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Background and Purpose: Peripheral sensitization contributes to pathological pain. While prostaglandin E2 (PGE2) and nerve growth factor (NGF) sensitize peptidergic C-nociceptors (TRPV1+), glial cell line-derived neurotrophic factor (GDNF) sensitizes non-peptidergic C-neurons (IB4+). Sigma-1 receptor (σ1R) is a Ca2+-sensing chaperone known to modulate analgesia induced by opioid drugs. This receptor binds both to TRPV1 and the µ-opioid receptor (MOPr), although the functional repercussions of these physical interactions in peripheral sensitization are unknown. Experimental Approach: We tested the effect of sigma-1 antagonism on PGE2-, NGF- and GDNF-induced mechanical and heat hyperalgesia in mice. We used immunohistochemistry to determine the presence of endomorphin-2, an endogenous MOPr agonist, on dorsal root ganglion (DRG) neurons. Recombinant proteins were used to study the interactions between σ1R, MOPr and TRPV1. We used calcium imaging to study the effects of sigma-1 antagonism on PGE2-induced sensitization of TRPV1+ nociceptors. Key Results: σ1R antagonists reversed PGE2- and NGF-induced hyperalgesia, but not GDNF-induced hyperalgesia. Endomorphin-2 was detected on TRPV1+ but not on IB4+ neurons. Peripheral opioid receptor antagonism by naloxone methiodide or administration of an anti-endomorphin-2 antibody to a sensitized paw, reversed the antihyperalgesia induced by sigma-1 antagonists. Sigma-1 antagonism transfers σ1R from TRPV1 to MOPr, suggesting that σ1R participate in TRPV1-MOPr crosstalk. Moreover, σ1R antagonism reversed, in a naloxone-sensitive manner, PGE2-induced sensitization of DRG neurons to the calcium flux elicited by capsaicin, the prototypic TRPV1 agonist. Conclusion and Implications: σ1R antagonism harnesses endogenous opioids produced by TRPV1+ neurons to reduce hyperalgesia by increasing MOPr activity.

Pain measures traditionally used in rodents record mere reflexes evoked by sensory stimuli; the results thus may not fully reflect the human pain phenotype. Alterations in physical and emotional functioning, pain-depressed behaviors and facial pain expressions were recently proposed as additional pain outcomes to provide a more accurate measure of clinical pain in rodents, and hence to potentially enhance analgesic drug development. We aimed to review how preclinical pain assessment has evolved since the development of the tail flick test in 1941, with a particular focus on a critical analysis of some nonstandard pain outcomes, and a consideration of how sex differences may affect the performance of these pain surrogates. We tracked original research articles in Medline for the following periods: 1973-1977, 1983-1987, 1993-1997, 2003-2007, and 2014-2018. We identified 606 research articles about alternative surrogate pain measures, 473 of which were published between 2014 and 2018. This indicates that preclinical pain assessment is moving toward the use of these measures, which may soon become standard procedures in preclinical pain laboratories., FPU grant from the Spanish Ministry of Education, Culture and Sports, Spanish Ministry of Economy and Competitiveness (MINECO, grant SAF2016-80540-R), Ramón Areces Foundation, Junta de Andalucía (grant CTS 109), Esteve Pharmaceuticals, European Regional Development Fund (ERDF)

Immune cells have a known role in pronociception, since they release a myriad of inflammatory algogens which interact with neurons to facilitate pain signaling. However, these cells also produce endogenous opioid peptides with analgesic potential. The sigma-1 receptor is a ligand-operated chaperone that modulates neurotransmission by interacting with multiple protein partners, including the µ-opioid receptor. We recently found that sigma-1 antagonists are able to induce opioid analgesia by enhancing the action of endogenous opioid peptides of immune origin during inflammation. This opioid analgesia is seen only at the inflamed site, where immune cells naturally accumulate. In this article we review the difficulties of targeting the opioid system for selective pain relief, and discuss the dual role of immune cells in pain and analgesia. Our discussion creates perspectives for possible novel therapeutic uses of sigma-1 antagonists as agents able to maximize the analgesic potential of the immune system, University of Granada, Martín Escudero postdoctoral program, FPU grants from the Spanish Ministry of Economy and Competitiveness (MINECO), Juan de la Cierva-Incorporación postdoctoral grant from MINECO, MINECO [grant number SAF2016-80540-R], Junta de Andalucía (grant CTS109), FEDER funds

Methoxycarbonyl-1-phenyl-2-cyclopropylmethyl based derivatives cis-(+)-1a [cis-(+)-MR200], cis-(-)-1a [cis-(-)-MR201], and trans-(±)-1a [trans-(±)-MR204], have been identified as new potent sigma (σ) receptor ligands. In the present paper, novel enantiomerically pure analogues were synthesized and optimized for their σ receptor affinity and selectivity. Docking studies rationalized the results obtained in the radioligand binding assay. Absolute stereochemistry was unequivocally established by X-ray analysis of precursor trans-(+)-5a as camphorsulfonyl derivative 9. The most promising compound, trans-(+)-1d, showed remarkable selectivity over a panel of more than 15 receptors as well as good chemical and enzymatic stability in human plasma. An in vivo evaluation evidenced that trans-(+)-1d, in contrast to trans-(-)-1d, cis-(+)-1d, or cis-(-)-1d, which behave as σ

Morphine induces peripherally μ-opioid-mediated antinociception to heat but not to mechanical stimulation. Peripheral sigma-1 receptors tonically inhibit μ-opioid antinociception to mechanical stimuli, but it is unknown whether they modulate μ-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 μ-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 μ-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.

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.

Grip strength deficit is a measure of pain-induced functional disability in rheumatic disease. We tested whether this parameter and tactile allodynia, the standard pain measure in preclinical studies, show parallels in their response to analgesics and basic mechanisms. Mice with periarticular injections of complete Freund's adjuvant (CFA) in the ankles showed periarticular immune infiltration and synovial membrane alterations, together with pronounced grip strength deficits and tactile allodynia measured with von Frey hairs. However, inflammation-induced tactile allodynia lasted longer than grip strength alterations, and therefore did not drive the functional deficits. Oral administration of the opioid drugs oxycodone (1–8 mg/kg) and tramadol (10–80 mg/kg) induced a better recovery of grip strength than acetaminophen (40–320 mg/kg) or the nonsteroidal antiinflammatory drugs ibuprofen (10–80 mg/kg) or celecoxib (40–160 mg/kg); these results are consistent with their analgesic efficacy in humans. Functional impairment was generally a more sensitive indicator of drug-induced analgesia than tactile allodynia, as drug doses that attenuated grip strength deficits showed little or no effect on von Frey thresholds. Finally, ruthenium red (a nonselective TRP antagonist) or the in vivo ablation of TRPV1-expressing neurons with resiniferatoxin abolished tactile allodynia without altering grip strength deficits, indicating that the neurobiology of tactile allodynia and grip strength deficits differ. In conclusion, grip strength deficits are due to a distinct type of pain that reflects an important aspect of the human pain experience, and therefore merits further exploration in preclinical studies to improve the translation of new analgesics from bench to bedside., This study was partially supported by the Spanish Ministry of Economy and Competitiveness (MINECO, grant SAF2013-47481P), the Junta de Andalucía (grant CTS 109), and funding from Esteve and the European Regional Development Fund (FEDER).

Sigma-1 (σ1) receptor inhibition ameliorates neuropathic pain by inhibiting central sensitization. However, it is unknown whether σ1 receptor inhibition also decreases inflammatory hyperalgesia, or whether peripheral σ1 receptors are involved in this process. The purpose of this study was to determine the role of σ1 receptors in carrageenan-induced inflammatory hyperalgesia, particularly at the inflammation site. The subcutaneous (s.c.) administration of the selective σ1 antagonists BD-1063 and S1RA to wild-type mice dose-dependently and fully reversed inflammatory mechanical (paw pressure) and thermal (radiant heat) hyperalgesia. These antihyperalgesic effects were abolished by the s.c. administration of the σ1 agonist PRE-084 and also by the intraplantar (i.pl.) administration of this compound in the inflamed paw, suggesting that blockade of peripheral σ1 receptors in the inflamed site is involved in the antihyperalgesic effects induced by σ1 antagonists. In fact, the i.pl. administration of σ1 antagonists in the inflamed paw (but not in the contralateral paw) was sufficient to completely reverse inflammatory hyperalgesia. σ1 knockout (σ1-KO) mice did not develop mechanical hyperalgesia but developed thermal hypersensitivity; however, the s.c. administration of BD-1063 or S1RA had no effect on thermal hyperalgesia in σ1-KO mice, supporting on-target mechanisms for the effects of both drugs. The antiedematous effects of σ1 inhibition do not account for the decreased hyperalgesia, since carrageenan-induced edema was unaffected by σ1 knockout or systemic σ1 pharmacological antagonism. σ1 receptors play a major role in inflammatory hyperalgesia. Targeting σ1 receptors in the inflamed tissue may be useful for the treatment of inflammatory pain.

We evaluated the effects of σ 1 -receptor inhibition on μ -opioid–induced mechanical antinociception and constipation. σ 1 -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 σ 1 antagonists BD-1063 [1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride] or S1RA [4-[2-[[5-methyl-1-(2-naphthalenyl)1 H -pyrazol-3-yl]oxy]ethyl] morpholine hydrochloride] potentiated μ -opioid antinociception; these effects were fully reversed by the σ 1 agonist PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate) hydrochloride], showing the selectivity of the pharmacological approach. The μ -opioid antinociception potentiated by σ 1 inhibition (by σ 1 -receptor knockout or σ 1 -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 σ 1 receptor cannot account for our results, since the former lacked affinity for σ 1 receptors (labeled with [ 3 H](+)-pentazocine). A peripheral role for σ 1 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, σ 1 -receptor inhibition did not alter fentanyl- or loperamide-induced constipation, a peripherally mediated nonanalgesic opioid effect. Therefore, σ 1 -receptor inhibition may be used as a systemic or local adjuvant to enhance peripheral μ -opioid analgesia without affecting opioid-induced constipation.

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.

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.