G6PD; Nrf2-ARE
Nrf2 (nuclear factor erythroid 2‑related factor 2) – RRx-001 activates Nrf2, leading to its nuclear translocation and subsequent ARE‑driven gene expression. No IC50/Ki values are reported. [2]
Not applicable (no direct enzyme target reported).

Glucose 6 phosphate dehydrogenase (G6PD), a crucial enzyme in the pentose phosphate pathway, is in charge of ensuring that adequate levels of the main cellular reductant, NADPH, are kept in the body. This is how RRx-001 exerts its anti-proliferative effect, at least in part. In three distinct cancer cell lines, Hep G2, CACO-2, and HT-29, RRx-001 alters the activity of the glucose and G6PD enzymes. The concentration-dependent effects of RRx-001 resulted in G6PD inhibition and increased glucose consumption. By producing ROS/RNS, RRx-001 activates p53 and PARP-1. It exerts anticancer activity, at least in part, by interfering with 3 crucial metabolic demands of rapidly proliferating cells: bioenergetics, macromolecular biosynthesis, and control of cytosolic redox homeostasis in living cells[1]. HO-1 and NQO1—two of Nrf2's downstream enzymes—are activated and translocated to the nucleus in tumor cells as a result of RRx-001. Other than epigenetic changes, RRx-001 works through pleiotropic mechanisms that involve redox signaling and redox-induced dysregulation of numerous signal pathways, including Nrf2, p53, PARP cleavage, HIF1 alpha, and G6PD activity. Along with deregulating cancer cell energetics and metabolism, it also activates p53 and p21 in response to double-stranded DNA breaks. By producing ROS and RNS, the drug RRx-001 effectively activates the Nrf2-ARE signaling pathway[3].

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Nrf2 activation in SCC VII cells: Treatment with RRx-001 (2 or 5 μM for 24 h) caused a 6‑fold increase in nuclear Nrf2 protein compared to baseline, while cytoplasmic Nrf2 levels remained unchanged. Western blot analysis showed dose‑ and time‑dependent increases in HO‑1 (∼2‑fold at 8 h, returning to basal level at 24 h) and NQO1 (2‑ to 4‑fold at 8 h, >6‑fold at 24 h). [2]
In SCC VII cells stably co‑expressing ARE‑Firefly luciferase (ARE‑FLUC) and CMV‑Renilla luciferase‑mRFP, RRx-001 induced dose‑ and time‑dependent activation of ARE‑FLUC, with maximum activation at 6 h after treatment with 2.3 μM RRx‑001. Higher doses (4.7‑18.7 μM) reduced signal due to cell killing. The known Nrf2 activator TBHQ (20 μM) served as positive control, and 2.3 μM RRx‑001 achieved ∼80% of the TBHQ signal. [2]
Knockdown of endogenous Nrf2 using Nrf2‑specific siRNA increased sensitivity of SCC VII cells to RRx-001; IC50 values: parental cells 1.09 μM, scrambled siRNA 1.04 μM, Nrf2 siRNA clones C2 and C3 0.74 and 0.75 μM respectively, as measured by WST‑8 cell proliferation assay. [2]
In SCC VII tumors in mice, a single intravenous dose of 10 mg/kg RRx-001 increased cytoplasmic Nrf2 by 2.0‑ and 2.4‑fold at 24 and 48 h, and nuclear Nrf2 by ∼1.6‑ and 2.3‑fold, compared to control. HO‑1 and NQO1 levels in tumors increased by 13% and 20% for HO‑1, and 12% and 27% for NQO1 at 8 h and 24 h post‑injection (p < 0.05 vs control, n=6 per time point). [2]
In SCC VII and U87 tumors, a single intravenous dose of 15 mg/kg RRx-001 caused redistribution of blood flow. In SCC VII tumors, the percent of perfused vessels (DiOC7(3) positive) increased from 21% (control) to 28% at 12 h post‑dose (p < 0.05), but no significant change in tissue oxygenation or hypoxia (pimonidazole binding) was observed. HIF1‑α‑positive tissue decreased at 90 min and 12 h (p < 0.05 at 12 h). In U87 tumors, a dramatic decrease in central vessel perfusion was seen at 90 min, with large unperfused areas (p < 0.05 vs control), but these areas did not become necrotic and recovered by 12 h; the percent of perfused vessels increased from <20% (control) to ∼28% at 12 h (not statistically significant). No significant change in tumor hypoxia was seen at 90 min or 12 h. These data indicate a vascular normalization effect. [3]

RRx-001 shows promise for short-term blood flow redistribution in tumors with a pericyte- and α-SMA-rich vasculature[2]. RRx-001 monotherapy is well tolerated and free of toxicities that would limit its dosage. It not only helps Nrf2 move into the nucleus, but it also increases endogenous Nrf2 expression in mouse SCC VII tumors[3].
Not described in the provided literature.

Cell viability assay (WST‑8): SCC VII cells were seeded, treated with various concentrations of RRx-001 for a specified period, and cell viability was measured using WST‑8 reagent according to the manufacturer's protocol. IC50 values were calculated from dose‑response curves. [2]
Western blot analysis for Nrf2, HO‑1, NQO1: Cells or tumor tissues were lysed in RIPA buffer or NE‑PER nuclear/cytoplasmic extraction buffer. Protein concentrations were quantified, equal amounts (20 μg) were separated by 10% SDS‑PAGE, transferred to PVDF membranes, blocked with 5% non‑fat milk, probed with primary antibodies (anti‑Nrf2, anti‑HO‑1, anti‑NQO1, anti‑β‑actin, anti‑lamin B), then HRP‑conjugated secondary antibodies, and detected by ECL chemiluminescence. Bands were quantified using ImageJ. [2]
Dual‑luciferase reporter assay: Cells stably expressing ARE‑FLUC and CMV‑RLUC‑mRFP were lysed in passive lysis buffer, cleared by centrifugation, and 20 μL of supernatant was mixed with LARII solution for FLUC measurement or with coelenterazine (1 μg in 100 μL PBS) for RLUC measurement using a luminometer. Total protein was used for normalization. [2]
Immunohistochemistry for tumor sections: Cryosections were fixed in acetone‑methanol (1:1), stained with anti‑CD31 for vasculature, anti‑pimonidazole for hypoxia, anti‑BrdUrd for proliferation, or anti‑HIF1‑α, followed by appropriate fluorescent secondary antibodies. Cellular DNA was counterstained with Hoechst 33342. Image acquisition used a robotic fluorescence microscope with tiling to capture whole tumor sections. Analysis involved manual removal of necrosis/artifacts, fixed thresholding for positive regions, and calculation of average intensities and distances from vessels. [3]

Cell growth and proliferation is assessed using the MTT proliferation assay kit. In a nutshell, 96-well plates with 5×103 cells in each well are used to treat or leave untreated cells. Up to 72 hours can pass during cell culture. Each well is given 10 μL of MTT reagent before being left to sit for 3 hours. Following incubation, the culture medium is taken out, 100 μL of Crystal Dissolving Solution is poured into each well, and the solution's absorbance at 570 nm is gauged. In triplicate, MTT assays are carried out on samples from the same conditions. Adding MTT solution (0.5 mg/mL), allowing cells to incubate for 4 hours at 37 degrees Celsius, and then measuring absorption at 540 nm with a microplate reader are the steps in short order.

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SCC VII murine squamous cell carcinoma cells were cultured in DMEM with 10% fetal calf serum, penicillin/streptomycin, at 37°C in 5% CO2. For Nrf2 knockdown, cells were transiently transfected with 120 pmol of Nrf2‑specific siRNA or scrambled siRNA using transfection reagent for 24 h, then replaced with normal medium for another 24 h before assays. [2]
Stable cell line generation: SCC VII cells were co‑transfected with pcPUR‑ARE‑FLUC and pcDNA‑CMV‑RLUC‑mRFP plasmids using lipofectamine. Double selection with puromycin and G418, followed by FACS sorting for RFP, yielded a single clone verified by dual‑luciferase assay. [2]
In vitro Nrf2 activation assay: SCC VII cells were treated with 0, 2, or 5 μM RRx-001 for 8 or 24 h, then lysed for Western blot. [2]
ARE‑FLUC reporter assay: Stable cells were treated with various doses of RRx-001 (0 to 18.7 μM) or 20 μM TBHQ, and FLUC signal was measured at different time points (e.g., 6 h) using luminometer after addition of D‑luciferin. [2]

5 mg/kg or 10 mg/kg; i.v. CB-17 SCID-mice

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For in vivo Nrf2 activation study (literature [2]): Female nude mice (7‑8 weeks, 20‑25 g) were subcutaneously inoculated with 5×10^5 SCC VII cells (parental or ARE‑FLUC/RLUC‑mRFP expressing) in both flanks. When tumors reached ∼150 mm³ (day 10), mice received a single intravenous (i.v.) injection of RRx-001 at 10 mg/kg (formulated by dissolving 10 mg in 0.5 mL DMA‑PEG400 1:2, then diluted with double distilled water to 2 mg/mL). For tumor response, mice were randomized to vehicle control or RRx‑001 groups (8 tumors per group). Tumor volumes measured with calipers three times weekly; tumor volume quadrupling time (TVQT) and tumor growth delay (TGD) were calculated. For Western blot analysis, mice were euthanized at 8, 24, or 48 h post‑dose, perfused with PBS, and tumors collected. [2]
For vascular normalization study (literature [3]): Female C3H/Hen mice (for SCCVII) and female NOD.CB17‑Prkdcscid mice (for U87) were subcutaneously implanted with 0.5×10^6 SCCVII cells or 5×10^6 U87 cells in sacral region. When tumors reached ∼150 mm³, mice received a single i.v. dose of RRx-001 at 15 mg/kg. At 90 min and 12 h post‑dose, mice were injected i.p. with BrdUrd (1000 mg/kg) and pimonidazole (60 mg/kg) 1 h before euthanasia; 5 min before euthanasia, DiOC7(3) (0.6 mg/mL in 25% DMSO, 50 μL) was administered. Tumors were excised, frozen, and sectioned for immunohistochemistry. [3]

Not described in the provided literature.

Not described in the provided literature (no LD50, organ toxicity, or protein binding data). However, in Phase I clinical trials (referenced in [2]), RRx-001 monotherapy was well tolerated with no dose‑limiting toxicities. [2]

[1]. Discov Med . 2016 Apr;21(116):251-65.

[2]. Clin Epigenetics . 2016 May 11:8:53.

[1]. Oncotarget . 2015 Aug 28;6(25):21547-56.

RRx-001 has been used in clinical trials for the treatment of various tumors, including lymphoma, brain metastases, cholangiocarcinoma, colorectal tumors, and malignant solid tumors. Nibuprofen, a dinitroazacyclobutane derivative, possesses potential radiosensitizing activity. After administration, nibuprofen dilates blood vessels, thereby increasing tumor blood flow and improving oxygenation at the tumor site. By enhancing oxygenation levels, these tumor cells may become more sensitive to radiotherapy. Tumor hypoxia is closely related to tumor invasiveness, metastasis, and radiotherapy resistance.

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RRx-001 is a cyclic nitro compound (1‑bromoacetyl‑3,3‑dinitroazetidine) with molecular formula C5H6BrN3O5 and molecular weight 268.02. It is a redox‑active agent that generates reactive oxygen and nitrogen species, leading to epigenetic alterations including DNA methylation and histone acetylation. It binds to hemoglobin and influences red blood cell rheology, causing selective homing to hypoxic tumor endothelium and redistribution of blood flow (vascular normalization). This normalization improves delivery of chemotherapy and oxygen for radiosensitization. RRx-001 has been evaluated in Phase I and Phase II clinical trials for various cancers, including advanced colorectal cancer. It is also known as ABDNAZ. The synthesis and characterization are reported elsewhere. [2][3]

C5H6BRN3O5

268.02

266.949

C, 22.41; H, 2.26; Br, 29.81; N, 15.68; O, 29.85

925206-65-1

15950826

Solid powder

0.457

0

5

1

14

273

0

BrC([H])([H])C(N1C([H])([H])C(C1([H])[H])([N+](=O)[O-])[N+](=O)[O-])=O

JODKFOVZURLVTG-UHFFFAOYSA-N

InChI=1S/C5H6BrN3O5/c6-1-4(10)7-2-5(3-7,8(11)12)9(13)14/h1-3H2

2-bromo-1-(3,3-dinitroazetidin-1-yl)ethanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 2.5 mg/mL (9.33 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 25.0 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: ≥ 2.5 mg/mL (9.33 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 25.0 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: ≥ 2.5 mg/mL (9.33 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 25.0 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.)