Bcr-Abl (IC50 = 1.0 nM); Src (IC50 = 0.5 nM); lck (IC50 = 0.4 nM); yes (IC50 = 0.5 nM); c-kit (IC50 = 5.0 nM); PDGFRβ (IC50 = 28 nM); p38 (IC50 = 100 nM); Her1 (IC50 = 180 nM); Her2 (IC50 = 710 nM); FGFR-1 (IC50 = 880 nM); MEK (IC50 = 1700 nM)

Against Bcr-Abl, Src, Lck, Yes, c-Kit, PDGFRβ, p38, Her1, Her2, FGFR-1, and MEK, dasatinib exhibits noteworthy action, with IC50 values of less than 1.0, 0.50, 0.40, 0.50, 5.0, 28, 100, 180, 720, 880, and 1700 nM, respectively[1]. With an IC50 of less than 1.0 nM and 9.4 nM, respectively, dasatinib demonstrated antiproliferative action when compared to the K562 chronic myelogenous leukemia (CML), PC3 human prostate tumor, MDA-MB-231 human breast tumor, and WiDr human colon carcinoma cell line. 52 nM and 12 nM[1].

Dasatinib (10 mg/kg) has a pharmacokinetic profile that is appropriate for further in vivo efficacy research. Dasatinib (5 mg/kg and 50 mg/kg, qd) has modest toxicity at different dose levels and is resolved [1]. When administered intravenously or orally, dasatinib (10 mg/kg) has a good half-life (t1/2s) of 3.3 and 3.1 hours, respectively. In this study, the oral bioavailability (Fpo) was 27% [1].

Kinase autophosphorylation assays with glutathione S-transferase–Abl kinase domains. [2]
Kinase assays using wild-type and mutant glutathione S-transferase (GST)–Abl fusion proteins (c-Abl amino acids 220-498) were done as described, with minor alterations (15). GST-Abl fusion proteins were released from glutathione-Sepharose beads before use; the concentration of ATP was 5 μmol/L. Immediately before use in kinase autophosphorylation and in vitro peptide substrate phosphorylation assays, GST-Abl kinase domain fusion proteins were treated with LAR tyrosine phosphatase according to the manufacturer's instructions. After 1-hour incubation at 30°C, LAR phosphatase was inactivated by addition of sodium vanadate (1 mmol/L). Immunoblot analysis comparing untreated GST-Abl kinase to dephosphorylated GST-Abl kinase was routinely done using phosphotyrosine-specific antibody 4G10 to confirm complete (>95%) dephosphorylation of tyrosine residues and c-Abl antibody CST 2862 to confirm equal loading of GST-Abl kinase. The inhibitor concentration ranges for IC50 determinations were 0 to 5,000 nmol/L (imatinib and AMN107) or 0 to 32 nmol/L (Dasatinib (BMS354825) ). The BMS-354825 concentration range was extended to 1,000 nmol/L for mutant T315I. These same inhibitor concentrations were used for the in vitro peptide substrate phosphorylation assays. The three inhibitors were tested over these same concentration ranges against GST-Src kinase and GST-Lyn kinase.

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In vitro peptide substrate phosphorylation assays with glutathione S-transferase–Abl kinase domains. [2]
he effects of imatinib (0-5,000 nmol/L), AMN107 (0-5,000 nmol/L), and Dasatinib (BMS354825) (0-32 nmol/L) on the catalytic activity of unphosphorylated GST-Abl kinase were assessed using a synthetic, NH2-terminal biotin-linked peptide substrate (biotin-EAIYAAPFAKKK-amide; ref. 16). Assays were carried out at 30°C for 5 minutes in 25 μL of reaction mixture consisting of kinase buffer [25 mmol/L Tris-HCl (pH 7.5), 5 mmol/L β-glycerophosphate, 2 mmol/L DTT, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2], 50 μmol/L peptide substrate, 10 nmol/L wild-type or mutant GST-Abl kinase, and 50 μmol/L ATP/[γ-32P]ATP (5,000 cpm/pmol). Reactions were terminated by addition of guanidine hydrochloride to a final concentration of 2.5 mol/L. A portion of each terminated reaction mixture was transferred to a streptavidin-coated membrane, washed, and dried according to the manufacturer's instructions; phosphate incorporation was determined by scintillation counting. Results were corrected for background binding to the membranes as determined by omitting peptide substrate from the kinase reaction. Time course experiments to establish the linear range of enzymatic activity preceded kinase assays. Similar in vitro peptide substrate phosphorylation assays were conducted with two Src family kinases: GST-Src kinase and GST-Lyn kinase. For Src family kinases, SignaTECT PTK biotinylated peptide substrate 2 was the peptide substrate; all other conditions were as described for the GST-Abl kinase assays.

Mastocytosis is associated with an activating mutation in the KIT oncoprotein (KITD816V) that results in autophosphorylation of the KIT receptor in a ligand-independent manner. This mutation is inherently resistant to imatinib and, to date, there remains no effective curative therapy for systemic mastocytosis associated with KITD816V. Dasatinib (BMS-354825) is a novel orally bioavailable SRC/ABL inhibitor that has activity against multiple imatinib-resistant BCR-ABL isoforms in vitro that is presently showing considerable promise in early-phase clinical trials of chronic myeloid leukemia (CML). Pharmacokinetic analysis suggests that high nanomolar concentrations of dasatinib can be achieved safely in humans. In this study, we demonstrate significant inhibitory activity of dasatinib against both wild-type KIT and the KITD816V mutation in the nanomolar range in in vitro and cell-based kinase assays. Additionally, dasatinib leads to growth inhibition of a KITD816V-harboring human masto-cytosis cell line. Significantly, dasatinib selectively kills primary neoplastic bone marrow mast cells from patients with systemic mastocytosis while sparing other hematopoietic cells. Computer modeling suggests that the KITD816V mutation destabilizes the inactive conformation of the KIT activation loop to which imatinib binds, but it is not predicted to impair binding of KIT by dasatinib. Based upon our results, further evaluation of dasatinib for the treatment of systemic masto-cytosis in clinical trials is warranted. Moreover, dasatinib may be of clinical utility in other disease settings driven by activating KIT mutations.[3]

Animal/Disease Models: Nude mice bearing K562 xenografts
Doses: 5 mg/kg and 50 mg/kg
Route of Administration: Oral administration on a 5 day on and 2 day off schedule.
Experimental Results: demonstrated partial tumor regressions after one treatment cycle and complete disappearance of the tumor mass by the end of drug treatment. No toxicity (animal deaths, lack of weight gain) was observed.

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Animal/Disease Models: SD (Sprague-Dawley) Rats
Doses: 10 mg/kg (pharmacokinetic/PK Analysis)
Route of Administration: Oral and iv
Experimental Results: Cmax of 13.2 and 0.5 μM for iv and po (oral gavage) respectively.

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Mouse 4 hour oral exposure assay [1]
The in vivo exposure of compounds were assessed in male Balb-c mice after administration of a single oral dose of 50 mg/kg. The vehicle used was propylene glycol:water (1:1). There were three mice per compound. The mice were fasted overnight and throughout the study. Serum concentrations in mice were collected at 30 min, 1 and 4 h after oral dosing. Samples were analyzed for each compound by LC/MS/MS. Composite serum concentration-time profiles were constructed for pharmacokinetic analysis.

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Rat pharmacokinetic study [1]
The pharmacokinetics of BMS-354825 were investigated in male Sprague-Dawley rats which were fasted overnight, following a single dose of 10 mg/kg either intravenously (IV) as a 10 minute infusion or orally by gavage. There were three rats per group The dosing vehicle used was propylene glycol:water (1:1). The rats were fed 4 h post dose. Blood samples were collected at 15, 30, 45 min, 1, 2, 4, 6, 8 and 10 h after IV and oral dosing. An additional 10 min sample was collected after IV dosing. Approximately 0.3 ml of blood was collected from the jugular vein in tubes containing EDTA, and plasma was obtained by centrifugation. Plasma samples were stored at -20ºC until analysis. Samples were analyzed for BMS-354825 by LC/MS/MS.

Absorption, Distribution and Excretion
Dasatinib has a dose-proportional pharmacokinetic profile and a linear elimination between 15 mg/day (0.15 times the lowest approved recommended dose) and 240 mg/day (1.7 times the highest approved recommended dose). At 100 mg once a day, dasatinib has a Cmax and AUC of 82.2 ng/mL and 397 ng/mL*hr, respectively. In healthy adult subjects given dasatinib as dispersed tablets in juice, the adjusted geometric mean ratio compared to intact tablets was 0.97 for Cmax, and 0.84 for AUC. The Tmax of dasatinib is between 0.5 and 6 hours following oral administration. Following a single dose of 100 mg, a high-fat meal increases the AUC of dasatinib by 14%.
Dasatinib is mainly eliminated via feces. Within 10 days, 4% of dasatinib is recovered in urine, while 85% is recovered in feces. Approximately 0.1% and 19% of the administered dasatinib dose was recovered unchanged in urine and feces, respectively, and the rest was recovered as metabolites.
Dasatinib has an apparent volume of distribution of 2505 L.
The clearance of dasatinib does not vary over time. Dasatinib has an apparent oral clearance of 363.8 L/hr.

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Metabolism / Metabolites
In humans, dasatinib is mainly metabolized by CYP3A4, although flavin-containing monooxygenase 3 (FMO3) and uridine diphosphate-glucuronosyltransferase (UGT) enzymes are also involved in the formation of dasatinib metabolites. Five pharmacologically active dasatinib metabolites have been identified: M4, M5, M6, M20 and M24. M4, M20, and M24 are mainly generated by CYP3A4, M5 is generated by FMO3, and M6 is generated by a cytosolic oxidoreductase. M4 is equipotent to dasatinib and represents approximately 5% of the AUC. However, it is unlikely to play a major role in the observed pharmacology of dasatinib. M5 and M6 are more than 10 times less active than dasatinib and are considered minor circulating metabolites.

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Biological Half-Life
The terminal half-life of dasatinib is 3-5 hours.

Hepatotoxicity
In large clinical trials, elevations in serum aminotransferase levels during dasatinib therapy occurred in up to 50% of patients, but were usually mild and self-limited. Elevations above 5 times the upper limit of normal (ULN) occurred in 1% to 9% of patients and generally responded to dose adjustment or temporary discontinuation and restarting at a lower dose, which is recommended if liver test results are markedly elevated (ALT or AST persistently greater than 5 times ULN or bilirubin more than 3 times ULN). While episodes of marked serum aminotransferase elevations with symptoms have been reported, there have been no published reports of clinically apparent liver injury with jaundice attributed to dasatinib therapy. Certainly other tyrosine kinase receptor inhibitors used in the therapy of CML such as imatinib, nilotinib and ponatinib have been associated with cases of acute liver injury with jaundice. With these agents, the liver injury typically arises after several months of therapy and the pattern of serum enzyme elevations is typically hepatocellular. Immunoallergic features (rash, fever and eosinophilia) and autoantibody formation are usually not present.
Reactivation of hepatitis B has been reported with dasatinib as well as imatinib and nilotinib therapy. Reactivation typically occurs in an HBsAg positive person treated with the tyrosine kinase inhibitor for 3 to 6 months, presenting with jaundice, marked serum aminotransferase elevations and an increase in HBV DNA levels. Reactivation of hepatitis B can be severe and fatal instances have been reported after imatinib and nilotinib therapy. Screening of patients for HBsAg and anti-HBc is sometimes recommended before starting cancer chemotherapy and those with HBsAg offered prophylaxis with oral antiviral agents, such as lamivudine, tenofovir or entecavir.
Likelihood score: D (possible cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Although one breastfed infant apparently experienced no adverse effects during maternal use of dasatinib, no long-term data are available. Because dasatinib and its metabolite are more than 90% bound to plasma proteins, the amounts in milk are likely to be low. However, there is little published experience with dasatinib during breastfeeding, and an alternate drug may be preferred, especially while nursing a newborn or preterm infant. National Comprehensive Cancer Network guidelines recommend avoiding breastfeeding during dasatinib therapy and the manufacturer recommends withholding breastfeeding until 2 weeks following the last dose.
◉ Effects in Breastfed Infants
A woman with chronic myeloid leukemia received dasatinib 100 mg daily throughout pregnancy and continuing postpartum, apparently while breastfeeding her infant (extent not stated). No adverse reactions were reported in her infant.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
_In vitro_, the binding of dasatinib to human plasma proteins is approximately 96%.

[1]. Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem. 2004 Dec 30;47(27):6658-61.
[1]. In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer Res. 2005 Jun 1;65(11):4500-5.
[2]. Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis. Blood. 2006 Jul 1;108(1):286-91.

Pharmacodynamics
Dasatinib is an orally available small-molecule multikinase inhibitor. During clinical trials, less than 1% of patients treated with dasatinib had QTc prolongation as an adverse reaction, and 1% experienced a QTcF higher than 500 ms. The use of dasatinib is also associated with myelosuppression, bleeding-related events, fluid retention, cardiovascular toxicity, pulmonary arterial hypertension, severe dermatologic reactions, tumor lysis syndrome and hepatotoxicity. It may also cause embryo-fetal toxicity and lead to adverse reactions associated with bone growth and development in pediatric patients.

C22H26CLN7O2S

488.01

487.155

C, 54.15; H, 5.37; Cl, 7.26; N, 20.09; O, 6.56; S, 6.57

302962-49-8

Dasatinib hydrochloride;854001-07-3;Dasatinib monohydrate;863127-77-9;Dasatinib-d8;1132093-70-9; 302962-49-8 (free); 2112837-79-1 (cabaldehyde); 910297-52-8 (N-oxide)

3062316

Typically exists as White to off-white solid at room temperature

1.4±0.1 g/cm3

275-286°C

1.688

2.24

3

9

7

33

642

0

O=C(C1=CN=C(S1)NC2=NC(C)=NC(N3CCN(CC3)CCO)=C2)NC4=C(C=CC=C4Cl)C

XHXFZZNHDVTMLI-UHFFFAOYSA-N

InChI=1S/C22H26ClN7O2S.H2O/c1-14-4-3-5-16(23)20(14)28-21(32)17-13-24-22(33-17)27-18-12-19(26-15(2)25-18)30-8-6-29(7-9-30)10-11-31;/h3-5,12-13,31H,6-11H2,1-2H3,(H,28,32)(H,24,25,26,27);1H2

N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide monohydrate.

Trade name: Sprycel; BMS-354825; BMS354825; Sprycel; BMS-354825; Dasatinib anhydrous; BMS 354825; Dasatinib (anhydrous); BMS354825. Dasatinib;

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.12 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (5.12 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (4.26 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 20.8 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 4: ≥ 2.08 mg/mL (4.26 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 20.8 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 5: ≥ 2.08 mg/mL (4.26 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O:5 mg/mL

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Solubility in Formulation 7: 6.67 mg/mL (13.67 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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