Molecule

Description

Glioma,Glioblastoma

Description

Tandutinib (MLN518, CT53518) is an ATP-competitive and highly selective inhibitor of Flt3, PDGFR and c-Kit with IC50 of 0.22 μM, 0.20 μM and 0.17 μM, respectively.

Targets

IC₅₀

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]

Clinical Trials

A Phase II study of Tandutinib in androgen-independent prostate cancer with bone metastases has been completed.

Features

Description

Tandutinib markedly reduces the amount of cytarabine or daunorubicin necessary to achieve any given amount of anti-proliferative effect, suggesting that the combination of tandutinib and cytarabine/daunorubicin is strongly synergistic. ^[3]

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 βPDGFR, chimeric protein βPDGFR/c-Kit, and βPDGFR/Flt3 which contain the extracellular and transmembrane domains of βPDGFR 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-βPDGFR 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 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

Methods

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 Models

Female athymic nude (nu/nu) mice injected with Ba/F3 cells expressing W51 FLT3-ITD mutant

Formulation

Suspended in a 0.5% methylcellulose (MC) in water solution

Doses

40-120 mg/kg/day

Administration

Orally by gavage