The proof of concept of 2-{3-[N-(1-benzylpiperidin-4-yl)propyl]amino}-6-[N-methyl-N-(prop-2-yn-1-yl)amino]-4-phenylpyridine-3,5-dicarbonitrile for the therapy of neuropathic pain.

In the search for new small molecules for the therapy of neuropathic pain, we found that 2-{3-[N-(1-benzylpiperidin-4-yl)propyl]amino}-6-[N-methyl-N-(prop-2-yn-1-yl)amino]-4-phenylpyridine-3,5-dicarbonitrile (12) induced a robust antiallodynic effect in capsaicin-induced mechanical allodynia, a behavioural model of central sensitization, through σ1R antagonism. Furthermore, administration of compound 12 to neuropathic animals, fully reversed mechanical allodynia, increasing its mechanical threshold to levels that were not significantly different from those found in paclitaxel-vehicle treated mice or from basal levels before neuropathy was induced. Ligand 12 is thus a promising hit-compound for the therapy of neuropathic pain.

Efficacy and Security of Tetrodotoxin in the Treatment of Cancer-Related Pain: Systematic Review and Meta-Analysis.

The pharmacological treatment of cancer-related pain is unsatisfactory. Tetrodotoxin (TTX) has shown analgesia in preclinical models and clinical trials, but its clinical efficacy and safety have not been quantified. For this reason, our aim was to perform a systematic review and meta-analysis of the clinical evidence that was available. A systematic literature search was conducted in four electronic databases (Medline, Web of Science, Scopus, and ClinicalTrials.gov) up to 1 March 2023 in order to identify published clinical studies evaluating the efficacy and security of TTX in patients with cancer-related pain, including chemotherapy-induced neuropathic pain. Five articles were selected, three of which were randomized controlled trials (RCTs). The number of responders to the primary outcome (≥30% improvement in the mean pain intensity) and those suffering adverse events in the intervention and placebo groups were used to calculate effect sizes using the log odds ratio. The meta-analysis showed that TTX significantly increased the number of responders (mean = 0.68; 95% CI: 0.19-1.16, p = 0.0065) and the number of patients suffering non-severe adverse events (mean = 1.13; 95% CI: 0.31-1.95, p = 0.0068). However, TTX did not increase the risk of suffering serious adverse events (mean = 0.75; 95% CI: -0.43-1.93, p = 0.2154). In conclusion, TTX showed robust analgesic efficacy but also increased the risk of suffering non-severe adverse events. These results should be confirmed in further clinical trials with higher numbers of patients.

Dual Sigma-1 receptor antagonists and hydrogen sulfide-releasing compounds for pain treatment: Design, synthesis, and pharmacological evaluation.

The development of σ1 receptor antagonists hybridized with a H2S-donor is here reported. We aimed to obtain improved analgesic effects when compared to σ1 receptor antagonists or H2S-donors alone. In an in vivo model of sensory hypersensitivity, thioamide 1a induced analgesia which was synergistically enhanced when associated with the σ1 receptor antagonist BD-1063. The selective σ1 receptor agonist PRE-084 completely reversed this effect. Four thioamide H2S-σ1 receptor hybrids (5a-8a) and their amide derivatives (5b-8b) were synthesized. Compound 7a (AD164) robustly released H2S and showed selectivity for σ1 receptor over σ2 and opioid receptors. This compound induced marked analgesia that was reversed by PRE-084. The amide analogue 7b (AD163) showed only minimal analgesia. Further studies showed that 7a exhibited negligible acute toxicity, together with a favorable pharmacokinetic profile. To the best of our knowledge, compound 7a is the first dual-acting ligand with simultaneous H2S-release and σ1 antagonistic activities.

Discovery of a σ1 receptor antagonist by combination of unbiased cell painting and thermal proteome profiling.

Phenotypic screening for bioactive small molecules is typically combined with affinity-based chemical proteomics to uncover the respective molecular targets. However, such assays and the explored bioactivity are biased toward the monitored phenotype, and target identification often requires chemical derivatization of the hit compound. In contrast, unbiased cellular profiling approaches record hundreds of parameters upon compound perturbation to map bioactivity in a broader biological context and may link a profile to the molecular target or mode of action. Herein we report the discovery of the diaminopyrimidine DP68 as a Sigma 1 (σ1) receptor antagonist by combining morphological profiling using the Cell Painting assay and thermal proteome profiling. Our results highlight that integration of complementary profiling approaches may enable both detection of bioactivity and target identification for small molecules.

Urinary bladder sigma-1 receptors: A new target for cystitis treatment.

No adequate treatment is available for painful urinary bladder disorders such as interstitial cystitis/bladder pain syndrome, and the identification of new urological therapeutic targets is an unmet need. The sigma-1 receptor (σ1-R) modulates somatic pain, but its role in painful urological disorders is unexplored. The urothelium expresses many receptors typical of primary sensory neurons (e.g. TRPV1, TRPA1 and P2X3) and high levels of σ1-R have been found in these neurons; we therefore hypothesized that σ1-R may also be expressed in the urothelium and may have functional relevance in this tissue. With western blotting and immunohistochemical methods, we detected σ1-R in the urinary bladder in wild-type (WT) but not in σ1-R-knockout (σ1-KO) mice. Interestingly, σ1-R was located in the bladder urothelium not only in mouse, but also in human bladder sections. The severity of histopathological (edema, hemorrhage and urothelial desquamation) and biochemical alterations (enhanced myeloperoxidase activity and phosphorylation of extracellular regulated kinases 1/2 [pERK1/2]) that characterize cyclophosphamide-induced cystitis was lower in σ1-KO than in WT mice. Moreover, cyclophosphamide-induced pain behaviors and referred mechanical hyperalgesia were dose-dependently reduced by σ1-R antagonists (BD-1063, NE-100 and S1RA) in WT but not in σ1-KO mice. In contrast, the analgesic effect of morphine was greater in σ1-KO than in WT mice. Together these findings suggest that σ1-R plays a functional role in the mechanisms underlying cyclophosphamide-induced cystitis, and modulates morphine analgesia against urological pain. Therefore, σ1-R may represent a new drug target for urinary bladder disorders.

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 nociceptor­specific 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.

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 [(3)H](+)-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.

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.

Tumor microenvironment and breast cancer progression: a complex scenario.

It is now widely accepted that the development and progression of a tumor toward the malignant phenotype is highly dependent on interactions between tumor cells and the tumor microenvironment. Different components of the tumor microenvironment may have stimulatory or inhibitory effects on tumor progression by regulating the gene expression repertoire in tumor cells and stromal cells. This review analyzes novel research findings on breast cancer progression, discussing acquisition of the metastatic phenotype in breast disease in relation to different aspects of cross-talk among components of the tumor microenvironment. Knowledge of the interaction of all of these factors would contribute to elucidating the mechanisms that disrupt regulatory/signaling cascades and downstream effects in breast cancer.