Inherent Fluorescence Demonstrates Immunotropic Properties for Novel Janus Kinase 3 Inhibitors.

There is a general question in small molecule pharmacology about how apparent compound concentrations in blood, plasma, and organs actually relate to actual amounts at the target site of a compound. In this study, we used inherently fluorescent JAK3 ligands and their macrolide conjugates to investigate the relationship between physical properties, apparent bulk concentration, and organ and subcellular distribution. In vitro uptake into immune cells suggested that much of the substance was associated with granules or organelles. Samples from murine pharmacokinetic studies were analyzed by both conventional mass spectrometry and cryofluorescence microscopy methods to show the distribution of a compound within organs and cells without artifacts of fixation. These observations confirm the uptake of granules observed in vitro. Data from macrolides carrying either a coumarin fluorophore or a JAK3 inhibitor were similar, suggesting that the distribution is directed by the properties of the larger macrolide. These data show a propensity for azalide macrolides to concentrate in the lung and gut epithelia and suggest that the plasma- or whole-blood-derived estimates of drug levels almost certainly underestimate concentrations of macrolides in the mucous membranes. Thus, their apparent efficacy at sub-bacteriostatic doses may reflect their higher levels in barrier layers.

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

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.

Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells.

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.

Synthesis and structure­activity­relationship of 3,4­Diaryl­1H­pyrrolo[2,3­b]pyridines as irreversible Inhibitors of mutant EGFR­L858R/T790M.

The epidermal growth factor receptor (EGFR) is a well­validated drug target for the treatment of non­small cell lung cancer. Here we present an optimization approach and preliminary structure­activity relationship for 1H­pyrrolo[2,3­b]pyridines as covalent irreversible mutant EGFR inhibitors. We synthesized a focused library to investigate the effect of different aromatic substituents in the 4­position of this scaffold, interacting with the gatekeeper. We determined the activity of the synthesized compounds mutant EGFR enzyme assays and determined the selectivity over the wild type.