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 has an IC50 of 25 nM and vigorously suppresses the growth of FGF-stimulated WT and F384L-FGFR3-expressing B9 cells. Furthermore, dovitinib stops B9 cells that express each of the different FGFR3 activation mutants from proliferating. Interestingly, with the IC50 ranging from 70 to 90 nM for each of the different mutations, there are very few differences observed in the sensitivity of the different FGFR3 mutations to dovitinib. Dovitinib's inhibitory effect can be resisted by IL-6-dependent B9 cells that solely contain vector (B9-MINV cells) at concentrations as high as 1 μM. With IC50 values of 90 nM (for KMS11 and OPM2) and 550 nM (for KMS18), respectively, dovitinib suppresses the growth of KMS11 (FGFR3-Y373C), OPM2 (FGFR3-K650E), and KMS18 (FGFR3-G384D) cells. In primary MM cells expressing FGFR3, dovitinib causes cytotoxicity and inhibits FGF-mediated ERK1/2 phosphorylation. With 44.6% growth inhibition for cells treated with 500 nM Dovitinib and cultured on stroma compared with 71.6% growth inhibition for cells grown without BMSCs, BMSCs do confer a modest degree of resistance. With a median effective concentration (EC50) of 220 nM, dovitinib prevents the proliferation of M-NFS-60, an M-CSF growth-driven mouse myeloblastic cell line. Dovitinib treatment of SK-HEP1 cells causes a dose-dependent decrease in the number of cells, a G2/M phase arrest with a decrease in the G0/G1 and S phases, an inhibition of growth that is not dependent on anchorage, and a blockage of cell motility induced by bFGF. Dovitinib has an IC50 of roughly 1.7 μM in SK-HEP1 cells. In both SK-HEP1 and 21-0208 cells, dovitinib also significantly lowers the basal phosphorylation levels of FGFR-1, FGFR substrate 2α (FRS2-α), and ERK1/2, but not Akt. Dovitinib significantly inhibits bFGF-induced phosphorylation of FGFR-1, FRS2-α, and ERK1/2 in 21-0208 HCC cells, but not Akt.

Dovitinib causes tumors that express FGFR3 to shrink in vivo by inducing both cytotoxic and cytostatic reactions. When Target Receptor Tyrosine Kinases (RTKs) are expressed in tumor xenografts, dovitinib inhibits them in a dose- and exposure-dependent manner. The tumor growth of six HCC lines is potently inhibited by dovitinib. FGFR/PDGFRβ/VEGFR2 signaling pathway inactivation was correlated with inhibition of angiogenesis. In an orthotopic model, dovitinib markedly increased mouse survival while potently inhibiting lung metastasis and primary tumor growth. Dovitinib treatment causes large, established tumors (500–1,000 mm³) as well as notable tumor regressions and growth inhibition.

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 represents the cell viability. Densities of 5 × 103 (B9 cells) or 2 × 104 (MM cell lines) cells per well are used for seeding cells in 96-well plates. To culture the cells, different concentrations of Dovitinib are added along with 30 ng/mL aFGF, 100 μg/mL heparin, or 1% IL-6 as needed. Aliquots of 10 μL of drug or DMSO diluted in culture medium are added for each concentration of dovitinib. Drug combination studies involve incubating cells with either 100 nM Dovitinib or 0.5 μM dexamethasone, or both at the same time if necessary. 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.

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]

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Dissolved in 5 mM citrate buffer; 10, 30, or 60 mg/kg; p.o.

Female BNX mice bearing KMS11 cells

[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-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.

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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.

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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.

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Purpose: There is no standard of therapy for the treatment of Waldenström macroglobulinemia (WM), therefore there is a need for the development of new agents. Fibroblast growth factor receptor 3 (FGFR3) was shown to play a major role in several types in cancer. Dovitinib, an inhibitor of FGFR3, was effective in hematologic malignancies. In this study, we tested FGFR3 as a therapeutic target in WM and tested the effect of dovitinib on cell proliferation and apoptosis of WM cells in the context of BM microenvironment.
Methods: The expression of FGFR3 in WM cells was tested using immunofluorescence and flow cytometry. Cell signaling in response to stimulation with FGF3 and stromal cells, and its inhibition by dovitinib was performed using immunoblotting. Cell survival and cell proliferation were assessed by MTT and BrdU assays. Apoptosis was measured by detection of APO-2.7 and cleavage of caspase-3 using flow cytometry. Cell cycle was performed by PI staining of cells and flow cytometry. The combinatory effect of dovitinib with other drugs was analyzed using Calcusyn software. The effect of dovitinib was tested in vivo.
Results: FGFR3 was overexpressed in WM cells and its activation induced cell proliferation. Inhibition of FGFR3 with dovitinib decreased cell survival, increased apoptosis, and induced cell cycle arrest. Inhibition of FGFR3 by dovitinib reduced the interaction of WM to bone marrow components, and reversed its proliferative effect. Dovitinib had an additive effect with other drugs. Moreover, dovitinib reduced WM tumor progression in vivo.
Conclusion: We report that FGFR3 is a novel therapeutic target in WM, and suggest dovitinib for future clinical trial the treatment of patients with WM.[Clin Cancer Res . 2011 Jul 1;17(13):4389-99]

C27H33FN6O7

572.59

572.239

C, 59.74; H, 5.64; F, 3.94; N, 17.42; O, 13.26

852433-84-2

Dovitinib lactate;692737-80-7;Dovitinib;405169-16-6;Dovitinib lactate hydrate;915769-50-5

135985126

Solid powder

2.444

7

12

4

41

737

0

O=C(C(C)O)O.O=C1C(C2NC3C(=CC=C(N4CCN(C)CC4)C=3)N=2)=C(N)C2C(=CC=CC=2F)N1

XXLPVQZYQCGXOV-UHFFFAOYSA-N

InChI=1S/C21H21FN6O.2C3H6O3/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;2*1-2(4)3(5)6/h2-6,11H,7-10H2,1H3,(H,24,25)(H3,23,26,29);2*2,4H,1H3,(H,5,6)

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

2934.99.9001

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)