Migraine is a common disorder that affects more than a billion of people worldwide, with an incidence in women three times higher than in men. The medical need for new therapeutic treatments is only partially satisfied by innovative approaches, and a significant percentage of migraineurs are still not satisfied with their therapy. Several lines of evidence links migraine to the activation of the NLRP3-IL-1β pathway. The aim of this research was to test whether NLRP3 blockers may represent innovative therapeutics for migraine. A well-established murine migraine model triggered by nitroglycerine (GTN) was used, together with NLRP3 knockout (Nlrp3^(-/-)) mice and the NLRP3 inhibitor MCC950. Both genetic and pharmacological blockade of NLRP3 were effective in reducing and preventing the onset of the periorbital mechanical allodynia (PMA) evoked by GTN in female mice, but not in males. The chronic administration of MCC950 was sufficient to induce PMA on its on, suggesting that the chronic treatment with a NLRP3 blocker may induce medication overuse headache as a side effect. Western blot analysis showed the activation of the NLRP3 pathway in the hippocampus of female mice treated with GTN, but not in their trigeminal ganglion, trigeminal nucleus caudalis and cortex. No activation of the NLRP3 pathway was detected in male mice. Collectively, this study strengthens the view that NLRP3 inhibitors may represent innovative migraine treatments. However, at the same time, it underlines for the first time some limitations that may affect NLRP3 inhibitors, namely sexual dimorphism in drug responsiveness and issues related to their chronic use.

Background and Purpose: Migraine is a neurovascular disorder largely mediated by calcitonin gene‐related peptide (CGRP). This study explores the role of the nociceptin/orphanin FQ (N/OFQ)–N/OFQ receptor (NOP) system in CGRP‐induced periorbital mechanical allodynia (PMA) in mice.Experimental Approach: Male or female wild type (NOP(+/+)) and NOP receptor knockout (NOP(−/−)) mice and CD‐1 mice were used. The brain penetrant, AT‐403, and the peripherally restricted, UFP‐112, NOP agonists were tested for PMA prevention. To identify a potential site of action at the cellular level, the ability of N/OFQ to signal at membrane and endosomal level in NOP‐expressing HEK293 cells, and to inhibit the increase in cyclic adenosine monophosphate (cAMP) induced by CGRP in human Schwann cells (hSCs) was investigated.Key Results: CGRP‐induced PMA was comparable in NOP(+/+) and NOP(−/−) mice. AT‐403 and UFP‐112 equally reduced CGRP‐evoked PMA in CD‐1 mice. In NOP‐expressing cells, activation of NOP resulted in the internalisation and movement of NOP away from the plasma membrane marker CAAX and to early endosomes marker Rab5a. N/OFQ stimulated G_αi recruitment to NOP at the plasma membrane and from the endosomal compartment. N/OFQ attenuated cAMP increase elicited by CGRP in hSCs.Conclusions and Implications: The peripherally restricted NOP agonist showed efficacy similar to the brain‐penetrant compound, indicating that peripheral NOP activation is sufficient to alleviate CGRP‐evoked PMA. Despite NOP ability to halt Gα_i recruitment and cAMP increase in cells, further studies are required to confirm that SCs are the cellular site where N/OFQ operates to attenuate the CGRP pro‐migraine action.

Exposure to physical and psychological stress modulates pain transmission in a dual manner. Stress-induced analgesia (SIA) refers to the reduction in pain sensitivity that can occur in response to acute stress. On the contrary, chronic stress exposure may lead to a phenomenon named stress-induced hyperalgesia (SIH). SIH is a clinically relevant phenomenon since it has been well documented that physical and psychological stress exacerbates pain in patients with several chronic pain syndromes, including migraine. The availability of animal models of SIA and SIH is of high importance for understanding the biological mechanisms leading to these phenomena and for the identification of pharmacological targets useful to alleviate the burden of stress-exacerbated chronic pain. Among these targets, the nociceptin/orphanin FQ (N/OFQ)–N/OFQ peptide (NOP) receptor system has been identified as a key modulator of both pain transmission and stress susceptibility. This review describes first the experimental approaches to induce SIA and SIH in rodents. The second part of the manuscript summarizes the scientific evidence that suggests the N/OFQ–NOP receptor system as a player in the stress–pain interaction and candidates NOP antagonists as useful drugs to mitigate the detrimental effects of stress exposure on pain perception.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) plays a key role in regulating several physiological functions and pathological states, which makes its receptor (NOP) a promising target for therapeutic interventions. In this study, we synthesized homodimeric N/OFQ-NH₂ derivatives linked by disulfide bonds between cysteines appropriately introduced in the addressing region of the native peptide in place of the original amino acids. The in vitro activity of the compounds was evaluated using both an NOP-G protein interaction BRET assay and a calcium mobilization assay. The most potent compound, 1h (pEC₅₀ > 9), was obtained by coupling two monomeric precursors via a Leu¹⁴-to-Cys substitution. In vivo, 1h demonstrated 3-fold greater potency than N/OFQ in eliciting loss of the righting reflex in mice and produced a long-lasting effect monitored for up to 7 h, supporting multimerization as a viable approach to developing long-acting peptide-based NOP ligands.

Ghrelin modulates several biological functions via selective activation of the growth hormone secretagogue receptor (GHSR). GHSR agonists may be useful for the treatment of anorexia and cachexia, while antagonists and inverse agonists may represent new drugs for the treatment of metabolic and substance use disorders. Thus, the identification and pharmacodynamic characterization of new GHSR ligands is of high interest. In the present work the label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of a panel of GHSR ligands. This includes the endogenous peptides ghrelin, desacyl-ghrelin and LEAP2(1-14). Among synthetic compounds, the agonists anamorelin and HM01, the antagonists HM04 and YIL-781, and the inverse agonist PF-05190457 have been tested, together with HM03, R011, and H1498 from patent literature. The DMR results have been compared to those obtained in parallel experiments with the calcium mobilization assay. Ghrelin, anamorelin, HM01, and HM03 behaved as potent full GHSR agonists. YIL-781 behaved as a partial GHSR agonist and R011 as antagonist in both the assays. LEAP2(1-14) resulted a GHSR inverse agonist in DMR but not in calcium mobilization assay. PF-05190457, HM04, and H1498 behaved as GHSR inverse agonists in DMR experiments, while they acted as antagonists in calcium mobilization studies. In conclusion, this study provided a systematic pharmacodynamic characterization of several GHSR ligands in two different pharmacological assays. It demonstrated that the DMR assay can be successfully used particularly to discriminate between antagonists and inverse agonists. This study may be useful for the selection of the most appropriate compounds to be used in future studies.

Stress is a major trigger of migraine, yet the underlying mechanisms remain unclear. Previous evidence supports the nociceptin/orphanin FQ (N/OFQ)/NOP receptor system as a potential target for novel anti-migraine strategies, although its role in stress-induced and persistent migraine-like states remains unclear. We evaluated the role of the N/OFQ/NOP receptor system in a mouse model where repeated restraint stress induces periorbital mechanical allodynia (PMA) and primes responses to a subthreshold dose of nitroglycerin (GTN, 0.1 mg/kg). Experiments were performed in male mice, as a first study showed that stress did not sensitize females to subthreshold GTN under our conditions. Stress-induced PMA resolved in young mice but persisted for weeks in older animals, inducing a persistent migraine-like phenotype. GTN, inactive in naïve mice, evoked PMA only in previously stressed animals. NOP knockout did not alter animal susceptibility, whereas NOP stimulation with the brain-penetrant NOP agonist AT-403 (30 μg/kg) or the peripherally restricted agonist UFP-112 (10 pmol) prevented GTN-evoked PMA and reversed stress-induced PMA, including persistent allodynia in older adult mice. Comparable efficacy of UFP-112 and AT-403 supports the contribution of peripheral NOP receptors. Together, our findings expand the evidence beyond acute pharmacological models, supporting the potential of NOP agonists for the treatment of both acute and persistent migraine.