Tandutinib (MLN518)

 

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Biological Activity

Description	Tandutinib (MLN518, CT53518) is a potent FLT3 antagonist with IC50 of 0.22 μM, also inhibits PDGFR and c-Kit, 15 to 20-fold higher potency for FLT3 versus CSF-1R and >100-fold selectivity for the same target versus FGFR, EGFR and KDR. Phase 1/2.
Targets	FLT3	PDGFRβ	c-Kit
IC50	0.22 μM	0.20 μM	0.17 μM [1]
In vitro	Tandutinib has little activity against EGFR, FGFR, KDR, InsR, Src, Abl, PKC, PKA and MAPKs. Tandutinib inhibits IL-3-independent cell growth and FLT3-ITD autophosphorylation with an IC50 of 10-100 nM. Tandutinib also inhibits the proliferation of human leukemia Ba/F3 cells containing FLT3-ITD mutations with IC50 values of 10-30 nM, and the FLT3-ITD-positive Molm-13 and Molm-14 cells with an IC50 of 10 nM. In FLT3-ITD-positive Molm-14 cells but not the FLT3-ITD-negative THP-1 cells, Tandutinib treatment leads to significant apoptosis by 51% and 78% at 24 and 96 hours, respectively, due to specific FLT3 inhibition. [1] Tandutinib preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML, without affecting colony formation by normal human progenitor cells. [2]
In vivo	Oral administration of Tandutinib at 60 mg/kg bid significantly increases the survival in mice bearing Ba/F3 cells expressing W51 FLT3-ITD mutant, and gives a significant reduction in mortality in a mouse bone marrow transplantation model. [1] Tandutinib treatment at 180 mg/kg twice daily has mild toxicity toward normal hematopoiesis, however, it is a dose at which Tandutinib is effective in treating FLT3 ITD-positive leukemia in mice. [2]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

Cell based receptor autophosphorylation assays

Autophosphorylation of PDGFR family kinase assays are cell-based enzyme-linked immunosorbent (ELISA) assays using CHO cells expressing wild-type PDGFRβ, chimeric protein PDGFRβ/c-Kit, and PDGFRβ/Flt3 which contain the extracellular and transmembrane domains of PDGFRβ and the cytoplasmic domain of c-Kit, and Flt-3. Cells are grown to confluency in 96-well microtiter plates under standard tissue culture conditions, followed by serum starvation for 16 hours. Briefly, quiescent cells are incubated at 37 °C with increasing concentrations of Tandutinib for 30 minutes followed by the addition of 8 nM PDGF-BB for 10 minutes. Cells are lysed in 100 mM Tris, pH 7.5, 750 mM NaCl, 0.5% Triton X-100, 10 mM sodium pyrophosphate, 50 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and the lysate is cleared by centrifugation at 15,000g for 5 minutes. Clarified lysates are transferred into a second microtiter plate in which the wells are previously coated with 500 ng/well of 1B5B11 anti-PDGFRβ mAb and then incubated for 2 hours at room temperature. After washing three times with binding buffer (0.3% gelatin, 25 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween 20), 250 ng/mL of rabbit polyclonal anti-phosphotyrosine antibody is added and plates are incubated at 37 °C for 60 minutes. Subsequently, each well is washed three times with binding buffer and incubated with 1 μg/mL of horseradish peroxidase-conjugated anti-rabbit antibody at 37 °C for 60 minutes. Wells are washed prior to adding 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), and the rate of substrate formation is monitored at 650 nm.

Cell Assay: ^[1]

Cell lines	Ba/F3, Molm-13, Molm-14, HL60, AML193, KG-1, KG-1a, THP-1, and RS4;11
Concentrations	Dissolved in DMSO, final concentrations ~30 μM
Incubation Time	~7 days
Method	Cells are exposed to increasing concentrations of Tandutinib (0.004-30 μM). Cells are grown for 3-7 days in tissue culture, and viable cells, determined by Trypan blue dye exclusion, are counted. At daily intervals, cells are harvested, washed, and resuspended in 100 uL binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Annexin V-FITC (100 ng) and propidium iodide (250 ng) are added to the cell suspension followed by incubation at room temperature for 15 minutes. Flow cytometry is performed immediately after staining on a FACSort flow cytometer with excitation at 488 nm. Fluorescence of annexin V-FITC and DNA propidium iodide staining are measured at 515 nm and 585 nm, respectively.

Animal Study: ^[1]

Animal Models	Female athymic nude (nu/nu) mice injected with Ba/F3 cells expressing W51 FLT3-ITD mutant
Dosages	40-120 mg/kg/day
Administration	Orally by gavage
Solubility	0.5% methylcellulose, 30 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00390468	Completed	Metastatic Cancer|Pain|Prostate Cancer	National Cancer Institute (NCI)	2006-10	Phase 2
NCT00408902	Completed	Clear Cell Renal Cell Carcinoma|Recurrent Renal Cell Cancer|Stage IV Renal Cell Cancer	National Cancer Institute (NCI)	2006-11	Phase 2
NCT00379080	Active, not recruiting	Adult Brain Tumor|Adult Giant Cell Glioblastoma|Adult Glioblastoma|Adult Gliosarcoma|Recurrent Adult Brain Tumor	National Cancer Institute (NCI)	2007-01	Phase 1|Phase 2
NCT00667394	Completed	Glioblastoma|Gliosarcoma|Anaplastic Astrocytoma|Anaplastic Oligodendroglioma|Anaplastic Mixed Oligoastrocytoma	National Institutes of Health Clinical Center (CC)|National Cancer Institute (NCI)	2008-04	Phase 2
NCT00904852	Withdrawn	Glioblastoma Multiforme	Millennium Pharmaceuticals, Inc.	2009-06	Phase 1

Chemical Information

Download Tandutinib (MLN518) SDF

Chemical Name	N-(4-isopropoxyphenyl)-4-(6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)piperazine-1-carboxamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.5627	5.627	-	-

Customer Reviews (1)

Click to enlarge	Rating
	Source	Eur J Pharm Sci. 2013 Jun 14;49(3):441-50 . Tandutinib (MLN518) purchased from Selleck
	Method	flow cytometry
	Cell Lines	S1 and S1-M1-80 cells
	Concentrations	0.75, 1.5 and 3.0 uM
	Incubation Time	3 h
	Results	Doxorubicin accumulation was signicantly higher (6.7-fold) in S1 cells than in S1-M1-80 cells, and tandutinib increased doxorubicin accumulation in S1-M1-80 cells in a dose dependent manner(A), Similarly, rhodamine 123 accumulation was also increased in S1-M1-80 cells by 1.5-, 1.9- and 2.9-fold,respectively(B).

Product Citations (3)

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  Vascular endothelial growth factor signals through platelet-derived growth factor receptor β in meningiomas in vitro. [Pfister C, et al. Br J Cancer 2012;107(10):1702-13]

  
  PubMed: 23047550


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  Tandutinib (MLN518/CT53518) targeted to stem-like cells by inhibiting the function of ATP-binding cassette subfamily G member 2. [Zhao XQ, et al. Eur J Pharm Sci 2013;49(3):441-50]

  
  PubMed: 23619284


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  Tandutinib (MLN518) reverses multidrug resistance by inhibiting the efflux activity of the multidrug resistance protein 7 (ABCC10). [Deng W, et al. Oncol Rep 2013;29(6):2479-85]

  
  PubMed: 23525656