Article Figures & Data
Figures
- Fig. 1.
Chemical structure of INCB040093 [(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-α]pyrimidin-5-one] and enzyme-inhibitory activity of INCB040093 against different members of the human class I PI3K family.
- Fig. 2.
INCB040093 is selective in inhibiting signaling by PI3K isoforms in cells. pAkt was induced by PDGF, LPA, C5a, and anti-IgM in NIH3T3, PC-3, RAW264.7, and Ramos cells, respectively, for PI3Kα-, PI3Kβ-, PI3Kγ-, and PI3Kδ-mediated signaling. The levels of pAkt and total Akt in the cell lysates were measured by Western blotting using an anti-pAkt (Ser473) and an anti-Akt antibody, respectively.
- Fig. 3.
INCB040093 potently inhibits constitutive PI3K signaling in Pfeiffer cells of DLBCL. The Pfeiffer cells were treated with different amount of INCB040093 for 2 hours. The levels of pAkt (Ser473) and pFOXO3a (T32)/pFOXO1 (T24) in the total cell lysates were then measured by Western blotting (A and B). The Pfeiffer cells were also spiked into human whole blood and treated with INCB040093. The level of pAkt (Ser473) in the Pfeiffer cells was evaluated by intracellular staining with an anti-pAkt (Ser473) antibody, followed by fluorescence-activated cell sorting analysis (C).
- Fig. 4.
INCB040093 induces apoptosis in DLBCL cell lines. SU-DHL-5, Pfeiffer cells, and WSU-NHL cells were treated with or without INCB040093 at 1 μM for 24 hours. The percentage of apoptotic cells was determined by annexin V-fluorescein isothiocyanate and propidium iodide staining, followed by two-color flow cytometric analysis. Percentages represent both annexin V single-positive and annexin V/propidium iodide double-positive cells.
- Fig. 5.
IL-10–induced Pfeiffer cell resistance to INCB040093 in viability assay can be overcome by inhibition of JAK. The Pfeiffer cells were treated with different amount of INCB040093 for 3 days in the absence or presence of IL-10 (10 ng/ml), INCB018424 (Ruxolitinib) (1 μM, JAK1/2 inhibitor) (A), or INCB039110 (itacitinib) (1 μM, JAK1 selective inhibitor) (B). The cell viability was assessed using a cell titer-grow assay. Phosphorylation of STAT3 induced by IL-10 (10 ng/ml), IL-6 (10 ng/ml), HS-5 stroma cell conditioned medium (SCM) in the Pfeiffer, and other DLBCL cell lines was evaluated by Western blotting (C).
- Fig. 6.
INCB040093 reduces TNF-β (A) and MIP-1β (B) production in a coculture of Hodgkin’s lymphoma cell line (HS-445) and a stroma cell line (HS-5). HS-445 and HS-5 cells were cocultured for 3 days in different ratios in the presence or absence of INCB040093 (1 μM). The levels of cytokines and chemokines in the supernatants were evaluated using a Luminex assay specific for a broad panel of human cytokines and chemokines.
- Fig. 7.
INCB040093 is effective in suppressing tumor growth in the Pfeiffer mouse model of DLBCL. The Pfeiffer cells were s.c. inoculated into the immune-compromised SCID mice. After 2–3 weeks, these cells formed tumors with volumes exceeding 100 mm3. The mice with inoculated Pfeiffer cells were randomized into treatment groups to receive twice-daily PO of vehicle or INCB040093 at 10, 30, 100, and 300 mg/kg for 16 days. Mean plasma concentration (nanomolar) of INCB040093 was measured at various times following repeated twice-daily PO in the Pfeiffer tumor xenograft model. (A) Mice bearing s.c. Pfeiffer tumors were also randomized into treatment groups that received vehicle, INCB040093 (100 mg/kg, oral twice daily), bendamustine (6 mg/kg, i.p., 4 consecutive days each week), or the combination of INCB040093 and bendamustine (B). Tumor volumes were ascertained approximately twice each week.
Tables
Primary Cell Stimulant Response IC50 (nM)a B (CD21+; canine) Anti-dog IgM Proliferation 2.1 ± 0.01 B (CD19+; human) Anti-human IgM Proliferation 2.4 ± 0.5 B (CD19+; human) LPS Proliferation 0.9 ± 0.3 B (CD19+; human) IL-4 Proliferation 3.8 ± 2.1 B (CD19+; human) IL-6 Proliferation 1.7 ± 0.3 B (CD19+; human) BAFF Proliferation 1.2 ± 0.8 B (CD19+; human) Anti-CD40 Proliferation 3.2 ± 1.0 B (CD19+; human) CD40 ligand Proliferation 2.8 ± 1.8 Basophilb Anti-Fcε receptor CD63 expression 35 ± 15 T (CD4+CD45RA+, human) Th17 cocktailc Th17 differentiation 3.9 ± 3 T (total T cellsd, human) Anti-CD3 Proliferation 2133 ± 1122 T (total T cellsd, human) Anti-CD28 Proliferation 130 ± 31 T (total T cellsd, human) Anti-CD28 and anti-CD3 Proliferation 1753 ± 632 T (CD3+, human) IL-2 Proliferation 3107 ± 1371 NK (CD56+, human) IL-2 Proliferation 708 ± 50 Neutrophil (human) fMLP Elastase exocytosis 1566 ± 545 PBMC (human) LPS TNF-α production 2704 (n = 1) Monocytes (human) CCL2 Chemotaxis >10,000 - TABLE 2
Inhibitory activity of INCB040093 against proliferation of cell lines derived from B cell malignancies
Cell Line Malignancy INCB040093a Idelalisiba Pfeiffer DLBCL 10 ± 7 8 ± 6 SU-DHL-6 DLBCL 15 ± 4 10 ± 2 WSU-NHL DLBCL 21 ± 9 12 ± 12 SU-DHL-8 DLBCL 69 ± 15 SU-DHL-5 DLBCL 71 ± 43 88 ± 4 SU-DHL-1 DLBCL >3000 >3000 SU-DHL-4 DLBCL >3000 >3000 SU-DHL-10 DLBCL >3000 SU-DHL-16 DLBCL >3000 Rec-1 MCL 23 ± 5 2.6 ± 1.7 Mino MCL 109 ± 82 13 ± 10 Jeko-1 MCL 145 ± 136 14 ± 8 HS-445 HL 90 ± 39 U-HO1 HL 407 ± 309 ↵a The data represent IC50 ± S.D. in nanomolar.
i.v. (5 mg/kg) Vss (l/kg) 1.52 ± 0.16 Clearance (l/h per kilogram) 1.42 ± 0.11 AUC0−∞ (μM × h) 8.37 ± 0.66 T1/2 (h) 5.3 ± 0.3 Dose excreted in urine (%) 0.447 ± 0.29 Renal clearance (l/h per kilogram) 0.00605 ± 0.0034 PO (10 mg/kg) Cmax (μM) 4.82 ± 1.5 Tmax (h) 0.5 AUC0−∞ (μM × h) 8.84 ± 1.5 T1/2 (h) 1.9 ± 1 % F 53.3 ± 13 AUC, area under the curve; T1/2 , half-life; Vss, mean volume of distribution.
In this issue
Characterization of the PI3Kδ Inhibitor INCB040093
