Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio

Abstract

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.

Inherent Fluorescence Demonstrates Immunotropic Properties for Novel Janus Kinase 3 Inhibitors

“…Compounds were selected for high in vitro potency (e.g., 1), pharmacokinetic properties (e.g., 2, 4, and 5), or for being macrolide conjugates of other compounds (7,8,9,10,11, and 12) (Figure 5). 7 Absorption spectra of the JAK3 inhibitor compounds and the positive control 13 were made at physiological pH 7.4 (Figure 4), 50 μM in phosphate-buffered saline.…”

“…The JAK3 ligands used here are highly selective and covalent binders. ,, In this regard, they may have served to label JAK3.…”

“…Next, we demonstrated their in vitro and in vivo efficacy in attenuating LPS-induced inflammatory reactions. 8 We now report their structure-dependent tendency to accumulate in cells of the immune system, in particular, in the lysosomal compartment and within subsections of organ tissues, e.g., epithelial layers.…”

“…The advantageous pharmacokinetic properties of macrolides (particularly azalides), including their abundance in immune cells, can be transferred to other pharmacologically active compounds by conjugation. Carriers based on modified macrolide scaffolds have already been successfully linked to active “warheads” that retained their activity, while now also possessing improved distribution profiles. ,,, This method is especially useful for anti-inflammatory drugs due to the aforementioned accumulation in acidic compartments and tissues. Furthermore, macrolides themselves are capable of nonspecific immune modulation, leading to potential synergistic effects. – …”