpaw of a rat in several models of acute and chronic nociception. Intraplantar (ED50=300 nmol) and intrathecal (ED50=30 nmol) injections of A-317491 produced dose-related antinociception in the CFA model of chronic thermal hyperalgesia. Administration of A-317491 by either route was much less effective to reduce thermal hyperalgesia in the carrageenan model of acute inflammatory hyperalgesia. Intrathecal, but not intraplantar, delivery of A-317491 attenuated mechanical allodynia in both the chronic constriction injury and L5-L6 nerve ligation models of neuropathy (ED50=10 nmol for both models). Intrathecal injections of A-317491 did not impede locomotor performance. Both routes of injection were effective in reducing the number of nocifensive events triggered by the injection of formalin into a hindpaw. Nocifensive behaviors were significantly reduced in both the first and second phases of the formalin assay (intrathecal ED50=10 nmol, intraplantar ED50>300 nmol). Nocifensive behaviors induced by the P2X receptor agonist alpha,beta-meATP were also significantly reduced by intraplantar injection of A-317491. These data indicate that both spinal and peripheral P2X3/P2X2/3 receptors have significant contributions to nociception in several animal models of nerve or tissue injury. Intrathecal administration of A-317491 appears to be more effective than intraplantar administration to reduce tactile allodynia following peripheral nerve injury.

Potent desensitization of human P2X3 receptors by diadenosine polyphosphates.

In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X3 (hP2X3) receptors were examined. Administration of Ap3A, Ap4A, Ap5A or Ap6A inhibited the hP2X3 receptor-mediated response to a subsequent application of 3 muM alphabeta-methyleneATP (alphabeta-meATP), in a concentration-dependent manner, with IC50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize alphabeta-meATP responses mediated by the slowly desensitizing heteromeric human P2X2/3 receptor. hP2X3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X3 receptor activation in vivo.

Increased WDR spontaneous activity and receptive field size in rats following a neuropathic or inflammatory injury: implications for mechanical sensitivity.

Spontaneous activity and receptive field size for spinal wide dynamic range (WDR) neurons were measured and related to the mechanical allodynia in both neuropathic (L5-L6 ligation, 14 days post-injury) and complete Freund's adjuvant-inflamed rats (CFA, 2 days post-injury). The size of the WDR receptive field located on the hindpaw expanded significantly (p<0.01) following both modes of injury, with no difference between CFA and neuropathic animals. Likewise, the spontaneous firing of WDR neurons was significantly elevated following both the CFA (4.4+/-0.6 spikes/s, p<0.01) and neuropathic (3.2+/-0.3 spikes/s, p<0.05) injuries compared to naive (2.1+/-0.2 spikes/s) and sham-neuropathic (1.9+/-0.3 spikes/s) rats. Furthermore, the spontaneous WDR activity recorded from CFA rats was also significantly greater (p<0.05) than neuropathic rats. Mechanical allodynia, as measured by application of a von Frey hair stimulus, was observed from both CFA and neuropathic rats, however, the degree of sensitivity was significantly greater (p<0.01) for the CFA animals. These data suggest that the differences in mechanical sensitivity between CFA and neuropathic rats may be related to their respective changes in WDR spontaneous activity, but not to the changes in receptive field size, and is further demonstration of the importance of spontaneous WDR activity in determining mechanical sensitivity following injury.

Comparison of the ability of adenosine kinase inhibitors and adenosine receptor agonists to attenuate thermal hyperalgesia and reduce motor performance in rats.

Inhibitors of adenosine kinase (AK) enhance extracellular concentrations of the inhibitory neuromodulator adenosine (ADO) at sites of tissue hyperexcitability and produce antinociceptive effects in animal models of pain and inflammation. The present study compared the ability of several novel and selective AK inhibitors and ADO receptor-selective agonists to attenuate carrageenan-induced thermal hyperalgesia and to impair motor performance as measured by effects on exploratory motor activity and rotorod performance. The prototypical nucleoside AK inhibitor, 5'deoxy-5-iodotubercidin (5'd-5IT), dose-dependently blocked thermal hyperalgesia (ED(50)=0.2 micromol/kg ip) and was 4- and 75-fold less potent in reducing exploratory motor activity and rotorod performance, respectively. The antihyperalgesic effects of 5'd-5IT were fully blocked by the A(1) antagonist, cyclopentyltheophylline (CPT) and the A(2A) antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). Novel nucleoside and non-nucleoside AK inhibitors (A-134974, A-286501 and ABT-702) also potently (ED(50)=0.7-2 micromol/kg ip) blocked carrageenan-induced thermal hyperalgesia and were significantly less potent than 5'd-5IT in impairing motor performance. The systemic administration of N(6)-cyclopentyladenosine (CPA), an A(1) receptor-selective agonist, CGS 21680, an A(2A) receptor-selective agonist, and N(6)-ethylcarboxamidoadenosine (NECA), a nonselective ADO receptor agonist potently reduced (ED(50)=0.3-1.0 micromol/kg ip) thermal hyperalgesia. Unlike the AK inhibitors, however, these ADO receptor agonists produced significant antinociception only at doses that also decreased motor performance. These data demonstrate that AK inhibitors produce specific antihyperalgesic effects via an interaction with ADO A(1) and A(2A) receptors at doses that lack detectable effects on exploratory motor activity and rotorod performance and offer an improved separation between antinociceptive and motor impairing effects as compared to ADO receptor agonists.

A peripherally acting, selective T-type calcium channel blocker, ABT-639, effectively reduces nociceptive and neuropathic pain in rats.

Activation of T-type Ca²âº channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²âº channels in chronic pain is supported by gene knockdown studies showing that decreased Ca(v)3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²âº channel blocker. ABT-639 blocks recombinant human T-type (Ca(v)3.2) Ca²âº channels in a voltage-dependent fashion (IC50 = 2 µM) and attenuates LVA currents in rat DRG neurons (IC50 = 8 µM). ABT-639 was significantly less active at other Ca²âº channels (e.g. Ca(v)1.2 and Ca(v)2.2) (IC50 > 30 µM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED50 = 2 mg/kg, p.o.). ABT-639 (10-100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced). [corrected]. ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100-300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca(v)3.2) channels in chronic pain states.

Mechanistic insights into the analgesic efficacy of A-1264087, a novel neuronal Ca(2+) channel blocker that reduces nociception in rat preclinical pain models.

UNLABELLED: Voltage-gated Ca(2+) channels play an important role in nociceptive transmission. There is significant evidence supporting a role for N-, T- and P/Q-type Ca(2+) channels in chronic pain. Here, we report that A-1264087, a structurally novel state-dependent blocker, inhibits each of these human Ca(2+) channels with similar potency (IC50 = 1-2 µM). A-1264087 was also shown to inhibit the release of the pronociceptive calcitonin gene-related peptide from rat dorsal root ganglion neurons. Oral administration of A-1264087 produces robust antinociceptive efficacy in monoiodoacetate-induced osteoarthritic, complete Freund adjuvant-induced inflammatory, and chronic constrictive injury of sciatic nerve-induced, neuropathic pain models with ED50 values of 3.0, 5.7, and 7.8 mg/kg (95% confidence interval = 2.2-3.5, 3.7-10, and 5.5-12.8 mg/kg), respectively. Further analysis revealed that A-1264087 also suppressed nociceptive-induced p38 and extracellular signal-regulated kinase 1/2 phosphorylation, which are biochemical markers of engagement of pain circuitry in chronic pain states. Additionally, A-1264087 inhibited both spontaneous and evoked neuronal activity in the spinal cord dorsal horn in complete Freund adjuvant-inflamed rats, providing a neurophysiological basis for the observed antihyperalgesia. A-1264087 produced no alteration of body temperature or motor coordination and no learning impairment at therapeutic plasma concentrations. PERSPECTIVE: The present results demonstrate that the neuronal Ca(2+) channel blocker A-1264087 exhibits broad-spectrum efficacy through engagement of nociceptive signaling pathways in preclinical pain models in the absence of effects on psychomotor and cognitive function.

Disturbances in slow-wave sleep are induced by models of bilateral inflammation, neuropathic, and postoperative pain, but not osteoarthritic pain in rats.

Preclinical assessment of pain has typically relied on measuring animal responses to evoked stimulation. Because of inherent limitations of these assays, there is a need to develop measures of animal pain/discomfort that are objective, not experimentally evoked, and mimic the human condition. Patients with chronic pain manifest a variety of co-morbidities, one of which is disturbances in sleep. We used electroencephalography to objectively assess 4 rat models of pain (inflammatory/complete Freund's adjuvant [CFA], neuropathic/chronic constriction injury [CCI], postoperative/skin incision, osteoarthritis/monosodium iodoacetate [MIA]) for the occurrence of sleep disturbances. Four different measures of slow-wave sleep (SWS) were examined: amplitude of 1- to 4-Hz waves, total time spent in SWS, time spent in SWS-1, and time spent in SWS-2. Bilateral injuries were more likely to induce a sleep disturbance than unilateral injuries in the CFA, CCI, and skin incision assays. Sleep disturbances occurred in the deeper stage of SWS, as the amplitude of 1- to 4-Hz waves and time spent in SWS-2 were significantly decreased in all models except the osteoarthritis model. Sleep disturbances lasted for approximately 3 to 14days, depending on the model, and were resolved despite continued hypersensitivity to evoked stimulation. Morphine, gabapentin, diclofenac, and ABT-102 (TRPV1 antagonist) all improved sleep in the bilateral CFA assay at doses that did not significantly alter SWS in uninjured rats. Preclinical assessment of compounds should follow the path of clinical studies and take into account diverse aspects of the "pain condition." This would include evaluating nociceptive thresholds as well as other endpoints, such as cognition and sleep, that may be affected by the pathological state.

Oral and cutaneous thermosensory profile of selective TRPV1 inhibition by ABT-102 in a randomized healthy volunteer trial.

The capsaicin receptor (TRPV1) antagonist ABT-102 demonstrates efficacy in multiple preclinical pain models. However, evolving clinical data for this compound class suggest potentially profound drug-induced thermosensory impairment. Safety and tolerability of ABT-102 were assessed in a multiple-dose, double-blind, placebo-controlled, randomized healthy volunteer trial. Thirty-six participants were randomized in a 2:1 ratio to ABT-102:placebo in 3 dose groups (1 mg, 2 mg, and 4 mg twice a day) and confined to an inpatient research unit for a 7-day treatment period and 3 follow-up days. Outcome measures included: oral and cutaneous cold detection, warm detection (WDT), and heat pain thresholds (HPT); oral perceived heat intensity (oral liquid test); time to hand withdrawal (water bath test); and cutaneous pain intensity (long thermal stimulus). Significant dose-dependent (placebo- and baseline-adjusted) increases in HPT and reduced painfulness of suprathreshold heat were present from days 1-7. For ABT-102 4 mg twice a day, model-based mean differences from placebo (95% confidence interval) were as follows: oral HPT, day 1=2.5°C (0.6-4.4), day 5=4.4°C (2.5-6.3); cutaneous HPT, day 2=3.3°C (1.4-5.3), day 5=5.3°C (3.3-7.2); oral WDT, day 1=2.6°C (0.5-4.7), day 5=2.7°C (0.6-4.9); cutaneous WDT, day 2=1.3 (0.0-2.6), day 5=1.6 (0.3-2.8) (all P<0.05). Oral liquid test and water bath test results followed a similar pattern. There was no effect on cutaneous cold detection. All effects were fully reversed by day 10. There were no other relevant safety findings. Core body temperature remained below 39°C in all participants. In conclusion, ABT-102 potently and reversibly increased HPT and reduced painfulness of suprathreshold oral/cutaneous heat.

TRPV1-related modulation of spinal neuronal activity and behavior in a rat model of osteoarthritic pain.

The TRPV1 receptor functions as a molecular integrator, and blockade of this receptor modulates enhanced somatosensitivity across several animal models of pathological pain, including models of osteoarthritic (OA) pain. In order to further characterize the contributions of TRPV1 to OA-related pain, we investigated the systemic effects of a selective TRPV1 receptor antagonist, A-889425, on grip force behavior, and on the evoked and spontaneous firing of spinal wide dynamic range (WDR) and nociceptive specific (NS) neurons in the monoiodoacetate (MIA) model of OA. Administration of A-889425 (10-300 µmol/kg, p.o.) alleviated grip force impairment in OA rats 3 weeks after the MIA injection. Also at 3 weeks post-MIA injection, the responses of WDR and NS neurons to 300 g von Frey hair stimulation of the knee joint were significantly reduced by A-889425 administration (10 and 30 µmol/kg, i.v.) in OA, but not sham-OA rats. Spontaneous firing of WDR neurons was elevated in the OA rats compared to sham-OA rats and may reflect ongoing discomfort in the OA animal. In addition to an effect on mechanotransmission, systemic administration of A-889425 reduced the elevated spontaneous firing of WDR neurons in OA rats but did not alter spontaneous firing in sham rats. The present data demonstrate that blockade of TRPV1 receptors modulates the firing of two important classes of spinal nociceptive neurons in a rat model of OA. The effect of A-889425 on neuronal responses to intense mechanical stimulation of the knee and on the spontaneous firing of WDR neurons adds to the growing appreciation for the role of TRPV1 receptors in pathological mechanotransmission and possibly non-evoked discomfort, respectively.