Luis R. Gardell

Reversal of experimental neuropathic pain by T-type calcium channel blockers

Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca2+ current is associated with increased neuronal activity and blockade of N‐ and P‐types, but not L‐type, calcium channels have been found to block experimental neuropathic pain. While T‐type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L5/L6 spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose‐dependent blockade of both tactile and thermal hypersensitivities in nerve‐injured rats; responses of sham‐operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T‐type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T‐type calcium channel blockers may have significant potential in the treatment of neuropathic pain states.

Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats

&NA; Neuropathic pain is often associated with the appearance of pain in regions not related to the injured nerve. One mechanism that may underlie neuropathic pain is abnormal, spontaneous afferent drive which may contribute to NMDA‐mediated central sensitization by the actions of glutamate and by the non‐opioid actions of spinal dynorphin. In the present study, injuries to lumbar or sacral spinal nerves elicited elevation in spinal dynorphin content which correlated temporally and spatially with signs of neuropathic pain. The increase in spinal dynorphin content was coincident with the onset of tactile allodynia and thermal hyperalgesia. Injury to the lumbar (L5/L6) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L5 and L6 spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S2 spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S2 spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S2 ligation blocked thermal hyperalgesia and tactile allodynia of the hindpaws suggesting that afferent drive was critical to maintenance of the pain state. Spinal injection of antiserum to dynorphin A(1–17) and of MK‐801 both blocked thermal hyperalgesia, but not tactile allodynia, of the hindpaw after S2 ligation. These data suggest that the elevated spinal dynorphin content consequent to peripheral nerve injury may drive sensitization of the spinal cord, in part through dynorphin acting directly or indirectly on the NMDA receptor complex. Furthermore, extrasegmental increases in spinal dynorphin content may partly underlie the development of extraterritorial neuropathic pain.

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.

Multiple actions of systemic artemin in experimental neuropathy

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury–induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

Role of NK-1 neurotransmission in opioid-induced hyperalgesia

&NA; Opiates are among the most important drugs for treatment of moderate to severe pain and prolonged opiate administration is often required to treat chronic pain states. We investigated the neurobiological actions of sustained opiate administration revealing paradoxical pronociceptive adaptations associated with NK‐1 receptor function. Sustained morphine delivered over 6 days elicited hyperalgesia in rats and mice during the period of opiate delivery. Sustained morphine administration increased substance P (SP) and NK‐1 receptor expression in the spinal dorsal horn. Sustained morphine treatment also enhanced capsaicin‐evoked SP release in vitro, and increased internalization of NK‐1 receptors in response to noxious stimulation. While NK‐1 receptor internalization was observed primarily in the superficial laminae of placebo‐treated rats, NK‐1 receptor internalization was seen in both superficial and deep lamina of the dorsal horn in morphine‐treated animals. Morphine‐induced hyperalgesia was reversed by spinal administration of an NK‐1 receptor antagonist in rats and mice, and was observed in wildtype (NK‐1+/+), but not NK‐1 receptor knockout (NK‐1−/−), mice. These data support a critical role for the NK‐1 receptor in the expression of sustained morphine‐induced hyperalgesia. Additionally, these data indicate that sustained opiate administration induces changes reminiscent of those associated with inflammatory pain. These opiate‐induced changes might produce unintended deleterious actions in the course of pain treatment in patients. Understanding of sustained morphine‐induced neurochemical changes will help identify approaches that limit the deleterious actions of opiates.

Dynorphin A activates bradykinin receptors to maintain neuropathic pain

Dynorphin A is an endogenous opioid peptide that produces non-opioid receptor-mediated neural excitation. Here we demonstrate that dynorphin induces calcium influx via voltage-sensitive calcium channels in sensory neurons by activating bradykinin receptors. This action of dynorphin at bradykinin receptors is distinct from the primary signaling pathway activated by bradykinin and underlies the hyperalgesia produced by pharmacological administration of dynorphin by the spinal route in rats and mice. Blockade of spinal B1 or B2 receptor also reverses persistent neuropathic pain but only when there is sustained elevation of endogenous spinal dynorphin, which is required for maintenance of neuropathic pain. These data reveal a mechanism for endogenous dynorphin to promote pain through its agonist action at bradykinin receptors and suggest new avenues for therapeutic intervention.

Enhanced Evoked Excitatory Transmitter Release in Experimental Neuropathy Requires Descending Facilitation

Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. Spinal nerve ligation (SNL) produced expected tactile and thermal hyperesthesias. Capsaicin-evoked calcitonin gene-related peptide (CGRP) release was markedly enhanced in lumbar spinal tissue from SNL rats when compared with sham-operated controls. Enhanced, evoked CGRP release from SNL rats was blocked by anti-dynorphin A(1-13) antiserum; this treatment did not alter evoked release in tissues from sham-operated rats. Dorsolateral funiculus lesion (DLF) or destruction of RVM neurons expressing μ-opioid receptors with dermorphin-saporin, blocked tactile and thermal hypersensitivity, as well as SNL-induced upregulation of spinal dynorphin. Spinal tissues from these DLF-lesioned or dermorphin-saporin-treated SNL rats did not exhibit enhanced capsaicin-evoked CGRP-IR release. These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.

Pharmacological and Behavioral Profile of N-(4-Fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl) Carbamide (2R,3R)-Dihydroxybutanedioate (2:1) (ACP-103), a Novel 5-Hydroxytryptamine2A Receptor Inverse Agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist ACP-103 competitively antagonized the binding of [3H]ketanserin to heterologously expressed human 5-HT2A receptors with a mean pKi of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC50 of 8.7. ACP-103 demonstrated lesser affinity (mean pKi of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC50 7.1 in R-SAT) at human 5-HT2C receptors, and lacked affinity and functional activity at 5-HT2B receptors, dopamine D2 receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT2A receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT2A receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.

Opioid receptor-mediated hyperalgesia and antinociceptive tolerance induced by sustained opiate delivery.

Opiates are commonly used to treat moderate to severe pain and can be used over prolonged periods in states of chronic pain such as those associated with cancer. In addition, to analgesic actions, studies show that opiate administration can paradoxically induce hyperalgesia. At the pre-clinical level, such hyperalgesia is associated with numerous pronociceptive neuroplastic changes within the primary afferent fibers and the spinal cord. In rodents, sustained opiate administration also induces antinociceptive tolerance. The mechanisms by which prolonged opiate exposure induces hyperalgesia and the relationship of this state to antinociceptive tolerance remain unclear. The present study was aimed at determining whether sustained opiate-induced hyperalgesia, associated neuroplasticity and antinociceptive tolerance are the result of specific opiate interaction at opiate receptors. Enantiomers of oxymorphone, a mu opioid receptor agonist, were administered to rats by spinal infusion across 7 days. Sustained spinal administration of (-)-oxymorphone, but not its inactive enantiomer (+)-oxymorphone or vehicle, upregulated spinal dynorphin content, produced thermal and tactile hypersensitivity, and produced antinociceptive tolerance. These results indicate that these pronociceptive actions of sustained opiate administration require specific interaction with opiate receptors and are unlikely to be the result of accumulation of potentially excitatory metabolic products. While the precise mechanisms, which may account for these pronociceptive changes remain to be unraveled, the present data point to plasticity initiated by opiate receptor interaction.

Constitutive activity at the cannabinoid CB1 receptor is required for behavioral response to noxious chemical stimulation of TRPV1: Antinociceptive actions of CB1 inverse agonists

The potential modulation of TRPV1 nociceptive activity by the CB1 receptor was investigated here using CB1 wild-type (WT) and knock-out (KO) mice as well as selective CB1 inverse agonists. No significant differences were detected in baseline thermal thresholds of ICR, CB1WT or CB1KO mice. Intraplantar capsaicin produced dose- and time-related paw flinch responses in ICR and CB1WT mice and induced plasma extravasation yet minimal responses were seen in CB1KO animals with no apparent differences in TRPV1 channel expression. Capsaicin-evoked CGRP release from spinal cord tissue and capsaicin-evoked action potentials on isolated skin-nerve preparation were significantly decreased in CB1KO mice. Pretreatment with intraplantar galanin and bradykinin, compounds known to sensitize TRPV1 receptors, restored capsaicin-induced flinching in CB1KO mice. The possibility that constitutive activity at the CB1 receptor is required to maintain the TRPV1 receptor in a “sensitized” state was tested using CB1 inverse agonists. The CB1 inverse agonists elicited concentration-related inhibition of capsaicin-induced calcium influx in F-11 cells and produced dose-related inhibition of capsaicin-induced flinching in ICR mice. These data suggest that constitutive activity at the CB1 receptor maintains the TRPV1 channel in a sensitized state responsive to noxious chemical stimuli. Treatment with CB1 inverse agonists may promote desensitization of the channel resulting in antinociceptive actions against chemical stimulus modalities. These studies propose possible therapeutic exploitation of a novel mechanism providing pain relief by CB1 inverse agonists.