Iain F. James

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

1 Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2 Capsazepine (10 μm) reversibly reduced or abolished the current response to capsaicin (500 nm) of voltage‐clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 μm γ‐aminobutyric acid (GABA) and 5 μm adenosine 5′‐triphosphate (ATP) were unaffected. 3 The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nm)‐induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 ± 46 nm (mean ± s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin‐like agonist was also inhibited. (Log concentration)‐effect curves for RTX (0.3 nm‐1 μm) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 ± 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nm (95% confidence limits, 57–400 nm). 4 Capsazepine also inhibited the capsaicin‐ and RTX‐evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nm (95% confidence limits, 30–332 nm) with capsaicin as the agonist and 107 nm (95% confidence limits, 49–162 nm) with RTX as agonist. 5 A similar competitive inhibition by capsazepine was seen for capsaicin‐induced [14C]‐guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nm; 95% confidence limits, 63 nm‐1.45 μm). No significant difference was noted in the apparent Kd estimates for capsazepine in assays on cultured DRG neurones and vagus nerve as shown by the overlap in the 95% confidence limits. 6 Capsazepine, at concentrations up to 10 μm, had no significant effects on the efflux of 86Rb+ from cultured DRG neurones evoked either by depolarization with high (50 mm) K+ solutions or by acidification of the external medium to pH 5.0–5.6. Similarly capsazepine had no significant effect on the depolarization (50 mm KCl)‐induced efflux of [14C]‐guanidinium from vagus nerve preparations. 7 Ruthenium Red was also tested for antagonism against capsaicin evoked [14C]‐guanidinium release from vague nerves and capsaicin induced 45Ca2+ uptake in cultures of DRG neurones. In contrast to capsazepine the inhibition by Ruthenium Red (10–500 nm in DRG and 0.5–10 μm in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex, non‐competitive inhibition.

The role of central and peripheral Cannabinoid1 receptors in the antihyperalgesic activity of cannabinoids in a model of neuropathic pain.

&NA; We have examined the effects of cannabinoid agonists on hyperalgesia in a model of neuropathic pain in the rat and investigated the possible sites of action. The antihyperalgesic activity of the cannabinoids was compared with their ability to elicit behavioural effects characteristic of central cannabinoid activity. WIN55,212‐2 (0.3–10 mg kg−1), CP‐55,940 (0.03–1 mg kg−1) and HU‐210 (0.001–0.03 mg kg−1) were all active in a ‘tetrad’ of tests consisting of tail‐flick, catalepsy, rotarod and hypothermia following subcutaneous administration, with a rank order of potency in each of HU‐210>CP‐55,940>WIN55,212‐2. The effects of WIN55,212‐2 in each assay were blocked by the Cannabinoid1 (CB1) antagonist SR141716A. In the partial sciatic ligation model of neuropathic pain WIN55,212‐2, CP‐55,940 and HU‐210 produced complete reversal of mechanical hyperalgesia within 3 h of subcutaneous administration with D50 values of 0.52, 0.08 and 0.005 mg kg−1, respectively. In this model WIN55,212‐2 was also effective against thermal hyperalgesia and mechanical allodynia. WIN55,212‐2 produced pronounced reversal of mechanical hyperalgesia following intrathecal administration that was blocked by the CB1 antagonist SR141716A. Following intraplantar administration into the ipsilateral hindpaw, WIN55,212‐2 produced up to 70% reversal of mechanical hyperalgesia, although activity was also observed at high doses following injection into the contralateral paw. The antihyperalgesic effect of WIN55,212‐2 injected into the ipsilateral paw was blocked by subcutaneously administered SR141716A, but was not affected by intrathecally administered SR141716A. These data show that cannabinoids are highly potent and efficacious antihyperalgesic agents in a model of neuropathic pain. This activity is likely to be mediated via an action in both the CNS and in the periphery.

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1).

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC50 values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90±0.20 μM; human=1.90±0.30 μM: CHO cells: rat=0.20±0.06 μM; human=0.19±0.08 μM). In CHO cell lines co‐expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC50 values for capsaicin‐induced responses were similar in both cell lines (rat=0.35±0.06 μM, human=0.53±0.03 μM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC50 pH 5.49±0.04 and pH 5.78±0.09, respectively). The threshold for heat activation was identical (42°C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC50 between 3 and 10 μM) but was virtually inactive at the human VR1 (EC50>10 μM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.

In vivo pharmacology of SDZ 249-665, a novel, non-pungent capsaicin analogue

&NA; Capsaicin and analogues are valuable analgesic agents when administered to mammals, including humans. However, their pungency and the effects on the cardiovascular and respiratory systems through their general activation of small calibre (nociceptive) primary afferents severely limit their use. Recently, structure activity analysis revealed that the initial pungent and general excitatory effects can be prevented by structural modifications in such a way that the analgesic activity is retained. In this paper we present SDZ 249‐665, a capsaicin analogue which produced analgesia in the mouse and anti‐hyperalgesic effects in the rat and guinea pig. SDZ 249‐665 was administered p.o., s.c. and i.v. in models of nociceptive pain, such as tail flick latency in response to a noxious thermal stimulus and acetic acid‐induced writhing in mice, and in models of inflammatory mechanical hyperalgesia induced by turpentine or carrageenan in the rat and guinea pig, respectively. SDZ 249‐665 was effective in the tail flick and the writhing assays and produced significant anti‐hyperalgesic effects in the inflammatory models. The efficacy of SDZ 245‐665 was similar to that of capsaicin, however, it was significantly more potent. SDZ 249‐665 did not produce any irritancy in a nose wipe assay in guinea pigs or an eye irritancy assay in rats, while capsaicin was clearly irritant in both cases. Furthermore, unlike capsaicin, SDZ 249‐665 did not produce unwanted side effects such as bronchoconstriction and blood pressure changes in the analgesic/anti‐hyperalgesic dose range. Thus, a clear analgesic therapeutic window exists for SDZ 249‐665. In summary, SDZ 249‐665 is a potent orally active, analgesic/anti‐hyperalgesic agent in mouse, rat and guinea pig. It lacks the excitatory effects associated with capsaicin and other close analogues, and therefore provides a clear therapeutic window for use in painful conditions. In addition to this favourable profile, no sign of tolerance was detected after a 5 day repeated dose treatment.

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells.

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cγ1 (PLCγ1). We used recombinant glutathione‐S‐transferase (GST)–PLCγ1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCγ1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology‐2 (SH2) domain of PLCγ1, but there was enhanced binding with tandemly expressed amino‐ and carboxy‐terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen‐activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.

The capsaicin analogue SDZ249-665 attenuates the hyper-reflexia and referred hyperalgesia associated with inflammation of the rat urinary bladder

&NA; This study assessed the effects of the systemically administered capsaicin analogue SDZ249‐665 in an animal model of visceral pain and hyper‐reflexia. The effects of prophylactic administration of SDZ249‐665 (in the dose range 0.05–1 mg/kg) on the viscero‐visceral hyper‐reflexia (VVH) and the referred viscero‐somatic hyperalgesia to mechanical stimuli (VSH) associated with turpentine inflammation of the rat urinary bladder were evaluated. SDZ249‐665 attenuated both the VVH and the VSH in a dose related fashion. In the VVH model, following solvent control administration, intra‐vesical turpentine administration was associated with a significant reduction in micturition threshold to 43.7% (SEM 6.3) of baseline, indicating the presence of a VVH. This effect was not observed when animals were prophylactically treated with SDZ249‐665 alone. At a dose of 0.1 mg/kg the micturition threshold was 90.7% (SEM 10.2) of baseline at 1 h after intra‐vesical instillation of turpentine. In the VSH model, curves were plotted of the difference in fore and hind limb withdrawal latencies from a mechanical stimulus and the area under these curves (AUCs) were compared between different treatment protocols. Intra‐vesical turpentine was associated with a negative deflection of the curve (AUC −5.2×103 SEM 1.7) in comparison with naïve animals (AUC −0.02×103 SEM 0.6), indicative of a referred hyperalgesia. This was prevented, in a dose‐related manner, by prophylactic administration of SDZ249‐665. For example, at a dose of 0.5 mg/kg the AUC was +0.4×103 (SEM 0.8). These findings support previous work indicating that capsaicin sensitive neurones participate in patho‐physiological events occurring following inflammation of the bladder, and provides evidence that systemically active capsaicin based compounds may be developed for use in the clinical setting.

Comparative, general pharmacology of SDZ NKT 343, a novel, selective NK1 receptor antagonist

The in vitro and in vivo pharmacology of SDZ NKT 343 (2‐nitrophenyl‐carbamoyl‐(S)‐prolyl‐(S)‐3‐(2‐naphthyl)alanyl‐N‐benzyl‐N‐methylamide), a novel tachykinin NK1 receptor antagonist was investigated. SDZ NKT 343 inhibited [3H]‐substance P binding to the human NK1 receptor in transfected Cos‐7 cell membranes (IC50=0.62±0.11 nM). In comparison, in the same assay Ki values for FK888, CP 99,994, SR 140,333 and RPR 100,893 were 2.13±0.04 nM, 0.96±0.20 nM, 0.15±0.06 nM and 1.77±0.41 nM, respectively. SDZ NKT 343 showed a markedly lower affinity at rat NK1 receptors in whole forebrain membranes (IC50=451±139 nM). SDZ NKT 343 caused an increase in EC50 as well as reduction in the number of binding sites (Bmax) determined for [3H]‐substance P, suggesting a non‐competitive interaction at the human NK1 receptor. SDZ NKT 343 also caused a reduction in the maximum elevation of [Ca2+]i evoked by substance P (SP) in human U373MG cells and depressed the maximum [Sar9]SP sulphone‐induced contraction of the guinea‐pig isolated ileum. The antagonism of SP effects on U373MG cells by SDZ NKT 343 was reversible. SDZ NKT 343 showed weak affinity to human NK2 and NK3 receptors in transfected Cos‐7 cells (Ki of 0.52±0.04 μM and 3.4±1.2 μM, respectively). SDZ NKT 343 was inactive in a broad array of binding assays including the bradykinin B2 receptor the histamine H1 receptor, opiate receptors and adrenoceptors. SDZ NKT 343 only weakly inhibited the voltage‐activated Ca2+ and Na+currents in guinea‐pig dorsal root ganglion neurones. The enantiomer of SDZ NKT 343, (R,R)‐SDZ NKT 343 was about 1000 times less active at human NK1 receptors expressed in Cos‐7 cell membranes. Contractions of the guinea‐pig ileum by [Sar9]SP sulphone were inhibited by SDZ NKT 343 in a concentration‐dependent manner, with an IC50=1.60±0.94 nM, while the enantiomer (R,R)‐SDZ NKT 343 was 100 times less active (IC50=162±26 nM). In comparison, in the same assay IC50 values for other NK1 receptor antagonists CP 99,994, SR 140,333, RPR 100,893 and FK 888 were 2.90±07 nM, 0.14±0.02 nM, 11.4±2.9 nM and 2.4±0.83 nM, respectively. In anaesthetized guinea‐pigs i.v. administered SDZ NKT 343 antagonized [Sar9]SP sulphone‐evoked bronchoconstriction (70% reduction at 0.4 mg kg−1, i.v.). Basal airway resistance, mean arterial blood pressure and heart rate were not affected. In conclusion, SDZ NKT 343 is a highly selective NK1 receptor antagonist with high potency at the human and guinea‐pig receptors. SDZ NKT 343 may be used as a potential novel therapeutic agent in human diseases where NK1 receptor hyperfunction is involved.

Opioid receptor ligands in the neonatal rat spinal cord: binding and in vitro depression of the nociceptive responses

1 Opioid receptors in the neonatal rat spinal cord have been characterized by measurements of ligand binding to crude membrane fractions and by functional tests on the nociceptive spinal response in a spinal cord‐tail preparation in vitro. 2 There were high affinity binding sites for [3H]‐[d‐Ala2, MePhe4, Gly(ol)5]enkephalin (DAGOL), [3H]‐U69593, and [3H]‐ethylketocyclazocine (EKC) on spinal cord membranes from neonatal rats. Hill slopes for binding of [3H]‐DAGOL and [3H]‐U69593 were close to unity. The Hill slope for binding of [3H]‐EKC was less than unity, even after its interactions at μ‐receptors had been blocked with 100 nm unlabelled DAGOL. Binding sites for [3H]‐[d‐Pen2, d‐Pen5]enkephalin (DPDPE) could not be detected. 3 In competition assays U50488 was as potent as PD117302 and U69593 in competition for either [3H]‐U69593 or [3H]‐EKC binding sites. Hill slopes for a range of competing ligands at [3H]‐DAGOL or [3H]‐U69593 sites were close to unity. Hill slopes for competition at [3H]‐EKC sites were less than one. 4 In the spinal cord‐tail preparation from neonatal rats, opioid receptor agonists depressed spinal nociceptive responses evoked by application of capsaicin or heat to the tail. The order of potency was DAGOL > U69593 = PD117302 > morphine > U50488 = [d‐Pen2, l‐Pen5]enkephalin (DPLPE). 5 The antagonist naloxone was about equally potent against DAGOL, morphine and DPLPE, and about ten times less potent against U69593 and PD117302. The effects of U50488 were much less sensitive to blockade by naloxone than the effects of PD11703 or U69593. The κ antagonist, nor‐binaltorphimine was equipotent against all three κ agonists. 6 The absence of δ‐binding sites, and the low potency and relatively high sensitivity to naloxone suggest that DPLPE could be working at μ‐receptors in the neonatal rat spinal cord. 7 The binding assays show that U50488 has the same affinity as PD117302 and U69593 for κ‐receptors, yet it was less effective in the depression of nociceptive responses. This may be because U50488 has a relatively low efficacy at κ‐receptors. It is possible that at high concentrations U50488 activates receptors not affected by other κ‐ligands. These additional receptors may be non‐opioid receptors (hence the insensitivity to naloxone), or they could be a subtype of κ‐opioid receptor.