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Original Research, Animal Studies, Human Studies, Pharmacology/Drug Development

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In Vivo Evaluation of Almotriptan malate Formulation through Intranasal Route for the Treatment of Migraine: Systematic Development and Pharmacokinetic Assessment.

Migraine headaches are usually intolerable, and a quick-relief treatment remains an unmet medical need. Almotriptan malate is a serotonin (5-HT) receptor agonist approved for the treatment of acute migraine in adults. It is currently available in an oral tablet dosage form and has a T of 1-3 h, and therefore, there is a medical need to develop a non-invasive rapidly acting formulation. We have developed an intranasal formulation of almotriptan malate using the quality-by-design (QbD) approach. A 2-factor 3-level full factorial design was selected to build up the experimental setting. The developed formulation was characterized for pH, viscosity, in vitro permeation, ex vivo permeation, and histopathological tolerance. To assess the potential of the developed formulation to produce a rapid onset of action following intranasal delivery, a pharmacokinetic study was performed in the Sprague-Dawley rat model and compared to the currently available marketed oral tablet formulation. For this, the LC-MS/MS bioanalytical method was developed and used for the determination of plasma almotriptan malate concentrations. Results of a pharmacokinetic study revealed that intranasal administration of optimized almotriptan malate formulation enabled an almost five-fold reduction in T and about seven-fold increase in bioavailability in comparison to the currently available oral tablet formulation, suggesting the potential of developed almotriptan malate intranasal formulation in producing a rapid onset of action as well as enhanced bioavailability.

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Lysophosphatidyl-choline 16: 0 mediates chronic joint pain associated to rheumatic diseases through acid-sensing ion channel 3.

Rheumatic diseases are often associated to debilitating chronic pain, which remains difficult to treat and requires new therapeutic strategies. We had previously identified lysophosphatidyl-choline (LPC) in the synovial fluids from few patients, and shown its effect as a positive modulator of Acid-Sensing Ion Channel 3 (ASIC3) able to induce acute cutaneous pain in rodents. However, the possible involvement of LPC in chronic joint pain remained completely unknown. Here we show, from two independent cohorts of patients with painful rheumatic diseases, that the synovial fluid levels of LPC are significantly elevated, especially the LPC16:0 species, compared to post mortem controls. Moreover, LPC16:0 levels are correlated with pain outcomes in a cohort of osteoarthritis (OA) patients. However, LPC16:0 but do not appear to be the hallmark of a particular joint disease, since similar levels are found in the synovial fluids of a second cohort of patients with various rheumatic diseases. The mechanism of action was next explored by developing a pathology-derived rodent model. Intra-articular injections of LPC16:0 is a triggering factor of chronic joint pain in both male and female mice, ultimately leading to persistent pain and anxiety-like behaviors. All these effects are dependent on ASIC3 channels, which drive sufficient peripheral inputs to generate spinal sensitization processes. This study brings evidences from mouse and human supporting a role for LPC16:0 via ASIC3 channels in chronic pain arising from joints, with potential implications for pain management in OA and possibly across other rheumatic diseases.

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Long-term intrathecal administration of morphine vs. baclofen: Differences in CSF glycoconjugate profiles using multiglycomics.

Opioid use for treatment of persistent pain has increased dramatically over the past two decades, but it has not resulted in improved pain management outcomes. To understand the molecular mechanisms of opioids, molecular signatures that arise from opioid exposure are often sought after, using various analytical methods. In this study, we performed proteomics, and multiglycomics via sequential analysis of polysialic acids, glycosaminoglycans, N-glycans and O-glycans, using the same cerebral spinal fluid (CSF) sample from patients that had long-term (>2 years), intrathecal morphine or baclofen administered via an indwelling pump. Proteomics and N-glycomics signatures between the two treatment groups were highly conserved, while significant differences were observed in polysialic acid, heparan sulfate glycosaminoglycan and O-glycan profiles between the two treatment groups. This represents the first study to investigate the potential relationships between diverse CSF conjugated glycans and long-term intrathecal drug exposure. The unique changes, observed by a sequential analytical workflow, reflect previously undescribed molecular effects of opioid administration and pain management.

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Site selective C-H functionalization of Mitragyna alkaloids reveals a molecular switch for tuning opioid receptor signaling efficacy.

Mitragynine (MG) is the most abundant alkaloid component of the psychoactive plant material "kratom", which according to numerous anecdotal reports shows efficacy in self-medication for pain syndromes, depression, anxiety, and substance use disorders. We have developed a synthetic method for selective functionalization of the unexplored C11 position of the MG scaffold (C6 position in indole numbering) via the use of an indole-ethylene glycol adduct and subsequent iridium-catalyzed borylation. Through this work we discover that C11 represents a key locant for fine-tuning opioid receptor signaling efficacy. 7-Hydroxymitragynine (7OH), the parent compound with low efficacy on par with buprenorphine, is transformed to an even lower efficacy agonist by introducing a fluorine substituent in this position (11-F-7OH), as demonstrated in vitro at both mouse and human mu opioid receptors (mMOR/hMOR) and in vivo in mouse analgesia tests. Low efficacy opioid agonists are of high interest as candidates for generating safer opioid medications with mitigated adverse effects.

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Pharmacological SARM1 inhibition protects axon structure and function in paclitaxel-induced peripheral neuropathy.

Axonal degeneration is an early and ongoing event that causes disability and disease progression in many neurodegenerative disorders of the peripheral and central nervous systems. Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of morbidity and the main cause of dose reductions and discontinuations in cancer treatment. Preclinical evidence indicates that activation of the Wallerian-like degeneration pathway driven by SARM1 is responsible for axonopathy in CIPN. SARM1 is the central driver of an evolutionarily conserved program of axonal degeneration downstream of chemical, inflammatory, mechanical or metabolic insults to the axon. SARM1 contains an intrinsic NADase enzymatic activity essential for its pro-degenerative functions, making it a compelling therapeutic target to treat neurodegeneration characterized by axonopathies of the peripheral and central nervous systems. Small molecule SARM1 inhibitors have the potential to prevent axonal degeneration in peripheral and central axonopathies and to provide a transformational disease-modifying treatment for these disorders. Using a biochemical assay for SARM1 NADase we identified a novel series of potent and selective irreversible isothiazole inhibitors of SARM1 enzymatic activity that protected rodent and human axons in vitro. In sciatic nerve axotomy (SNA), we observed that these irreversible SARM1 inhibitors decreased a rise in nerve cADPR and plasma neurofilament light chain (NfL) released from injured sciatic nerves in vivo. In a mouse paclitaxel model of CIPN we determined that Sarm1 KO mice prevented loss of axonal function, assessed by sensory nerve action potential (SNAP) amplitudes of the tail nerve, in a gene dosage-dependent manner. In that CIPN model, the irreversible SARM1 inhibitors prevented loss of intraepidermal nerve fibers induced by paclitaxel and provided partial protection of axonal function assessed by SNAP amplitude and mechanical allodynia.

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A ligand-receptor interactome platform for discovery of pain mechanisms and therapeutic targets.

In the peripheral nervous system, ligand-receptor interactions between cells and neurons shape sensory experience, including pain. We set out to identify the potential interactions between sensory neurons and peripheral cell types implicated in disease-associated pain. Using mouse and human RNA sequencing datasets and computational analysis, we created interactome maps between dorsal root ganglion (DRG) sensory neurons and an array of normal cell types, as well as colitis-associated glial cells, rheumatoid arthritis-associated synovial macrophages, and pancreatic tumor tissue. These maps revealed a common correlation between the abundance of heparin-binding EGF-like growth factor (HBEGF) in peripheral cells with that of its receptor EGFR (a member of the ErbB family of receptors) in DRG neurons. Subsequently, we confirmed that increased abundance of HBEGF enhanced nociception in mice, likely acting on DRG neurons through ErbB family receptors. Collectively, these interactomes highlight ligand-receptor interactions that may lead to treatments for disease-associated pain and, furthermore, reflect the complexity of cell-to-neuron signaling in chronic pain states.

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Mambalgin-3 potentiates human acid-sensing ion channel 1b under mild to moderate acidosis: Implications as an analgesic lead.

Acid-sensing ion channels (ASICs) are expressed in the nervous system, activated by acidosis, and implicated in pain pathways. Mambalgins are peptide inhibitors of ASIC1 and analgesic in rodents via inhibition of centrally expressed ASIC1a and peripheral ASIC1b. This activity has generated interest in mambalgins as potential therapeutics. However, most mechanism and structure-activity relationship work on mambalgins has focused on ASIC1a, and neglected the peripheral analgesic target ASIC1b. Here, we compare mambalgin potency and mechanism of action at heterologously expressed rat and human ASIC1 variants. Unlike the nanomolar inhibition at ASIC1a and rodent ASIC1b, we find mambalgin-3 only weakly inhibits human ASIC1b and ASIC1b/3 under severe acidosis, but potentiates currents under mild/moderate acidosis. Our data highlight the importance of understanding the activity of potential ASIC-targeting pharmaceuticals at human channels.

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Computational and Functional Mapping of Human and Rat α6β4 Nicotinic Acetylcholine Receptors Reveals Species-Specific Ligand-Binding Motifs.

Nicotinic acetylcholine receptors (nAChRs) are pharmacological targets for the treatment of neuropathic pain, and the α6β4 subtype has been identified as particularly promising. Rat α6β4 nAChRs are less sensitive to some ligands than the human homologue potentially complicating the use of rodent α6β4 receptors for screening therapeutic compounds. We used molecular dynamics simulations coupled with functional assays to study the interaction between α-conotoxin PeIA and α6β4 nAChRs and to identify key ligand-receptor interactions that contribute to species differences in α-conotoxin potency. Our results show that human and rat α6β4 nAChRs have distinct ligand-binding motifs and show markedly different sensitivities to α-conotoxins. These studies facilitated the creation of PeIA-5667, a peptide that shows 270-fold higher potency for rat α6β4 nAChRs over native PeIA and similar potency for the human homologue. Our results may inform the design of therapeutic ligands that target α6β4 nAChRs for the treatment of neuropathic pain.

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Co-expression gene modules involved in cisplatin-induced peripheral neuropathy according to sensitivity, status and severity.

Chemotherapy-induced peripheral neuropathy (CIPN) is among the most disabling and frustrating problems for cancer survivors. The neurotoxicity caused by cisplatin varies greatly among patients, and few predictors of appearance, duration of symptoms, susceptibility, or severity are available. A deeper understanding of the mechanisms underlying individual differences in status, severity, or sensitivity in response to cisplatin treatment is therefore required. By analyzing the GSE64174 gene expression profile and constructing a weighted gene co-expression network analysis (WGCNA) network, we screened gene modules and hub genes related to CIPN status, severity and sensitivity. We first identified the transcriptome profile of mouse dorsal root ganglion (DRG) samples and transformed their genes to human DRG counterparts. We then constructed WGCNA gene modules via optimal soft-threshold power-identification and module-preservation analysis. Comprehensive analysis and identification of module hub genes were performed via functional-enrichment analysis and significant common hub genes were identified, including "Cytoscape_cytoHubba," "Cytoscape_MCODE," and "Metascape_MCODE." Brown, green, and blue modules were selected to represent CIPN sensitivity, status and severity, resepectively, via trait-module correlational analysis. Additionally, functional enrichment analysis results indicated that these three modules were associated with some crucial biological functions, such as neutrophil migration, chemokine-mediated signaling pathway, and PI3K-Akt signaling pathway. We then identified seven common hub genes via three methods, including CXCL10, CCL21, CCR2, CXCR4, TLR4, NPY1R, and GALR2, related to CIPN status, severity and sensitivity. Our results provide possible targets and mechanism insights into the development and progress of CIPN, which can guide further transformation and pre-clinical research.

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Development of AITC-induced dermal blood flow as a translational in vivo biomarker of TRPA1 activity in human and rodent skin.

Develop a translational assay of Transient Receptor Potential Ankyrin 1 (TRPA1) activity for use as a preclinical and clinical biomarker.

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