Amynah Aa Pradhan

Background: The Delta-opioid receptor (DOR) has gained attention as a promising target for the treatment of migraine and headache disorders. This is largely attributed to its unique pharmacological profile, which suggests that DOR-targeting treatment offers effective therapeutic benefit with a lower risk of medication overuse headache (MOH), reduced abuse liability, and minimal potential for physical dependence. These advantages have driven the development of a novel DOR agonist PN6047 (3-[[4-(dimethylcarbamoyl) phenyl]-[1-(thiazol-5-ylmethyl)-4-piperidylidene] methyl]benzamide), which has completed Phase I clinical trial and showed a favorable safety and tolerability profile. Although PN6047 has shown promising effects in neuropathic pain models, its efficacy in preclinical models of headache-associated pain remains to be evaluated. Here, we investigated the effects of PN6047 in models of migraine-associated pain and aura as well as post-traumatic headache (PTH) and MOH. Methods: C57BL6/J mice were used to examine the effects of PN6047 in the following migraine models: chronic intermittent nitroglycerin (NTG)-induced migraine-associated pain, PTH, KCl-induced cortical spreading depression (CSD), and optogenetic evoked CSD in a freely behaving transgenic mice expressing ChR2-eYFP. In addition, we tested whether chronic PN6047 induced MOH and whether it could prevent the development of MOH induced by sumatriptan. Results: A single injection of PN6047 blocked chronic cephalic allodynia established by chronic intermittent NTG and PTH. Moreover, chronic PN6047 treatment prevented the development of MOH induced by sumatriptan, without causing MOH itself. In addition, PN6047 significantly reduced the number of CSD events in the KCl-induced CSD model, and delayed CSD onset triggered in freely behaving mice along with subsequent CSD-evoked allodynia. Conclusion: PN6047, a novel DOR agonist, strikingly blocks headache-associated mechanism and symptoms in preclinical models of chronic migraine, migraine aura, PTH, and MOH. Importantly, prolonged PN6047 treatment did not induce MOH or analgesic tolerance. Together, these data demonstrate that despite the distinct mechanisms underlying migraine and headache disorder, PN6047 exhibits robust efficacy without inducing MOH, and displays a favorable safety and tolerability profile.

Migraine affects over 1 billion people worldwide and is a leading cause of disability. Targeting the cannabinoid system offers a promising approach for pain and migraine relief. This study evaluated a novel monoacylglycerol lipase (MAGL) inhibitor to prolong endocannabinoid action in acute and chronic mouse models of migraine. It also examined MAGL and cannabinoid receptor 1 (CB1) mRNA expression in key head pain-processing regions. C57BL6/J male and female mice received the human migraine trigger nitroglycerin (NTG) acutely or every other day for 9 days. Allodynia was assessed by von Frey hair stimulation of the periorbital area. A single dose of MAGL inhibitor (ABD-1970) was tested in acute and chronic NTG models. Additionally, ABD-1970 was given daily for 5 days to assess tolerance. In situ hybridization measured transcript expression of MAGL, CB1, and neuronal marker Rbfox3 in trigeminal ganglia (TG) and trigeminal nucleus caudalis (TNC). A single injection of ABD-1970 blocked cephalic allodynia induced by acute NTG. ABD-1970 also blocked chronic allodynia established by chronic intermittent NTG. Repeated administration did not induce tolerance, and ABD-1970 continued to block NTG-induced allodynia after 5 days of administration. There was high expression of MAGL and CB1 in the TG and TNC, present in Rbfox3 positive and negative cells. MAGL inhibitor effectively blocked acute and chronic migraine-associated pain, likely through prolonged endocannabinoid action. This effect may be mediated through action at peripheral or central sites considering the high MAGL and CB1 expression in the TG and TNC, respectively. The endocannabinoid system appears to modulate migraine mechanisms, and MAGL may be a promising target for this disorder.

Chronic pain and opioid overdose deaths highlight the need for non-addictive analgesics with novel mechanisms. The δ opioid receptor (δOR) is a promising target, as it lacks the respiratory depression associated with µ opioid receptor (µOR) agonists. However, early δOR full agonists caused seizures, limiting their clinical use. Partial δOR agonists may offer more controlled receptor activation than full agonists, but their development has been hindered by uncertainty regarding the molecular mechanism of partial agonism. Here we show that C6-Quino, a bitopic ligand developed through structure-based design, acts as a selective δOR partial agonist. Functional studies reveal that C6-Quino shows differential activity at G-protein and arrestin pathways and interacts with the sodium binding pocket, confirmed through cryo-EM analysis. C6-Quino demonstrates oral activity, analgesic activity in chronic pain models without causing δOR-related seizures and µOR-related adverse effects which have limited opioid usage in recent times. This discovery outlines a new strategy for developing δOR-targeted analgesics and provides a framework for optimizing signaling profiles of other Class A GPCRs.

The current opioid overdose epidemic highlights the urgent need to develop safer and more effective treatments for chronic pain¹. Cannabinoid receptor type 1 (CB1) is a promising non-opioid target for pain relief, but its clinical use has been limited by centrally mediated psychoactivity and tolerance. We overcame both issues by designing peripherally restricted CB1 agonists that minimize arrestin recruitment. We achieved these goals by computationally designing positively charged derivatives of the potent CB1 agonist MDMB-Fubinaca². We designed these ligands to occupy a cryptic pocket identified through molecular dynamics simulations—an extended binding pocket that opens rarely and leads to the conserved signalling residue D^2.50 (ref. ³). We used structure determination, pharmacological assays and molecular dynamics simulations to verify the binding modes of these ligands and to determine the molecular mechanism by which they achieve this dampening of arrestin recruitment. Our lead ligand, VIP36, is highly peripherally restricted and demonstrates notable efficacy in three mouse pain models, with 100-fold dose separation between analgesic efficacy and centrally mediated side effects. VIP36 exerts analgesic efficacy through peripheral CB1 receptors and shows limited analgesic tolerance. These results show how targeting a cryptic pocket in a G-protein-coupled receptor can lead to enhanced peripheral selectivity, biased signalling, desired in vivo pharmacology and reduced adverse effects. This has substantial implications for chronic pain treatment but could also revolutionize the design of drugs targeting other G-protein-coupled receptors.

Background and purpose: Pituitary adenylate cyclase activating polypeptide (PACAP) is a human migraine trigger that is being targeted for migraine. The δ‐opioid receptor (δ‐receptor) is a novel target for the treatment of migraine, but its mechanism remains unclear. The goals of this study were to develop a mouse PACAP‐headache model using clinically significant doses of PACAP; determine the effects of δ‐receptor activation in this model; and investigate the co‐expression of δ‐receptors, PACAP and PACAP‐PAC1 receptor.Experimental approach: Cephalic allodynia to low doses of acute and chronic PACAP were tested. A triptan (sumatriptan) and a CGRP receptor antagonist (olcegepant) were tested in this model. The δ‐receptor agonist SNC80 was tested in PACAP and CGRP‐induced headache models. Expression of PACAP, PAC1, CRLR and δ‐receptors was determined using in situ hybridisation.Key results: Low doses of PACAP produced dose‐dependent acute and chronic cephalic allodynia, blocked by sumatriptan but not by olcegepant. The PAC1 antagonist (M65) did not inhibit CGRP‐induced allodynia. There was moderate co‐expression of PAC1 and CRLR transcripts in migraine‐related regions. SNC80 blocked PACAP‐ and CGRP‐induced allodynia. There was low co‐expression of PACAP and δ‐receptors in brain regions measured. However, there was high co‐expression of PAC1 and δ‐receptors in somatosensory cortex, hippocampus and trigeminal nucleus caudalis.Conclusion and implications: We developed a translationally significant model of PACAP‐induced headache, which was mechanistically distinct from CGRP. Activation of δ‐receptors blocked PACAP‐ and CGRP‐induced allodynia, and δ‐receptors were highly co‐expressed with the PACAP‐ergic system. Future studies will examine the functional relationship between δ‐receptors and PAC1.

Objective: To identify and disseminate research priorities for the headache field that should be areas of research focus during the next 10 years. Background: Establishing research priorities helps focus and synergize the work of headache investigators, allowing them to reach the most important research goals more efficiently and completely. Methods: The Headache Research Priorities organizing and executive committees and working group chairs led a multistakeholder and international group of experts to develop headache research priorities. The research priorities were developed and reviewed by clinicians, scientists, people with headache, representatives from headache organizations, health‐care industry representatives, and the public. Priorities were revised and finalized after receiving feedback from members of the research priorities working groups and after a public comment period. Results: Twenty‐five research priorities across eight categories were identified: human models, animal models, pathophysiology, diagnosis and management, treatment, inequities and disparities, research workforce development, and quality of life. The priorities address research models and methods, development and optimization of outcome measures and endpoints, pain and non‐pain symptoms of primary and secondary headaches, investigations into mechanisms underlying headache attacks and chronification of headache disorders, treatment optimization, research workforce recruitment, development, expansion, and support, and inequities and disparities in the headache field. The priorities are focused enough that they help to guide headache research and broad enough that they are widely applicable to multiple headache types and various research methods. Conclusions: These research priorities serve as guidance for headache investigators when planning their research studies and as benchmarks by which the headache field can measure its progress over time. These priorities will need updating as research goals are met and new priorities arise.

The persistence of chronic pain and continuing overdose deaths from pain-relieving opioids targeting µ opioid receptor (µOR) have fueled the need for reliable long-term analgesics which use different targets and mechanisms. The δ opioid receptor (δOR) is a potential alternative target for non-addictive analgesics to alleviate chronic pain, made more attractive by its lack of respiratory depression associated with µOR agonists. However, early δOR full agonists were found to induce seizures, precluding clinical use. Partial δOR agonists may offer more controlled activation of the receptor compared to full agonists, but the development of such ligands has been hindered by uncertainty over the molecular mechanism mediating partial agonism. Using a structure-based approach, we explored the engagement of the sodium binding pocket in δOR and developed a bitopic ligand, C6-Quino, predicted to be a selective δOR partial agonist. Functional studies of C6-Quino revealed that it displayed δOR partial agonist activity at both G-protein and arrestin pathways. Its interaction with the sodium pocket was confirmed and analyzed using a single particle cryo-EM. Additionally, C6-Quino demonstrated favorable chemical and physiological properties like oral activity, and analgesic activity in multiple chronic pain models. Notably, µOR-related hyperlocomotion and respiratory depression, and δOR-related convulsions, were not observed at analgesic doses of C6-Quino. This fundamentally new approach to designing δOR ligands provides a blueprint for the development of partial agonists as safe analgesics and acts as a generic method to optimize signaling profiles of other Class A GPCRs.

Objective: Our aim was to survey astrocyte and microglial activation across four brain regions in a mouse model of chronic migraine. Background: Chronic migraine is a leading cause of disability, with higher rates in females. The role of central nervous system neurons and glia in migraine pathophysiology is not fully elucidated. Preclinical studies have shown abnormal glial activation in the trigeminal nucleus caudalis of male rodents. No current reports have investigated glial activation in both sexes in other important brain regions involved with the nociceptive and emotional processing of pain. Methods: The mouse nitroglycerin model of migraine was used, and nitroglycerin (10 mg/kg) or vehicle was administered every other day for 9 days. Prior to injections on days 1, 5, and 9, cephalic allodynia was determined by periorbital von Frey hair testing. Immunofluorescent staining of astrocyte marker, glial fibrillary protein (GFAP), and microglial marker, ionized calcium binding adaptor molecule 1 (Iba1), in male and female trigeminal nucleus caudalis, periaqueductal gray, somatosensory cortex, and nucleus accumbens was completed. Results: Behavioral testing demonstrated increased cephalic allodynia in nitroglycerin‐ versus vehicle‐treated mice. An increase in the percent area covered by GFAP+ cells in the trigeminal nucleus caudalis and nucleus accumbens, but not the periaqueductal gray or somatosensory cortex, was observed in response to nitroglycerin. No significant differences were observed for Iba1 staining across brain regions. We did not detect significant sex differences in GFAP or Iba1 quantification. Conclusions: Immunohistochemical analysis suggests that, at the time point tested, immunoreactivity of GFAP+ astrocytes, but not Iba1+ microglia, changes in response to chronic migraine‐associated pain. Additionally, there do not appear to be significant differences between males and females in GFAP+ or Iba1+ cells across the four brain regions analyzed.