J.K. Webb

Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn

The present study investigated the regional distribution of the N-methyl-D-aspartate (NMDA) receptor containing the NR2B subunit protein in rat lumbar spinal cord and examined whether selective NR2B antagonists would exhibit antinociception with reduced side-effect liability than subtype non-selective NMDA antagonists and anticonvulsants. Immunocytochemical studies showed the NR2B subunit had a restricted distribution, with moderate labelling of fibres in laminas I and II of the dorsal horn suggesting a presynaptic location on primary afferent fibers and possible involvement in pain transmission. In the in vivo studies, the NMDA/glycine antagonists (MK-801, 0.02-1 mg/kg i.p., L-687,414 10-300 mg/kg i.p., and L-701,324 1-10 mg/kg i.p.) and the anticonvulsant, gabapentin (10-500 mg/kg p.o.), induced rotarod deficits at antinociceptive doses. In contrast, the selective NR2B antagonists, (+/-)-CP-101,606 (1-100 mg/kg p.o.) and (+/-)-Ro 25-6981 (3-100 mg/kg i.p.) showed a significant dose window. (+/-)-CP-101,606 caused no motor impairment or stimulation in rats at doses up to 100 mg/kg p.o., which is far in excess of those inhibiting allodynia in neuropathic rats (ID50 4.1 mg/kg, p.o.). (+/-)-Ro 25-6981 also showed a significant separation (ID50 allodynia 3.8 mg/kg, i.p.), however, some disruption of rotarod performance was observed at 100 mg/kg. The anticonvulsant lamotrigine (3-500 mg/kg p.o.) also showed a good dose window. These findings demonstrate that NR2B antagonists may have clinical utility for the treatment of neuropathic and other pain conditions in man with a reduced side-effect profile than existing NMDA antagonists.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

DFU (5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl)phenyl‐2(5H)‐furanone) was identified as a novel orally active and highly selective cyclo‐oxygenase‐2 (COX‐2) inhibitor. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid‐dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX‐2 (IC50=41±14 nM) over COX‐1 (IC50>50 μM). Indomethacin was a potent inhibitor of both COX‐1 (IC50=18±3 nM) and COX‐2 (IC50=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX‐1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore‐challenged human platelets (IC50>50 μM and 4.1±1.7 nM, respectively). DFU caused a time‐dependent inhibition of purified recombinant human COX‐2 with a Ki value of 140±68 μM for the initial reversible binding to enzyme and a k2 value of 0.11±0.06 s−1 for the first order rate constant for formation of a tightly bound enzyme‐inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s−1, respectively, were obtained for indomethacin. The enzyme‐inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t1/2=1–3 h) with recovery of intact inhibitor and active enzyme. The time‐dependent inhibition by DFU was decreased by co‐incubation with arachidonic acid under non‐turnover conditions, consistent with reversible competitive inhibition at the COX active site. Inhibition of purified recombinant human COX‐1 by DFU was very weak and observed only at low concentrations of substrate (IC50=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX‐2, inhibition was time‐independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX‐1. DFU inhibited lipopolysaccharide (LPS)‐induced PGE2 production (COX‐2) in a human whole blood assay with a potency (IC50=0.28±0.04 μM) similar to indomethacin (IC50=0.68±0.17 μM). In contrast, DFU was at least 500 times less potent (IC50>97 μM) than indomethacin at inhibiting coagulation‐induced TXB2 production (COX‐1) (IC50=0.19±0.02 μM). In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 μM), DFU inhibited COX‐1 with an IC50 value of 13±2 μM as compared to 20±1 nM for indomethacin. CGP 28238, etodolac and SC‐58125 were about 10 times more potent inhibitors of COX‐1 than DFU. The order of potency of various inhibitors was diclofenac>indomethacin∼naproxen>nimesulide∼ meloxicam∼piroxicam>NS‐398∼SC‐57666>SC‐58125>CGP 28238∼etodolac>L‐745,337>DFU. DFU inhibited dose‐dependently both the carrageenan‐induced rat paw oedema (ED50 of 1.1 mg kg−1 vs 2.0 mg kg−1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg−1 vs 1.5 mg kg−1 for indomethacin). The compound was also effective at reversing LPS‐induced pyrexia in rats (ED50=0.76 mg kg−1 vs 1.1 mg kg−1 for indomethacin). In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg−1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg−1), meloxicam (3 mg kg−1) or etodolac (10–30 mg kg−1). A 5 day administration of DFU in squirrel monkeys (100 mg kg−1) did not affect chromium leakage in contrast to diclofenac (1 mg kg−1) or naproxen (5 mg kg−1). The results indicate that COX‐1 inhibitory effects can be detected for all selective COX‐2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX‐1, a consistent high selectivity of inhibition of COX‐2 over COX‐1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX‐1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti‐inflammatory effect to gastropathy can be achieved with a selective COX‐2 inhibitor.

Effects of the bradykinin B1 receptor antagonist des-Arg9[Leu8]bradykinin and genetic disruption of the B2 receptor on nociception in rats and mice

Abstract The contributions of B1 and B2 bradykinin receptors to acute and chronic inflammatory hyperalgesia were examined using the peptide B1 receptor antagonist des‐Arg9[Leu8]bradykinin and transgenic Bk2r‐/‐ mice. In normal rats and mice, des‐Arg9[Leu8]bradykinin (30 nmol/kg i.v. or s.c.) inhibited carrageenan‐induced hyperalgesia and the late phase nociceptive response to formalin. The active dose range was narrow, suggesting partial agonist activity of this peptide. In rats with monoarthritis, des‐Arg9[Leu8]bradykinin (up to 30 nmol/kg i.v.) failed to reduce the number of vocalisations elicited by gentle flexion and extension of the inflamed limb; however, hyperalgesia was exacerbated by administration of the B1 receptor agonist des‐[Arg9]bradykinin (100 nmol/kg i.v.), consistent with other evidence for local induction of B1 receptors during adjuvant‐induced arthritis. The nociceptive response to intraplantar injection of bradykinin (10 nmol) and hyperalgesia induced by carrageenan (0.6 mg) were absent in Bk2r‐/‐ mice, indicating that stimulation of B2 receptors is an essential step in the initiation of some nociceptive and inflammatory reactions. However, the nociceptive response to formalin (2.5% intraplantar), including inhibition of the late phase by des‐Arg9[Leu8]bradykinin (0.3 nmol), and induction of thermal hyperalgesia by Freunds adjuvant (0.1%) appeared intact in Bk2r‐/‐ mice. These findings support other evidence for an involvement of B1 receptors in inflammatory hyperalgesia and suggest that B1 receptor antagonists may be clinically useful as anti‐inflammatory and analgesic drugs.

Functional Properties of the High-Affinity TRPV1 (VR1) Vanilloid Receptor Antagonist (4-Hydroxy-5-iodo-3-methoxyphenylacetate ester) Iodo-Resiniferatoxin

We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC50 = 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC50 = 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [3H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.

Enantioselective inhibition of the formalin paw late phase by the NK1 receptor antagonist L-733,060 in gerbils

&NA; Intravenous administration of the NK1 receptor antagonist l‐733,060 to gerbils 3 h before intraplantar injection of formalin caused a dose‐dependent and complete inhibition of the late, but not the early, phase nociceptive response (paw licking). The ID50 for l‐733,060 (0.17 mg/kg) revealed a greater than 50‐fold separation in potency over its less active enantiomer l‐733,061 (ID50 ≥ 10 mg/kg). In contrast, the non‐brain penetrant quaternary ketone NK1 receptor antagonist, l‐743,310 (3 mg/kg), did not attenuate the response to formalin, indicating that the antinociceptive effect of blockade of NK1 receptors by l‐733,060 in this assay is centrally‐mediated. These findings add to the preclinical evidence that NK1 receptor antagonists may be of therapeutic use as centrally‐acting analgesics.

L-745,337: A selective inhibitor of cyclooxygenase-2 elicits antinociception but not gastric ulceration in rats

L-745,337 [5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indan one] a selective cyclooxygenase-2 inhibitor reversed hyperalgesia induced by carrageenan in rats without causing gastric ulceration at doses 100 times those causing antinociception. In contrast, piroxicam and indomethacin produced ulcerations at antinociceptive doses. These findings demonstrate that L-745,337 possesses antinociceptive activity but has a reduced liability for gastric ulceration.

Analgesic and toxic effects of ABT-594 resemble epibatidine and nicotine in rats

&NA; The present study directly compared the antinociceptive and toxic effects of the neuronal nicotinic receptor agonist ABT‐594 ((R)‐5‐(2‐azetidinylmethoxy)‐2‐chloropyridine) with (−)‐nicotine and (+)‐epibatidine. Like (−)‐nicotine (0.8 and 1.6 mg/kg s.c.) and (+)‐epibatidine (0.005 and 0.01 mg/kg s.c.), ABT‐594 (0.05 and 0.1 mg/kg s.c.) increased response latencies in the hot‐plate test in rats, indicating that it has antinociceptive activity. In contrast to (−)‐nicotine and (+)‐epibatidine, ABT‐594 did not cause rotarod impairment at antinociceptive doses but did cause hypothermia and life‐threatening adverse effects including seizures. ABT‐594 (0.01 and 0.1 mg/kg i.v.) also produced a dose‐dependent increase in blood pressure resembling that observed with (−)‐nicotine (0.03, 0.1 and 0.03 mg/kg i.v.) and (+)‐epibatidine (0.001 and 0.003 mg/kg i.v.). Both the antinociceptive and toxic effects (convulsions and hypertension) were abolished by pretreatment with the brain penetrant neuronal nAChR antagonist mecamylamine (1 mg/kg s.c.; i.v. for cardiovascular studies), demonstrating that these actions of ABT‐594 were mediated via activation of neuronal nicotinic receptors. Continuous infusion of ABT‐594 (0.2 mg/kg per day s.c.) to rats for 7 days followed by challenge with mecamylamine (1 mg/kg i.p.) induced a nicotine‐like abstinence syndrome suggesting that ABT‐594 has nicotine‐like dependence liability. These findings indicate that the acute safety profile of ABT‐594 is not significantly improved over other nicotinic analgesics.

SAR in the alkoxy lactone series: the discovery of DFP, a potent and orally active COX-2 inhibitor.

Extensive SAR has been established in the alkoxy lactone series and this has lead to the discovery of DFP (5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanon e), a potent COX-2 inhibitor exhibiting in vivo efficacy in all models studied.

Effects of a partial agonist and a full antagonist acting at the glycine site of the NMDA receptor on inflammation-induced mechanical hyperalgesia in rats.

1 NMDA receptor antagonists have previously been shown to have antinociceptive effects in behavioural experiments, but controversy remains as to the role of NMDA receptors in mechanical hyperalgesia. We have studied the effects on mechanical nociceptive thresholds in rats with carrageenin‐induced paw inflammation of L‐687,414, a low efficacy partial agonist which acts as a functional antagonist at the glycine modulatory site of the NMDA receptor and of L‐701,324, a structurally novel, highly selective, full antagonist at this site. 2 Mechanical thresholds were measured for both hind paws 1 h before and 3 h after carrageenin or saline was injected into 1 hind paw. Dose‐response curves were constructed for each test compound in separate experiments, with test compound or vehicle being given i.p. 1 h before the final test. 3 Both compounds produced selective dose‐dependent and statistically significant reversal of mechanical hyperalgesia, with minimum effective doses of 100 mg kg−1 L‐687,414 and 3 mg kg−1 L‐ 701,324. Neither L‐687,414 nor L‐701,324 affected the response threshold of the contralateral non‐inflamed paw over the dose‐range producing reversal of carrageenin‐induced hyperalgesia. Neither compound had any effect on the paw oedema produced by carrageenin injection. 4 These results show that both a full antagonist and a low efficacy partial agonist at the glycine modulatory site of the NMDA receptor complex reverse inflammation‐induced mechanical hyperalgesia, thus supporting the argument that maximal activation of the glycine site is required for transmission via NMDA receptors, and showing that NMDA receptor‐mediated actions are important in mechanical hyperalgesia induced by inflammation

The substance P (NK1) receptor antagonist L-760735 inhibits fear conditioning in gerbils.

The ability of the substance P (NK(1) receptor) antagonist (SPA) L-760735 to inhibit conditioned fear was assessed in gerbils using a four plate apparatus. Animals that had been treated with diazepam (3 mg/kg) or L-760735 (3 mg/kg) 30 min before a 3 min conditioning session in the apparatus exhibited a release of plate crossings during the retest session approximately 3 h later. Plate crossings were also increased when animals received diazepam or L-760735 30 min before the retest session. In contrast, fluoxetine and venlafaxine (30 mg/kg) did not exhibit anxiolytic-like effects. During the retest session, gerbils drummed their hind feet on the floor; this behaviour was not observed spontaneously in gerbils that were naïve to the apparatus. Foot drumming was abolished by pretreatment with L-760735 or diazepam (3 mg/kg) but was markedly increased following administration of fluoxetine or venlafaxine (30 mg/kg). Foot drumming elicited by aversive conditioning alone or in combination with fluoxetine was abolished by administration of L-760735 and by amygdala lesions involving the basolateral and lateral nuclei, indicating that this behaviour is an alarm signal or fear response mediated via release of substance P in brain circuits involving the amygdala. The observations provide further evidence for an anxiolytic-like profile of SPAs in preclinical assays and demonstrate a clear difference between the actions of SPAs and established antidepressant drugs.