Evofosfamide (TH-302)

H460, Calu-6, PC-3, H82, A375, Stew2, 786-O, PLC/PRF/5, Hs766t, BxPC-3, and SU.86.86 xenografts are established in NCI SCID female mice.

(1-methyl-2-nitro-1H-imidazol-5-yl)methyl N, N-bis(2-bromoethyl)phosphordiamidate

0.01 -1 uM

50 mg/kg

19 nM [1], 19 nM [1], 19 nM [1], 19 nM [1], 19 nM [1], 19 nM [1]

TH302 inhibits primary tumor growth by 41% on day 25 after implantation, whereas TH302 plus gemcitabine (a nucleoside analog) inhibits primary tumor growth by 96% on day 25. [1] When TH-302 is administered at 6.25, 12.5, 25, or 50 mg/kg in the H460 NSCLC xenograft model QD x 5/wk x 2 wks (once a day for 5 days per week for 2 weeks) i.p., the tumor growth inhibition at Day 22 is 43%, 51%, 75%, and 89%, respectively. TH-302 at 100 mg/kg shows a decrease in blood cell counts 3 days after treatment end, but is totally recovered 7 days post-treatment. TH-302 under all tested regimens exhibits efficacy metrics ranging from 58% to 89% tumor growth inhibition. TH-302 induced cell killing is breathing oxygen concentration dependent, with the greatest cytotoxicity occurring when the tumor-bearing mice are exposed to low oxygen concentrations. Tumor growth is significantly reduced by TH-302 in animals breathing 10% O2 compared with 95% O2 breathing. After TH-302 treatment, the pimonidazole-positive area is significantly decreased at 48 hours after dosing (6.3 +/- 1.2% in vehicle vs. 1.8 +/- 1.1% in the TH-302 treatment group). [4]

Exponentially growing human H460 or HT29 cells are seeded into 60 mm notched glass plates at 3 x 105 cells per plate and grown in RPMI medium supplemented with 10% fetal bovine serum for 2 days prior to initiating treatment. On the day of the test, TH-302 stocks of known concentrations are prepared in complete medium and 2 mL of the desired stock is added to each plate. The plates are placed in either an anaerobic chamber or a standard tissue-culture incubator. The anaerobic chamber is evacuated and gassed with the anaerobic gas mixture (90% N2/5% CO2/5% H2) to create a hypoxic environment. Cells are then incubated with TH-302 for 2 hours at 37 C. At the end of treatment, plates are removed from each vessel and washed with phosphate-buffered saline and a solution of trypsin-EDTA and then trypsinized for 5 min at 37 C. Detached cells are neutralized with medium plus serum and spun for 5 min at 100g. Cells are resuspended at approximately 1 x 106 cells/mL and diluted 10-fold for plating. The exact concentration of each stock is determined. Known numbers of cells are plated and placed undisturbed in an incubator for between 9 and 13 days. Colonies are fixed and stained with a solution of 95% ethanol with 0.25% crystal violet stain. Colonies of greater than 50 cells are counted, and the surviving fraction was determined. Plating efficiencies (PEs) are determined by dividing the number of colonies by the actual number of cells plated. Surviving fractions are calculated by dividing the PEs of treated cells by the PEs of untreated cells.

TH-302 Chemical Structure