Tasidotin hydrochloride (ILX651)

Kinase experiment:

MDA-MB-435 and HS 578-T breast carcinoma cells are grown in 25 mL of medium in Corning 75-cm2 culture flasks. For monolayer cells, when the cells are at near confluence, the medium is exchanged for 15 mL of fresh medium containing the indicated concentration of 0.1 μM [3H]Tasidotin, and the incubation continues for the indicated time. Monolayer cells are harvested by scraping them from the surface of the culture flask. For suspension cells, when the cells are in log phase and at approximately 106 cells/mL, the [3H]Tasidotin is added at the indicated concentration, and the incubation continued for the indicated time as specified in individual experiments. Suspension cells are harvested by centrifugation. Both types of cells are washed twice with PBS, pH 7.2. The cells are resuspended in 0.5 mL of water and disrupted by 10 s periods of sonication at 28 W, followed by 10 s pauses, for a total of 1 min. Protein content and radiolabel of all cell extracts are determined[1].

Cell experiment:

Growth inhibition is determined by the microculture tetrazolium method. Briefly, cells are seeded in 96 well flat-bottomed microtiter plates at a density of 500 cells per well in 100 μL of medium. After overnight incubation, 100 μL of medium containing Tasidotin are added to achieve specified final concentrations and a final volume of 200 μL/well. At 120 h, the relative metabolic activities of treated and untreated cells are measured by mitochondrial conversion of MTT to formazine. At the completion of the drug treatment, 250 μg of MTT are added to each well and incubated at 37°C, 5% CO2 for 6 h. Formazine crystals are dissolved in DMSO and absorbance at 595 nm is measured on a VERSAmax spectrophotometer. Absorbance values are normalized to the values obtained for the vehicle-treated cells to determine the percentage of survival. The IC50 is defined as the concentration at which absorbance of the treated cells is 50% that of the controls[2].

Animal experiment:

Mice[2] CB17 female scid-/- mice are treated with Tasidotin daily for 5 days via i.p. injection starting on day 1 every 21 days for two cycles. Five non–tumor-bearing mice are assigned to each treatment group. Mice are treated with either 70, 80, 90, or 100 mg/kg/d Tasidotin. A toxic event is defined as weight loss ≥20% of the animal's weight at the time of randomization or death. The maximally tolerated dose (MTD) is defined as the highest dose at which no toxicity occurred.

References:

[1]. Bai R, et al. Intracellular activation and deactivation of Tasidotin, an analog of dolastatin 15: correlation with cytotoxicity. Mol Pharmacol. 2009 Jan;75(1):218-26.
[2]. Garg V, et al. Preclinical analysis of Tasidotin HCl in Ewing's sarcoma, rhabdomyosarcoma, synovial sarcoma, and osteosarcoma. Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5446-54.