Hemoglobin (no IC50/Ki/EC50/DC50 values reported in this paper). [1]

Efaproxiral, a synthetic allosteric modifier of hemoglobinoxygen binding affinity, has been shown to bind reversibly to hemoglobin, stabilizing the deoxyhemoglobin tetramer conformation to reduce its affinity for oxygen.

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Efaproxiral plus oxygen breathing reduces the radiobiological hypoxic fraction of EMT6 tumors from the value of 24% found in both air-breathing and oxygen-breathing mice to 9% and improves the response of the tumors to radiation. Carboplatin (100 mg/kg) slowes tumor growth in air-breathing mice, producing a growth delay of 3.3 days. Efaproxiral plus oxygen increases the growth delay to 5.7 days; this is 2.4 days (71%) greater than that for carboplatin alone and 2.1 days (57%) greater than that for carboplatin plus oxygen breathing. Efaproxiral plus oxygen breathing, therefore, improves the tumor growth delay obtained with 100 mg/kg carboplatin to or beyond that obtained with the highly toxic dose of 150 mg/kg carboplatin, but does so without increasing the toxicity beyond that seen with 100 mg/kg carboplatin in air-breathing mice. Efaproxiral significantly increases tumor oxygenation by 8.4 to 43.4 mmHg within 5 days in C3H mice with RIF-1 tumors, with maximum increases at 22-31 min after treatment. Efaproxiral plus oxygen plus Radiation produces tumor growth inhibition throughout the treatment duration in C3H mice with RIF-1 tumors, and inhibition is significantly different from radiation plus oxygen from day 3 to day 5.
In EMT6 mouse mammary tumors treated with 150 mg/kg carboplatin, Efaproxiral (three doses of 150 mg/kg: 15 min before, 1.5 h and 3.5 h after carboplatin) plus oxygen breathing for 5 h after carboplatin significantly decreased tumor cell survival (surviving fraction ~0.011) compared to air-breathing mice (surviving fraction ~0.10) or oxygen alone (surviving fraction ~0.025) (P<0.01 and P<0.05 respectively). The three-dose Efaproxiral plus oxygen breathing for 5.25 h had no significant effect on tumor cell viability in the absence of carboplatin. [1]
In tumor growth delay assay, treatment with 100 mg/kg carboplatin plus Efaproxiral (three doses of 150 mg/kg) and oxygen breathing produced a tumor growth delay of 5.7 ± 0.7 days (time to reach 4× initial volume), which was significantly greater than carboplatin alone (3.3 ± 0.7 days, P<0.001) and carboplatin plus oxygen alone (3.6 ± 0.7 days, P<0.05). The relative effect ratio for carboplatin plus Efaproxiral plus oxygen was 1.6 vs. carboplatin plus oxygen, 1.9 vs. carboplatin only, and 4.4 vs. control by log-rank analysis. [1]

Colony formation assay (clonogenic assay) for tumor cell survival: Tumors were explanted and single cell suspensions were prepared by mincing and trypsinization. Cells were counted under phase contrast microscopy using trypan blue to identify damaged cells. Cells were plated at low densities into petri dishes containing Waymouth's medium supplemented with 15% serum (1:1 mixture of fetal bovine serum and fetal clone), penicillin/streptomycin, fungizone, and gentamycin. Cultures were incubated at 37°C in a humidified atmosphere of 95% air/5% CO2 for 14 days. Cultures were fixed with methanol and stained with crystal violet. Colonies containing more than 50 cells were scored. Surviving fractions were calculated using the plating efficiencies of cells from untreated control tumors plated the same day. [1]

Mice

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Tumor implantation: EMT6 tumors were implanted by injecting 2×10^5 cells intradermally into the flank of male BALB/c Rw mice (2.5-3.5 months old). Tumors were treated approximately 2 weeks later when volumes reached about 100 mm³. [1]
Drug preparation and administration: Efaproxiral (RSR13) and carboplatin were dissolved in sterile, pyrogen-free physiologic saline and injected intraperitoneally (ip). For prolonged treatment with Efaproxiral plus oxygen, 150 mg/kg Efaproxiral was administered 15 min before, as well as 1.5 h and 3.5 h after carboplatin injection (450 mg/kg cumulative dose). Carboplatin dose was 150 mg/kg for tumor cell survival assays and 100 mg/kg for tumor growth delay assays. [1]
Oxygen breathing: Mice receiving oxygen were placed in gassing boxes flushed continuously with 100% oxygen for 15 min before and for 2 or 5 h after carboplatin injection. For efaproxiral plus oxygen groups, oxygen breathing was maintained from 15 min before carboplatin until assay (2 or 5 h after injection). [1]
Tumor growth delay assay: Mice were randomized by tumor volume into treatment groups when tumors were ~100 mm³. Tumors were measured three times per week by a blinded observer using vernier calipers to measure length (L), width (W), and height (H); volume was calculated as (L×W×H×π)/6. Mice were euthanized when tumor volume reached 1000 mm³. Time to reach 4× initial treatment volume was analyzed by Kaplan-Meier. Lung metastases were assessed: lungs were removed, cleaned, fixed in Bouin's, washed with 95% ethanol, stored in ethanol, and tumor nodules on lung surfaces were counted under a dissecting microscope. [1]

Host toxicity monitoring: Mice were weighed three times per week. Body weight measurements over the course of experiments showed no statistically significant differences between carboplatin-treated groups (air breathing, oxygen breathing, and Efaproxiral plus oxygen breathing), all showing average weight losses of ~1 g at nadir. Observations of animal condition (behavior, grooming, general appearance) revealed no significant differences between the three carboplatin-treated groups. Necropsy findings at euthanasia were generally unremarkable with no differences between groups. [1]
Carboplatin dose-limiting toxicity: A dose of 150 mg/kg carboplatin produced unacceptable toxicity: two of seven mice were euthanized at hematologic crises, and surviving mice showed severe weight loss (4.6±0.5 g), lethargy, failure to groom, and other signs of toxicity. The dose of 100 mg/kg carboplatin was the highest that could be given without producing severe toxicities. Efaproxiral plus oxygen breathing did not increase the toxicity of 100 mg/kg carboplatin. [1]

Exp Biol Med (Maywood).2006 Mar;231(3):317-21;Radiat Res.2007 Aug;168(2):218-25.

Efaproxiral sodium is the sodium salt of efrapand, a propionic acid derivative and an allosteric modulator of hemoglobin with radiosensitizing activity. Efrapand binds non-covalently to hemoglobin tetramers, thereby reducing the affinity of hemoglobin for oxygen. This leads to an increase in oxygen content in hypoxic tumor tissues; enhanced tumor oxygenation can reduce tumor radioresistance.

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Efaproxiral is being tested in clinical trials as an adjuvant to radiotherapy and may also be used in combination with chemotherapy and combined modality therapy. In human patients, Efaproxiral is generally given as an infusion over 30-60 min and has a half-life of 3-4.5 h (reference 12 cited in paper, but not from this study). The three-injection regimen in mice models a clinical regimen achievable with a single treatment because mice have a much shorter drug half-life. Efaproxiral combined with oxygen breathing increases tumor and tissue oxygenation in several rodent models and increases whole blood p50 in humans. [1]

C20H22NNAO4

363.3828

363.144

170787-99-2

Efaproxiral;131179-95-8

2725048

White to off-white solid powder

554.5ºC at 760mmHg

289.2ºC

3.93E-13mmHg at 25°C

2.465

1

4

6

26

464

0

SWDPIHPGORBMFR-UHFFFAOYSA-M

InChI=1S/C20H23NO4.Na/c1-13-9-14(2)11-16(10-13)21-18(22)12-15-5-7-17(8-6-15)25-20(3,4)19(23)24;/h5-11H,12H2,1-4H3,(H,21,22)(H,23,24);/q;+1/p-1

Sodium 2-[4-[2-[(3,5-Dimethylphenyl)amino]-2-oxoethyl]phenoxy]-2-methylpropanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.72 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)