Evofosfamide (TH-302) is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM) conjugated with 2-nitroimidazole, with potential antineoplastic activity. When exposed to hypoxic conditions, such as those found in hypoxic tumors, the 2-nitroimidazole moiety of evofosfamide is reduced. This releases the DNA-alkylating Br-IPM moiety, which introduces intra- and inter-strand DNA crosslinks in nearby cells; the crosslinks inhibit both DNA replication and cell division and may lead to apoptosis of cells in the tumor. The inactive form of the prodrug is stable under normoxic conditions, which may limit systemic toxicity.

Hypoxia (N2):10 μM, Normoxia (21% O2):1000 μM

Evofosfamide在低氧条件下选择性强，对肝微粒体稳定。3b中磷酸芥上氯与溴的替换增强了10倍的活性，并保持了高低氧选择性[Hypoxia cytotoxicity ratio (HCR) = 270]。在人类肺癌H460细胞和人类结肠癌HT29细胞中，Evofosfamide在N2环境下显示出强大的细胞毒性。Evofosfamide分别以IC90 0.1 μM和0.2 μM抑制H460细胞和HT29细胞。[1] 相比于常氧条件下的H460单层细胞，Evofosfamide在H460球状细胞中展现出极大增强的活性。[2] Evofosfamide对MM细胞表现出具有低氧选择性和剂量依赖性的强大细胞毒性。Evofosfamide可在低氧条件下诱导G0/G1期细胞周期阻滞，其对细胞周期机制的影响通过下调cyclin D1/2/3、CDK4/6、p21cip-1、p27kip-1和pRb表达实现，而CDK2表达不受影响。Evofosfamide能在低氧条件下诱导人类和小鼠MM细胞的剂量依赖性凋亡。Evofosfamide激活的凋亡通过下调抗凋亡蛋白BCL-2和BCL-xL，以及上调cleaved proapoptotic protein caspase-3、-8和-9以及poly ADP-ribose polymerase表达实现。与特异性低氧毒性相反，Evofosfamide在常氧条件下即使在高浓度下也显示出极低的毒性。[3]

Evofosfamide通过在植入后第25天抑制原发性肿瘤生长41%，而Evofosfamide加上吉西他滨（一种核苷类似物）在第25天可抑制原发性肿瘤生长96%。[1] 当TH-302在H460 NSCLC异种移植模型中以6.25、12.5、25或50 mg/kg剂量每周5天每天一次，连续2周内腹腔注射（i.p.），第22天时的肿瘤生长抑制率分别为43%、51%、75%和89%。TH-302在100 mg/kg剂量下，治疗结束后3天血细胞计数下降，但在治疗后7天完全恢复。在所有测试方案下，TH-302展示的肿瘤生长抑制效果介于58%至89%之间。TH-302诱导的细胞杀伤依赖于呼吸氧浓度，当肿瘤携带的小鼠暴露于低氧浓度时，最大的细胞毒性发生。与吸入95% O2相比，吸入10% O2的动物中TH-302显著减少了肿瘤生长。TH-302治疗后，在给药48小时后，pimonidazole阳性区域显著减少（对照组为6.3%，TH-302治疗组为1.8%）。[4]

Exponentially growing human H460 or HT29 cells are seeded into 60 mm notched glass plates at 3 × 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 100 g. Cells are resuspended at approximately 1 × 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 is 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 untreate(Only for Reference)