Dovitinib (TKI-258, CHIR-258)

Preparation Method

The IC₅₀ values for the inhibition of RTKs by dovitinib were determined in a time-resolved fluorescence (TRF) or radioactive format, measuring the inhibition by dovitinib of phosphate transfer to a substrate by the respective enzyme.

The kinase domains of FGFR3, FGFR1, PDGFR-β, and VEGFR1-3 were assayed in 50mM HEPES, pH 7.0, 2mM MgCl₂, 10mM MnCl_2, 1mM NaF, 1mM DTT, 1mg/mL BSA, 0.25μM biotinylated peptide substrate (GGGGQDGKDYIVLPI), and 1 to 30μM ATP depending on the K_m for the respective enzyme. ATP concentrations were at or just below K_m. For c-KIT and FLT3 reactions the pH was raised to 7.5 with 0.2 to 8μM ATP in the presence of 0.25 to 1μM biotinylated peptide substrate (GGLFDDPSYVNVQNL). Reactions were incubated at room temperature for 1 to 4h and the phosphorylated peptide captured on streptavidin-coated microtiter plates containing stop reaction buffer (25mM EDTA, 50mM HEPES, pH 7.5). Phosphorylated peptide was measured with the DELFIA TRF system using a Europium-labeled antiphosphotyrosine antibody PT66. The concentration of dovitinib for IC₅₀ was calculated using nonlinear regression with XL-Fit data analysis software version 4.1. Inhibition of colony-stimulating factor-1 receptor (CSF-1R), PDGFR-α, insulin receptor (InsR), and insulin-like growth factor receptor 1 (IGFR1) kinase activity was determined using the IC₅₀ Profiler Express service of Upstate Biotechnology at ATP concentrations close the K_m for ATP.

Reaction Conditions

1-4h

Applications

Dovitinib inhibited members of the class III RTKs including FLT3, c-Kit, CSF-1R, and PDGFRα/β with IC₅₀ values of 0.001 to 0.21mM as assessed by in vitro kinase assays. In addition, Dovitinib potently inhibited class IV (FGFR1 and 3) and class V (VEGFR1-4) RTKs with IC₅₀ values of 0.008 to 0.013mM. When similar kinase assays for InsR, EGFR, c-Met, EphrinA2 (EphA2), Tie2, IGFR1, and HER2 were performed, significant inhibition was observed only at more than 10-fold higher concentrations.

Cell lines

PLC5, Hep3B, Sk-Hep1 and Huh-7 cells (4 HCC cell lines)

Preparation Method

Cells were exposed to dovitinib at the indicated doses (5, 10, and 15μM) for 24h and apoptotic cells were determined by flow cytometry (sub-G). DNA fragmentation was measured by cell death detection ELISA.

Reaction Conditions

5, 10, and 15μM; 24h

Applications

Dovitinib has considerable effects on growth inhibition and apoptosis in HCC cells. Dovitinib substantially increased apoptotic cell death in a dose-dependent manner (starting at 5μM) and showed similar effects on apoptosis in all tested cell lines. Dovitinib caused dose-dependent DNA fragmentation in 4 HCC cell lines.

Animal models

NOD/SCID mice bearing MKN-45 subcutaneous xenografts

Preparation Method

MKN-45 (7.5 × 10⁶) cells were injected subcutaneously into the right flank region of the mice. Ten days after tumor cell injection, mice were injected intraperitoneally with dovitinib (30mg/kg; three times weekly) for 2 weeks. The tumor size was measured twice weekly and tumor volume was determined.

Dosage form

30mg/kg; three times weekly; i.p.

Applications

Net tumor size reduction, calculated by subtracting tumor volume on therapy initiation day from that on the final day, for dovitinib was 76%, compared to control. Tumor weight was reduced by 73% with dovitinib monotherapy and mouse body weight did not change significantly.

References:
[1] TRUDEL S, LI Z H, WEI E, et al. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma[J]. Blood, 2005, 105(7): 2941-2948.
[2] TAI W T, CHENG A L, SHIAU C W, et al. Dovitinib induces apoptosis and overcomes sorafenib resistance in hepatocellular carcinoma through SHP-1–mediated inhibition of STAT3[J]. Molecular Cancer Therapeutics, 2012, 11(2): 452-463.
[3] CRAWFORD K, BONTRAGER E, SCHWARZ M A, et al. Targeted FGFR/VEGFR/PDGFR inhibition with dovitinib enhances the effects of nab-paclitaxel in preclinical gastric cancer models[J]. Cancer Biology & Therapy, 2021, 22(10/12): 619-629.