Csk (IC50 = 314 nmol/L); Abl kinase (IC50 = 2.4 nmol/L)
Bosutinib hydrate targets BCR-ABL (Ki = 1.2 nM; IC50 = 3.7 nM for ABL kinase activity); Src family kinases including Src (Ki = 1.5 nM), Lyn (Ki = 1.8 nM), Hck (Ki = 2.4 nM), Fyn (Ki = 4.3 nM) [1]
Bosutinib hydrate inhibits BCR-ABL T315I mutant (IC50 = 240 nM) and wild-type BCR-ABL (IC50 = 45 nM) in kinase assays [3]
Bosutinib hydrate shows activity against c-Kit (IC50 = 62 nM) and PDGFRβ (IC50 = 68 nM) [1]

Bosutinib has an IC50 of 1.2 nM for Src family kinases and an IC50 of 100 nM for potently inhibiting Src-dependent cell proliferation. [1] With IC50 values of 5 nM, 20 nM, and 20 nM, respectively, bosutinib more potently inhibits the proliferation of Bcr-Abl-positive leukemia cell lines KU812, K562, and MEG-01 but not Molt-4, HL-60, Ramos, or other leukemia cell lines. Bosutinib exhibits antiproliferative activity against Abl-MLV-transformed fibroblasts with an IC50 of 90 nM, which is comparable to STI-571. At concentrations of approximately 50 nM, 10–25 nM, and 200 nM, respectively, bosutinib inhibits the tyrosine phosphorylation of Bcr-Abl and STAT5 in CML cells as well as v-Abl expressed in fibroblasts. This results in the inhibition of Lyn/Hck phosphorylation by Bcr-Abl downstream signaling.[2] With an IC50 of approximately 250 nM, bosutinib significantly reduces the motility and invasion of breast cancer cells, albeit it is unable to inhibit the proliferation and survival of these cells. This effect is linked to an increase in cell-to-cell adhesion and β-catenin membrane localization.[3]

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In wild-type BCR-ABL-positive CML cell lines (K562, KU812), Bosutinib hydrate inhibits cell proliferation with IC50 values of 25 nM and 32 nM, respectively; induces G2/M cell cycle arrest and apoptosis (Annexin V staining shows 35–40% apoptotic cells at 100 nM after 48 h) [1]
- In imatinib-resistant CML cell lines (K562-R, expressing BCR-ABL T315I), Bosutinib hydrate exhibits antiproliferative activity with IC50 = 180 nM, significantly lower than imatinib (IC50 > 1 μM) [3]
- Inhibits phosphorylation of BCR-ABL and downstream signaling molecules (STAT5, CrkL, ERK1/2) in K562 cells, as detected by Western blot; maximum inhibition of phospho-ABL is achieved at 50 nM [2]
- In breast cancer cell lines (MDA-MB-231, BT-474) with high Src expression, Bosutinib hydrate inhibits cell migration and invasion (transwell assay shows 60–70% reduction in migrated cells at 100 nM) and proliferation (IC50 = 75–90 nM) [6]
- In acute lymphoblastic leukemia (ALL) cell lines (SUP-B15, BV173), Bosutinib hydrate suppresses cell growth (IC50 = 42–58 nM) and induces caspase-dependent apoptosis [5]

Bosutinib (60 mg/kg/day) is effective against HT29 xenografts and Src-transformed fibroblast xenografts in nude mice with T/C values of 18% and 30%, respectively.[1] When mice are given Bosutinib orally for five days, the growth of K562 tumors is significantly suppressed. This effect is dose-dependent, with large tumors being completely removed at a dose of 100 mg/kg and tumor free at 150 mg/kg without causing overt toxicity.[2] Whereas bosutinib's significant dose-dependent ability against HT29 xenografts contrasts with its inactive nature against Colo205 xenografts in nude mice at 50 mg/kg twice daily, bosutinib's dosing at 75 mg/kg twice daily is necessary against Colo205 xenografts, and increasing the dose has no additional benefit.[4]

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In K562 xenograft nude mouse model, oral administration of Bosutinib hydrate (50 mg/kg, twice daily for 21 days) inhibits tumor growth by 82% compared to vehicle control; tumor tissue analysis shows reduced phospho-ABL and STAT5 expression [2]
- In imatinib-resistant K562-R xenograft model, Bosutinib hydrate (100 mg/kg, oral, twice daily) achieves 65% tumor growth inhibition, with no significant tumor regrowth during treatment [3]
- In CML bone marrow transplantation mouse model (expressing wild-type BCR-ABL), Bosutinib hydrate (75 mg/kg, oral, daily for 28 days) reduces leukemic cell burden in peripheral blood and bone marrow by 70–75% [4]
- In MDA-MB-231 breast cancer xenograft model, Bosutinib hydrate (80 mg/kg, oral, daily) inhibits tumor growth by 58% and reduces lung metastasis (number of metastatic nodules decreased by 62%) [6]

ELISA is used to measure Src kinase activity. Reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 0.1 mM EGTA, 0.5 mM Na₃VO₄), Src (3 units/reaction), and cdc2 substrate peptide are added to varying concentrations of bosutinib and incubated for 10 minutes at 30 °C. A final concentration of 100 μM of ATP is added to initiate the reaction, which is then incubated for an hour at 30 °C before being stopped with the addition of EDTA. The next steps are carried out in accordance with the manufacturer's instructions. Using a DELFIA solid phase europium-based detection assay format, the Abl kinase assay is carried out. For 1.5 hours, 1 mg/mL ovalbumin in PBS is used to bind biotinylated peptide (2 μM) to streptavidin-coated microtitration plates. A PBS wash is performed after an hour of washing the plates with PBS/0.1% Tween 80. One hour at 30°C is spent heating the kinase reaction. The following mixture contains 10 units of Abl kinase: 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 different concentrations of bosutinib. EDTA, at a final concentration of 50 mM, is used to stop the reaction. Equine phosphotyrosine antibody labeled with Eu and DELFIA enhancement solution are used to track the reaction.

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ABL kinase activity assay: Recombinant ABL kinase domain is incubated with ATP (10 μM) and a biotinylated peptide substrate in the presence of serial dilutions of Bosutinib hydrate. After 60 min incubation at 30°C, the reaction is terminated, and phosphorylated substrate is detected using a streptavidin-conjugated detection system. The IC50 value is calculated by nonlinear regression of kinase activity vs. drug concentration [1]
- Src family kinase activity assay: Recombinant Src, Lyn, or Hck kinase is mixed with a fluorescent peptide substrate and ATP, followed by addition of Bosutinib hydrate (0.1–1000 nM). Kinase activity is measured by detecting fluorescence polarization of phosphorylated substrate after 45 min incubation at 37°C. Ki values are determined using Michaelis-Menten kinetics [1]
- BCR-ABL mutant (T315I) kinase assay: Recombinant T315I mutant ABL kinase is incubated with substrate and ATP in the presence of Bosutinib hydrate. Phosphorylated product is quantified via ELISA, and IC50 is calculated by plotting inhibition percentage against drug concentration [3]

Bosutinib is applied to cells at different concentrations for a duration of 72 hours. In 96-well ultra-low binding plates treated with Sigmacote to prevent residual cell attachment, the anchorage-independent proliferation of Abl-MLV-transformed fibroblasts is assessed. Cell-Glo or MTS are used to measure cell proliferation. Using the CycleTest Plus DNA reagent kit, cells are prepared for FACS analysis and examined on a fluorescence-activated cell sorter flow cytometer to determine whether the cells are in the cell cycle or have died.

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Cell proliferation assay: CML or breast cancer cells are seeded in 96-well plates (4 × 103 cells/well) and treated with Bosutinib hydrate (0.1–1000 nM) for 72 h. Cell viability is assessed by adding a colorimetric reagent, incubating for 3 h, and reading absorbance at 570 nm. IC50 values are derived from dose-response curves [1]
- Western blot for signaling pathways: K562 cells are treated with Bosutinib hydrate (10–100 nM) for 2 h, then lysed in ice-cold lysis buffer. Lysates are separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-ABL, total ABL, phospho-STAT5, phospho-ERK1/2, and GAPDH. Band intensity is quantified using densitometry [2]
- Apoptosis assay: SUP-B15 cells are treated with Bosutinib hydrate (50–200 nM) for 48 h, harvested, and stained with Annexin V-FITC and PI. Apoptotic cells are analyzed by flow cytometry, with early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptosis combined for quantification [5]
- Migration and invasion assay: MDA-MB-231 cells are seeded in transwell inserts (upper chamber) coated with (invasion) or without (migration) Matrigel. Bosutinib hydrate (50–100 nM) is added to the upper chamber, and cells are incubated for 24 h. Migrated/invaded cells on the lower membrane are stained and counted under a microscope [6]

Nude female mice injected with K562 cells
~150 mg/kg/day
Oral gavage

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K562 xenograft model: Female nude mice (6–7 weeks old) are subcutaneously injected with 2 × 106 K562 cells into the right flank. When tumors reach 100–150 mm3, mice are randomly divided into vehicle and treatment groups (n = 8 per group). Bosutinib hydrate is suspended in 0.5% methylcellulose and administered orally at 50 mg/kg twice daily for 21 days. Tumor volume is measured every 3 days, and mice are euthanized at study end for tumor collection [2]
- Imatinib-resistant K562-R xenograft model: Nude mice are implanted with 2 × 106 K562-R cells. Once tumors reach 120–180 mm3, Bosutinib hydrate (100 mg/kg, oral, twice daily) or vehicle is administered for 28 days. Body weight and tumor volume are monitored weekly, and tumor lysates are analyzed for phospho-ABL expression [3]
- CML bone marrow transplantation model: Syngeneic mice are transplanted with bone marrow cells expressing wild-type BCR-ABL. Two weeks post-transplantation, mice are treated with Bosutinib hydrate (75 mg/kg, oral, daily) for 28 days. Peripheral blood and bone marrow are collected to quantify leukemic cell burden via flow cytometry [4]
- Breast cancer metastasis model: Nude mice are injected with 5 × 105 MDA-MB-231 cells via tail vein. One day later, Bosutinib hydrate (80 mg/kg, oral, daily) is administered for 4 weeks. Mice are euthanized, and lungs are harvested to count metastatic nodules [6]

In rats, the bioavailability of oral bosutinib hydrate (25 mg/kg) was 34 ± 5%, reaching Cmax = 1.2 ± 0.2 μM 2 hours after administration [1]
- The plasma half-life (t1/2) in rats was 6.8 ± 1.1 hours, and the AUC0–24h was 9.7 ± 1.5 μM·h [1]
- In mice, the tissue distribution of oral bosutinib hydrate (50 mg/kg) showed high concentrations in the liver (tissue/plasma ratio of 4.2 ± 0.6), moderate concentrations in the kidneys (2.8 ± 0.4), and low concentrations in the brain (0.3 ± 0.1) [4]
- Studies on human liver microsomal metabolism showed that bosutinib hydrate is mainly metabolized through the following pathways: CYP3A4 is the main enzyme, while CYP2C9 and CYP2C19 contribute less [7]

In a 28-day repeated-dose toxicity study in rats (oral doses of 25, 50, and 100 mg/kg/day), bosutinib hydrate at a dose of 100 mg/kg caused mild weight loss (≤8%), with no significant changes in ALT, AST, BUN, or creatinine levels. Histopathological examination showed mild hepatocyte vacuolation at a dose of 100 mg/kg [4] - The plasma protein binding rate of bosutinib hydrate in human plasma was 94 ± 2% and that in rat plasma was 92 ± 3% as determined by equilibrium dialysis [1] - In vitro microsomal studies showed no significant drug interaction between bosutinib hydrate and warfarin (CYP2C9 substrate) or omeprazole (CYP2C19 substrate); due to competitive inhibition, bosutinib had a moderate interaction with midazolam (CYP3A4 substrate) [7] - In mice, the maximum tolerated dose (MTD) of oral bosutinib hydrate was 150 mg/kg/day (twice daily), and the median lethal dose (LD50) was > 200 mg/kg [3]

[1]. J Med Chem . 2001 Nov 8;44(23):3965-77.

[2]. Cancer Res . 2003 Jan 15;63(2):375-81.

[3]. Mol Cancer Ther . 2008 May;7(5):1185-94.

[4]. Cancer Res . 2005 Jun 15;65(12):5358-64.

[5]. Leukemia . 2010 Jun;24(6):1223-7.

[6]. Oncol Rep . 2011 Mar;25(3):661-7.

[7]. Oncotarget . 2017 Jan 3;8(1):1469-1480.

Bosutinib hydrate is the monohydrate form of anhydrous bosutinib. It is an antitumor drug and a tyrosine kinase inhibitor. It contains the bosutinib molecule. Bosutinib monohydrate is the monohydrate form of bosutinib, a synthetic quinolone derivative and a dual kinase inhibitor that simultaneously targets Abl and Src kinases, possessing potential antitumor activity. Unlike imatinib, bosutinib inhibits the autophosphorylation of both Abl and Src kinases, thereby inhibiting cell growth and inducing apoptosis. Due to its dual mechanism of action, this drug may be effective against drug-resistant chronic myeloid leukemia (CML), other myeloid malignancies, and solid tumors. Abl kinase expression is upregulated in the presence of the aberrant Bcr-abl fusion protein, which is commonly associated with chronic myeloid leukemia (CML). Overexpression of specific Src kinases is also associated with the imatinib-resistant CML phenotype. See also: Bosutinib (with active moiety).

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Drug Indications
Bosutinib is indicated for the treatment of the following adult patients: newly diagnosed chronic phase (CP) Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML). Bosutinib hydrate is recommended in this study for chronic phase (CP), accelerated phase (AP), and blast crisis (BP) Ph+ chronic myeloid leukemia (CML) patients who have previously received one or more tyrosine kinase inhibitors (TKIs) and for whom imatinib, nilotinib, and dasatinib are not applicable. Bosutinib hydrate is a dual BCR-ABL/Src family tyrosine kinase inhibitor designed to overcome imatinib resistance in CML by targeting BCR-ABL and Src-mediated signaling pathways [1]. - Resistance to bosutinib hydrate in CML cells is associated with BCR-ABL mutations (e.g., E255K, Y253H) or increased expression of the ABCG2 efflux pump [3]. - Bosutinib hydrate and dasatinib showed synergistic antiproliferative activity in imatinib-resistant CML cells. Combined index (CI) < 1 [7] - In preclinical studies, bosutinib hydrate showed efficacy against chronic phase and blast crisis chronic myeloid leukemia (CML) and Src-driven solid tumors (breast cancer, prostate cancer) [6]

C26H31CL2N5O4

548.465

547.175

C, 56.94; H, 5.70; Cl, 12.93; N, 12.77; O, 11.67

918639-08-4

Bosutinib;380843-75-4;Bosutinib-d8

11990828

White to off-white solid powder

4.423

2

9

9

37

734

0

N#CC1C(NC2C(Cl)=CC(Cl)=C(OC)C=2)=C2C(C=C(C(=C2)OC)OCCCN2CCN(C)CC2)=NC=1.O

BXPOSPOKHGNMEP-UHFFFAOYSA-N

InChI=1S/C26H29Cl2N5O3.H2O/c1-32-6-8-33(9-7-32)5-4-10-36-25-13-21-18(11-24(25)35-3)26(17(15-29)16-30-21)31-22-14-23(34-2)20(28)12-19(22)27;/h11-14,16H,4-10H2,1-3H3,(H,30,31);1H2

4-(2,4-dichloro-5-methoxyanilino)-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile;hydrate

KIN-001160; SK606; KIN001-160; SKI 606; KIN 001160; KIN001160; SKI606; SK-I606; SK-606; SK 606; KIN 001-160; KIN-001-160; trade name: Bosulif.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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