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Research Area
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Description
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Solid tumours,Breast cancer |
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Biological Activity
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Description
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Bosutinib (SKI-606) is a novel, dual Src/Abl inhibitor with IC50 of 1.2 nM and 1 nM, respectively. |
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Targets
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Src |
Abl |
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IC50 |
1.2 nM [1] |
1 nM [2] |
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In Vitro
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Bosutinib is selective for Src over non-Src family kinases with an IC50 of 1.2 nM, and potently inhibits Src-dependent cell proliferation with an IC50 of 100 nM. [1] Bosutinib significantly inhibits the proliferation of Bcr-Abl-positive leukemia cell lines KU812, K562, and MEG-01 but not Molt-4, HL-60, Ramos, and other leukemia cell lines, with IC50 of 5 nM, 20 nM and 20 nM, respectively, more potently than that of STI-571. Similar to STI-571, Bosutinib displays antiproliferative activity against the Abl-MLV-transformed fibroblasts with IC50 of 90 nM. Bosutinib ablates tyrosine phosphorylation of Bcr-Abl and STAT5 in CML cells and of v-Abl expressed in fibroblasts at the concentration of ~50 nM, 10-25 nM and 200 nM, respectively, leading to the Bcr-Abl downstream signaling inhibition of Lyn/Hck phosphorylation. [2] Although unable to inhibit the proliferation and survival of breast cancer cells, Bosutinib significantly decreases the motility and invasion of breast cancer cells with IC50 of ~250 nM, involved with an increase in cell-to-cell adhesion and membrane localization of β-catenin. [3] |
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In Vivo
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Bosutinib (60 mg/kg/day) is active against Src-transformed fibroblasts xenografts and HT29 xenografts in nude mice with T/C of 18% and 30%, respectively. [1] Oral administration of Bosutinib for 5 days significantly suppresses K562 tumor growth in mice in a dose-dependent manner, with the large tumors eradicated at dose of 100 mg/kg and tumor free at 150 mg/kg without overt toxicity. [2] As being inactive against Colo205 xenografts in nude mice at 50 mg/kg twice daily, Bosutinib dosing at 75 mg/kg twice daily is necessary against Colo205 xenografts, and increasing the dose of Bosutinib has no additional benefit, in contrast to the significant dose-dependent ability against HT29 xenografts. [4] |
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Clinical Trials
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A Phase I study to compare the Bosutinib clinical tablet and clinical capsule and to investigate food effect on Bosutinib commercial formulation in healthy subjects has been completed. |
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Features
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Combination Therapy
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Description
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The combination of Bosutinib and Imatinib produces a synergistic inhibitory effect on proliferation of Bcr-Abl-positive cell lines resistant to imatinib, although to a lesser degree than observed in imatinib-sensitive lines. [5] Bosutinib in combination with ABT-263 has synergistic effects on inducing the anoikis of LC-KJ and HCC827 cells. [6] |
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Protocol
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Kinase Assay
[1]
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| The Src and Abl kinase assays |
The Src kinase activity is measured in an ELISA format. Src (3 units/reaction), reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM EGTA, 0.5 mM Na3VO4) and cdc2 substrate peptide are added to various concentration of Bosutinib and incubated at 30 °C for 10 minutes. The reaction is started by the addition of ATP to a final concentration of 100 μM, incubated at 30 °C for 1 hour and stopped by addition of EDTA. Instructions from the manufacturer are followed for subsequent steps. The Abl kinase assay is performed in a DELFIA solid phase europium-based detection assay format. Biotinylated peptide (2 μM) is bound to streptavidin-coated microtitration plates for 1.5 hours in 1 mg/mL ovalbumin in PBS. The plates are washed for 1 hour with PBS/0.1% Tween 80, followed by a PBS wash. The kinase reaction is incubated for 1 hour at 30°C. Abl kinase (10 units) is mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 80 μM EGTA, 100 μM ATP, 0.5 mM Na3VO4, 1% DMSO, 1 mM HEPES (pH 7.0), 200 μg/mL ovalbumin and various concentration of Bosutinib. The reaction is stopped with EDTA at a final concentration of 50 mM. The reaction is monitored with Eu-labeled phosphotyrosine antibody and DELFIA enhancement solution. |
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Cell Assay
[2]
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| Cell Lines |
Abl-MLV, Rat 2, KU812, K562, and MEG-01 cells |
| Concentrations |
Dissolved in DMSO, final concentrations ~1 μM |
| Incubation Time |
72 hours |
| Methods |
Cells are exposed to various concentrations of Bosutinib for 72 hours. Anchorage-independent proliferation of Abl-MLV-transformed fibroblasts is measured in 96-well ultra-low binding plates treated with Sigmacote to block residual cell attachment. Cell proliferation is measured with MTS or Cell-Glo. For the determination of cell cycle or cell death, cells are prepared for FACS analysis in the CycleTest Plus DNA reagent kit and analyzed on a fluorescence-activated cell sorter flow cytometer. |
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Animal Study
[2]
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| Animal Models |
Nude female mice injected with K562 cells |
| Formulation |
Suspended in 0.5% methocel/0.4% Tween 80 |
| Doses |
~150 mg/kg/day |
| Administration |
Oral gavage |
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References |
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[1] Boschelli DH, et al. J Med Chem, 2001, 44(23), 3965-3977.
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[2] Golas JM, et al. Cancer Res, 2003, 63(2), 375-381.
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[3] Vultur A, et al. Mol Cancer Ther, 2008, 7(5), 1185-1194.
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[4] Golas JM, et al. Cancer Res, 2005, 65(12), 5358-5364.
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[5] Redaelli S, et al. Leukemia, 2010, 24(6), 1223-1227.
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[6] Sakuma Y, et al. Oncol Rep, 2011, 25(3), 661-667.
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