(T1530-03-15) Dual Immunomodulatory and Neuroprotective Effects of Urea-Based Soluble Epoxide Hydrolase Inhibitor

Purpose: Chronic inflammation is increasingly recognized as a key driver in the progression of neurological disorders such as multiple sclerosis (MS) and Alzheimer’s disease (AD) [1]. Targeting inflammatory pathways within the brain microenvironment offers a promising therapeutic strategy for mitigating disease progression and symptom severity. Soluble epoxide hydrolase (sEH) is a critical enzyme involved in the degradation of anti-inflammatory epoxyeicosatrienoic acids (EETs) and its inhibition promotes inflammation resolution [2]. Meanwhile, restoring dopaminergic signaling, for example, via dopamine transporter (DAT) inhibition, has also been linked to reduced neuroinflammation. In this context, we have evaluated UR 1-1-8, a novel dual acting DAR/sEH inhibitor, designed for CNS application. Previous studies demonstrated that UR 1-1-8 exhibits strong anti-inflammatory effects, attenuates motor deficits, and reduces pain behaviors in the experimental autoimmune encephalomyelitis (EAE) model that mimics clinical features of MS [3]. In this current study we further evaluate anti-inflammatory and immunomodulatory effects of UR-1-1-8 in vitro. Based in its dual mechanism, we hypothesize it promotes M2 polarization, while protecting cells from stress-induced damage. Ongoing experiments assess changes in cytokine profiles, macrophage phenotype markers, and neuronal health following LPS stimulation and/or oxygen-glucose deprivation (OGD), providing insight into the compound’s dual mechanism in regulating immune responses and protecting neural tissue.

Methods: Inhibition of human sEH was confirmed using a cell-free fluorescence enzymatic assay. We employed murine macrophage cell lines (RAW 264.7) to evaluate the effects on macrophage polarization, immune resolution, and cytokine production. Macrophages were pretreated with compounds for 0.5 hr and then polarized toward either M1 phenotype using lipopolysaccharide LPS (1 µg/mL) or M2 phenotype using IL-4 and IL-13 (20 ng/mL each) for 3–24 hours. Polarization was evaluated via flow cytometry and qPCR for markers such as CD86, CD206, IL-6, TNF-α, IL-1β, iNOS, Arg-1, YM1, and IL-10. For neuroprotective activity of our compound, we used the human pluripotent stem cells (iPSC) derived neurons. The iPSCs were differentiated into neuronal lineage over 14 days using Neurobasal medium supplemented with BDNF and additional neurotrophic factors. Following differentiation, cells were treated with UR-1-1-8 (1 µM) for 22 hours prior to 2 hours of oxygen-glucose deprivation (OGD). Neuronal marker βIII-tubulin expression was assessed by immunocytochemistry to evaluate neuronal maturation and structural preservation post-treatment. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test and Student's t-test.

Results: UR-1-1-8 effectively inhibited sEH activity, with a Ki of less than 50 nanomolar. The compound suppressed M1 macrophage polarization, promoting a resolving, anti-inflammatory phenotype. Moreover, pro-inflammatory cytokines were significantly reduced, highlighting the compound’s anti-inflammatory potential.
Treatment with UR-1-1-8 (1 µM) significantly enhanced βIII-tubulin expression in iPSC-derived neurons compared to control (vehicle: DMSO). Immunocytochemistry revealed increased neurite density and length, suggesting improved neuronal differentiation and structural integrity under ischemic-like conditions. These findings indicate a protective and pro-neurogenic effect of UR-1-1-8 following OGD.

Conclusion: These findings highlight UR-1-1-8 as a promising dual DAT/sEH inhibitor with potent anti-inflammatory, immunomodulatory, and neuroprotective properties. By modulating macrophage polarization and enhancing neuronal survival under stress, UR-1-1-8 demonstrates therapeutic potential in neuroinflammatory contexts. Future studies will focus on evaluating its effects on microglial cells and further assess its efficacy in in vivo models of MS and Alzheimer’s disease, supporting its development as a next-generation treatment for CNS disorders driven by chronic inflammation.

References: 1. Adamu, A., Li, S., Gao, F., & Xue, G. (2024). The role of neuroinflammation in neurodegenerative diseases: Current understanding and future therapeutic targets. Frontiers in aging neuroscience, 16, 1347987.
2. Shan J, Hashimoto K. Soluble Epoxide Hydrolase as a Therapeutic Target for Neuropsychiatric Disorders. Int J Mol Sci. 2022 Apr 29;23(9):4951. doi: 10.3390/ijms23094951. PMID: 35563342; PMCID: PMC9099663.
3. Ashraf-Uz-Zaman M, Ji G, Tidwell D, Yin L, Thakolwiboon S, Pan J, Junell R, Griffin Z, Shahi S, Barthels D, Sajib MS, Trippier PC, Mikelis CM, Das H, Avila M, Neugebauer V, German NA. Correction to "Evaluation of Urea-Based Inhibitors of the Dopamine Transporter Using the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis". ACS Chem Neurosci. 2022 Nov 2;13(21):3138. doi: 10.1021/acschemneuro.2c00459. Epub 2022 Oct 20. Erratum for: ACS Chem Neurosci. 2022 Jan 19;13(2):217-228. doi: 10.1021/acschemneuro.1c00647. PMID: 36264675.


Figure 1. UR-1-1-8 effect on macrophage polarization: A.) Flow cytometry data of M1 marker (CD-86): RAW 264.7 cells were pretreated for 0.5 hr with UR-1-1-8 (1 µM) or DMSO (control and LPS); followed by LPS (1ug/mL) exposure for 24 hrs. B,C.) Gene expression of M1 markers of RAW 264.7 cells pretreated (B-0.5 hr pretreatment, 3 hrs LPS exposure) and post-treatment (C-3hrs LPS followed by 24 hrs compound exposure). (*P < 0.05, **P < 0.01,***P < 0.001 , mean ± SEM, n = 3).


Figure 2. UR-1-1-8 increased neurite outgrowth and βIII-tubulin expression: (A) Immunofluorescence images of iPSC-derived neurons stained for βIII-tubulin (green) and nuclei (DAPI, blue) after vehicle or UR-1-1-8 (1 µM) treatment 22 hrs prior to 2 hrs of OGD. (B) Quantification of βIII-tubulin intensity (mean ± SEM, n=3). *p < 0.05. scale bar = 100 µm.