PI3K-δ and PI3K-γ Inhibition by IPI-145 Abrogates Immune Responses and Suppresses Activity in Autoimmune and Inflammatory Disease Models

SummaryPhosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and in vivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and T cell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored the therapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multiple inflammatory, autoimmune, and hematologic diseases

IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway

The transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-kappaB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IkappaB kinase homologs, IkappaB kinase-epsilon (IKKepsilon) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-kappaB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKepsilon and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response

Impact of the Smoothened inhibitor, IPI-926, on smoothened ciliary localization and Hedgehog pathway activity.

A requisite step for canonical Hedgehog (Hh) pathway activation by Sonic Hedgehog (Shh) ligand is accumulation of Smoothened (Smo) to the primary cilium (PC). Activation of the Hh pathway has been implicated in a broad range of cancers, and several Smo antagonists are being assessed clinically, one of which is approved for the treatment of advanced basal cell carcinoma. Recent reports demonstrate that various Smo antagonists differentially impact Smo localization to the PC while still exerting inhibitory activity. In contrast to other synthetic small molecule Smo antagonists, the natural product cyclopamine binds to and promotes ciliary accumulation of Smo and "primes" cells for Hh pathway hyper-responsiveness after compound withdrawal. We compared the properties of IPI-926, a semi-synthetic cyclopamine analog, to cyclopamine with regard to potency, ciliary Smo accumulation, and Hh pathway activity after compound withdrawal. Like cyclopamine, IPI-926 promoted accumulation of Smo to the PC. However, in contrast to cyclopamine, IPI-926 treatment did not prime cells for hyper-responsiveness to Shh stimulation after compound withdrawal, but instead demonstrated continuous inhibition of signaling. By comparing the levels of drug-induced ciliary Smo accumulation with the degree of Hh pathway activity after compound withdrawal, we propose that a critical threshold of ciliary Smo is necessary for "priming" activity to occur. This "priming" appears achievable with cyclopamine, but not IPI-926, and is cell-line dependent. Additionally, IPI-926 activity was evaluated in a murine tumor xenograft model and a pharmacokinetic/pharmacodynamic relationship was examined to assess for in vivo evidence of Hh pathway hyper-responsiveness. Plasma concentrations of IPI-926 correlated with the degree and duration of Hh pathway suppression, and pathway activity did not exceed baseline levels out to 96 hours post dose. The overall findings suggest that IPI-926 possesses unique biophysical and pharmacological properties that result in Hh pathway inhibition in a manner that differentiates it from cyclopamine

Hyper-responsiveness to paracrine Hh signaling was not observed in IPI-926 treated pancreatic xenografts.

<p>(A) In the MiaPaCa pancreatic xenograft model, 50% tumor growth inhibition was observed with daily oral administration of IPI-926 at 40 mg/kg (N = 12 mice per group). (B) Plasma levels of IPI-926 correlate with the degree and duration of stromal mGli1 inhibition after a single dose of IPI-926 at 40 mg/kg. * indicates a p-value <0.003. Modulation of tumor-derived hGli1 was not observed (data not shown).</p

Smo antagonists differentially localize endogenous Smo protein (red) to the PC (green).

<p>In both NIH-3T3 (A) and HEPM cells (C), 24 h treatment with either cyclopamine (CYC, 5 µM) or IPI-926 (500 nM) in the absence of Shh, results in Smo accumulation to the PC while treatment with GDC-0449 (500 nM) does not. Shh treatment results in Smo accumulation to the PC in both NIH-3T3 cells (B) and HEPM cells (D), which can be blocked by GDC-0449 but not with cyclopamine or IPI-926.</p

IPI-926 is significantly more potent than cyclopamine in cell based assays.

<p>IPI-926 inhibits Shh-induced Gli-Luciferase activity with an EC₅₀ value of 9 nM in NIH-3T3 cells (A) and 2 nM in HEPM cells (B) compared to cyclopamine which had an EC₅₀ value of 315 nM in NIH-3T3 cells (A) and 59 nM in HEPM cells (B) (N = 3). (C) IPI-926 inhibits alkaline phosphatase production resulting from Shh-induced C3H10T1/2 cell differentiation with an EC₅₀ value of 12 nM compared to 399 nM for cyclopamine (N = 2). (D) IPI-926 inhibits the binding of BODIPY-labeled cyclopamine to C3H10T1/2 cells overexpressing wild-type human Smo with an EC₅₀ value of 1 nM compared to an EC₅₀ value of 114 nM for unlabeled cyclopamine.</p

Treatment of NIH-3T3 cells with cyclopamine but not IPI-926 results in increased ciliary Smo accumulation.

<p>In the absence of Shh, NIH-3T3 cells were treated for 24 h with either cyclopamine (A) or IPI-926 (B) and the mean fluorescence intensity of ciliary Smo accumulation was calculated. A positive correlation was observed between increasing doses of cyclopamine and mean ciliary Smo fluorescence intensity. Dose responsive Smo accumulation is not observed with IPI-926. Ten µM cyclopamine yielded the maximal Smo fluorescence intensity (63%) and all other data points were normalized to this max value. Dose responsive Smo accumulation is not observed in HEPM cells treated for 24 h with either cyclopamine (C) or IPI-926 (D).</p

Treatment of NIH-3T3 cells with cyclopamine but not IPI-926 confers hyper-responsiveness to Shh stimulation.

<p>Cells were pre-treated with either cyclopamine or IPI-926 for 24 h followed by extensive washing and subsequent treatment with either media alone or media containing 100 nM rShh. Gli1 mRNA levels were assessed by qRT-PCR 21 h post stimulation. Hyper-responsiveness to rShh stimulation is observed in NIH-3T3 cells treated with high doses of cyclopamine (A) but not IPI-926 (B). Note different Y-axis scales between the 2 graphs. Identical experiments carried out in HEPM cells treated with either cyclopamine (C) or IPI-926 (D) does not result in Shh hyper-responsiveness. No Shh hyper-responsiveness was observed in C3H10T1/2 cells (E), pancreatic xenograft tumor stroma (F) or SW-872 cells (G) when pre-treated with 5 µM cyclopamine or 500 nM IPI-926.</p