Fig 1.
Molecular structure and In Vitro profile of INCB054828.
(A) Molecular structure of INCB054828. (B) Potency of INCB054828 against FGFR kinases. Activity of recombinant human enzymes was assayed as described at the Michaelis–Menten constant (Km) ATP for each enzyme. The mean IC50 and S.D. for 6 (FGFR4) or 8 (FGFR1, FGFR2, FGFR3, KDR) independent experiments using multiple lots of inhibitor is reported. (C) Selectivity profile of INCB054828. Biochemical IC50 values of INCB054828 for 56 kinases. Small gray circles indicate tested kinases with IC50 >10,000 nM. Among non-FGFRs, KDR and c-KIT were the only kinases inhibited with an IC50 value <1,000 nM.
Fig 2.
INCB054828 inhibits FGFR-dependent signaling pathways.
(A) KG1a or (B) RT-4 cells were treated with INCB054828 for 2 hours, lysed and subjected to immunoblotting for phospho- and total proteins in the FGFR signal transduction pathway including FGFR, ERK, FRS2, and STAT5. (C) Concentration-dependent inhibition of phospho-FGFR3 by INCB054828 in RT-112 cells was determined using a proximity ligation assay with a mouse monoclonal anti-phospho-FGFR (Y653/Y654) and rabbit anti-FGFR. Original Western blot images are shown in S1 File (S1 Raw images).
Table 1.
Growth inhibition of tumor cell lines with activation of FGFR signaling by INCB054828.
Table 2.
In vitro ADME and pharmacokinetics of INCB054828.
Fig 3.
Pharmacokinetics and pharmacodynamics of INCB054828 in the mouse.
(A) Pharmacokinetic profile of INCB054828 in the mouse. Plasma was collected over time from mice administered a single oral dose of INCB054828 for determination of total INCB054828 concentration. (B) Pharmacokinetic-pharmacodynamic analyses in KATO III tumors. Tumors and plasma were harvested from KATO III tumor-bearing mice following a single oral administration of INCB054828. Plasma was subjected to analytical analysis and phospho-FGFR2 in tumor homogenate was determined by ELISA. (C) Phosphate levels in the serum of mice following administration of INCB054828. 24 hours after a single oral administration of INCB054828 to C57BL/6 mice, blood was collected and the plasma submitted for analysis of inorganic phosphate.
Fig 4.
Efficacy of INCB054828 in tumor models with FGFR alterations.
(A) KATO III (FGFR2-amplified) gastric cancer model. Severe combined immunodeficiency mice bearing KATO III tumors were administered INCB054828 (0.03, 0.1, 0.3, or 1 mg/kg) or vehicle by gavage once daily for 10 days. The mean tumor size is plotted for each group of 8 mice. ***P < 0.001 vs vehicle. (B) KG1 (FGFROP2-FGFR1 fusion positive) AML model. Humanized NSG mice bearing KG1 tumors were administered INCB054828 (0.3 mg/kg) or vehicle by gavage once daily for 14 days. The mean tumor size is plotted for each group of 6 mice. *P < 0.05 vs vehicle by paired t-test. (C) RT-112 (FGFR3-TACC3 fusion positive) bladder carcinoma model. RNU immunocompromised rats bearing RT-112 tumors were administered INCB054828 (0.3 or 1 mg/kg) or vehicle by gavage once daily for 14 days. The mean tumor size is plotted for each group of 7 mice. **P < 0.01 vs vehicle. (D) CTG-0997 (FGFR2-TRA2B fusion positive) cholangiocarcinoma PDX model. Tumor bearing nu/nu mice were administered INCB054828 (0.3 or 1 mg/kg) or vehicle by gavage once daily for 42 days. The mean tumor size is plotted for each group of 12 mice. **P < 0.01 vs vehicle. S.E.M., standard error of the mean.