Evofosfamide (TH-302)

Catalog No.S2757 Batch:S275701

Technical Data

Formula	C₉H₁₆Br₂N₅O₄P
Molecular Weight	449.04	CAS No.	918633-87-1
Solubility (25°C)*	In vitro	DMSO	90 mg/mL (200.42 mM)
		Ethanol	90 mg/mL (200.42 mM)
		Water	10 mg/mL (22.26 mM)
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description	Evofosfamide (TH-302) is a selective hypoxia-activated prodrug targeting hypoxic regions of solid tumors with IC50 of 19 nM, demonstrates 270-fold enhanced cytotoxicity under hypoxia versus their potency under aerobic conditions, stable to cytochrome P450 metabolism.
In vitro	TH-302 is selectively potent under hypoxia and stable to liver microsomes. Substitution of the chlorine with bromine on the phosphorus mustard in 3b increases the potency by 10-fold and maintaines the high hypoxic selectivity [Hypoxia cytotoxicity ratio (HCR) = 270]. In both human lung cancer H460 cells and human colon cancer HT29 cells, potent cytotoxicity of TH-302 is observed under N₂. TH-302 inhibits H460 cells and HT29 cells with IC90 of 0.1 μM and 0.2 μM, respectively. ^[1] TH-302 shows much enhanced potency in H460 spheroids compared to H460 monolayer cells under normoxia. ^[2] TH-302 exhibits potent cytotoxicity to MM cells with hypoxic selectivity and dose dependency. TH-302 can induce G0/G1 cell-cycle arrest under hypoxic conditions. The effect of TH-302 on cell-cycle machinery is mediated by down-regulating cyclin D1/2/3, CDK4/6, p21cip-1, p27kip-1, and pRb expression, whereas CDK2 expression remained undisturbed. TH-302 can induce dose-dependent apoptosis in both human and murine MM cells in hypoxic conditions. TH-302-activated apoptosis is mediated through down-regulating the antiapoptotic proteins BCL-2 and BCL-xL, as well as up-regulating the expression of cleaved proapoptotic protein caspase-3, -8, and -9 and poly ADP-ribose polymerase. In contrast to the hypoxia-specific toxicity, TH-302 shows very low toxicity in normoxic condition, even at high concentrations. ^[3]
In vivo	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 × 5/wk × 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 % in vehicle vs. 1.8 % in the TH-302 treatment group). ^[4]

Protocol (from reference)

Cell Assay:^{^[1]}	Cell lines Human H460 or HT29 cells Concentrations 0.01 -1 μM Incubation Time 2 hours Method 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 100g. 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
Animal Study:^{^[4]}	Animal Models 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. Dosages 50 mg/kg Administration Intraperitoneally

Cell Assay:^{^[1]}

Cell lines
Human H460 or HT29 cells
Concentrations
0.01 -1 μM
Incubation Time
2 hours
Method
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 100g. 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

Animal Study:^{^[4]}

Animal Models
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.
Dosages
50 mg/kg
Administration
Intraperitoneally

References

[4] Sun JD, et al. Clin Cancer Res, 2012, 18(3), 758-770.

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Selleck's Evofosfamide (TH-302) has been cited by 3 publications

Predicting response to combination evofosfamide and immunotherapy under hypoxic conditions in murine models of colon cancer [ Math Biosci Eng, 2023, 20(10):17625-17645]	PubMed: 38052529
18F-FMISO PET Imaging Identifies Hypoxia and Immunosuppressive Tumor Microenvironments and Guides Targeted Evofosfamide Therapy in Tumors Refractory to PD-1 and CTLA-4 Inhibition [ Clin Cancer Res, 2021, clincanres.2394.2021]	PubMed: 34615724
Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition [Meng F BMC Cancer, 2015, 15:422]	PubMed: 25994202

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