Assessing carrageenan-induced locomotor activity impairment in rats: comparison with evoked endpoint of acute inflammatory pain.

BACKGROUND: Most animal models currently used to evaluate antinociceptive efficacy of analgesics rely on the assessment of evoked pain behaviours as primary endpoints. METHODS: Here, we have developed and characterized the carrageenan-induced locomotor activity impairment (CLAIM) model to objectively assess non-evoked inflammatory pain behaviour in rats. In this model, 100 µL of 1% carrageenan was subcutaneously injected into the plantar aspect of the right hind paw and exploratory behaviour in the novel testing chamber was recorded using an automated locomotor activity system. RESULTS: Carrageenan-injected animals exhibited an exploratory behavioural deficit 2-7 h following injection compared to saline-injected animals. The severity of impairment was carrageenan dose related, and sensitive to the light intensity in the testing room. The effects of standard analgesics on CLAIM were examined 2 or 3 h following carrageenan injection. Diclofenac and ibuprofen, in a dose range exerting no effect on locomotor activity in naïve rats, exhibited dose-related reversal of CLAIM (ED(50) = 1.5 and 5.0 mg/kg, respectively), with comparable efficacy on carrageenan-induced thermal hyperalgesia (ED(50) = 2.0 and 6.0 mg/kg, respectively). Gabapentin and duloxetine produced no reversal of CLAIM, or attenuation of thermal hyperalgesia. Efficacy discrepancy was noted for morphine on thermal hyperalgesia and CLAIM. Additionally, amphetamine dose dependently reversed CLAIM, and similarly increased locomotor activity in normal animals. DISCUSSION AND CONCLUSION: The results presented here demonstrate that CLAIM provides an objective assessment of non-evoked pain behaviours for acute inflammatory pain. The pharmacological profile of standard analgesics supports that CLAIM model can be used to identify agents to treat acute inflammatory pain in the clinic.

Peripheral and central sites of action for the non-selective cannabinoid agonist WIN 55,212-2 in a rat model of post-operative pain.

BACKGROUND AND PURPOSE: Activation of cannabinoid (CB) receptors decreases nociceptive transmission in inflammatory or neuropathic pain states. However, the effects of CB receptor agonists in post-operative pain remain to be investigated. Here, we characterized the anti-allodynic effects of WIN 55,212-2 (WIN) in a rat model of post-operative pain. EXPERIMENTAL APPROACH: WIN 55,212-2 was characterized in radioligand binding and in vitro functional assays at rat and human CB(1) and CB(2) receptors. Analgesic activity and site(s) of action of WIN were assessed in the skin incision-induced post-operative pain model in rats; receptor specificity was investigated using selective CB(1) and CB(2) receptor antagonists. KEY RESULTS: WIN 55,212-2 exhibited non-selective affinity and agonist efficacy at human and rat CB(1) versus CB(2) receptors. Systemic administration of WIN decreased injury-induced mechanical allodynia and these effects were reversed by pretreatment with a CB(1) receptor antagonist, but not with a CB(2) receptor antagonist, given by systemic, intrathecal and supraspinal routes. In addition, peripheral administration of both CB(1) and CB(2) antagonists blocked systemic WIN-induced analgesic activity. CONCLUSIONS AND IMPLICATIONS: Both CB(1) and CB(2) receptors were involved in the peripheral anti-allodynic effect of systemic WIN in a pre-clinical model of post-operative pain. In contrast, the centrally mediated anti-allodynic activity of systemic WIN is mostly due to the activation of CB(1) but not CB(2) receptors at both the spinal cord and brain levels. However, the increased potency of WIN following i.c.v. administration suggests that its main site of action is at CB(1) receptors in the brain.

Blockade of mGluR1 receptor results in analgesia and disruption of motor and cognitive performances: effects of A-841720, a novel non-competitive mGluR1 receptor antagonist.

BACKGROUND AND PURPOSE: To further assess the clinical potential of the blockade of metabotropic glutamate receptors (mGluR1) for the treatment of pain. EXPERIMENTAL APPROACH: We characterized the effects of A-841720, a novel, potent and non-competitive mGluR1 antagonist in models of pain and of motor and cognitive function. KEY RESULTS: At recombinant human and native rat mGluR1 receptors, A-841720 inhibited agonist-induced calcium mobilization, with IC50 values of 10.7+/-3.9 and 1.0 +/- 0.2 nM, respectively, while showing selectivity over other mGluR receptors, in addition to other neurotransmitter receptors, ion channels, and transporters. Intraperitoneal injection of A-841720 potently reduced complete Freund's adjuvant-induced inflammatory pain (ED50 = 23 micromol kg(-1)) and monoiodoacetate-induced joint pain (ED50 = 43 micromol kg(-1)). A-841720 also decreased mechanical allodynia observed in both the sciatic nerve chronic constriction injury and L5-L6 spinal nerve ligation (SNL) models of neuropathic pain (ED50 = 28 and 27 micromol kg(-1), respectively). Electrophysiological studies demonstrated that systemic administration of A-841720 in SNL animals significantly reduced evoked firing in spinal wide dynamic range neurons. Significant motor side effects were observed at analgesic doses and A-841720 also impaired cognitive function in the Y-maze and the Water Maze tests. CONCLUSIONS AND IMPLICATIONS: The analgesic effects of a selective mGluR1 receptor antagonist are associated with motor and cognitive side effects. The lack of separation between efficacy and side effects in pre-clinical models indicates that mGluR1 antagonism may not provide an adequate therapeutic window for the development of such antagonists as novel analgesic agents in humans.

Comparison of antinociceptive actions of standard analgesics in attenuating capsaicin and nerve-injury-induced mechanical hypersensitivity.

Intradermal capsaicin injection produces immediate spontaneous pain behaviors, and a secondary mechanical hypersensitivity (SMH) that is employed in the clinic as a model potentially predictive of human neuropathic pain. Presently, we have characterized capsaicin-induced SMH in rats, and compared pharmacological actions of standard analgesics in this and two nerve injury models, the L5/L6 spinal nerve ligation (SNL) and sciatic nerve chronic constriction injury (CCI) models. Intraplantar capsaicin produced dose-related SMH (enhanced paw withdrawal response to von Frey monofilament stimulation at an area away from injection site) that lasted for over 4 h. While pretreatment with a potent selective transient receptor potential vanilloid receptor-1 (TRPV1) antagonist A-425619 (1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea) prevented development of acute nocifensive (flinching) behavior immediately following capsaicin injection (ED(50)=4.9 mg/kg), the compound failed to attenuate the SMH when administered 2 h following capsaicin (10 microg/10 microl). Additional standard analgesics were also tested 3 h following intraplantar capsaicin in the SMH model. Comparison of their potencies in attenuating mechanical hypersensitivity in capsaicin, SNL and CCI models revealed similar ED(50)s for morphine (2.3 mg/kg, 1.6 mg/kg and 3.2 mg/kg, respectively), gabapentin (33.1 mg/kg, 33.9 mg/kg and 26.3 mg/kg, respectively) and lamotrigine (9.1 mg/kg, 8.9 mg/kg and 15.5 mg/kg, respectively). Duloxetine produced 50-65% effect at the highest tested dose (50 mg/kg), whereas the highest tested doses of morphine (10 mg/kg), gabapentin (85.5 mg/kg) and lamotrigine (30 mg/kg) all produced >70% efficacy in capsaicin SMH, SNL and CCI models. In contrast, celecoxib and ibuprofen showed weak effects in all three models. All standard analgesics generally had weak efficacy in attenuating capsaicin-induced immediate acute flinching behavior when administered before capsaicin. These results provide further support to the suggestions that distinct pharmacological mechanisms underlie capsaicin-induced acute nocifensive and SMH behaviors, and certain neuronal mechanisms underlying neuropathic pain states are also contributory to capsaicin-induced SMH.

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 Freund's 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.

The therapeutic potential of nicotinic acetylcholine receptor agonists for pain control.

Due to the limitations of currently available analgesics, a number of novel alternatives are currently under investigation, including neuronal nicotinic acetylcholine receptor (nAChR) agonists. During the 1990s, the discovery of the antinociceptive properties of the potent nAChR agonist epibatidine in rodents sparked interest in the analgesic potential of this class of compounds. Although epibatidine also has several mechanism-related toxicities, the identification of considerable nAChR diversity suggested that the toxicities and therapeutic actions of the compound might be mediated by distinct receptor subtypes. Consistent with this view, a number of novel nAChR agonists with antinociceptive activity and improved safety profiles in preclinical models have now been identified, including A-85380, ABT-594, DBO-83, SIB-1663 and RJR-2403. Of these, ABT-594 is the most advanced and is currently in Phase II clinical evaluation. Nicotinically-mediated antinociception has been demonstrated in a variety of rodent pain models and is likely mediated by the activation of descending inhibitory pathways originating in the brainstem with the predominant high-affinity nicotine site in brain, the alpha4beta2 subtype, playing a critical role. Thus, preclinical findings suggest that nAChR agonists have the potential to be highly efficacious treatments in a variety of pain states. However, clinical proof-of-principle studies will be required to determine if nAChR agonists are active in pathological pain.

Spinal mechanisms underlying A-85380-induced effects on acute thermal pain.

Systemic administration of nicotinic receptor (nAChR) agonists is antinociceptive in models of acute pain whereas their intrathecal (i. t.) administration has been reported to be antinociceptive, nociceptive or without effect. It has been hypothesized that the action induced is dependent upon the subtype and location of the nAChR activated. In addition, there is considerable evidence that nAChR ligand-induced antinociception is mediated by other neurotransmitter systems via descending pathways from the brainstem to the spinal cord. The present study investigated the effects of i. t. and systemic administration of A-85380, a novel nAChR agonist, in the paw withdrawal model of acute thermal pain in the rat. Given i.t. , A-85380 (1 and 10 nmol/rat) decreased the latency to paw withdrawal by 2-4 s. This pronociception was accompanied by a spontaneous flinching behavior. Both of these effects were differentially blocked by i.t. pretreatment with the nAChR antagonists mecamylamine (10 nmol)>MLA (100 nmol)>DHbetaE (50% with 1000 nmol) but not by alpha-bungarotoxin (0% at 0.63 nmol). Given systemically, A-85380 (0.56 micromol/kg, i.p.) induced antinociception as indicated by an increased latency to paw withdrawal, an effect differentially altered by i.t. pretreatment with monoaminergic antagonists (100 nmol/rat). While mecamylamine and prazosin had no effect, scopolamine, methysergide and MDL 72222 partially antagonized and idazoxan completely antagonized A-85380-induced antinociception. Finally, as measured by in vivo microdialysis, levels of 5-HT, but not NE, in the i.t. space of the lumber region of the spinal cord were significantly increased following the systemic administration of A-85380. Together these data suggest that the nociceptive properties of spinally administered nAChR agents are not mediated by either an alpha(4)beta(2) or an alpha(7) subtype nAChR, whereas the antinociceptive properties of systemically-administered nAChR agents are mediated by descending noradrenergic, serotonergic and muscarinic inhibitory pathways.

Reduced nicotinic receptor-mediated antinociception following in vivo antisense knock-down in rat.

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain alpha4 and beta2 subunits. The purpose of the present study was to investigate the role of alpha4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the alpha4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the alpha4 nAChR subunit in the dorsal raphe nucleus. The expression of alpha4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific alpha4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of alpha4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the alpha7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that alpha4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

The identification of novel structural compound classes exhibiting high affinity for neuronal nicotinic acetylcholine receptors and analgesic efficacy in preclinical models of pain.

Neuronal nicotinic acetylcholine receptors represent a new and potentially useful target for the development of novel non-opioid, non-NSAID (nonsteroidal antiinflammatory drug) analgesic agents. A variety of nicotinic acetylcholine receptor agonists such as nicotine, epibatidine and the azetidinyl ether, (R)-5-(2-azetidinylmethoxy-2-chloropyridine (ABT-594) possesses significant efficacy in preclinical models of pain. A preponderance of evidence suggests that nicotinic acetylcholine receptor agonists produce their analgesic effects predominantly via activation of descending inhibitory pain pathways originating in the key brainstem regions of the nucleus raphe magnus, dorsal raphe, and locus coeruleus, and that alpha4-containing nicotinic acetylcholine receptor subunits mediate these effects. Although these studies may provide a pharmacological target for the development of nicotinic acetylcholine receptor analgesics, the rational design of selective ligands based on the protein structure of the binding site is hampered by insufficient structural information. Using an approach based upon homology to known high-affinity ligands for the alpha4beta2 binding site, a four-point model is proposed which defines distance and directionality parameters common to this set of nicotinic acetylcholine receptor ligands.

Therapeutic potential of neuronal nicotinic acetylcholine receptor agonists as novel analgesics.

Pharmacological treatments for pain have come largely from two classes of compounds--the opioids and the nonsteroidal anti-inflammatory drugs (NSAIDs). Because of deficiencies associated with these two classes of compounds, exploration of novel approaches to pain relief has intensified of late. Nicotine, a neuronal nicotinic acetylcholine receptor (nAChR) agonist, has long been known to have antinociceptive effects in both experimental animals and humans. The relatively modest antinociceptive effects and the toxicities associated with nicotine preclude its development as an analgesic agent. However, recent discoveries in the nAChR field have stimulated interest in nAChR-targeted compounds as potential analgesic agents. Epibatidine, a potent nAChR agonist, was found to have full efficacy relative to opioids in preclinical pain models. Although epibatidine is toxic, these observations demonstrated that modest efficacy is not a general limitation of nAChR agonists. Moreover, exploration of the molecular biology of nAChRs revealed evidence of receptor diversity, suggesting that nAChR subtype-selective agents less toxic than nicotine might be discovered; and early medicinal chemistry efforts already have resulted in compounds with improved safety profiles. For example, ABT-594 is a nAChR agonist with the antinociceptive efficacy of epibatidine, but with an improved safety profile. This commentary reviews recent findings with nAChR-targeted compounds, explores potential mechanisms responsible for nAChR-mediated antinociception, and raises issues that must be addressed in developing compounds of this class as analgesics.

Enhancement of sustained attention performance by the nicotinic acetylcholine receptor agonist ABT-418 in intact but not basal forebrain-lesioned rats.

RATIONALE: Loss of telencephalic cholinergic projections has been postulated to contribute significantly to the cognitive decline associated with aging and dementia. OBJECTIVE: The effects of the nicotinic acetylcholine receptor agonist ABT-418, a potential therapeutic drug for the treatment of the age- and dementia-associated cognitive disorders, were tested in an animal model of the cortical cholinergic deafferentation-induced impairments in sustained attention. METHODS: Animals were trained in an operant task designed to test sustained attention performance. A partial loss of cortical cholinergic inputs was produced by infusions of 192 IgG-saporin into the basal forebrain. The effects of the systemic administration of ABT-418 (0.04, 0.13, 0.39 mg/kg) and the psychostimulant methylphenidate (0.2, 0.4, 0.8 mg/kg) were assessed. RESULTS: Compared with sham-lesioned animals, this lesion resulted in a decrease in the relative number of hits while the relative number of correct rejections remained unaffected. Administration of ABT-418 significantly improved the relative number of hits. Furthermore, this effect of ABT-418 interacted with the effects of the lesion. Unexpectedly, this interaction was based on a significant enhancement of the performance of sham-lesioned animals while no effects were found in 192 IgG-saporin-lesioned animals. Administration of methylphenidate did not affect performance. CONCLUSIONS: While these data do not support the hypothesis that administration of ABT-418 attenuates the impairments in attentional performance that result from loss of cortical cholinergic inputs, they support previous notions about this drug's ability to enhance cognitive processes in intact subjects.

The analgesic potential of compounds acting at acetylcholine receptors.

Compounds acting at both nicotinic and muscarinic acetylcholine receptors appear to have antinociceptive activity, and acetylcholine release in the spinal cord may be involved in endogenous pain control. The therapeutic potential of most cholinergic agonists or of agents that increase synaptic acetylcholine is limited by side effect liabilities. Recent studies, however, have identified some compounds with improved safety profiles. Multiple subtypes of nicotinic and muscarinic receptors exist, and molecular and pharmacological studies are just beginning to identify which subtypes are involved in the antinociceptive effects of cholinergic receptor activation. Further advances in this area will be necessary to determine if the rational design of subtype selective cholinergic agonists will provide novel analgesic agents.

Differences between the antinociceptive effects of the cholinergic channel activators A-85380 and (+/-)-epibatidine in rats.

(+/-)-Epibatidine (EPIB) and A-85380 are nicotinic acetylcholine receptor (nAChR) agonists that bind to the agonist ([3H]cytisine) binding site with 40 to 50 pM affinity but have different affinities in nAChR subtype selective functional receptor assays. In vivo EPIB was more (23-fold) potent than A-85380 in reducing open field activity and more (12-fold) potent in reducing nociception in the formalin test of persistent chemical pain. In the rat hot box test of thermal acute pain, both compounds produced antinociception, as indicated by an increase in the paw withdrawal latency, however EPIB was a approximately 33-fold more potent than A-85380 (ED50 = 0.004 and 0.11 micromol/kg, i.p., respectively). The systemic effects of both nAChR agonists were blocked by central (i.c.v.) administration of the nAChR antagonist chlorisondamine suggesting a central site of action for these compounds. Injections of EPIB (0.0013 to 0.013 nmol) and A-85380 (0.013 to 0.13 nmol) directly into the nucleus raphe magnus (NRM) were also effective in the hot box and could be blocked by coadministration of the nAChR antagonists chlorisondamine (0.23 nmol) or mecamylamine (0.8 nmol). The NRM was found to be critical for the antinociceptive effects of systemic EPIB but not for A-85380 in that NRM injections of either mecamylamine (0.8 nmol) or lidocaine (74 nmol) blocked the antinociceptive effects of systemic (i.p.) EPIB but not those of A-85380. These results suggest that A-85380 may act at multiple sites both within and outside the NRM, whereas EPIB acts largely via descending inhibitory pathways arising from the NRM.

The role of neuronal nicotinic acetylcholine receptors in antinociception: effects of ABT-594.

ABT-594, a nicotinic acetylcholine receptor agonist, has antinociceptive effects in rat models of acute thermal, persistent chemical, and neuropathic pain. Direct injection of ABT-594 into the nucleus raphe magnus (NRM) is antinociceptive in a thermal threshold test and destruction of serotonergic neurons in the NRM attenuates the effect of systemic ABT-594. However, lidocaine-inactivation of the NRM prevents the antinociceptive effect of systemic (-)-nicotine but not that of systemic ABT-594.

ABT-594, a novel cholinergic channel modulator, is efficacious in nerve ligation and diabetic neuropathy models of neuropathic pain.

A novel cholinergic channel modulator, ABT-594, was tested in two established and distinct models of neuropathic pain; the Chung model (i.e., tight ligation of L5 and L6 spinal nerves) and a diabetic neuropathy model (i.e., streptozotocin-induced diabetes). Tactile allodynia and mechanical hyperalgesia were assessed in the Chung and diabetic neuropathy models, respectively. ABT-594 produced a significant antiallodynic effect following both oral (0.1-1 micromol/kg) and intraperitoneal (i.p.) (0.3 micromol/kg) administration. Equal efficacy was observed following both routes of administration. ABT-594 (0.3 micromol/kg, i.p.) maintained efficacy following repeated dosing (5 days; twice daily) in the Chung model, but the effect of morphine (21 micromol/kg, i.p.) was significantly reduced after repeated dosing. In the diabetic neuropathy model, ABT-594 (0.3 micromol/kg, i.p.) effectively reduced mechanical hyperalgesia. Morphine (21 micromol/kg, i.p.) was not effective in this model. Overall, these results suggest development of ABT-594 may provide a novel pharmacotherapy for the chronic treatment of neuropathic pain.

Role of the nucleus raphe magnus in antinociception produced by ABT-594: immediate early gene responses possibly linked to neuronal nicotinic acetylcholine receptors on serotonergic neurons.

Recently, a novel cholinergic channel modulator, (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), was shown to produce potent analgesia in a variety of rodent pain models when administered either systemically or centrally into the nucleus raphe magnus (NRM). The purpose of the present study was to investigate the possible supraspinal contribution of ABT-594 by assessing its ability to induce expression of the immediate early gene c-fos, a biochemical marker of neuronal activation, in the NRM of rats. Putative serotonergic neurons in the NRM, a medullary nucleus proposed to be involved in descending antinociceptive pathways, were identified immunohistochemically using a monoclonal antibody (mAb) against tryptophan hydroxylase. ABT-594 (0.03-0.3 micromol/kg, i.p.) produced a dose-dependent induction of Fos protein that was blocked by the central nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (5 micromol/kg, i.p.) but not by the peripheral nAChR antagonist hexamethonium (15 micromol/kg, i.p.). Immunohistological studies using mAb 299 revealed the expression of alpha4-containing nAChRs in the NRM. The alpha4 immunostaining was dramatically reduced by pretreating (30 d) animals with the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which was previously shown to substantially attenuate the antinociceptive actions of ABT-594. In a double immunohistochemical labeling experiment, coexpression of the serotonin marker tryptophan hxdroxylase and the alpha4 nAChR subunit in NRM neurons was observed. These results suggest that the analgesic mechanism of ABT-594 may in part involve the activation of the NRM, a site where alpha4-containing nAChRs are expressed by serotonergic neurons.