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Treatment of fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-hydroxytriptamine (5-HT) release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic α 2 and postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4 to 5 days after the cessation of treatments. The repeated mirtazapine treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of small interfering RNA (siRNA) adrenergic α 2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of α 2 receptor gene expression after ICS, reversed the hyperalgesia but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular α 2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of α 2 receptor in unidentified brain regions. SIGNIFICANCE STATEMENT: This study reports possible mechanisms underlying the complete reversal of hyperalgesia and recovery of morphine analgesia by mirtazapine, a unique antidepressant with adrenergic α 2 and serotonergic receptor antagonist properties, in a type of intermittently repeated stress (ICS)-induced fibromyalgia-like pain model. Habenula, a brain region which is related to the control of emotional pain, was found to play key roles in the antihyperalgesia, whereas other brain regions appeared to be involved in the recovery of morphine analgesia in the ICS model., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration, continues to be a dose-limiting and debilitating side effect during and/or after cancer chemotherapy. Paclitaxel, a taxane commonly used to treat breast, lung, and ovarian cancers, causes CIPN in 59-78% of cancer patients. Novel interventions are needed due to the current lack of effective CIPN treatments. Our studies were designed to investigate whether nicotine can prevent and/or reverse paclitaxel-induced peripheral neuropathy in a mouse model of CIPN, while ensuring that nicotine will not stimulate lung tumor cell proliferation or interfere with the antitumor properties of paclitaxel. Male C57BL/6J mice received paclitaxel every other day for a total of four injections (8 mg/kg, i.p.). Acute (0.3-0.9 mg/kg, i.p.) and chronic (24 mg/kg per day, s.c.) administration of nicotine respectively reversed and prevented paclitaxel-induced mechanical allodynia. Blockade of the antinociceptive effect of nicotine with mecamylamine and methyllycaconitine suggests that the reversal of paclitaxel-induced mechanical allodynia is primarily mediated by the α 7 nicotinic acetylcholine receptor subtype. Chronic nicotine treatment also prevented paclitaxel-induced intraepidermal nerve fiber loss. Notably, nicotine neither promoted proliferation of A549 and H460 non-small cell lung cancer cells nor interfered with paclitaxel-induced antitumor effects, including apoptosis. Most importantly, chronic nicotine administration did not enhance Lewis lung carcinoma tumor growth in C57BL/6J mice. These data suggest that the nicotinic acetylcholine receptor-mediated pathways may be promising drug targets for the prevention and treatment of CIPN., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

The prostaglandin (PG) EP(4) receptor subtype is expressed by peripheral sensory neurons. Although a potential role of EP(4) receptor in pain has been suggested, a limited number of selective ligands have made it difficult to explore the physiological functions of EP(4) or its potential as a new analgesic target. Here, we describe the in vitro and in vivo pharmacology of a novel EP(4) receptor antagonist, N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo [4,5-c] pyridin-1-yl) phenyl]ethyl}amino) carbonyl]-4-methylbenzenesulfonamide (CJ-023,423). In vitro, CJ-023,423 inhibits [(3)H]PGE(2) binding to both human and rat EP(4) receptors with K(i) of 13 +/- 4 and 20 +/- 1 nM, respectively. CJ-023,423 is highly selective for the human EP(4) receptor over other human prostanoid receptor subtypes. It also inhibits PGE(2)-evoked elevation in intracellular cAMP at the human and rat EP(4) receptors with pA(2) of 8.3 +/- 0.03 and 8.2 +/- 0.2 nM, respectively. In vivo, oral administration of CJ-023,423 significantly reduces thermal hyperalgesia induced by intraplantar injection of PGE(2) (ED(50) = 12.8 mg/kg). CJ-023,423 is also effective in models of acute and chronic inflammatory pain. CJ-023,423 significantly reduces mechanical hyperalgesia in the carrageenan model. Furthermore, CJ-023,423 significantly reverses complete Freund's adjuvant-induced chronic inflammatory pain response. Taken together, the present data indicate that CJ-023,423, a highly potent and selective antagonist of both human and rat EP(4) receptors, produces antihyperalgesic effects in animal models of inflammatory pain. Thus, specific blockade of the EP(4) receptor signaling may represent a novel therapeutic approach for the treatment of inflammatory pain.

This study investigated three possible mechanisms by which the antinociceptive effects of the mu-opioid receptor (MOR) agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and the delta-opioid receptor (DOR) agonist [d-Ala(2),Glu(4)]-deltorphin (deltorphin II) (DELT), microinjected into the rostral ventromedial medulla (RVM), are enhanced in rats with persistent inflammatory injury. Radioligand binding determined that neither the B(max) nor the K(d) values of [(3)H]DAMGO differed in RVM membranes from rats that received an intraplantar injection of saline or complete Freund's adjuvant (CFA) in one hindpaw 4 h, 4 days, or 2 weeks earlier. Likewise, neither the EC(50) nor the E(max) value for DAMGO-induced stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding differed in the RVM of saline- or CFA-treated rats at any time point. Microinjection of fixed dose combinations of DAMGO and DELT in the RVM of naive rats indicated that these agonists interact synergistically to produce antinociception when DAMGO is present in equal or greater amounts than DELT and, additively, when DELT is the predominant component. Thus, unlike the periphery or spinal cord, potentiation of MOR-mediated antinociception does not entail an increase in MOR number, affinity, or coupling. Rather, the data are concordant with our proposal that potentiation results from a synergistic interaction of exogenous MOR agonist with DOR-preferring enkephalins whose levels are increased in CFA-treated rats (J Neurosci 21:2536-2545, 2001). Virtually no specific [(3)H]DELT binding nor stimulation of [(35)S]GTPgammaS binding by DELT was obtained in RVM membranes from CFA- or saline-treated rats at any time point. The mechanisms responsible for the potentiation of DELT-mediated antinociception remain to be elucidated.

The present study characterizes the involvement of the N-methyl-D-aspartic acid receptors (NMDARs) in mediating thermal hyperalgesia induced by activation of group I metabotropic glutamate receptors (mGluRs). Intrathecal administration of the mGluR1/5 agonist (S)-3,5-DHPG [(S)-3,5-dihydroxyphenylglycine] to mice resulted in significant hyperalgesia as assessed by the tail immersion test. The pretreatment of mice i.t. with CGS 19755 (selective antagonist of the NMDAR), CGP 78608 [[(1S)-1-[[(7-bromo-1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]amino]ethyl]phosphonic acid] (selective antagonist at the glycine-binding site of the NMDAR), ifenprodil and Ro 25-6981 (selective antagonists of the NR2B subunit of the NMDAR), bisindolylmaleimide I and Go-7874 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (inhibitors of protein kinase C), or PKI-(14-22)-amide [Myr-N-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH(2)] (inhibitor of protein kinase A) dose-dependently inhibited the hyperalgesia induced by i.t. administration of the mGluR1/5 receptor agonist (S)-3,5-DHPG. In contrast, i.t. pretreatment of mice with NVP-AAM077 [[(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid] (selective antagonist of the NR2A subunit of the NMDAR) or DT-3 [H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Leu-Arg-Lys-Lys-Lys-Lys-Lys-His-OH] (inhibitor of protein kinase G) had no effect on (S)-3,5-DHPG-mediated hyperalgesia. We also show for the first time that i.t. injection of pSM2 (pShag Magic version 2)-grin2b (coding for an short-hairpin RNA to the NR2B subunit of the NMDAR) resulted in a dose-dependent decrease in the NR2B protein and blockade of hyperalgesia induced by activation of the mGluR1/5 in (S)-3,5-DHPG-treated mice. Taken together, our results suggest the hypothesis that mGluRs are coupled to the NMDAR channels through the NR2B subunit in the spinal cord and that this coupling involves the activation of protein kinase C and protein kinase A.

Pain may become intractable as tolerance develops to opioids and the opioids, paradoxically, induce pain. We examined the hypothesis that the analgesia produced by the novel analgesic and high-efficacy 5-hydroxytryptamine (5-HT)(1A) receptor agonist (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl)-amino]methyl]piperidin-1-yl]methanone, fumaric acid salt (F 13640) may counteract opioid-induced pain. In studies of the somatosensory quality of pain in infraorbital nerve-injured rats, morphine infusion (5 mg/day) by means of osmotic pumps initially caused analgesia (i.e., decreased the behavioral response to von Frey filament stimulation), followed by hyperallodynia and analgesic tolerance. Infusion of F 13640 (0.63 mg/day) prevented the development of opioid hyperallodynia and reversed opioid hyperallodynia once established. In studies of the affective/motivational quality of pain, F 13640 both prevented and reversed the conditioned place aversion induced by naloxone (0.04 mg/kg i.p.) in morphine-infused rats; F 13640 also prevented and reversed the conditioned place preference induced by morphine injections (7.5 mg/kg i.p.). The data confirm that opioids produce bidirectional hypo- and proalgesic actions, and offer initial evidence that high-efficacy 5-HT(1A) receptor activation counteracts both the sensory and the affective/motivational qualities of opioid-induced pain. The data also indicate that F 13640 may be effective with opioid-resistant pain. It further is suggested that opioid addiction may represent self-therapy of opioid-induced pathological pain.

Agmatine is an endogenous decarboxylation product of arginine that has been previously shown to antagonize the N-methyl-d-aspartate (NMDA) receptor and inhibit nitric-oxide synthase. Many neuropharmacological studies have shown that exogenous administration of agmatine prevents or reverses biological phenomena dependent on central nervous system glutamatergic systems, including opioid-induced tolerance, opioid self-administration, and chronic pain. However, the central nervous system (CNS) pharmacokinetic profile of agmatine remains minimally defined. The present study determined the spinal cord pharmacokinetics and acute pharmacodynamics of intrathecally administered agmatine in mice. After a single bolus intrathecal injection, agmatine concentrations in spinal cord (cervical, thoracic, and lumbosacral) tissue and serum were quantified by an isocratic high-performance liquid chromatography fluorescence detection system. Agmatine persisted at near maximum concentrations in all levels of the spinal cord for several hours with a half-life of approximately 12 h. Initial agmatine concentrations in serum were 10% those in CNS. However, the serum half-life was less than 10 min after intrathecal injection of agmatine, consistent with previous preliminary pharmacokinetic reports of systemically administered agmatine. The pharmacodynamic response to agmatine in the NMDA-nociceptive behavior and thermal hyperalgesia tests was assessed. Whereas MK-801 (dizocilpine maleate) inhibits these two responses with equal potency, agmatine inhibits the thermal hyperalgesia with significantly increased potency compared with the nociceptive behavior, suggesting two sites of action. In contrast to the pharmacokinetic results, the agmatine inhibition of both behaviors had a duration of only 10 to 30 min. Collectively, these results suggest the existence of a currently undefined agmatinergic extracellular clearance process in spinal cord.

We determined whether the i.t. administration of an 18-mer phosphodiester antisense oligodeoxynucleotide (ODN) that reduces the expression of the rat NMDAR1 subunit of the N-methyl-d-aspartate (NMDA) receptor would affect nociceptive behaviors and prevent the development of morphine tolerance. Rats received 5 microl of i.t. saline, 30 nM antisense, or mismatch ODN twice a day for 5 days (NMDA-induced nociception, NMDA-induced thermal hyperalgesia, NR1 mRNA, and ligand binding studies) or for 3 days (formalin study). For the tolerance study, 5 days of ODNs or saline were followed by 3 days of concurrent administration of ODNs or saline (twice a day) and i.t. morphine (three times a day). Antisense, but not mismatch, results in the reduction of formalin phase 2 flinching by 50%, the spinal cord dorsal horn levels of NMDAR1 mRNA by 30%, and ligand binding by 50%. The i.t. ED(50) for NMDA-induced nociceptive behaviors is doubled, and thermal hyperalgesia is blocked by antisense treatment. The effects of antisense on NMDA-induced nociception and thermal hyperalgesia are completely reversed by discontinuing antisense. The coadministration of antisense with increasing doses of i.t. morphine for 3 days attenuates the development of morphine tolerance. These results demonstrate that an in vivo antisense targeting of the NMDAR1 subunit results in antihyperalgesic effects and a partial blockade of spinal morphine tolerance. They provide additional support for the critical role of the NMDA receptor in these forms of spinal nociception and in the development of morphine tolerance and suggest the potential therapeutic utility of this approach.

3-[2-Cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59-3074) is a novel, selective cannabinoid CB(1)/CB(2) receptor ligand (K(i) = 55.4, 48.3, and 45.5 nM at rat and human cannabinoid CB(1) and human CB(2) receptors, respectively), with partial agonist properties at these receptors in guanosine 5-[gamma(35)S]-thiophosphate triethyl-ammonium salt ([(35)S]GTPgammaS) binding assays. In rats, generalization of BAY 59-3074 to the cue induced by the cannabinoid CB(1) receptor agonist (-)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 38-7271) in a drug discrimination procedure, as well as its hypothermic and analgesic effects in a hot plate assay, were blocked by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). BAY 59-3074 (0.3-3 mg/kg, p.o.) induced antihyperalgesic and antiallodynic effects against thermal or mechanical stimuli in rat models of chronic neuropathic (chronic constriction injury, spared nerve injury, tibial nerve injury, and spinal nerve ligation models) and inflammatory pain (carrageenan and complete Freund's adjuvant models). Antiallodynic efficacy of BAY 59-3074 (1 mg/kg, p.o.) in the spared nerve injury model was maintained after 2 weeks of daily administration. However, tolerance developed rapidly (within 5 days) for cannabinoid-related side effects, which occur at doses above 1 mg/kg (e.g., hypothermia). Uptitration from 1 to 32 mg/kg p.o. (doubling of daily dose every 4th day) prevented the occurrence of such side effects, whereas antihyperalgesic and antiallodynic efficacy was maintained/increased. No withdrawal symptoms were seen after abrupt withdrawal following 14 daily applications of 1 to 10 mg/kg p.o. It is concluded that BAY 59-3074 may offer a valuable therapeutic approach to treat diverse chronic pain conditions.

The specific involvement of the delta-opioid receptor in the control of nociception was explored by investigating the pharmacological activity in vivo of a selective, orally active, and centrally penetrant delta-opioid agonist. [8R-(4bS*,8aalpha,8abeta,12bbeta)]7,10-dimethyl-1-methoxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride (SB-235863) is a new pyrrolomorphinan with high affinity (Ki = 4.81 +/- 0.39 nM) for the delta-opioid receptor, full agonist activity, and binding selectivity versus the mu- and kappa-opioid receptors of 189-fold and 52-fold, respectively. Perorally administered SB-236863 was inactive in the rat tail-flick and hot-plate tests of acute pain response, but potently reversed thermal hyperalgesia in rats resulting from a carrageenan-induced inflammatory response. This activity could be blocked by the delta-opioid antagonist naltrindole (3 mg/kg s.c.), but selective mu- and kappa-opioid antagonists were ineffective. Naltrindole (1 microg i.c.v.) also blocked the activity of 10 mg/kg (p.o.) SB-235863, showing that the compound activates delta-opioid receptor sites in the central nervous system. SB-235863 was additionally effective at reversing chronic hyperalgesia in the Seltzer rat model of partial sciatic nerve ligation after peroral administration. These data show that the delta-opioid receptor plays a selective role in regulating evoked and lasting changes in nociceptive pain signaling. Classical side effects of mu- and kappa-opioid receptor activation (slowing of gastrointestinal transit and motor incoordination, respectively) were not observed after administration of 70 mg/kg (p.o.) SB-235863, nor was evoked seizure activity affected. These results suggest a selective and limited role of delta-opioid receptors in the modulation of nociception.

Activation of ionotropic glutamate receptors has been shown previously to be essential for the development of secondary thermal hyperalgesia. The present study assessed involvement of group I metabotropic glutamate receptors (mGlu) in both the induction and maintenance phases of secondary thermal hyperalgesia initiated by knee joint inflammation in rats. The dose dependence of each drug in antagonism of thermal hypersensitivity was demonstrated in pre- and post-treatment paradigms. Knee joint inflammation was induced by injection of kaolin and carrageenan. Four hours later the paw withdrawal latencies were significantly shorter than baseline values. Rats were pretreated by spinal microdialysis infusion of group I mGlu receptor antagonists, LY393053 [(+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid], LY367385 [(S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid], or AIDA [(R,S)-1-aminoindan-1,5-dicarboxylic acid/UPF 523] before knee joint injection. The paw withdrawal latencies measured 4 h after the injection were significantly longer in the presence of group I mGlu receptor antagonists than those of the artificial cerebrospinal fluid-treated arthritic control group. Post-treatment with the group I mGlu receptor antagonists LY367385 and AIDA allowed significant recovery of the paw withdrawal latencies after the onset of the knee joint inflammation. The knee joint inflammation itself was not affected by either treatment. The results of the present study indicate that secondary thermal hyperalgesia can be effectively attenuated during both the development and maintenance phases of acute knee joint inflammation by spinal application of specific group I mGlu receptor antagonists.

The effect of selective neurokinin receptor (NKR) antagonists for the NK1R (SR140,333), NK2R (SR48,968), and NK3R (SR142,801) on the visceromotor response to noxious colorectal distension (CRD) was examined. NKR antagonists or vehicle were given intrathecally (i.th.) to rats made hyperalgesic by intracolonic instillation of zymosan or after intracolonic instillation of saline (control). Given alone, the NK1R (up to 3 microg of SR140,333) and NK2R (up to 60 microg of SR48,968) antagonists tested failed to significantly affect responses to the noxious visceral stimulus. However, coadministration of 3 microg of SR140,333 and 60 microg of SR48,968 (both i.th.) significantly reduced responses to noxious CRD (p < 0.05 versus vehicle). The NK3R antagonist (60 microg of SR142,801) significantly reduced responses to noxious CRD when given alone to either hyperalgesic (zymosan-treated) or normal (saline-treated) rats (p < 0.05 versus vehicle for both groups). Responses of rats receiving the NK3R antagonist in combination with either the NK1R or the NK2R antagonist were not different from rats receiving the NK3R antagonist alone. These results suggest that activation of spinal NK1R and NK2R, presumably by their endogenous ligands (substance P and neurokinin A), maintain visceral hyperalgesia and support the notion that activation of NK3R (presumably by neurokinin B) is pronociceptive.

The present study was undertaken to determine whether propentofylline, a glial modulating agent, could both prevent the induction of mechanical allodynia and attenuate existing mechanical allodynia in a rodent L5 spinal nerve transection model of neuropathic pain. In a preventative paradigm, propentofylline (1 and 10 mg/kg intraperitoneally) was administered systemically daily, beginning 1 day prior to nerve transection. This regimen produced a dose-dependent decrease in mechanical allodynia (p < 0.01). In another preventative paradigm, propentofylline (0.1, 1, or 10 microg) was administered daily intrathecally via direct lumbar puncture. Intrathecal administration of propentofylline was more effective than systemic administration at dose dependently reducing mechanical allodynia (p < 0.01). The effect of systemic propentofylline on existing allodynia was examined with 0.1-, 1-, and 10-mg/kg intraperitoneal administration initiated on day 4 post L5 spinal nerve transection. Systemic propentofylline was found to be equally effective in the attenuation of existing allodynia (p < 0.01) as in the prevention of allodynia in this rodent model of neuropathic pain. Spinal cords (L4-L6 segments) were removed for immunohistochemical analysis on day 10 or 20 post-transection. Microglial and astrocytic activation was decreased by both peripheral and central administration of propentofylline in both preventative and existing allodynia paradigms. This research supports a growing body of literature highlighting the importance of glial activation in the development of persistent neuropathic pain states, and the potential to therapeutically modulate glial activation in the treatment of neuropathic pain.

The effects of SNC80 and other structurally related delta-opioid receptor agonists were assessed under conditions of chemically induced hypersensitivity to thermal stimuli in four rhesus monkeys. The shaved tail of each monkey was exposed to warm water (38, 42, 46, and 50 degrees C), and the tail-withdrawal latency from each temperature was recorded. The effects of drugs on the temperature that produced a 10-s tail-withdrawal latency (the T(10) value) were examined. Capsaicin (0.01-0.32 mg) injected into the tail of monkeys dose dependently decreased the T(10), indicating that capsaicin increased sensitivity to thermal stimuli. A dose of 0.1 mg of capsaicin decreased the T(10) from 48.0 to 42.1 degrees C (a -5.9 degrees C change) 15 min after injection. SNC80 (1.0-10.0 mg/kg s.c.) dose dependently blocked the capsaicin-induced decrease in the T(10), and 10.0 mg/kg SNC80 fully blocked the effects of capsaicin. The delta-selective antagonist naltrindole (0.1-1.0 mg/kg) dose dependently antagonized the effects of SNC80, whereas a mu-selective dose of the opioid antagonist quadazocine (0.1 mg/kg) did not. Two other delta-selective agonists, SNC162 (1.0-10.0 mg/kg) and SNC243A (1.0-10.0 mg/kg), also dose dependently blocked capsaicin-induced thermal hypersensitivity. In contrast, neither SNC67 (10.0 mg/kg), which is the (-)-enantiomer of SNC80, nor the nonsteroidal anti-inflammatory drug (NSAID) ketorolac (1.0-10.0 mg/kg) modified the effects of capsaicin. SNC80 was also effective in reversing thermal hypersensitivity induced by prostaglandin E(2) (0.0158 mg) and Freund's complete adjuvant (10% concentration). These findings suggest that delta-agonists have antinociceptive effects in primates under conditions of chemically induced thermal hypersensitivity and might be effective under a broader range of conditions than clinically available NSAIDs.

The present study investigated 1) antihyperalgesic actions of a novel and selective adenosine kinase (AK) inhibitor, A-134974 (IC(50) = 60 pM), in the carrageenan model of thermal hyperalgesia; 2) effects of A-134974 on locomotor activity; and 3) relative contributions of supraspinal, spinal, and peripheral sites to the actions of A-134974. Systemic A-134974 (i.p.) dose dependently reduced hyperalgesia (ED(50) = 1 micromol/kg) and at higher doses, reduced locomotor activity (ED(50) = 16 micromol/kg). Administration of A-134974 intrathecally (i.t.) was more potent (ED(50) = 6 nmol) at producing antihyperalgesia than delivering the compound by intracerebralventricular (ED(50) = 100 nmol, i.c.v.) or intraplantar (ED(50) >300 nmol) routes. In contrast, i.c.v. administration of A-134974 was more effective in reducing locomotor activity than i.t. administration (ED(50) values were 1 and >100 nmol, respectively). Increasing the pretreatment time for i.t.-delivered A-134974 caused a greater reduction in locomotor activity (ED(50) = 10 nmol). This was due to diffusion of A-134974 (i.t.) to supraspinal sites. The antihyperalgesic effects of systemic A-134974 were antagonized by the adenosine receptor antagonist theophylline (THEO, 30-500 nmol) administered i.t., but not i.c.v. In the locomotor assay, i.t.-injected THEO did not antagonize hypomobility caused by systemic or i.t. administration of A-134974. However, i.c.v. infusion of THEO did block the hypomotive actions of i.c.v.-, i.t.-, and i.p.-administered A-134974. These data demonstrate that the novel AK inhibitor A-134974 potently reduces thermal hyperalgesia primarily through interactions with spinal sites, whereas its ability to depress locomotor activity is predominantly mediated by supraspinal sites.

The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50) = 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50) = 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50) > 50 microM in U937 cells and IC(50) > 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50) = 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50) = 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50) = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50) = 0.24 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.

Previous in vitro and in vivo studies have determined that the d isomer of methadone has N-methyl-D-aspartate (NMDA) receptor antagonist activity. The present studies examined the ability of d-methadone to attenuate the development of morphine tolerance in mice and rats and to modify NMDA-induced hyperalgesia in rats. A decrease in the percentage of mice analgesic (tail-flick response) after 5 days of once-daily morphine (7 mg/kg s.c.) was completely blocked by coadministration of d-methadone given s.c. at 10 mg/kg. Morphine given s.c. to mice on an escalating three times per day dosing schedule resulted in a nearly 3-fold increase in the tail-flick ED50 dose of morphine which was prevented by s.c. coadministered d-methadone at 15 mg/kg. In rats, intrathecal (i.t.) morphine produced a 38-fold increase in the ED50, which was completely prevented by the coadministration of i.t. d-methadone at 160 micrograms/rat. A decrease in thermal paw withdrawal latency induced by the i.t. administration of 1.64 micrograms/rat NMDA was completely blocked by pretreatment with 160 micrograms/rat d-methadone. Thus, systemically coadministered d-methadone prevents systemically induced morphine tolerance in mice, i.t. d-methadone attenuates tolerance produced by i.t. morphine in rats, and i.t. d-methadone, at the same dose which modulates morphine tolerance, blocks NMDA-induced hyperalgesia. These results support the conclusion that d-methadone affects the development of morphine tolerance and NMDA-induced hyperalgesia by virtue of its NMDA receptor antagonist activity.

Among nonsteroidal anti-inflammatory drugs (NSAIDs), 2-arylpropionic acids exist as a racemic mixture of its enantiomeric forms, with S-enantiomers primarily responsible for inhibition of prostaglandin synthesis and of inflammatory events. The aim of this study was to compare the anti-inflammatory effects of R- and S-ketoprofen in vitro and in vivo. S-Ketoprofen efficiently inhibited carrageenan-induced edema formation, but it could also amplify the LPS-induced production of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), in close correlation with its ability to inhibit prostaglandin synthesis. Because these inflammatory cytokines are among the factors involved in carrageenan-induced inflammation and also are possibly involved in gastric damage, enhanced cytokine production could partially mask the analgesic effect of S-ketoprofen, and it can be associated with the clinical evidence of its gastric toxicity. On the other hand, R-ketoprofen contributes to the overall activity of the racemate, by playing the main role in ketoprofen-induced analgesia. Unlike the S-isomer, R-ketoprofen did not induce a significant increase of cytokine production even at cyclooxygenase-blocking concentrations. It is concluded that the R-isomer directly contributes to the anti-inflammatory effects of ketoprofen, being more analgesic, and because it does not amplify inflammatory cytokine production.

High voltage calcium channels are implicated in nociceptive transmission after nerve injury, capsaicin or formalin injection. The purpose of this study was to investigate the role of calcium channels in secondary heat hyperalgesia associated with acute joint inflammation. After induction of acute inflammation (knee joint injection of kaolin and carrageenan), decreased paw withdrawal latency (PWL) to radiant heat (i.e., secondary heat hyperalgesia), increased guarding of the limb and increased joint circumference occurs. Spinal administration (through a microdialysis fiber placed in dorsal horn) of an N-type calcium channel blocker (MVIIA, SNX 111, ziconotide, 0.001-0.1 mM), before induction of inflammation, prevents the decrease in PWL. Treatment with SNX 111 4 hr after inflammation reverses heat hyperalgesia. A small reduction in spontaneous pain-related behaviors (guarding of the limb) occurs after pre- or post-treatment with SNX 111. Spinal blockade of P/Q-type calcium channels (with omega-agatoxin IVA) had no effect on the decrease in PWL to radiant heat when administered after induction of inflammation. However, pre-treatment with omega-agatoxin IVA prevents secondary heat hyperalgesia. omega-Agatoxin IVA has no effect on spontaneous pain-related behaviors whether administered before or after induction of inflammation. In contrast, pre or post-treatment with nifedipine (L-type calcium channel blocker, 0.01-1.0 mM), had no effect on heat hyperalgesia or spontaneous pain-related behaviors induced by acute inflammation. There were no differences in joint circumference between groups with any treatment. Thus, N-type calcium channels contribute to both the development and maintenance of secondary heat hyperalgesia while P-type calcium channels are only involved during development of hyperalgesia.

Intrathecal administration of nonsteroidal anti-inflammatory drugs in the rat blocks the thermal hyperalgesia induced by tissue injury, which suggests a role for spinal cyclooxygenase (COX) products in this facilitated state. Two isozymes of the COX enzyme have been reported, COX-1 and COX-2, but the agents thus far examined are not isozyme selective. We examined the effects of intrathecally (i.t.) or systemically (i.p.) administered S(+)-ibuprofen (a nonselective COX inhibitor) or 1-[(4-methysulfonyl)phenyl]-3-tri-fluoromethyl-5-(4-fluorophenyl) pyrazole (SC58125; a COX-2 selective inhibitor) on carrageenan-induced thermal hyperalgesia (reduced hindpaw-withdrawal latency). The following observations were made: 1) Thermal hyperalgesia otherwise observed during the first 170 min was blocked in a dose-dependent manner by S(+)-ibuprofen or SC58125 administered i.t. or i.p. before carrageenan treatment. 2) Intraperitoneal, but not i.t., administration of either inhibitor after the establishment of hyperalgesia (170 min after carrageenan injection) reversed thermal hyperalgesia in a dose-dependent manner. Thus, the initial component of thermal hyperalgesia after tissue injury was blocked by systemic or spinal administration of both COX inhibitors, whereas established hyperalgesia was reversed only by systemic inhibitors. This study demonstrates that at least spinal COX-2, if not both COX-1 and COX-2, are necessary for the initiation of thermal hyperalgesia, whereas nonspinal sources of prostanoids (synthesized by COX-2 and perhaps also COX-1) are important for the maintenance of thermal hyperalgesia associated with tissue injury and inflammation.

PGs derived from cyclooxygenase-2 (COX-2), in particular PGE2, play important roles in the initiation of inflammation and pain. In the present study, we evaluated the role of COX-2-derived PGE2 in an animal model of established hyperalgesia. Inflammation and hyperalgesia were first induced by injection of carrageenan into rat footpads. Then we investigated the effects of subsequent therapeutic treatment with a selective COX inhibitor, with a nonsteroidal anti-inflammatory drug and with anti-PGE2 antibody. Test compounds were administered 1 to 3 hr after carrageenan challenge, and inhibition of pain (hyperalgesia, measured by withdrawal from a thermal stimulus), and changes in paw edema and PG levels were evaluated. The i.v. administration of a nonselective COX inhibitor, ketorolac, caused a rapid reduction in hyperalgesia in the inflamed footpad, returning it to near-normal values within 1 hr. Normal (control) paw response times were not affected. Therapeutic administration of ketorolac prevented most further swelling caused by carrageenan but did not reverse edema already present at the time of dosing. Administered p.o., a selective COX-2 inhibitor (SC-58635) was as efficacious as ketorolac in reducing inflammatory hyperalgesia. Footpad PG levels returned to base line or below within 5 min of dosing with ketorolac, which suggests rapid turnover of PG in the inflamed tissue. Therapeutic treatment with a monoclonal anti-PGE2 antibody also fully reversed the hyperalgesia response. These studies suggest that continuous production of PGE2 by the COX-2 enzyme is a critical element in sustaining the hyperalgesic response at sites of tissue inflammation.

It has been demonstrated that kappa-2 opioid receptor agonists can inhibit the current that flows through the N-methyl-o-aspartate (NMDA) subclass of excitatory amino acid receptor. NMDA receptor antagonists have been shown to be effective antihyperalgesic agents when administered intrathecally into rats. Antihyperalgesia is defined as the ability to block enhanced sensitivity, usually produced by nerve injury or inflammation, to nociceptive stimuli. Thus, the hypothesis was proposed that kappa-2 opioid receptor agonists would be antihyperalgesic when injected intrathecally into rats with an inflamed hind paw. The kappa agonists bremazocine and GR89,696 were effective at reversing the hyperalgesia associated with the inflamed hind paw but did not influence the sensitivity of the noninflamed hind paw to noxious heat. The kappa-1-selective agonist U69,593 had no effect on the heat sensitivity of either the inflamed paw or the noninflamed paw. Intrathecal injection of the mu-selective agonist [D-Ala2,N-MePhe4,Gly5-ol]enkephalin or the delta-selective agonist [D-Pen(2,5)]enkephalin elevated paw withdrawal latencies to heat in both hind paws. These findings indicate that activation of presumed kappa-2 receptors in the rat spinal cord results in suppression of the hyperalgesic state without influencing normal sensitivity to noxious stimuli. It is proposed that the antihyperalgesic effect of kappa-2 receptor activation is mediated by the ability of the opioid receptor to reduce the flow of current through the NMDA receptor ionophore.

The role of spinal delta opioid receptors in mediating antinociception was studied by using the carrageenan-induced model of thermal hyperalgesia. Intrathecal administration of [D-Ala2, Glu4]-deltorphin (DELT), a delta-2 receptor agonist, or DPDPE, a delta-1 receptor agonist, produced a dose-dependent increase in paw-flick latency (PFL) with an ED50 of 14.0 micrograms for DELT and 30.4 micrograms for DPDPE. DAMGO, a mu receptor agonist, also increased the PFL in a dose-dependent manner when administered intrathecally with an ED50 of 0.02 microgram. Each opioid agonist increased the PFL to values that exceeded base-line latencies determined before the injection of carrageenan. However, DELT and DPDPE increased the PFL to a greater extent than did DAMGO. Coadministration of 30 micrograms of naltrindole shifted the dose-effect line of DELT to the right by 3.5-fold and that of DPDPE to the right by 2.5-fold, consistent with its characterization as a mixed delta-1/delta-2 receptor antagonist. Coadministration of 3 micrograms of naltriben (NTB) shifted the dose-effect line of DELT to the right by 3.2-fold, whereas 10 micrograms of NTB shifted the dose-effect line of DELT at least 15-fold to the right. Neither dose of NTB antagonized the effects of DPDPE. These data are consistent with characterization of NTB as a selective delta-2 receptor antagonist. The antinociception produced by DAMGO was noncompetitively antagonized by 30 micrograms of naltrindole and it was competitively antagonized by 10 micrograms of NTB. Thus, although NTB distinguishes between delta-1 and delta-2 opioid receptors, high doses may not effectively distinguish between delta and mu receptors.(ABSTRACT TRUNCATED AT 250 WORDS)