Abstract
Dimethyl fumarate (DMF) is approved as a treatment for multiple sclerosis (MS), however, its mode of action remains unclear. One hypothesis proposes that Michael addition to thiols by DMF, notably glutathione is immunomodulatory. The alternative proposes that monomethyl fumarate (MMF), the hydrolysis product of DMF, is a ligand to the fatty acid receptor GPR109A found in the lysosomes of immune cells. We prepared esters of MMF and macrolides derived from azithromycin, which were tropic to immune cells by virtue of lysosomal trapping. We tested the effects of these substances in an assay of response to Lipopolysaccharide (LPS) in freshly isolated human peripheral blood mononuclear cells (PBMCs). In this system, we observed that the 4′′ ester of MMF (compound 2 and 3) reduced levels of Interleukins (IL)-1β, IL-12 and tumor necrosis factor alpha (TNFα) significantly at a concentration of 1 µM, while DMF required about 25 µM for the same effect. The 2′ esters of MMF (compound 1 and 2) were, like MMF itself, inactive in vitro. The 4′′ ester formed glutathione conjugates rapidly while the 2′ conjugates did not react with thiols but did hydrolyze slowly to release MMF in these cells. We then tested the substances in vivo using the imiquimod/isostearate model of psoriasis where the 2′ ester was the most active at 0.06–0.12 mg/kg (approximately 0.1 µmol/kg), improving skin score, body weight and cytokine levels (TNFα, IL-17A, IL-17F, IL-6, IL-1β, NLRP3 and IL-23A). In contrast, the thiol reactive 4′′ ester was less active than the 2′ ester while DMF was ca. 300-fold less active. The thiol reactive 4′′ ester was not easily recovered from either plasma or organs while the 2′ ester exhibited conventional uptake and elimination. The 2′ ester also reduced levels of IL-6 in acute monosodium urate (MSU) induced inflammation. These data suggest that mechanisms that are relevant in vivo center on the release of MMF. Given that GPR109A is localized to the lysosome, and that lysosomal trapping increases 2′ ester activity by > 300 fold, these data suggest that GPR109A may be the main target in vivo. In contrast, the effects associated with glutathione (GSH) conjugation in vitro are unlikely to be as effective in vivo due to the much lower dose in use which cannot titrate the more concentrated thiols. These data support the case for GPR109A modulation in autoimmune diseases.
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Acknowledgements
We would like to thank colleagues from Synovo GmbH and the University of Tübingen who assisted in this research. Special thanks to the members of the in vivo facility and the team of the analytics/bioanalytics department at both institutions.
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All authors have read and agreed to the published version of the manuscript. Conceptualization, SS, TLS and MB; methodology, SS, NC, TLS and MB; validation, SS and TLS; formal analysis, SS and TLS; investigation, JG, SS, TLS, LDO, NS, SG and AS; resources, MB and SL; writing—original draft preparation, SS; writing—review and editing, TLS, MB and JHG; visualization, SS; supervision, MB, JHG and SL; project administration, MB; funding acquisition, MB.
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S.S., J.G., N.C., M.M., S.G., A.S., N.S., L.D.O., J.H.G., T.L.S. are employees of Synovo GmbH. M.B. is general manager of Synovo GmbH. Synovo GmbH holds patent rights to the published structures.
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All study protocols were approved by the local Animal Care and Ethics Committee (Federal government ethics committee, Tübingen, Germany under the licenses 35/9185.81-7/SYN 06/20; 35/9185.81-7/SYN 07/18; and 35/9185.81-7/SYN 11/19). Human blood products used in the in vitro assays (used for cell stimulation, viability, stability and uptake assays) were obtained from the center for transfusion medicine in Tübingen, Germany (Zentrum für Klinische Transfusionmedizin Tübingen GmbH, (ethical approval number ZKT-FoPro202106-2305-01 and ZKT-FoPro202012-2211)).
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Straß, S., Geiger, J., Cloos, N. et al. Immune cell targeted fumaric esters support a role of GPR109A as a primary target of monomethyl fumarate in vivo. Inflammopharmacol 31, 1223–1239 (2023). https://doi.org/10.1007/s10787-023-01186-0
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DOI: https://doi.org/10.1007/s10787-023-01186-0