Seletalisib (UCB-5857)

Seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate–stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. From 239 kinases screened, seletalisib at a concentration of 10 μM showed no inhibitory activity greater than 47% (MAP4K4) against non-PI3K kinase enzymes. Against nonkinase enzymes, seletalisib showed weak activities against phosphodiesterase (PDE)3A, PDE2A1, and PDE4D2, with inhibition varying between 32 and 74% at 10 μM. When screened at a concentration of 10 μM against 55 receptors and ion channels, the highest inhibitory activity of seletalisib observed was 20%. One receptor, neuropeptide Y receptor (Y1) showed 54% activation. In vitro receptor binding and enzyme assays across a broad range of target classes showed that seletalisib is selective for PI3Kδ. Seletalisib potently inhibited the phosphorylation of AKT following anti-IgM stimulation of the BCR on Ramos cells with an IC50 of 15 nM^[1]. When profiled in a wide range of primary cell assay systems, including fibroblasts, epithelial, endothelial and vascular smooth muscle cells, seletalisib showed significant activity only in those systems containing lymphocytes, demonstrating its functional selectivity towards PI3Kδ-expressing cells^[2].

Seletalisib shows dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Analysis of the relationship between inhibition of IL-2 release and seletalisib blood concentration, using combined data across experiments, demonstrated that seletalisib has potent in vivo effects with an estimated IC50 value of <10 nM^[1]. In first-in-man studies, mean seletalisib plasma concentration-time profiles increased with increasing dose after single and multiple dosing, with no major deviations from dose proportionality. There was no unexpected accumulation or loss of exposure after multiple dosing (time-independent pharmacokinetic (PK) profile) and apparent t1/2 values (approx. 20h) were supportive of once-daily dosing^[2].

• In Vitro Kinase Activity Assays and Off-Target Profiling

  The activities of seletalisib and the pan-PI3K inhibitor UCB1370037 were tested in biochemical kinase assays of the PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ (manufactured in-house) isoforms. Competitive time-resolved (TR) fluorescence resonance energy transfer (FRET) assays using a four-step PI3K assay reagent kit were performed. In this assay, PI3K activity is measured by the detection of phosphatidylinositol triphosphate (PIP3) as follows: A complex is formed between biotinylated PIP3 and a glutathione S-transferase (GST)-tagged pleckstrin homology–domain protein, which are detected by streptavidin-allophycocyanin (APC) and europium-labeled anti-GST antibody, respectively. Proximity of the two fluorophores results in a stable TR-FRET signal. The production of unlabeled PIP3 by PI3K in the kinase reaction results in competitive inhibition of the complex and therefore the FRET signal. Briefly, 2 μl of compound was added to wells containing 8 μl of a mixture containing phosphatidylinositol bisphosphate (PIP2) substrate and ATP, before addition of 10 μl of PI3K (prediluted to the required concentration). The final assay concentrations of ATP and PIP2 substrate were 2 μM and 10 μM, respectively. The final assay concentration of PI3K enzymes was 1–8 nM depending on batch activity. The final assay concentration of DMSO was 2.0%. The plate was incubated at room temperature for 30 minutes before addition of stop buffer and detection buffer according to manufacturer instructions, and the plate was then incubated for a further 6 hours at room temperature. TR-FRET measurements were performed on an Analyst GT plate reader. The ATP concentration dependence of the activity of seletalisib against PI3Kδ was established by running the assay in the presence of varying ATP concentrations (2, 40, 200, and 1000 μM). The final assay concentrations of PI3Kδ and PIP2 substrate in these assays were 1 nM and 25 μM, respectively.

• Cell lines

  Ramos B-cell line

• Concentrations

  0.1 nM-10 μM

• Incubation Time

  10 min

• Method

  Serially diluted seletalisib and goat anti-human F(ab)2 IgM in serum-free RPMI 1640 were added to Ramos cells plated in serum-free RPMI 1640. The plate was incubated for 10 minutes, chilled on ice, and the cells pelleted by centrifugation. Cellular phosphorylated AKT was detected by MSD assay.

• Animal Models

  Adult Lewis rats (male, 6-8 weeks of age)

• Dosages

  0.1-10 mg/kg in 500 μl volume

• Administration

  via oral gavage