FLT3 (IC50 = 1 nM); c-Kit (IC50 = 2 nM); FGFR1 (IC50 = 8 nM); FGFR3 (IC50 = 9 nM); VEGFR3 (IC50 = 8 nM); VEGFR1 (IC50 = 10 nM); VEGFR2 (IC50 = 13 nM); PDGFRβ (IC50 = 27 nM); PDGFRα (IC50 = 210 nM); CSF-1R (IC50 = 36 nM)

Dovitinib causes FGFR3-expressing tumors to shrink in vivo by inducing both cytostatic and cytotoxic reactions.[1] Dovitinib inhibits target receptor tyrosine kinases (RTKs) expressed in tumor xenografts in a dose- and exposure-dependent manner. Six HCC lines exhibit a potent inhibition of tumor growth by dovitinib. The deactivation of the FGFR/PDGFRβ/VEGFR2 signaling pathways is associated with the inhibition of angiogenesis. Dovitinib markedly increased mouse survival in an orthotopic model by potently inhibiting lung metastasis and primary tumor growth.[2] When dovitinib is administered, there is a noticeable reduction in the growth of tumors and a regression of large, established tumors (500-1,000 mm3).[3]

In a time-resolved fluorescence (TRF) or radioactive format, the inhibitory concentration of 50% (IC50) values for the inhibition of RTKs by dovitinib are calculated, measuring the inhibition of phosphate transfer to a substrate by the corresponding enzyme caused by dovitinib. The assay conditions for the kinase domains of FGFR3, FGFR1, PDGFRβ, and VEGFR1-3 are 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.0, 2 mM MgCl2, 10 mM MnCl2, 1 mM NaF, 1 mM dithiothreitol (DTT), 1 mg/mL of bovine serum albumin (BSA), 0.25 μM biotinylated peptide substrate (GGGGQDGKDYIVLPI), and 1 to 30 μM adenosine triphosphate (ATP), contingent on the Km corresponding to each enzyme. The concentration of ATP is at or slightly below Km. The pH is increased to 7.5 for the c-KIT and FLT3 reactions, and 0.2 to 8 μM ATP is added along with 0.25 to 1 μM biotinylated peptide substrate (GGLFDDPSYVNVQNL). The phosphorylated peptide is captured on streptavidin-coated microtiter plates containing stop reaction buffer (25 mM EDTA [ethylenediaminetetraacetic acid], 50 mM HEPES, pH 7.5) after reactions are incubated at room temperature for one to four hours. The DELFIA TRF system measures phosphorylated peptide using an antiphosphotyrosine antibody (PT66) labeled with europium. Using XL-Fit data analysis software version 4.1 (IDBS), nonlinear regression is used to calculate the concentration of dovitinib for IC50. At ATP concentrations near the ATP Km, the kinase activity of insulin receptor (InsR), PDGFRα, colony-stimulating factor-1 receptor (CSF-1R), and insulin-like growth factor receptor 1 (IGFR1) is inhibited.

The 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) dye absorbance is used to measure the viability of cells. In 96-well plates, 5 × 10³ (B9 cells) or 2 × 10⁴ (MM cell lines) cells are seeded per well. Increasing concentrations of Dovitinib are incubated with cells along with 30 ng/mL aFGF, 100 μg/mL heparin, or 1% IL-6 where indicated. Ten microliter aliquots of the drug or DMSO diluted in culture medium are added for every Dovitinib concentration. Cells are cultured with 100 nM Dovitinib, 0.5 μM dexamethasone, or both at once when specified for drug combination studies. In order to assess the impact of Dovitinib on the growth of MM cells adherent to BMSCs, 10⁴ KMS11 cells are cultured in the presence or absence of Dovitinib on 96-well plates coated with BMSCs. The incubation period for plates is 48–96 hours. 5 × 10³ M-NFS-60 cells/well are cultured with serial dilutions of Dovitinib with 10 ng/mL M-CSF and without granulocyte-macrophage colony-stimulating factor (GM-CSF) in order to evaluate the growth of M-CSF-mediated macrophage colony-growth. Using the Cell Titer-Glo Assay, cell viability is assessed after 72 hours. Every experimental condition is run through three times.

8-week-old female BNX mice bearing KMS11 cells
10, 30, or 60 mg/kg
Gavage

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Xenograft mouse model[1]
The xenograft mouse model was prepared as previously described. Briefly, 6- to 8-week-old female BNX mice obtained from Frederick Cancer Research and Development Centre were inoculated subcutaneously into the right flank with 3 × 107 KMS11 cells in 150 μL IMDM, together with 150 μL Matrigel basement membrane matrix . Treatment was initiated when tumors reached volumes of 200 mm3 at which time mice were randomized to receive 10, 30, or 60 mg/kg Dovitinib (CHIR-258) or 5 mM citrate buffer. Dosing was performed daily for 21 days by gavage. Eight to 10 mice were included in each treatment group. Caliper measurements were performed twice weekly to estimate tumor volume, using the formula: 4π/3 × (width/2)2 × (length/2). One-way analysis of variance was used to compare differences between vehicle- and CHIR-258-treated groups.

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21-0208 and SK-HEP1 cells as well as patient-derived HCC models were employed to study the antitumor effect of dovitinib. Changes of biomarkers relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry.
Results: Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced cell migration. Dovitinib inhibited basal expression and FGF-induced phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo, dovitinib potently inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated with inactivation of FGFR/PDGFR-β/VEGFR-2 signaling pathways. Dovitinib also caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, dovitinib potently inhibited primary tumor growth and lung metastasis and significantly prolonged mouse survival.
Conclusions: Dovitinib demonstrated significant antitumor and antimetastatic activities in HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.[2]

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The pharmacologic activity of Dovitinib (CHIR-258) was characterized by monitoring target modulation as well as by evaluating the antitumor and antiangiogenic effects in human colon xenograft models.
Results: CHIR-258 inhibits vascular endothelial growth factor receptor 1/2, fibroblast growth factor receptor 1/3, and platelet-derived growth factor receptor beta (PDGFRbeta) and shows both antitumor and antiangiogenic activities in vivo. Treatment of KM12L4a human colon cancer cells with CHIR-258 resulted in a dose-dependent inhibition of vascular endothelial growth factor receptor 1 and PDGFRbeta phosphorylation and reduction of phosphorylated extracellular signal-regulated kinase (ERK) levels, indicating modulation of target receptors and downstream signaling. In vivo administration of CHIR-258 resulted in significant tumor growth inhibition and tumor regressions, including large, established tumors (500-1,000 mm(3)). Immunohistochemical analysis showed a reduction of phosphorylated PDGFRbeta and phosphorylated ERK in tumor cells after oral dosing with CHIR-258 compared with control tumors. These changes were accompanied by decreased tumor cell proliferation rate and reduced intratumoral microvessel density. CHIR-258 inhibited the phosphorylation of PDGFRbeta and ERK phosphorylation in tumors within 2 hours following dosing and the inhibitory activity was sustained for >24 hours. Significant antitumor activity was observed with intermittent dosing schedules, indicating a sustained biological activity.
Conclusion: These studies provide evidence that biological activity of CHIR-258 in tumors correlates with efficacy and aids in the identification of potential biomarkers of this multitargeted receptor tyrosine kinase inhibitor. CHIR-258 exhibits properties that make it a promising candidate for clinical development in a variety of solid and hematologic malignancies.[3]

[1]. Blood . 2005 Apr 1;105(7):2941-8.

[2]. J Hepatol . 2012 Mar;56(3):595-601.

[3]. Clin Cancer Res . 2005 May 15;11(10):3633-41.

[4]. Clin Cancer Res . 2011 Jul 1;17(13):4389-99.

4-amino-5-fluoro-3-[5-(4-methyl-1-piperazinyl)-1,3-dihydrobenzimidazol-2-ylidene]-2-quinolinone is a N-arylpiperazine.
Dovitinib is an orally active small molecule that exhibits potent inhibitory activity against multiple RTKs involved in tumor growth and angiogenesis. Preclinical data show that dovitinib works to inhibit multiple kinases associated with different cancers, including acute myeloid leukemia (AML) and multiple myeloma. Chiron currently has three ongoing Phase I clinical trials for dovitinib.
Dovitinib Lactate is the orally bioavailable lactate salt of a benzimidazole-quinolinone compound with potential antineoplastic activity. Dovitinib strongly binds to fibroblast growth factor receptor 3 (FGFR3) and inhibits its phosphorylation, which may result in the inhibition of tumor cell proliferation and the induction of tumor cell death. In addition, this agent may inhibit other members of the RTK superfamily, including the vascular endothelial growth factor receptor; fibroblast growth factor receptor 1; platelet-derived growth factor receptor type 3; FMS-like tyrosine kinase 3; stem cell factor receptor (c-KIT); and colony-stimulating factor receptor 1; this may result in an additional reduction in cellular proliferation and angiogenesis, and the induction of tumor cell apoptosis. The activation of FGFR3 is associated with cell proliferation and survival in certain cancer cell types.
Dovitinib is a benzimidazole-quinolinone compound and receptor tyrosine kinase (RTK) inhibitor with potential antineoplastic activity. Dovitinib binds to and inhibits the phosphorylation of type III-V RTKs, such as vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) that promote tumor cell proliferation and survival in certain cancer cells. In addition, this agent also inhibits other members of the RTK superfamily, including fibroblast growth factor receptor 1 and 3, FMS-like tyrosine kinase 3, stem cell factor receptor (c-KIT), and colony stimulating factor receptor 1. This may further lead to a reduction of cellular proliferation and angiogenesis, and an induction of tumor cell apoptosis.

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Drug Indication
Investigated for use/treatment in multiple myeloma and solid tumors.

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Mechanism of Action
Unlike many kinase inhibitors that only target vascular endothelial growth factor (VEGF), Dovitinib inhibits receptors in the fibroblast growth factor (FGF ) pathway, as well as VEGF and platelet-derived growth factor (PDGF). FGF receptor tyrosine kinase inhibition is potentially of therapeutic significance to a group of myeloma patients whose cancer cells express high levels of surface FGF receptors.

C21H21FN6O

392.43

392.176

C, 64.27; H, 5.39; F, 4.84; N, 21.42; O, 4.08

405169-16-6

Dovitinib lactate;692737-80-7;Dovitinib dilactic acid;852433-84-2;Dovitinib-d8;1246819-84-0;Dovitinib lactate hydrate;915769-50-5

135398510

Light yellow to green yellow solid powder

1.4±0.1 g/cm3

1.691

1.59

3

6

2

29

678

0

FC1=C([H])C([H])=C([H])C2=C1C([H])=C(C(N2N([H])[H])=O)C1=NC2C([H])=C([H])C(=C([H])C=2N1[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]

PIQCTGMSNWUMAF-UHFFFAOYSA-N

InChI=1S/C21H21FN6O/c1-27-7-9-28(10-8-27)12-5-6-14-16(11-12)25-20(24-14)18-19(23)17-13(22)3-2-4-15(17)26-21(18)29/h2-6,11H,7-10H2,1H3,(H,24,25)(H3,23,26,29)

4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-quinolin-2-one

TKI-258; CHIR-258; TKI258; TKI-258; Dovitinib; 405169-16-6; CHIR-258; TKI-258; 4-Amino-5-fluoro-3-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-one; Dovitinib [INN]; Dovitinib (TKI-258, CHIR-258); Dovitinib lactate; TKI 258; CHIR258; CHIR-258; CHIR 258; Dovitinib lactate

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/kg

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 (Please use freshly prepared in vivo formulations for optimal results.)