research use only

Evofosfamide (TH-302)

Name: Evofosfamide (TH-302)
Brand: Selleck Chemicals
SKU: S2757
Availability: InStock
Rating: 5 (4 reviews)

Cat.No.S2757

Evofosfamide (TH-302) is a selective hypoxia-activated prodrug targeting hypoxic regions of solid tumors with IC50 of 19 nM. It demonstrates 270-fold enhanced cytotoxicity under hypoxia versus their potency under aerobic conditions, and is stable to cytochrome P450 metabolism.

Chemical Structure

Molecular Weight: 449.04

Jump to Mechanism of Action Cell Culture & Working Concentration Solubility Chemical Information, Storage & Stability

Quality Control

Batch: Purity: 99.82%

99.82

Related Targets	Dyes Antineoplastic and Immunosuppressive Antibiotics ADC Cytotoxin Selection Antibiotics for Transfected Cell PROTAC Antibiotics for Mammalian Cell Culture ADC Linker Antibiotics for Plant Cell Culture PROTAC Linker Hydrotropic Agents Antibody-Drug Conjugate E3 ligase Ligand Target Protein Ligand Molecular Glues Enzyme Substrate Drug-Linker Conjugates for ADC Chelating Agent
Related Products	BAPTA-AM Rat IgG2b isotype control-InVivo Blebbistatin Methyl-β-cyclodextrin (MβCD) AP-III-a4 (ENOblock) Carbazochrome sodium sulfonate (AC-17) Ursolic Acid Osthole 4-Methylumbelliferone (4-MU) Dioscin Ethidium bromide Pitstop 2 Sesamin Bilobalide Formononetin Hesperidin Azeliragon (TTP488) L-Mimosine Carbenoxolone Sodium Vitexin N6022 Dp44mT Chlorogenic Acid Sphingosine Chrysin Coenzyme Q10 (CoQ10) Spermine Tetrahydrochloride 2-NBDG D-Lin-MC3-DMA (MC3) Morin Hydrate

Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Incubation Time	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

Chemical Information, Storage & Stability

Molecular Weight	449.04	Formula	C₉H₁₆Br₂N₅O₄P	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	918633-87-1	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent

Solubility

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

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
=	×	×

Dilution Calculator Molecular Weight Calculator

In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	30%propylene glycol 1 5%Tween80 2 65%D5W Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	30.000mg/ml (66.81mM)	Taking the 1 mL working solution as an example, add 300 μL of 100 mg/ml clarified propylene glycol stock solution to 50 μL of Tween 80, mix evenly to clarify it; then continue to add 650 μL of D5W to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Mechanism of Action

In vitro	Evofosfamide (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 this compound is observed under N₂. It inhibits H460 cells and HT29 cells with IC90 of 0.1 μM and 0.2 μM, respectively. ^[1] It shows much enhanced potency in H460 spheroids compared to H460 monolayer cells under normoxia. ^[2] This compound exhibits potent cytotoxicity to MM cells with hypoxic selectivity and dose dependency. It can induce G0/G1 cell-cycle arrest under hypoxic conditions. The effect of it 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. It 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, it shows very low toxicity in normoxic condition, even at high concentrations. ^[3]
In vivo	Evofosfamide (TH-302) inhibits primary tumor growth by 41% on day 25 after implantation, whereas this compound plus a nucleoside analog inhibits primary tumor growth by 96% on day 25. ^[1] When 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., it shows tumor growth inhibition at Day 22 of 43%, 51%, 75%, and 89%, respectively. At 100 mg/kg, it causes a decrease in blood cell counts 3 days after treatment end, but these are totally recovered 7 days post-treatment. Under all tested regimens, it exhibits efficacy metrics ranging from 58% to 89% tumor growth inhibition. Its 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 this compound in animals breathing 10% O₂ compared with 95% O₂ breathing. After 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]
References	https://pubmed.ncbi.nlm.nih.gov/18257544/ https://pubmed.ncbi.nlm.nih.gov/22147748/ https://pubmed.ncbi.nlm.nih.gov/20530289/ https://pubmed.ncbi.nlm.nih.gov/22184053/

Clinical Trial Information

(data from https://clinicaltrials.gov, updated on 2024-05-22)

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

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

C1=C0/X	C1:	LOG(C1):
C2=C1/X	C2:	LOG(C2):
C3=C2/X	C3:	LOG(C3):
C4=C3/X	C4:	LOG(C4):
C5=C4/X	C5:	LOG(C5):
C6=C5/X	C6:	LOG(C6):
C7=C6/X	C7:	LOG(C7):
C8=C7/X	C8:	LOG(C8):