Immune cell targeted fumaric esters support a role of GPR109A as a primary target of monomethyl fumarate in vivo.

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)-1ß, 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-1ß, 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.

Isostearic acid is an active component of imiquimod formulations used to induce psoriaform disease models.

Topical imiquimod based creams are indicated as immune stimulants for papillomas and various skin neoplasms. Imiquimod is considered a TLR7 ligand. These creams are also used in research to induce skin inflammation in mice as a model for psoriasis. We observed that this inflammatory response was not strictly imiquimod dependent and we set out to establish which components drive the proinflammatory effects. To this end, we examined the induction response in a BALB/cJRj mouse model, in which 50 mg of cream is applied to 2 cm2 of skin (125 mg/kg imiquimod-5% W/V, and/or 625 mg/kg isostearic acid-25% W/V). Comparing cream formulations containing isostearic acid, imiquimod and the combination, we observed that isostearic acid causes skin inflammation within 2 days, whereas imiquimod requires up to 5 days for initial signs. Isostearic acid activated an inflammasome response, stimulated release of proinflammatory cytokines and upregulated the IL-23/17 axis. Animals treated with isostearic acid had enlarged livers (+ 40% weight), which was not observed with imiquimod alone. Imiquimod was readily metabolized and cleared from plasma and liver, but was maintained at high levels in the skin throughout the body (200 mM at area of application; 200 µM in untreated skin). Imiquimod application was associated with splenomegaly, cytokine induction/release and initial body weight loss over 3 days. Despite high imiquimod skin levels throughout the animal, inflammation was only apparent in the treated areas and was less severe than in isostearic acid groups. As the concentrations in these areas are well above the 10 µM required for TLR7 responses in vitro, there is an implication that skin inflammation following imiquimod is due to effects other than TLR7 agonism (e.g., adenosine receptor agonism). In brain, isostearic caused no major changes in cytokine expression while imiquimod alone sightly stimulated expression of IL-1ß and CCL9. However, the combination of both caused brain induction of CCL3, -9, CXCL10, -13, IL-1ß and TNFα. The implication of these data is that isostearic acid facilitates the entry of imiquimod or peripherally secreted cytokines into the brain. Our data suggest that psoriaform skin responses in mice are more driven by isostearic acid, than generally reported and that the dose and route used in the model, leads to profound systemic effects, which may complicate the interpretation of drug effects in this model.

Synthesis, Characterization, and in†vivo Distribution of Intracellular Delivered Macrolide Short-Chain Fatty Acid Derivatives.

Short-chain fatty acids (SCFAs) have a range of effects in metabolism and immune regulation. We have observed that delivery of SCFAs to lysosomes has potent immune regulatory effects, possibly as a surrogate signal for the presence of anaerobic organisms. To better understand the pharmacology of lysosomal SCFA donors, we investigated the distribution and metabolism of propionate and butyrate donors. Each analog (1 a and 2 a) can donate three SCFA equivalents via ester hydrolysis through six intermediate metabolites. The compounds are stabilized by low pH, and stability in cells is usually higher than in medium, but is cell-type specific. Butyrate derivatives were found to be more stable than propionates. Tri-esters were more stable than di- or mono-esters. The donors were surprisingly stable in vivo, and hydrolysis of each position was organ specific. Jejunum and liver caused rapid loss of 4'' esters. The gut metabolite pattern by i. v. differed from that of p.o. application, suggesting luminal and apical enzyme effects in the gut epithelium. Central organs could de-esterify the 11-position. Levels in lung relative to other organs were higher by p.o. than via i. v., suggesting that delivery route can influence the observed pharmacology and that gut metabolites distribute differently. The donors were largely eliminated by 24 h, following near linear decline in organs. The observed levels and distribution were found to be consistent with pharmacodynamic effects, particularly in the gut.