The main objective of this work is to assess whether ibogalogs decrease headache-like behaviors in rats using the cortical spreading depression (CSD) and medication overuse headache (MOH) models. The CSD and MOH results showed that tabernanthalog (TBG), ibogainalog (IBG), and ibogaminalog (DM506) significantly decreased allodynia induced by acute injection of KCl or repetitive administration of sumatriptan. The effect of ibogalogs in the CSD model was sex-independent. The observed effects lasted 6-8 h, with TBG showing comparatively higher efficacy in both models. We also assessed the potential role of serotonin type 2 receptor (5-HT 2 R) and 5-HT 1B/1D R subtypes the effects of ibogalogs using selective antagonists such as ketanserin and GR127935, respectively. The antagonist effects were headache type-dependent: in acute headache, GR127935 inhibited ibogalogs' activity during the first hour but subsequently enhanced their effects, whereas ketanserin enhanced their activities; in chronic headache, GR127935 abolished ibogalog's effects during the first 3 h, whereas ketanserin inhibited IBG's effect during the first 2 h but enhanced TBG's effect. These results suggested that the antiheadache effects of ibogalogs are mediated by co-modulation of 5-HT 1 R and 5-HT 2 R subtypes. The functional results showed the ibogalogs activate 5-HT 1 R subtypes with the following selectivity: 5-HT 1F R ~ 5-HT 1D R > 5-HT 1B R > > 5-HT 1E R. Thus, we compared the activity of ibogalogs between wild type (WT) and 5-HT 1F R knockout (KO) mice in the MOH model. The results in WT mice showed that ibogalogs increase the facial withdrawal threshold with the following efficacy: TBG > IBG > DM506. Moreover, ibogalog antiallodynic effects were reduced in KO mice after 4 h, supporting a role for the 5-HT 1F R in the actions of ibogalogs in headache attenuation. In conclusion, ibogalogs significantly reduce acute and chronic headache-like behaviors by a co-modulatory mechanism between 5-HT 1 R and 5-HT 2 R subtypes.

A rapid and highly sensitive LC-MS/MS method was developed and validated for the determination of the nitrosamine drug substance-related impurity N-nitroso-desmethyl sumatriptan (NNDS) in sumatriptan succinate injection. Separation was achieved on an Inertsil ODS-3 column using gradient elution with 0.1% formic acid in water and acetonitrile, followed by positive electrospray ionization in multiple reactions monitoring mode. The method was validated according to ICH Q2(R2) and USP <1225> guidelines, demonstrating excellent specificity, linearity (R[2] = 1.0000), precision, accuracy, robustness, and solution stability. The limits of detection and quantification were 0.05 ppm and 0.15 ppm, respectively, providing sensitivity well below the regulatory specification limit for NNDS. Analysis of commercial injection batches confirmed NNDS levels within acceptable limits. The validated method is suitable for routine quality control, stability assessment, and regulatory monitoring of nitrosamine impurities in pharmaceutical products.

Pain perception is initiated upon activation of nociceptors of the dorsal root ganglia (DRG) and trigeminal ganglia. We identify G-protein-coupled receptors (GPCRs) expressed in CGRP[+] mouse and human DRG neurons and find that agonists of several identified Gi/o-coupled and orphan GPCRs attenuate neuronal excitability. Experiments focusing on the Gi/o-coupled serotonin receptor Htr1b, which is expressed in mouse and human CGRP[+] DRG neurons, reveal that Htr1b/1d agonists, the triptans sumatriptan and zolmitriptan, attenuate CGRP[+] neuron excitability in vitro and exhibit analgesia across several pain models, including neuropathic pain. Conditional genetic deletion experiments show that triptan-induced analgesia is mediated by Htr1b expressed in A-fiber mechanonociceptors. Also, triptan-associated adverse effects are partially mediated by Htr1b-independent targets. Further testing identifies the GPCR Gpr19 as an additional promising target for treating pain. These findings establish a preclinical screening platform for identifying analgesic candidates and reveal nociceptor GPCRs that may be targeted to treat pain.

Pain disorders such as neuropathic pain and headache remain areas of considerable unmet need and considered high risk by pharma. Human-induced pluripotent stem cells (iPSC)-derived sensory neurons have already been used to accelerate translational research but the current differentiation protocols produce non-peptidergic nociceptors. We demonstrate for the first time the robust differentiation of hiPSC into peptidergic nociceptor lineage with high yield. These nociceptors express CGRP and TRPV1 and show functional maturity including the expression of TTX-resistant currents and responding to TRPV1 and TRPA1 agonists. Importantly, they were able to release CGRP basally and upon stimulation by inflammatory soup, which was inhibited upon the application of the 5-HT1B/1D/1F agonist, sumatriptan, a migraine prophylactic drug. We report the successful generation of a novel in vitro functional peptidergic nociceptor model which will allow investigation of disease mechanisms in pain and translational phenotypic drug screening for new effective pain therapies.

Growing evidence suggests that esculetin, a 5-lipoxygenase inhibitor, has pharmacotherapeutic potential due to its various pharmacological properties, such as potent anti-inflammatory, anti-nociceptive, and γ-aminobutyric acid type A (GABAA) receptor partial agonist activities. However, the effects of this promising agent on migraine remain unexplored. This study therefore examined the impact of esculetin on relevant mechanisms in migraine-like conditions in rats. The systemic effects of esculetin at three distinct doses (5, 10, and 20 mg/kg) were tested in a nitroglycerin (NTG)-induced migraine model using in vivo experimental sets. The direct action of esculetin on the release of calcitonin gene-related peptide (CGRP) from critical structures of the trigeminovascular system (trigeminal ganglion, trigeminal nucleus, and meningeal afferents) was also tested in ex vivo experimental sets. Sumatriptan was used as a positive control in both sets of experiments. The in vivo results showed that esculetin reduced NTG-induced mechanical hyperalgesia and decreased trigeminal CGRP and cellular Fos proto-oncogene (c-Fos) levels. It also decreased degranulation and meningeal mast cell numbers. The ex vivo results revealed that esculetin reduced NTG-stimulated CGRP release from trigeminovascular explants, with the exception of meningeal explants. Sumatriptan reversed the NTG-induced changes in both experimental sets. Our findings suggest that esculetin exhibits anti-nociceptive activities in experimental migraine conditions, alleviating trigeminovascular CGRP concentrations and the degranulation of meningeal mast cells. Esculetin may thus represent a therapeutic option for relieving migraine headaches, although further research is needed to confirm this.