Neutrophil Formyl Peptide Receptor-1 Activation Triggers Endogenous Opioid Release for Local, Non-addictive Analgesia 2260008

Abstract

Abstract Introduction Pain remains a major clinical challenge; while opioid analgesics are effective, their use is constrained by tolerance, dependence, and respiratory depression. Immune cells both sense injury and secrete analgesic mediators at inflamed sites, enabling local modulation with minimal systemic exposure. Among these mediators, endogenous opioid peptides (e.g., Met-enkephalin) can provide potent peripheral analgesia without central nervous system toxicity. Activation of the Formyl Peptide Receptor-1 (FPR1) on neutrophils by bacterial peptides such as fMLF initiates a Phosphoinositide 3-Kinase (PI3K)-driven signaling cascade that mobilizes intracellular Ca²+, triggering Met-enkephalin release. We hypothesized that activating FPR1 on neutrophils would physiologically trigger Met-enkephalin release, enabling localized, non-addictive analgesia. Methods A library of 140 small-molecule FPR1 agonists was designed and synthesized to identify candidates capable of inducing endogenous opioid release. Compounds were screened in HL60 cells differentiated toward a neutrophil-like phenotype and in primary human neutrophils. Functional responses were quantified by intracellular Ca²+ mobilization (FPR1 signaling readout) and Met-enkephalin release measured by ELISA and LC—MS/MS. Results Lead compounds produced robust, concentration-dependent calcium flux in differentiated HL-60 cells with EC50 values ranging from 1.46—13 µM. Agonist stimulation promoted Met-enkephalin release to ∼100 pg/mL in cell-based assays. Preliminary studies in primary human neutrophils confirm agonist-evoked Ca²+ mobilization, with opioid peptide-release. Conclusion Small-molecule activation of neutrophil FPR1 can elicit endogenous opioid peptide secretion, revealing a neuroimmune pathway for site-specific analgesia. By leveraging innate immune trafficking and secretory programs, this approach may enable peripherally acting, opioid-mediated pain relief while minimizing systemic adverse effects, motivating in vivo efficacy and safety studies. Funding Source NIH Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)