Sex Differences in Kappa Opioid Receptor Agonist Mediated Attenuation of Chemotherapy-Induced Neuropathic Pain in Mice

Chemotherapy-induced neuropathic pain is a common side effect for cancer patients which has limited effective treatment options. Kappa opioid receptor (KOR) agonists are a promising alternative to currently available opioid drugs due to their low abuse potential. In the current study, we have investigated the effects of Salvinorin A (SalA) analogues, 16-Ethynyl SalA, 16-Bromo SalA and ethyoxymethyl ether (EOM) SalB, and in a preclinical model of paclitaxel-induced neuropathic pain in male and female C57BL/6J mice. Using an acute dose-response procedure, we showed that compared to morphine, 16-Ethynyl SalA was more potent at reducing mechanical allodynia; and SalA, 16-Ethynyl SalA, and EOM SalB were more potent at reducing cold allodynia. In the mechanical allodynia testing, U50,488 was more potent in males and SalA was more potent in females. There were no sex differences in the acute cold allodynia testing. In the chronic administration model, treatment with U50,488 (10 mg/kg) reduced the mechanical and cold allodynia responses to healthy levels over 23 days of treatment. Overall, we have shown that KOR agonists are effective in a model of chemotherapy-induced neuropathic pain, indicating that KOR agonists could be further developed to treat this debilitating condition

Nalfurafine Reduces Neuroinflammation and Drives Remyelination in Models of CNS Demyelinating Disease

Objectives: Multiple sclerosis (MS) is a neurodegenerative disease characterised by inflammation and damage to the myelin sheath, resulting in physical and cognitive disability. There is currently no cure for MS, and finding effective treatments to prevent disease progression has been challenging. Recent evidence suggests that activating kappa opioid receptors (KOR) has a beneficial effect on the progression of MS. Although many KOR agonists like U50,488 are not suitable for clinical use because of a poor side-effect profile, nalfurafine is a potent, clinically used KOR agonist with a favorable side-effect profile. Methods: Using the experimental autoimmune encephalomyelitis (EAE) model, the effect of therapeutically administered nalfurafine or U50,488 on remyelination, CNS infiltration and peripheral immune responses were compared. Additionally, the cuprizone model was used to compare the effects on non-immune demyelination. Results: Nalfurafine enabled recovery and remyelination during EAE. Additionally, it was more effective than U50,488 and promoted disease reduction when administered after chronic demyelination. Blocking KOR with the antagonist, nor‐BNI, impaired full recovery by nalfurafine, indicating that nalfurafine mediates recovery from EAE in a KOR‐dependent fashion. Furthermore, nalfurafine treatment reduced CNS infiltration (especially CD4+ and CD8+ T cells) and promoted a more immunoregulatory environment by decreasing Th17 responses. Finally, nalfurafine was able to promote remyelination in the cuprizone demyelination model, supporting the direct effect on remyelination in the absence of peripheral immune cell invasion. Conclusions: Overall, our findings support the potential of nalfurafine to promote recovery and remyelination and highlight its promise for clinical use in MS

Evaluation of Biased and Balanced Salvinorin A Analogs in Preclinical Models of Pain

This work is licensed under a Creative Commons Attribution 4.0 International License.In the search for safer, non-addictive analgesics, kappa opioid receptor (KOPr) agonists are a potential target, as unlike mu-opioid analgesics, they do not have abuse potential. Salvinorin A (SalA) is a potent and selective KOPr agonist, however, clinical utility is limited by the short duration of action and aversive side effects. Biasing KOPr signaling toward G-protein activation has been highlighted as a key cellular mechanism to reduce the side effects of KOPr agonists. The present study investigated KOPr signaling bias and the acute antinociceptive effects and side effects of two novel analogs of SalA, 16-Bromo SalA and 16-Ethynyl SalA. 16-Bromo SalA showed G-protein signaling bias, whereas 16-Ethynyl SalA displayed balanced signaling properties. In the dose-response tail-withdrawal assay, SalA, 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail-withdrawal assay, and 16-Ethynyl had greater antinociceptive effect in the hot-plate assay, compared to SalA. In the intraplantar 2% formaldehyde test, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced both nociceptive and inflammatory pain-related behaviors. Moreover, 16-Ethynyl SalA and 16-Bromo SalA had no anxiogenic effects in the marble burying task, and 16-Bromo SalA did not alter behavior in the elevated zero maze. Overall, 16-Ethynyl SalA significantly attenuated acute pain-related behaviors in multiple preclinical models, while the biased KOPr agonist, 16-Bromo SalA, displayed modest antinociceptive effects, and lacked anxiogenic effects.Health Research Council – Explorer grant (16/646)National Institute on Drug Abuse (DA018151)Victoria University of Wellington doctoral scholarshi

Evaluation of neurological changes in secondary progressive multiple sclerosis patients treated with immune modulator MIS416: results from a feasibility study

Abstract Background While disease progression can be readily monitored in early stage relapsing multiple sclerosis (MS), it is more challenging for secondary progressive multiple sclerosis (SPMS). This advanced stage of disease has distinct pathophysiology due to compartmentalization of neuroinflammatory activity within the central nervous system, resulting in increased incidence and severity of cognitive dysfunction. The shift in the dominant disease pathways is underscored by the failure of relapsing therapies to benefit SPMS patients, highlighting the need for novel treatment strategies and clinical trial endpoints that are well-aligned with potential benefits. The Expanded Disability Status Scale (EDSS) is widely used but is weighted towards ambulatory ability, lacking sensitivity to other aspects of neurological impairment experienced in more severely disabled SPMS patients, so may not effectively capture their clinical status. To investigate the feasibility of an alternative clinical trial endpoint model for a phase 2B trial of an immune modulator for SPMS, the potential for treatment efficacy-based patient-centered outcomes was assessed within the context of a before and after, 12-week clinical trial of safety and tolerability. Methods Patients treated with MIS416 for 12 weeks were evaluated for clinical status at baseline and end of dosing, using the established Multiple Sclerosis Functional Composite, Short Form Health Survey, and Expanded Disability Status Scale. Responder status was determined for eight outcome measures based on minimally important change, defined using published studies. To evaluate the patients’ immune response to MIS416, blood plasma samples collected at baseline and pre- and 24-h post doses 1–4 were analyzed using multiplex cytokine quantification assays. Results Using a combination of patient-centered outcomes, MIS416 treatment was associated with improved clinical status for 10/11 patients: eight patients showed improvement on two to five outcome measures, five of which also showed improvement by EDSS. Multi-dimensional scaling analysis of MIS416-induced factors quantified in individual patients, revealed immune response patterns which had a strong concordance with the extent of the patients’ clinical response. Conclusions The data support the feasibility of using patient-centered outcomes as additional clinical trial endpoints, for determining the efficacy of disease-modifying therapies, in secondary progressive multiple sclerosis patients. Trial registration ClinicalTrial.gov, NCT0119199

Central Role for Interleukin-4 in Regulating Nitric Oxide-Mediated Inhibition of T-Cell Proliferation and Gamma Interferon Production in Schistosomiasis

Schistosoma mansoni-infected wild-type (WT) mice develop a Th2 response and chronic disease. In contrast, infected interleukin-4 double-deficient (IL-4(−/−)) mice develop a Th1-like response and an acute, lethal syndrome. Disease severity in these animals correlates with excessive and prolonged production of nitric oxide (NO) associated with enhanced antigen-driven gamma interferon (IFN-γ) production in the absence of IL-4. Strikingly, splenic lymphocytes from infected IL-4(−/−) mice failed to proliferate as well as those from infected WT mice following stimulation in vitro with antigen or anti-CD3 antibody. Contrary to antigen-driven IFN-γ responses, anti-CD3 antibody stimulation of splenocytes resulted in significantly less IFN-γ being produced by CD8 cells from infected IL-4(−/−) mice than by those from infected WT mice or normal mice. NO is largely responsible for the impaired T-cell functions in infected IL-4(−/−) mice, as inhibition of iNOS significantly enhanced proliferation and IFN-γ production

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

INTRODUCTION:In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent to which platelets can modulate neutrophil function in response to in vitro stimulation with TLR4, TLR2/1, and TLR2/6 agonists. METHODS:Neutrophils from 10 healthy individuals were cultured alone or with autologous platelets. Neutrophils ± platelets were left unstimulated or were stimulated with 1 or 100 ng/mL lipopolysaccharide (LPS; a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist) and fibroblast-stimulating lipopeptide (FSL)-1 (a TLR2/6 agonist). Neutrophil activation and phagocytic activity were assessed by flow cytometry, and elastase and interleukin-8 secretion were assessed by ELISA. RESULTS:The addition of platelets attenuated neutrophil CD66b and CD11b expression in response to various doses of Pam3CSK4 and FSL-1. Furthermore, platelet co-culture was associated with higher CD62L expression (indicating reduced CD62L shedding) in response to these TLR agonists. Platelets also reduced elastase secretion in unstimulated cultures and in response to low-dose TLR stimulation. Conversely, platelet co-culture increased neutrophil phagocytosis in unstimulated cultures and in response to low-dose Pam3CSK4 and FSL-1. Platelets also increased IL-8 secretion in response to low-dose LPS. CONCLUSION:Platelets are complex immunomodulators that can attenuate some, and simultaneously augment other, neutrophil functions. This modulation can occur both in the absence and presence of TLR stimulation

The effects of aspirin and ticagrelor on Toll-like receptor (TLR)-mediated platelet activation: results of a randomized, cross-over trial

<p>Platelet activation underlies the pathology of an acute myocardial infarction (AMI), and dual antiplatelet therapy (DAPT) is administered post-AMI to limit this activation. Platelets express Toll-like receptors (TLRs) 1, 2, and 4 and become potently activated in response to TLR2/1 and TLR4 stimulation. However, it is unknown whether antiplatelet agents can protect against platelet activation via these TLR pathways. This study aimed to determine the extent to which TLR-mediated platelet activation can be inhibited by currently used antiplatelet agents. Ten healthy subjects were enrolled into a single-blinded randomized cross-over trial. Subjects received either aspirin monotherapy or DAPT (aspirin in combination with ticagrelor) for 1 week, were washed out, and crossed over to the other drug regimen. Platelet activation was assessed in response to Pam3CSK4 (a TLR2/1 agonist) and lipopolysaccharide (LPS; a TLR4 agonist) at baseline and after each antiplatelet drug regimen. Platelet-surface expression of CD62p and PAC1 by flow cytometry was measured as markers of platelet activation. At baseline, expression of CD62p and PAC1 increased significantly in response to high-dose LPS and in a dose-dependent manner in response to Pam3CSK4. Aspirin monotherapy did not inhibit platelet activation in response to any TLR agonist tested. DAPT with aspirin and ticagrelor only modestly inhibited expression of both activation markers in response to high doses of Pam3CSK4 and LPS. However, incubation with these TLR agonists led to substantial platelet activation despite treatment with these anti-platelet agents. Platelet-TLR2/1 and platelet-TLR4 represent intact on-treatment platelet activation pathways, which may contribute to on-going platelet activation post-AMI.</p