Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

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Purpose Loss of therapeutic response (LOR) due to anti-drug antibodies (ADA) against tumor necrosis factor (TNF) inhibitors is common in patients with inflammatory bowel disease (IBD). We aimed to investigate whether immunomodulator comedication can reverse the immunogenic LOR to TNF inhibitors in IBD. Methods In this real-world retrospective cohort study, 123 IBD patients with neutralizing ADA to infliximab or adalimumab and concomitant subtherapeutic trough levels were screened for clinical LOR. Subsequent ADA and trough level measurements and clinical outcomes were analyzed for patients who received either immunomodulator comedication or dose intensification of infliximab or adalimumab to overcome LOR. Results Following immunogenic LOR, the initial anti-TNF regimen was optimized in 33 patients. In univariable and multivariable logistic regression analyses, immunomodulator comedication was identified as the crucial factor for regaining clinical remission and ADA clearance. Detectable trough levels (≥ 0.98 or ≥ 1.00 mg/L, respectively) had optimal predictive performance for both endpoints in receiver operating characteristics curves [area under the curve 0.86 (95% confidence interval 0.68–1.00) for regaining clinical remission, 0.87 (0.71–1.00) for ADA clearance]. Furthermore, 11/20 patients (55%) on a comedication with azathioprine or methotrexate and 2/13 patients (15%) receiving anti-TNF dose intensification exclusively (P = 0.032) exhibited ADA elimination, regain of therapeutic trough levels, and clinical remission. Regain of clinical remission alone was achieved in 17/20 (85%) patients receiving comedication and 2/13 (15%) patients receiving anti-TNF dose intensification (P = 1.6 × 10−4). Conclusion Immunogenic LOR to infliximab or adalimumab in IBD can be successfully reversed using immunomodulator comedication.

Short chain fatty acids (SCFAs) are known as metabolites produced from gut microbiome fermenting dietary fibers and resistant starch. They are regulators of the interplay between the microbiome and its host and may have a role in the pathogenesis of inflammatory bowel disease (IBD). Free SCFAs influence intestinal epithelial and immune cells (e.g. macrophages and primary monocytes) through activation of free fatty acid receptors FFAR2 and FFAR3, inhibition of histone deacetylases (HDAC) and as a source of energy. SCFAs are known to impact cytokine production and differentiation of immune cells (e.g. Tumor necrosis factor alpha (TNFa), Interleukin (IL)-10, IL-6, IL-18, IL-1ß). We investigated the role of SCFAs in dextran sulfate sodium-induced colitis and their effect on the cytokine production by primary human immune cells. Treatment with SCFAs (acetate, propionate or butyrate) stimulated release of IL-1ß and IL-18 by buffy coat leukocytes or U937 cells without causing increased expression of corresponding genes. This raises the possibility of activation via the NLRP3 (NOD-, LRR- and pyrin domain- containing protein 3) inflammasome. NLRP3 is a multimeric inflammasome complex. Once activated, NLRP3 inflammasome releases caspase 1 leading to formation of mature IL-1ß and IL-18. Recent studies have shown that IL-1ß promotes phagocytosis and clearance of bacteria and aids the gut in eliciting an effective response in early stages of IBD. We asked whether this effect is mediated via surface or lysosomal (phagosomal) receptors? To answer this question, we prepared a series (SYD010) of novel compounds which are able to accumulate in the phagolysosome of immune cells through their macrolide backbone and deliver SCFAs, bound as esters to the lumen of the activated lysosome. In vitro, together with LPS stimulation, the substances modulated secretion of TNFa, IL-1ß, IL-10 and IL-6 at concentrations about 100x lower than free SCFAs (Figure 1). When tested in a DSS colitis mouse model, the SYD010 series caused a decrease in diarrhea scoring compared to the vehicle-treated control group at a concentration of 0.1 mg/kg (Figure 2) which corresponds to a total dose of ca. 100 nmol/kg (the compounds are systemically distributed). This is lower than the known luminal concentrations of SCFAs which is in the range of 40 mM. Our underlying hypothesis is that lysosomal reception of SCFAs leads to beneficial immune modulation in colitis in so far as stimulation of IL-1ß release promotes bacterial clearance. Furthermore, that concentrative uptake to the phagolysosome leads to enhanced stimulation of these receptors leading to responses at lower ambient concentrations or doses. We are assessing this substance class as potential IBD therapeutics. Figure 1. Effect of sodium butyrate or CSY4286 (lysosomal butyrate donor) on cytokine production by U937 cells. Supernatants were harvested after 24 h (IL-6) and 48 h (IL-1ß, IL-6) incubation with the test substances and cytokines determined by ELISA. The dotted line represents levels with LPS stimulation alone. SEM was applied for error bars. Figure 2. Results from DSS-induced (2.5% in drinking water) IBD study in BALB/c mice (8 mice per group). Scoring of body weight and diarrhea score over 8 days (a and b show data from final day of study). SEM was applied for error bars.