Evofosfamide (TH-302)

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.

Evofosfamide (TH-302) Chemical Structure

Evofosfamide (TH-302) Chemical Structure

CAS: 918633-87-1

Selleck's Evofosfamide (TH-302) has been cited by 2 publications

Purity & Quality Control

Batch: Purity: 99.82%
99.82

Evofosfamide (TH-302) Related Products

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
H460 Cytotoxicity assay 2 hrs Cytotoxicity against human H460 cells under hypoxic condition after 2 hrs by Alamar blue staining assay, IC50=0.019μM. 18257544
H460 Cytotoxicity assay 2 hrs Cytotoxicity against human H460 cells under hypoxic condition after 2 hrs by clonogenic assay, IC90=0.1μM. 18257544
HT29 Cytotoxicity assay 2 hrs Cytotoxicity against human HT29 cells under hypoxic condition after 2 hrs by clonogenic assay, IC90=0.2μM. 18257544
H460 Cytotoxicity assay 2 hrs Cytotoxicity against human H460 cells under normoxic condition after 2 hrs by Alamar blue staining assay, IC50=5.1μM. 18257544
H460 Cytotoxicity assay 2 hrs Cytotoxicity against human H460 cells under normoxic condition after 2 hrs by clonogenic assay, IC90=30μM. 18257544
HT29 Cytotoxicity assay 2 hrs Cytotoxicity against human HT29 cells under normoxic condition after 2 hrs by clonogenic assay, IC90=40μM. 18257544
NCI-H460 Cytotoxicity assay 24 hrs Cytotoxicity against human NCI-H460 cells pretreated for 24 hrs under hypoxic condition followed by compound washout measured after 72 hrs by MTT assay, IC50=9.08μM. 28350997
HT-29 Cytotoxicity assay 24 hrs Cytotoxicity against human HT-29 cells pretreated for 24 hrs under hypoxic condition followed by compound washout measured after 72 hrs by MTT assay, IC50=49.51μM. 28350997
NCI-H460 Cytotoxicity assay 24 hrs Cytotoxicity against human NCI-H460 cells after 24 hrs under hypoxic condition by luminescence-based Cell-Titer Glo assay, IC50=0.0093μM. 29079474
NCI-H460 Cytotoxicity assay 24 hrs Cytotoxicity against human NCI-H460 cells after 24 hrs under normoxic condition by luminescence-based Cell-Titer Glo assay, IC50=6.65μM. 29079474
DU145 Cytotoxicity assay 2 hrs Cytotoxicity against human DU145 cells incubated for 2 hrs under hypoxic condition measured after 72 hrs by Alamar blue assay, IC50=4.14μM. 29259746
PC3 Cytotoxicity assay 2 hrs Cytotoxicity against human PC3 cells incubated for 2 hrs under hypoxic condition measured after 72 hrs by Alamar blue assay, IC50=6.49μM. 29259746
HEMC-SS Antiproliferative assay 24 hrs Antiproliferative activity against human HEMC-SS cells treated for 24 hrs under hypoxic condition followed by compound wash-out and incubated under normoxic condition for 48 hrs by alamar blue assay, IC50=0.13μM. 30199705
HEMC-SS Antiproliferative assay 24 hrs Antiproliferative activity against human HEMC-SS cells treated for 24 hrs under normoxic condition followed by compound wash-out and incubated under normoxic condition for 48 hrs by alamar blue assay, IC50=2.9μM. 30199705
NCI-H460 Antiproliferative assay Antiproliferative activity against human NCI-H460 cells in presence of N2, IC50=2μM. 30295477
NCI-H460 Antiproliferative assay Antiproliferative activity against human NCI-H460 cells in presence of O2, IC50=25μM. 30295477
MDA-MB-468 Cytotoxicity assay 4 hrs Cytotoxicity against human MDA-MB-468 cells incubated for 4 hrs under hypoxic condition followed by compound washout and measured after 5 days by SRB assay, IC50=0.0062μM. 30885680
SW620 Cytotoxicity assay 4 hrs Cytotoxicity against human SW620 cells incubated for 4 hrs under hypoxic condition followed by compound washout and measured after 5 days by SRB assay, IC50=0.052μM. 30885680
MDA-MB-468 Cytotoxicity assay 4 hrs Cytotoxicity against human MDA-MB-468 cells incubated for 4 hrs under aerobic condition followed by compound washout and measured after 5 days by SRB assay, IC50=4.403μM. 30885680
SW620 Cytotoxicity assay 4 hrs Cytotoxicity against human SW620 cells incubated for 4 hrs under aerobic condition followed by compound washout and measured after 5 days by SRB assay, IC50=20.6μM. 30885680
Click to View More Cell Line Experimental Data

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
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]
Cell Research 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
In Vivo
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]
Animal Research 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
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT02020226 Unknown status
Solid Tumors
Threshold Pharmaceuticals
November 2013 Phase 1
NCT01833546 Completed
Solid Tumor|Pancreatic Cancer
Merck KGaA Darmstadt Germany|Threshold Pharmaceuticals
April 18 2013 Phase 1

Chemical Information & Solubility

Molecular Weight 449.04 Formula

C9H16Br2N5O4P

CAS No. 918633-87-1 SDF Download Evofosfamide (TH-302) SDF
Smiles CN1C(=CN=C1[N+](=O)[O-])COP(=O)(NCCBr)NCCBr
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 90 mg/mL ( (200.42 mM); Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 90 mg/mL

Water : 10 mg/mL


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In vivo
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