Janus kinase (JAK) inhibitors act at low doses (e.g., tofacitinib, 0.2–0.4 μmol/kg bid) in clinical use, suggesting an efficient underlying mode of action. We hypothesized that their effectiveness is due to their ability to raise the ratio of IL-10 to TNFα. Unlike other JAK isoforms, JAK3 is expressed mainly in hematopoietic cells and is essential for immune function. We used JAK3 selective inhibitors with preferential distribution to immune cells. Inhibition of JAK3 in human leukocytes reduced TNFα and IL-6 but maintained levels of IL-10, while pan-JAK inhibitors increased TNFα, IL-6, and IL-10. JAK1 is required for IL-10 receptor signaling, which suggests that, at exposure above the IC50 (55 nM for tofacitinib on JAK1), there is less feedback control of TNFα levels. This leads to self-limiting effects of JAK1 inhibitors and could place an upper limit on appropriate doses. In vivo, treating mice with JAK3 inhibitors before LPS administration decreased plasma TNFα and increased IL-10 above vehicle levels, suggesting that JAK3 inhibition may limit TNFα release by increasing IL-10 while leaving the IL-10 receptor functional. This mechanism should have general utility in controlling autoimmune diseases and can be conveniently observed by measuring the ratio of IL-10 to TNFα. In summary, our targeted, “leukotropic” inhibitors more effectively increased IL-10/TNFα ratios than unselective control compounds and could, therefore, be ideal for autoimmune therapy.

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 cm 2 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-1β and CCL9. However, the combination of both caused brain induction of CCL3, -9, CXCL10, -13, IL-1β 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.

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 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 substances 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.

TLR Agonists have promising activity in preclinical models of viral infection and cancer. However, clinical use is only in topical application. Systemic uses of TLR-ligands such as Resiquimod, have failed due to adverse effects that limited dose and thus, efficacy. This issue could be related to pharmacokinetic properties that include fast elimination leading to low AUC with simultaneously high cmax at relevant doses. The high cmax is associated with a sharp, poorly tolerated cytokine pulse, suggesting that a compound with a higher AUC/cmax-ratio could provide a more sustained and tolerable immune activation. Our approach was to design TLR7/8-agonist Imidazoquinolines intended to partition to endosomes via acid trapping using a macrolide-carrier. This can potentially extend pharmacokinetics and simultaneously direct the compounds to the target compartment. The compounds have hTLR7/8-agonist activity (EC50 of the most active compound in cellular assays: 75-120 nM hTLR7, 2.8-3.1 µM hTLR8) and maximal hTLR7 activation between 40 and 80% of Resiquimod. The lead candidates induce secretion of IFNα from human Leukocytes in the same range as Resiquimod but induce at least 10-fold less TNFα in this system, consistent with a higher specificity for human TLR7. This pattern was reproduced in vivo in a murine system, where small molecules are thought not to activate TLR8. We found that Imidazoquinolines conjugated to a macrolide or, substances carrying an unlinked terminal secondary amine, had longer exposure compared with Resiquimod. The kinetics of pro-inflammatory cytokine release for these substances in vivo were slower and more extended (for comparable AUCs, approximately half-maximal plasma concentrations). Maximal IFNα plasma levels were reached 4 h post application. Resiquimod-treated groups had by then returned to baseline from a peak at 1 h. We propose that the characteristic cytokine profile is likely a consequence of altered pharmacokinetics and, potentially, enhanced endosomal tropism of the novel substances. In particular, our substances are designed to partition to cellular compartments where the target receptor and a distinct combination of signaling molecules relevant to IFNα-release are located. These properties could address the tolerability issues of TLR7/8 ligands and provide insight into approaches to fine-tune the outcomes of TLR7/8 activation by small molecules.