Rafael González-Cano

Tetrodotoxin (TTX) as a Therapeutic Agent for Pain

Tetrodotoxin (TTX) is a potent neurotoxin that blocks voltage-gated sodium channels (VGSCs). VGSCs play a critical role in neuronal function under both physiological and pathological conditions. TTX has been extensively used to functionally characterize VGSCs, which can be classified as TTX-sensitive or TTX-resistant channels according to their sensitivity to this toxin. Alterations in the expression and/or function of some specific TTX-sensitive VGSCs have been implicated in a number of chronic pain conditions. The administration of TTX at doses below those that interfere with the generation and conduction of action potentials in normal (non-injured) nerves has been used in humans and experimental animals under different pain conditions. These data indicate a role for TTX as a potential therapeutic agent for pain. This review focuses on the preclinical and clinical evidence supporting a potential analgesic role for TTX. In addition, the contribution of specific TTX-sensitive VGSCs to pain is reviewed.

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.

Modulation of Peripheral μ-Opioid Analgesia by σ1 Receptors

We evaluated the effects of σ1-receptor inhibition on μ-opioid–induced mechanical antinociception and constipation. σ1-Knockout mice exhibited marked mechanical antinociception in response to several μ-opioid analgesics (fentanyl, oxycodone, morphine, buprenorphine, and tramadol) at systemic (subcutaneous) doses that were inactive in wild-type mice and even unmasked the antinociceptive effects of the peripheral μ-opioid agonist loperamide. Likewise, systemic (subcutaneous) or local (intraplantar) treatment of wild-type mice with the selective σ1 antagonists BD-1063 [1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride] or S1RA [4-[2-[[5-methyl-1-(2-naphthalenyl)1H-pyrazol-3-yl]oxy]ethyl] morpholine hydrochloride] potentiated μ-opioid antinociception; these effects were fully reversed by the σ1 agonist PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate) hydrochloride], showing the selectivity of the pharmacological approach. The μ-opioid antinociception potentiated by σ1 inhibition (by σ1-receptor knockout or σ1-pharmacological antagonism) was more sensitive to the peripherally restricted opioid antagonist naloxone methiodide than opioid antinociception under normal conditions, indicating a key role for peripheral opioid receptors in the enhanced antinociception. Direct interaction between the opioid drugs and σ1 receptor cannot account for our results, since the former lacked affinity for σ1 receptors (labeled with [3H](+)-pentazocine). A peripheral role for σ1 receptors was also supported by their higher density (Western blot results) in peripheral nervous tissue (dorsal root ganglia) than in several central areas involved in opioid antinociception (dorsal spinal cord, basolateral amygdala, periaqueductal gray, and rostroventral medulla). In contrast to its effects on nociception, σ1-receptor inhibition did not alter fentanyl- or loperamide-induced constipation, a peripherally mediated nonanalgesic opioid effect. Therefore, σ1-receptor inhibition may be used as a systemic or local adjuvant to enhance peripheral μ-opioid analgesia without affecting opioid-induced constipation.

σ1 receptors are involved in the visceral pain induced by intracolonic administration of capsaicin in mice.

Background:Visceral pain is an important and prevalent clinical condition whose treatment is challenging. Sigma-1 (&sgr;1) receptors modulate somatic pain, but their involvement in pure visceral pain is unexplored. Methods:The authors evaluated the role of &sgr;1 receptors in intracolonic capsaicin-induced visceral pain (pain-related behaviors and referred mechanical hyperalgesia to the abdominal wall) using wild-type (WT) (n = 12 per group) and &sgr;1 receptor knockout (&sgr;1-KO) (n = 10 per group) mice, selective &sgr;1 receptor antagonists (BD-1063, S1RA, and NE-100), and control drugs (morphine and ketoprofen). Results:The intracolonic administration of capsaicin (0.01–1%) induced concentration-dependent visceral pain-related behaviors and referred hyperalgesia in both WT and &sgr;1-KO mice. However, the maximum number of pain-related behaviors induced by 1% capsaicin in &sgr;1-KO mice (mean ± SEM, 22 ± 2.9) was 48% of that observed in WT animals (46 ± 4.2). Subcutaneous administration of the &sgr;1 receptor antagonists BD-1063 (16–64 mg/kg), S1RA (32–128 mg/kg), and NE-100 (8–64 mg/kg) dose-dependently reduced the number of behavioral responses (by 53, 62, and 58%, respectively) and reversed the referred hyperalgesia to mechanical control threshold (0.53 ± 0.05 g) in WT mice. In contrast, these drugs produced no change in &sgr;1-KO mice. Thus, the effects of these drugs are specifically mediated by &sgr;1 receptors. Morphine produced an inhibition of capsaicin-induced visceral pain in WT and &sgr;1-KO mice, whereas ketoprofen had no effect in either mouse type. Conclusion:These results suggest that &sgr;1 receptors play a role in the mechanisms underlying capsaicin-induced visceral pain and raise novel perspectives for their potential therapeutic value.

Visceral and somatic pain modalities reveal NaV1.7‐independent visceral nociceptive pathways

Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.

Effects of Tetrodotoxin in Mouse Models of Visceral Pain

Visceral pain is very common and represents a major unmet clinical need for which current pharmacological treatments are often insufficient. Tetrodotoxin (TTX) is a potent neurotoxin that exerts analgesic actions in both humans and rodents under different somatic pain conditions, but its effect has been unexplored in visceral pain. Therefore, we tested the effects of systemic TTX in viscero-specific mouse models of chemical stimulation of the colon (intracolonic instillation of capsaicin and mustard oil) and intraperitoneal cyclophosphamide-induced cystitis. The subcutaneous administration of TTX dose-dependently inhibited the number of pain-related behaviors in all evaluated pain models and reversed the referred mechanical hyperalgesia (examined by stimulation of the abdomen with von Frey filaments) induced by capsaicin and cyclophosphamide, but not that induced by mustard oil. Morphine inhibited both pain responses and the referred mechanical hyperalgesia in all tests. Conditional nociceptor‑specific Nav1.7 knockout mice treated with TTX showed the same responses as littermate controls after the administration of the algogens. No motor incoordination after the administration of TTX was observed. These results suggest that blockade of TTX-sensitive sodium channels, but not Nav1.7 subtype alone, by systemic administration of TTX might be a potential therapeutic strategy for the treatment of visceral pain.

Sigma-1 receptors control immune-driven peripheral opioid analgesia during inflammation in mice

Significance New pain medications with novel mechanisms of action are needed. Here we show that sigma-1 antagonism decreases inflammatory pain hypersensitivity by enhancing the actions of endogenous opioid peptides produced by leukocytes in mice. Sigma-1 antagonism results in opioid analgesia only at the inflamed site, where immune cells naturally accumulate. This mechanism, which maximizes the analgesic potential of immune cells in painful inflamed sites, differs from that of conventional analgesics. Sigma-1 antagonism potentiates the antinociceptive effects of opioid drugs, so sigma-1 receptors constitute a biological brake to opioid drug-induced analgesia. The pathophysiological role of this process is unknown. We aimed to investigate whether sigma-1 antagonism reduces inflammatory pain through the disinhibition of the endogenous opioidergic system in mice. The selective sigma-1 antagonists BD-1063 and S1RA abolished mechanical and thermal hyperalgesia in mice with carrageenan-induced acute (3 h) inflammation. Sigma-1–mediated antihyperalgesia was reversed by the opioid antagonists naloxone and naloxone methiodide (a peripherally restricted naloxone analog) and by local administration at the inflamed site of monoclonal antibody 3-E7, which recognizes the pan-opioid sequence Tyr–Gly–Gly–Phe at the N terminus of most endogenous opioid peptides (EOPs). Neutrophils expressed pro-opiomelanocortin, the precursor of β-endorphin (a known EOP), and constituted the majority of the acute immune infiltrate. β-endorphin levels increased in the inflamed paw, and this increase and the antihyperalgesic effects of sigma-1 antagonism were abolished by reducing the neutrophil load with in vivo administration of an anti-Ly6G antibody. The opioid-dependent sigma-1 antihyperalgesic effects were preserved 5 d after carrageenan administration, where macrophages/monocytes were found to express pro-opiomelanocortin and to now constitute the majority of the immune infiltrate. These results suggest that immune cells harboring EOPs are needed for the antihyperalgesic effects of sigma-1 antagonism during inflammation. In conclusion, sigma-1 receptors curtail immune-driven peripheral opioid analgesia, and sigma-1 antagonism produces local opioid analgesia by enhancing the action of EOPs of immune origin, maximizing the analgesic potential of immune cells that naturally accumulate in painful inflamed areas.

Mild Social Stress in Mice Produces Opioid-Mediated Analgesia in Visceral but Not Somatic Pain States

Visceral pain has a greater emotional component than somatic pain. To determine if the stress-induced analgesic response is differentially expressed in visceral versus somatic pain states, we studied the effects of a mild social stressor in either acute visceral or somatic pain states in mice. We show that the presence of an unfamiliar conspecific mouse (stranger) in an adjacent cubicle of a standard transparent observation box produced elevated plasma corticosterone levels compared with mice tested alone, suggesting that the mere presence of a stranger is stressful. We then observed noxious visceral or somatic stimulation-induced nociceptive behavior in mice tested alone or in mildly stressful conditions (ie, beside an unfamiliar stranger). Compared with mice tested alone, the presence of a stranger produced a dramatic opioid-dependent reduction in pain behavior associated with visceral but not somatic pain. This social stress-induced reduction of visceral pain behavior relied on visual but not auditory/olfactory cues. These findings suggest that visceral pain states may provoke heightened responsiveness to mild stressors, an effect that could interfere with testing outcomes during simultaneous behavioral testing of multiple rodents. PERSPECTIVE In mice, mild social stress due to the presence of an unfamiliar conspecific mouse reduces pain behavior associated with noxious visceral but not somatic stimulation, suggesting that stress responsiveness may be enhanced in visceral pain versus somatic pain states.

Modality-specific peripheral antinociceptive effects of μ-opioid agonists on heat and mechanical stimuli: Contribution of sigma-1 receptors

&NA; Morphine induces peripherally &mgr;‐opioid‐mediated antinociception to heat but not to mechanical stimulation. Peripheral sigma‐1 receptors tonically inhibit &mgr;‐opioid antinociception to mechanical stimuli, but it is unknown whether they modulate &mgr;‐opioid heat antinociception. We hypothesized that sigma‐1 receptors might play a role in the modality‐specific peripheral antinociceptive effects of morphine and other clinically relevant &mgr;‐opioid agonists. Mechanical nociception was assessed in mice with the paw pressure test (450 g), and heat nociception with the unilateral hot plate (55 °C) test. Local peripheral (intraplantar) administration of morphine, buprenorphine or oxycodone did not induce antinociception to mechanical stimulation but had dose‐dependent antinociceptive effects on heat stimuli. Local sigma‐1 antagonism unmasked peripheral antinociception by &mgr;‐opioid agonists to mechanical stimuli, but did not modify their effects on heat stimulation. TRPV1+ and IB4+ cells are segregated populations of small neurons in the dorsal root ganglia (DRG) and the density of sigma‐1 receptors was higher in IB4+ cells than in the rest of small nociceptive neurons. The in vivo ablation of TRPV1‐expressing neurons with resiniferatoxin did not alter IB4+ neurons in the DRG, mechanical nociception, or the effects of sigma‐1 antagonism on local morphine antinociception in this type of stimulus. However, it impaired the responses to heat stimuli and the effect of local morphine on heat nociception. In conclusion, peripheral opioid antinociception to mechanical stimuli is limited by sigma‐1 tonic inhibitory actions, whereas peripheral opioid antinociception to heat stimuli (produced in TRPV1‐expressing neurons) is not. Therefore, sigma‐1 receptors contribute to the modality‐specific peripheral effects of opioid analgesics. Highlights&mgr;‐opioid agonists induce peripheral antinociception to heat stimulus.&mgr;‐opioid agonists do not induce peripheral antinociception to mechanical stimulus.&sgr;1 receptors do not modulate peripheral &mgr;‐opioid antinociception to heat stimulus.&sgr;1 receptors limit peripheral &mgr;‐opioid antinociception to mechanical stimulus.&sgr;1 receptors contribute to the modality‐specific peripheral effects of opioids.

245 ROLE OF SIGMA-1 RECEPTORS IN COLD ALLODYNIA INDUCED BY PACLITAXEL

This substance regulates various systems including excitatory glutamatergic nervous system activation of the N-methyl-Daspartate (NMDA) receptor related to nitric oxide (NO) production. Hyperalgesia caused by peripheral tissue damage is involved in glutamate-related neuronal plasticity of the spinal cord. However, its mechanism of action still is unknown. Therefore, we determined the analgesic effect of agmatine in relation to modification of glutamate-related neuronalplasticity using a rat inflammatory pain model. Methods: SD rats implanted with an intrathecal catheter were subjected to formalin-induced hyperalgesia. Pain behavior was assessed by counting spontaneous flinches per min after formalin injection into paws under halothane anesthesia. The rats were separated into six groups as follows, (1) Non-treated (saline, it. inj.); (2) Agmatine (30ug, it. inj.); (3) MK-801 (15ug, it. inj.); (4) AVS (OH radical scavenger, 100ug, it. inj); (5) Agmatine + MK-801; (6) Agmatine + AVS. Results: Rats showed biphasic pain behavior – the sum of the first (0–5 min) is 14/min and the sum of the second (20–60 min) is 147/min. All drugs diminished the sum of the second phase 32–68% compared with non-treated animals but did not affect the first phase. An additive effect of agmatine was obtained with MK801 but not with AVS. Conclusion: We suggest that the synergistic analgesia effect of agmatine may involve the inhibition of NMDA receptor(s) related to NO production, and that agmatine acts on multiple sites of glutamatergic neuronal activation in developing hyperalgesia.