1. Apoptosis
  2. Apoptosis
  3. Evofosfamide

Evofosfamide  (Synonyms: TH-302)

Cat. No.: HY-10535 Purity: ≥98.0%
COA Handling Instructions

Evofosfamide (TH-302) is a hypoxia-activated proagent with IC50 of 10 μM and 1000 μM in hypoxia (N2) and normoxia (21% O2), respectively.

For research use only. We do not sell to patients.

Evofosfamide Chemical Structure

Evofosfamide Chemical Structure

CAS No. : 918633-87-1

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10 mM * 1 mL in DMSO
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Customer Review

Based on 4 publication(s) in Google Scholar

Top Publications Citing Use of Products
  • Biological Activity

  • Protocol

  • Purity & Documentation

  • References

  • Customer Review

Description

Evofosfamide (TH-302) is a hypoxia-activated proagent with IC50 of 10 μM and 1000 μM in hypoxia (N2) and normoxia (21% O2), respectively.

IC50 & Target

Hypoxia-activated prodrug[1]

In Vitro

Evofosfamide (TH-302) induces γH2AX and apoptosis. Evofosfamide displays hypoxia-selective and concentration-dependent cytotoxic activity that is comparable in both p53-proficient and -deficient cells. Treatment with Evofosfamide (TH-302) alone causes an accumulation of G2/M cells. Inhibition of Chk1 by PF47736 in cells treated with Evofosfamide reduces Evofosfamide (TH-302)-mediated G2/M arrest under both normoxia and hypoxia[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

In Vivo

Evofosfamide (TH-302) is a hypoxia-activated prodrug known to activate selectively under the hypoxic conditions commonly found in solid tumors. The mean values of normalized Ktrans decrease 69.2% for Evofosfamide (TH-302)-treated mice in Hs766t tumors, decrease 46.1% for Mia PaCa-2 tumors and increase 4.9% in SU.86.86 tumors. Both changes for Hs766t and Mia PaCa-2 treatment groups are statistically significant (P<0.01) when compare to their own control group[2]. A significant reduction in the hypoxic fraction (HF) to 2.1%±4.7% is seen after 95% oxygen breathing (P<0.001), whereas 7% oxygen breathing significantly increase the HF to 29.5%±14.7% (P=0.029). Exposing rhabdomyosarcoma-bearing rats to increasing oxygen conditions abolish the effect of TH-302 and reduce the T4×SV from 20.4±3.5 to 15.3±2.5 days (P=0.007), whereas control animals have an increased T4×SV. Upon combination with radiotherapy, the T4×SV of TH-302-treated tumors decrease from 30.8±5.9 (Evofosfamide (TH-302)+radiotherapy) to 25.7±2.9 days (Evofosfamide (TH-302)+radiotherapy+95% O2)[3].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Clinical Trial
Molecular Weight

449.04

Formula

C9H16Br2N5O4P

CAS No.
Unlabeled CAS

Appearance

Solid

Color

White to yellow

SMILES

O=P(NCCBr)(OCC1=CN=C(N1C)[N+]([O-])=O)NCCBr

Shipping

Room temperature in continental US; may vary elsewhere.

Storage
Powder -20°C 3 years
4°C 2 years
In solvent -80°C 6 months
-20°C 1 month
Solvent & Solubility
In Vitro: 

DMSO : 94 mg/mL (209.34 mM; Need ultrasonic and warming; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

H2O : 4.35 mg/mL (9.69 mM; ultrasonic and warming and heat to 60°C)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.2270 mL 11.1349 mL 22.2697 mL
5 mM 0.4454 mL 2.2270 mL 4.4539 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

  • Molarity Calculator

  • Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Mass
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Concentration
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Volume
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Molecular Weight *

Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

Concentration (start)

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In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: ≥ 2.5 mg/mL (5.57 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: ≥ 2.5 mg/mL (5.57 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.

For the following dissolution methods, please prepare the working solution directly. It is recommended to prepare fresh solutions and use them promptly within a short period of time.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  50% PEG300    50% Saline

    Solubility: 10 mg/mL (22.27 mM); Clear solution; Need ultrasonic

In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

g

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO +
+
%
Tween-80 +
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).
The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation

Purity: ≥98.0%

References
Cell Assay
[1]

Cells are treated with 0.1 μM of either PF477736 or AZD7762 and Evofosfamide (TH-302) for 2 h under either normoxia (21% O2) or hypoxia (N2). Following wash, cells are cultured for additional 22 h in the presence of Chk1 inhibitor under normoxia. Cells are fixed in 75% ethanol and cell cycle distribution is determined using cell cycle reagent and Guava flow cytometry. HT-29 cells are exposed to Evofosfamide (TH-302)e (8 nM, 40 nM, 200 nM, 1 μM, and 5 μM) and 0.1 μM of AZD7762 for 2 h under either normoxia (21% O2) or hypoxia (N2). After wash, cells are continuously cultured for additional 46 h in the presence of 0.1 μM of AZD7762. Luminescence-based caspase activity assay is performed[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Administration
[2][3]

Mice[2]
Female SCID mice of age 5-6 weeks are inoculated with SU.86.86, Hs766t or Mia-PaCa2 cells (5×106) subcutaneously on the left hind leg. Tumors are allowed to grow for an average of three weeks to an average size of ~150 mm3. Mice are then randomized and placed into cohorts and treated with saline (control) or Evofosfamide (TH-302) (50 mg/kg) injected intraperitoneally. A total of 34 mice underwent MR imaging studies. The SU.86.86 group consist of 5 TH-302 treated and 5 control animals; Mia-PaCa2 consist of 6 Evofosfamide treated and 5 control animals; Hs766t consist of 7 Evofosfamide treated and 6 control animals. Animals are sacrificed when tumors reach 2000 mm3.
Rats[2]
Syngeneic rhabdomyosarcoma R1 tumors (1 mm3) are implanted subcutaneously in the lateral flank of adult WAG/Rij rats. Experiments are started upon a mean tumor volume of 4.2 cm3(range, 2.0-8.1) to ensure a stable HF. Treatment is administered on 4 consecutive days and consist of an intraperitoneal injection (i.p.; QD×4) with either NaCl or Evofosfamide (TH-302) (25, 50, or 75 mg/kg). Before the start of treatment, a PET scan is made using [18F]HX4. Radiotherapy is applied in a single dose of 0, 4, 8, or 12 Gy on day 3 of the treatment, 3 hours after NaCl or Evofosfamide (TH-302) injection, 1 hour after oxygen modification. During both PET imaging and radiotherapy, rats are anesthetized using a mixture of ketamine/xylazine (i.p; 66.7 and 6.7 mg/kg, respectively). During the 5 days of treatment (1 day PET imaging, 4 days of injections with Evofosfamide or vehicle), animals are exposed to modified oxygen concentrations for 4 hours per day in order to alter the HF of the tumor. The combination oxygen modification of nicotinamide (i.p. 500 mg/kg) and carbogen (95% oxygen, 5% CO2; 5 L/minute) consist of a nicotinamide injection and 30 minutes later the exposure to carbogen breathing for 3.5 hours. In the middle of the nicotinamide/carbogen treatment, NaCl/Evofosfamide is administered. Reduced oxygen breathing (7%, residual N2; 2.5 L/minute) is given for 4 hours with the NaCl/Evofosfamide injection after the first 2 hours. The injection of the [18F]HX4 PET tracer [mean 18.8 MBq, range 7.1-25.1 MBq; lateral tail vein using an intravenous line (Venoflux 0.4 mm G27) flushed with 10% heparine)] is given 2 hours before the end of the oxygen modification. PET imaging is performed 3 hours after tracer injection.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
H2O / DMSO 1 mM 2.2270 mL 11.1349 mL 22.2697 mL 55.6743 mL
5 mM 0.4454 mL 2.2270 mL 4.4539 mL 11.1349 mL
DMSO 10 mM 0.2227 mL 1.1135 mL 2.2270 mL 5.5674 mL
15 mM 0.1485 mL 0.7423 mL 1.4846 mL 3.7116 mL
20 mM 0.1113 mL 0.5567 mL 1.1135 mL 2.7837 mL
25 mM 0.0891 mL 0.4454 mL 0.8908 mL 2.2270 mL
30 mM 0.0742 mL 0.3712 mL 0.7423 mL 1.8558 mL
40 mM 0.0557 mL 0.2784 mL 0.5567 mL 1.3919 mL
50 mM 0.0445 mL 0.2227 mL 0.4454 mL 1.1135 mL
60 mM 0.0371 mL 0.1856 mL 0.3712 mL 0.9279 mL
80 mM 0.0278 mL 0.1392 mL 0.2784 mL 0.6959 mL
100 mM 0.0223 mL 0.1113 mL 0.2227 mL 0.5567 mL

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

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    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

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