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.
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.
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