José M. Baeyens

Sigma-1 receptors regulate activity-induced spinal sensitization and neuropathic pain after peripheral nerve injury

ABSTRACT Sigma‐1 receptor (σ1R) is expressed in key CNS areas involved in nociceptive processing but only limited information is available about its functional role. In the present study we investigated the relevance of σ1R in modulating nerve injury‐evoked pain. For this purpose, wild‐type mice and mice lacking the σ1R gene were exposed to partial sciatic nerve ligation and neuropathic pain‐related behaviors were investigated. To explore underlying mechanisms, spinal processing of repetitive nociceptive stimulation and expression of extracellular signal‐regulated kinase (ERK) were also investigated. Sensitivity to noxious heat of homozygous σ1R knockout mice did not differ from wild‐type mice. Baseline values obtained in σ1R knockout mice before nerve injury in the plantar, cold‐plate and von Frey tests were also indistinguishable from those obtained in wild‐type mice. However, cold and mechanical allodynia did not develop in σ1R null mice exposed to partial sciatic nerve injury. Using isolated spinal cords we found that mice lacking σ1R showed reduced wind‐up responses respect to wild‐type mice, as evidenced by a reduced number of action potentials induced by trains of C‐fiber intensity stimuli. In addition, in contrast to wild‐type mice, σ1R knockout mice did not show increased phosphorylation of ERK in the spinal cord after sciatic nerve injury. Both wind‐up and ERK activation have been related to mechanisms of spinal cord sensitization. Our findings identify σ1R as a constituent of the mechanisms modulating activity‐induced sensitization in pain pathways and point to σ1R as a new potential target for drugs designed to alleviate neuropathic pain.

Sigma-1 receptors are essential for capsaicin-induced mechanical hypersensitivity: Studies with selective sigma-1 ligands and sigma-1 knockout mice

ABSTRACT We evaluated the role of σ1 receptors on capsaicin‐induced mechanical hypersensitivity and on nociceptive pain induced by punctate mechanical stimuli, using wild‐type and σ1 receptor knockout (σ1‐KO) mice and selective σ1 receptor‐acting drugs. Mutation in σ1‐KO mice was confirmed by PCR analysis of genomic DNA and, at the protein level, by [3H](+)‐pentazocine binding assays. Both wild‐type and σ1‐KO mice not treated with capsaicin showed similar responses to different intensities of mechanical stimuli (0.05–8 g force), ranging from innocuous to noxious, applied to the hind paw. This indicates that σ1 gene inactivation does not modify the perception of punctate mechanical stimuli. The intraplantar (i.pl.) administration of capsaicin induced dose‐dependent mechanical allodynia in wild‐type mice (markedly reducing both the threshold force necessary to induce paw withdrawal and the latency to paw withdrawal induced by a given force). In contrast, capsaicin was completely unable to induce mechanical hypersensitivity in σ1‐KO mice. The high‐affinity and selective σ1 antagonists BD‐1063, BD‐1047 and NE‐100, administered subcutaneously (s.c.), dose‐dependently inhibited mechanical allodynia induced by capsaicin (1 μg,i.pl.), yielding ED50 (mg/kg) values of 15.80 ± 0.93, 29.31 ± 1.65 and 40.74 ± 7.20, respectively. The effects of the σ1 antagonists were reversed by the σ1 agonist PRE‐084 (32 mg/kg, s.c.). None of the drugs tested modified the responses induced by a painful mechanical punctate stimulus (4 g force) in nonsensitized animals. These results suggest that σ1 receptors are essential for capsaicin‐induced mechanical hypersensitivity, but are not involved in mechanical nociceptive pain.

Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization

BACKGROUND AND PURPOSE The sigma‐1 (σ1) receptor is a ligand‐regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ1 receptor ligands used as pharmacological tools are unclear and the demonstration that σ1 receptor antagonists have efficacy in reversing central sensitization‐related pain sensitivity is still missing.

Tetrodotoxin inhibits the development and expression of neuropathic pain induced by paclitaxel in mice

&NA; We evaluated the effect of low doses of systemically administered tetrodotoxin (TTX) on the development and expression of neuropathic pain induced by paclitaxel in mice. Treatment with paclitaxel (2 mg/kg, i.p., once daily during 5 days) produced long‐lasting (2–4 weeks) heat hyperalgesia (plantar test), mechanical allodynia (electronic Von Frey test) and cold allodynia (acetone drop method), with maximum effects observed on days 7, 10 and 10–14, respectively. Acute subcutaneous treatment with 1 or 3 μg/kg of TTX reduced the expression of mechanical allodynia, whereas higher doses (3 or 6 μg/kg) were required to reduce the expression of cold allodynia and heat hyperalgesia. In contrast, TTX (3 or 6 μg/kg, s.c.) did not affect the response to the same thermal and mechanical stimuli in control animals, which indicates that the antihyperalgesic and antiallodynic effects of TTX were not due to unspecific inhibition of the perception of these stimuli. Administration of TTX (6 μg/kg, s.c.) 30 min before each of the 5 doses of paclitaxel did not modify the development of heat hyperalgesia produced by the antineoplastic, but abolished the development of mechanical and cold allodynia. Coadministration of a lower dose of TTX (3 μg/kg) also prevented the development of mechanical allodynia. No signs of TTX‐induced toxicity or motor incoordination were observed. These data suggest that low doses of TTX can be useful to prevent and treat paclitaxel‐induced neuropathic pain, and that TTX‐sensitive subtypes of sodium channels play a role in the pathogenesis of chemotherapy‐induced neuropathic pain.

Subgroups among μ-opioid receptor agonists distinguished by ATP-sensitive K+ channel-acting drugs

1 We evaluated the effects of the i.e.v. administration of different K+ channel blockers (gliquidone, 4‐aminopyridine and tetraethylammonium) and an opener of K+ channels (cromakalim) on the antinociception induced by several μ‐opioid receptor agonists in a tail flick test in mice.

Selective Sigma-1 (σ1) Receptor Antagonists: Emerging Target for the Treatment of Neuropathic Pain

A large number of therapeutic roles have been proposed for sigma(1) receptors but the involvement of sigma(1) receptor in non-acute pain had not been well explored up to now. sigma(1) receptor knock-out mice became available offering us the possibility to study the role of sigma(1) receptor in nociception, particularly in models where central sensitization processes play a significant role. Given the attractive therapeutic potential, we have developed a chemical program aimed at the discovery of novel and selective sigma(1) ligands. Herein we discuss the rational basis of this approach and report preliminary pharmacological results of several chemical series and aspects of their structure-activity relationship on sigma(1) receptor. Functional data in pain models are presented mainly on one series that provide evidence to consider selective sigma(1) receptor antagonists an innovative and alternative approach for treating neuropathic pain.

Role of Sigma-1 Receptors in Paclitaxel-Induced Neuropathic Pain in Mice

UNLABELLED Sigma-1 (σ(1)) receptors play a role in different types of pain and in central sensitization mechanisms; however, it is unknown whether they are involved in chemotherapy-induced neuropathic pain. We compared the ability of paclitaxel to induce cold (acetone test) and mechanical (electronic Von Frey test) allodynia in wild-type (WT) and σ(1) receptor knockout (σ(1)-KO) mice. We also tested the effect on paclitaxel-induced painful neuropathy of BD-1063 (16-64 mg/kg, subcutaneously) and S1RA (32-128 mg/kg, subcutaneously), 2 selective σ(1) receptor antagonists that bind to the σ(1) receptor with high affinity and competitively. The responses to cold and mechanical stimuli were similar in WT and σ(1)-KO mice not treated with paclitaxel; however, treatment with paclitaxel (2 mg/kg, intraperitoneally, once per day during 5 consecutive days) produced cold and mechanical allodynia and an increase in spinal cord diphosphorylated extracellular signal-regulated kinase (pERK) in WT but not in σ(1)-KO mice. The administration of BD-1063 or S1RA 30 minutes before each paclitaxel dose prevented the development of cold and mechanical allodynia in WT mice. Moreover, the acute administration of both σ(1) receptor antagonists dose dependently reversed both types of paclitaxel-induced allodynia after they had fully developed. These results suggest that σ(1) receptors play a key role in paclitaxel-induced painful neuropathy. PERSPECTIVE Antagonists of the σ(1) receptor may have therapeutic value for the treatment and/or prevention of paclitaxel-induced neuropathic pain. This possibility is especially interesting in the context of chemotherapy-induced neuropathy, where the onset of nerve damage is predictable and preventive treatment could be administered.

Differential effects of K+ channel blockers on antinociception induced by α2-adrenoceptor, GABAB and κ-opioid receptor agonists

1 The effects of several K+ channel blockers (sulphonylureas, 4‐aminopyridine and tetraethylammonium) on the antinociception induced by clonidine, baclofen and U50,488H were evaluated by use of a tail flick test in mice. 2 Clonidine (0.125–2 mg kg−1, s.c.) induced a dose‐dependent antinociceptive effect. The ATP‐dependent K+ (KATP) channel blocker gliquidone (4–8 μg/mouse, i.c.v.) produced a dose‐dependent displacement to the right of the clonidine dose‐response line, but neither 4‐aminopyridine (4‐AP) (25–250 ng/mouse, i.c.v.) nor tetraethylammonium (TEA) (10–20 μg/mouse, i.c.v.) significantly modified clonidine‐induced antinociception. 3 The order of potency of sulphonylureas in antagonizing clonidine‐induced antinociception was gliquidone > glipizide > glibenclamide > tolbutamide, which is the same order of potency as these drugs block KATP channels in neurones of the CNS. 4 Baclofen (2–16 mg kg−1, s.c.) also induced a dose‐dependent antinociceptive effect. Both 4‐AP (2.5–25 ng/mouse, i.c.v.) and TEA (10–20 μg/mouse, i.c.v.) dose‐dependently antagonized baclofen antinociception, producing a displacement to the right of the baclofen dose‐response line. However, gliquidone (8–16 μg/mouse, i.c.v.) did not significantly modify the baclofen effect. 5 None of the K+ channel blockers tested (gliquidone, 8–16 μg/mouse; 4‐AP, 25–250 ng/mouse and TEA, 10–20 μg/mouse, i.c.v.), significantly modified the antinociception induced by U50,488H (8 mg kg−1, s.c). 6 These results suggest that the opening of K+ channels is involved in the antinoceptive effect of α2 and GABAB, but not κ‐opioid, receptor agonists. The K+ channels opened by α2‐adrenoceptor agonists seem to be ATP‐dependent channels, whereas those opened by GABAB receptor agonists are not.

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [3H](+)‐pentazocine binding to σ1 receptors in SH‐SY5Y human neuroblastoma cells and guinea pig brain P1, P2 and P3 subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ1 antagonists or (−)‐sulpiride), [3H](+)‐pentazocine binding to brain membranes was markedly decreased. Recovery of σ1 receptor density to steady state after HP‐induced inactivation required more than 30 days. HP‐metabolite II (reduced HP, 4‐(4‐chlorophenyl)‐α‐(4‐fluorophenyl)‐4‐hydroxy‐1‐piperidinebutanol), but not HP‐metabolite I (4‐(4‐chlorophenyl)‐4‐hydroxypiperidine), irreversibly blocked σ1 receptors in guinea pig brain homogenate and P2 fraction in vitro. We found similar results in SH‐SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ1 receptors when it was incubated with brain homogenate and SH‐SY5Y cells, but not when incubated with P2 fraction membranes, which suggests that HP is metabolized to inactivate σ1 receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP‐metabolite II, completely prevented HP‐induced inactivation of σ1 receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ1 receptors in guinea pig and human cells, probably after metabolism to reduced HP.

Potentiation of morphine-induced mechanical antinociception by σ1 receptor inhibition: Role of peripheral σ1 receptors

We studied the modulation of morphine-induced mechanical antinociception and side effects by σ₁ receptor inhibition. Both wild-type (WT) and σ₁ receptor knockout (σ₁-KO) mice showed similar responses to paw pressure (100-600 g). The systemic (subcutaneous) or local (intraplantar) administration of σ₁ antagonists (BD-1063, BD-1047, NE-100 and S1RA) was devoid of antinociceptive effects in WT mice. However, σ₁-KO mice exhibited an enhanced mechanical antinociception in response to systemic morphine (1-16 mg/kg). Similarly, systemic treatment of WT mice with σ₁ antagonists markedly potentiated morphine-induced antinociception, and its effects were reversed by the selective σ₁ agonist PRE-084. Although the local administration of morphine (50-200 μg) was devoid of antinociceptive effects in WT mice, it induced dose-dependent antinociception in σ₁-KO mice. This effect was limited to the injected paw. Enhancement of peripheral morphine antinociception was replicated in WT mice locally co-administered with σ₁ antagonists and the opioid. None of the σ₁ antagonists tested enhanced morphine-antinociception in σ₁-KO mice, confirming a σ₁-mediated action. Morphine-induced side-effects (hyperlocomotion and inhibition of gastrointestinal transit) were unaltered in σ₁-KO mice. These results cannot be explained by a direct interaction of σ₁ ligands with μ-opioid receptors or adaptive changes of μ-receptors in σ₁-KO mice, given that [(3)H]DAMGO binding in forebrain, spinal cord, and hind-paw skin membranes was unaltered in mutant mice, and none of the σ₁ drugs tested bound to μ-opioid receptors. These results show that σ₁ receptor inhibition potentiates morphine-induced mechanical analgesia but not its acute side effects, and that this enhanced analgesia can be induced at peripheral level.