Synovo (Germany)

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 gluta- thione 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 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 IL-1β, IL-12 and 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 sub-stances in vivo using the imiquimod/isostearate model of psoriasis where the 2’ ester was the most active at 0.06 to 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 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 GSH conjugation in vitro are unlikely to be as effective in vivo due the much lower dose in use which cannot titrate the more concentrated thiols. These data support the case for GPR109A modulation in autoimmune diseases.

Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.