Prisca Honore

Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons.

The aim of this investigation was to determine whether murine models of inflammatory, neuropathic and cancer pain are each characterized by a unique set of neurochemical changes in the spinal cord and sensory neurons. All models were generated in C3H/HeJ mice and hyperalgesia and allodynia behaviorally characterized. A variety of neurochemical markers that have been implicated in the generation and maintenance of chronic pain were then examined in spinal cord and primary afferent neurons.Three days after injection of complete Freunds adjuvant into the hindpaw (a model of persistent inflammatory pain) increases in substance P, calcitonin gene-related peptide, protein kinase C gamma, and substance P receptor were observed in the spinal cord. Following sciatic nerve transection or L5 spinal nerve ligation (a model of persistent neuropathic pain) significant decreases in substance P and calcitonin gene-related peptide and increases in galanin and neuropeptide Y were observed in both primary afferent neurons and the spinal cord. In contrast, in a model of cancer pain induced by injection of osteolytic sarcoma cells into the femur, there were no detectable changes in any of these markers in either primary afferent neurons or the spinal cord. However, in this cancer-pain model, changes including massive astrocyte hypertrophy without neuronal loss, increase in the neuronal expression of c-Fos, and increase in the number of dynorphin-immunoreactive neurons were observed in the spinal cord, ipsilateral to the limb with cancer. These results indicate that a unique set of neurochemical changes occur with inflammatory, neuropathic and cancer pain in C3H/HeJ mice and further suggest that cancer induces a unique persistent pain state. Determining whether these neurochemical changes are involved in the generation and maintenance of each type of persistent pain may provide insight into the mechanisms that underlie each of these pain states.

Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord.

Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted ‘decoy’ receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.

A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat

P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22–92 nM) and was highly selective (IC50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 μmol/kg s.c.) reduced complete Freunds adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Nav1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC50 = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Nav1.8 (IC50 = 8 nM) and was >100-fold selective vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.7 (IC50 values ≥1 μM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED50 = 47 mg/kg, i.p.), sciatic nerve injury (ED50 = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED50 ≈ 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freunds adjuvant injection (ED50 = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Nav1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

A-740003 [N-(1-{[(Cyanoimino)(5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a Novel and Selective P2X7 Receptor Antagonist, Dose-Dependently Reduces Neuropathic Pain in the Rat

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including glial cells in the central nervous system. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1β (IL-1β), and following prolonged agonist exposure, cytolytic plasma membrane pore formation. P2X7 knockout mice show reduced inflammation as well as decreased nociceptive sensitivity following peripheral nerve injury. A-740003 (N-(1-{[(cyanoimino)(5-quinolinylamino) methyl] amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) is a novel competitive antagonist of P2X7 receptors (IC50 values = 40 nM for human and 18 nM for rat) as measured by agonist-stimulated changes in intracellular calcium concentrations. A-740003 showed weak or no activity (IC50 > 10 μM) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes. A-740003 potently blocked agonist-evoked IL-1β release (IC50 = 156 nM) and pore formation (IC50 = 92 nM) in differentiated human THP-1 cells. Systemic administration of A-740003 produced dose-dependent antinociception in a spinal nerve ligation model (ED50 = 19 mg/kg i.p.) in the rat. A-740003 also attenuated tactile allodynia in two other models of neuropathic pain, chronic constriction injury of the sciatic nerve and vincristine-induced neuropathy. In addition, A-740003 effectively reduced thermal hyperalgesia observed following intraplantar administration of carrageenan or complete Freunds adjuvant (ED50 = 38–54 mg/kg i.p.). A-740003 was ineffective in attenuating acute thermal nociception in normal rats and did not alter motor performance at analgesic doses. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists.

Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. Oral administration of either A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide) (good CNS penetration) or A-795614 (N-1H-indazol-4-yl-N′-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea) (poor CNS penetration) blocked capsaicin-induced acute pain with the same potency. In complete Freunds adjuvant (CFA)-induced chronic inflammatory pain, oral administration of either compound blocked thermal hyperalgesia with similar potency. Furthermore, intraplantar or intrathecal administration of A-784168 blocked CFA-induced thermal hyperalgesia, suggesting that both peripheral and CNS TRPV1 receptors may play a role in inflammatory thermal hyperalgesia. The effects of the two TRPV1 antagonists were further assessed in models presumably mediated by central sensitization, including CFA- and capsaicin-induced mechanical allodynia and osteoarthritic pain. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.

P2X7-related modulation of pathological nociception in rats

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

Analgesic profile of intrathecal P2X3 antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats

&NA; Extracellular adenosine triphosphate (ATP), acting at P2X ionotropic receptors, is implicated in numerous sensory processes. Exogenous ATP has been shown to be algogenic in both animals and humans. Research focus has been directed towards the P2X3 receptor, as it is preferentially expressed on nociceptive C‐fibers and its implication in pain processing is supported by an altered nociceptive phenotype in P2X3 knock‐out mice. In order to further characterize the role of P2X3 receptor activation in nociception, we evaluated the effects of continuous intrathecal administration of P2X3 antisense oligonucleotides for 7 days in the rat. P2X3 receptor antisense oligonucleotide treatment significantly decreased nociceptive behaviors observed after injection of complete Freunds adjuvant (CFA), formalin or &agr;&bgr;‐methylene ATP into the rats hind paw. The anti‐hyperalgesic effects of the antisense treatment in the CFA model of inflammatory pain were dose related. Similar effects were observed with two distinct P2X3 antisense oligonucleotides. These behavioral effects were significantly correlated with a decrease in P2X3 receptor protein expression in the dorsal root ganglia (DRG). In contrast, a decrease in P2X3 receptor protein expression in the DRG did not affect nociceptive behavior in the carrageenan model of acute thermal hyperalgesia. P2X3 receptor antisense oligonucleotide treatment also significantly reduced mechanical allodynia observed after spinal nerve ligation. Overall, the present data demonstrate that activation of P2X3 receptors contribute to the expression of chronic inflammatory and neuropathic pain states and that relief form these forms of chronic pain might be achieved by selective blockade of P2X3 receptor expression or activation.

Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs. inflammatory pain

&NA; Pain is the cancer related event that is most disruptive to the cancer patients quality of life. Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast, prostate and lung cancers, relatively little is known about the mechanisms that generate and maintain this pain. Recently, we developed a mouse model of bone cancer pain and 16 days following tumor implantation into the intramedullary space of the femur, significant bone destruction and bone cancer pain‐related behaviors were observed. A critical question is how closely this model mirrors human bone cancer pain. In the present study we show that, as in humans, pain‐related behaviors are diminished by systemic morphine administration in a dose dependent fashion that is naloxone‐reversible. Humans suffering from bone cancer pain generally require significantly higher doses of morphine as compared to individuals with inflammatory pain and in the mouse model, the doses of morphine required to block bone cancer pain‐related behaviors were ten times that required to block peak inflammatory pain behaviors of comparable magnitude induced by hindpaw injection of complete Freunds adjuvant (CFA) (1–3 mg/kg). As these animals were treated acutely, there was not time for morphine tolerance to develop and the rightward shift in analgesic efficacy observed in bone cancer pain vs. inflammatory pain suggests a fundamental difference in the underlying mechanisms that generate bone cancer vs. inflammatory pain. These results indicate that this model may be useful in defining drug therapies that are targeted for complex bone cancer pain syndromes.

Electrophysiological and in vivo characterization of A-317567, a novel blocker of acid sensing ion channels.

&NA; Acid Sensing Ion Channels (ASICs) are a group of sodium‐selective ion channels that are activated by low extracellular pH. The role of ASIC in disease states remains unclear partly due to the lack of selective pharmacological agents. In this report, we describe the effects of A‐317567, a novel non‐amiloride blocker, on three distinct types of native ASIC currents evoked in acutely dissociated adult rat dorsal root ganglion (DRG) neurons. A‐317567 produced concentration‐dependent inhibition of all pH 4.5‐evoked ASIC currents with an IC50 ranging between 2 and 30 μM, depending upon the type of ASIC current activated. Unlike amiloride, A‐317567 equipotently blocked the sustained phase of ASIC3‐like current, a biphasic current akin to cloned ASIC3, which is predominant in DRG. When evaluated in the rat Complete Freuds Adjuvant (CFA)‐induced inflammatory thermal hyperalgesia model, A‐317567 was fully efficacious at a dose 10‐fold lower than amiloride. A‐317567 was also potent and fully efficacious when tested in the skin incision model of post‐operative pain. A‐317567 was entirely devoid of any diuresis or natriuresis activity and showed minimal brain penetration. In summary, A‐317567 is the first reported small molecule non‐amiloride blocker of ASIC that is peripherally active and is more potent than amiloride in vitro and in vivo pain models. The discovery of A‐317567 will greatly help to enhance our understanding of the physiological and pathophysiological role of ASICs.