E. coli nitroreductase (NTR); human NOR1 (oxidored-nitro domain containing protein 1). [1]

The monofunctional alkylating agent CB1954 can be transformed into a toxic form by overexpressing nitroreductase oxidored nitro domain containing protein 1 (NOR1), which reduces the potent cytotoxin CB1954's 4 nitro group. When used on the NPC cell line CNE1, toxic CB1954 increases cell death. Grb2 expression is upregulated and MAPK signal transduction is activated in the HepG2 cell line by the NOR1 gene, which increases CB1954-mediated cell cytotoxicity [1].

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In HepG2 cells overexpressing NOR1, CB1954 induced cell killing in a concentration-dependent manner. Cell viability was determined after 2 days using Trypan Blue exclusion. NOR1 overexpression markedly enhanced CB1954-induced cell killing compared to control cells (P<0.05). The tyrosine kinase inhibitor genistein (5 μmol/l) reduced CB1954-induced cell killing in pcDNA3.1(+)-NOR1-HepG2 cells to the level of untreated control cells. Stable transfection of Grb2 shRNA (pU6+27-shGrb2) decreased Grb2 protein expression by 4-fold and decreased CB1954-mediated activation of MAPK by approximately 3-fold, and inhibited CB1954-induced cell killing to the level of untreated control group. The MEK inhibitor PD98059 (1 μM) inhibited CB1954-induced cell killing in pcDNA3.1(+)-NOR1-HepG2 cells. CB1954 increased phosphorylated-MAPK levels in pcDNA3.1(+)-NOR1-HepG2 cells. Western blot analysis showed that Grb2 downregulation reduced MAPK activity in CB1954-stimulated cells. [1]

In vivo, the NTR/CB1954 system is used to specifically ablate cells. This inducible ablation system has a dose-dependent effect[3]. Cell proliferation is not necessary for CB1954-mediated NTR-mediated cell killing. DNA cross-linking caused by the activated CB1954 likely starts the apoptosis cascade and swiftly kills off cells. Functional p53 is not necessary for NTR-CB1954 to kill cells in a targeted and efficient manner[2].

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In transgenic mice expressing NTR in mammary luminal epithelial cells, administration of CB1954 resulted in rapid cell ablation. Cell death was observed as early as 7 hours after a single injection of the prodrug. By 24 hours post-injection, the effects were quite well advanced. This rapid response is advantageous compared to HSV1tk-based systems, which take days or weeks. [2]
- In p53-deficient mice (p53−/−, NTR+), CB1954 induced cell death in the mammary gland at a level morphologically indistinguishable from that in p53 wild-type mice, indicating that functional p53 is not required for CB1954-NTR-mediated cell death. However, the degree of ablation in p53 null mice was generally lower than in p53 wild-type littermates. Among wild-type p53 mice, five out of six showed extensive ablation (>80%), whereas among p53 null mice, four out of five showed only moderate (<40%) or restricted (<10%) cell death after 72 hours. The variance was attributed to mosaic expression of the transgene rather than p53 dependence. [2]

- In lactating transgenic mice given single or triple doses of CB1954 at 50 mg/kg body weight, considerably more cell death was observed in the mammary gland compared to animals given 10 mg/kg. At a dosage of 2 mg/kg, little or no effect was observed. [2]

The human hepatocellular carcinoma cells, HepG2, are kept in a humidified culture incubator at 37˚C with 5% CO2 and 95% air, supplemented with 10% fetal calf serum (FCS). Cell cytotoxicity tests are carried out in accordance with earlier guidelines. After reaching approximately 80% confluence, HepG2 cells are treated with CB1954 or a signal transduction inhibitor after being rinsed with PBS. In each experiment, measurements are taken from ten to twelve different microscopic fields, and data are compiled from three to five experiments.

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Human hepatocellular carcinoma HepG2 cells (NOR1 stably-transfected, empty vector-transfected, or wild-type) were maintained in RPMI-1640 with 10% fetal calf serum at 37°C in 5% CO2. For cell cytotoxicity assays, cells grown to approximately 80% confluence were washed with PBS and treated with various concentrations of CB1954 (4-10 μmol/l) and/or signal transduction inhibitors (genistein at 5 μmol/l, PD98059 at 1 μM) or shRNA constructs. Cell viability was determined after 2 days using Trypan Blue exclusion. Measurements were collected from 10-12 individual microscopic fields per experiment, and data were summarized from 3-5 experiments. For stable transfection, HepG2 cells were seeded at 1×10^5 cells/well in 6-well plates until ~75% confluence, then transfected with 3 μg of linearized pU6+27-shGrb2 or pU6+27-shControl plasmid using Lipofectamine 2000. After two days, cells were incubated with neomycin (0.5-1 mg/ml). Neomycin-resistant clones were isolated, grown, and examined by western blot analysis to select clones expressing minimal endogenous Grb2 (HepG2-shGrb2). For western blot analysis, cells were lysed in ice-cold lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1 mM MgCl2, 1 mM PMSF, 1% Triton X-100, 0.5% sodium deoxycholate, 2 mM sodium orthovanadate, 25 mM sodium fluoride, 1% aprotinin, 10 μg/ml leupeptin). Protein concentration was determined. Equal amounts of total protein (5-10 μg) were separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in TBST with 5% non-fat dry milk, then incubated with primary antibodies (anti-Grb2 1:5000; anti-phosphorylated MAPK 1:5000; anti-MAPK 1:1000; anti-β-actin 1:7500), followed by HRP-conjugated secondary antibodies. Protein bands were detected via enhanced chemiluminescence and scanned using a densitometer. [1]

RED 40 female mice expressing high levels of BLG-NTR transgene in the mammary gland and nontransgenic control mice on lactation day 6
50 mg/kg
i.p.

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For the time-course study, adult female RED40 transgenic mice (expressing NTR) were injected intraperitoneally (i.p.) with a single dose of CB1954 at 50 mg/kg body weight or mock-injected on lactation day 6. The mice were killed at 7, 17, and 24 hours after injection, and mammary tissues were collected. Non-transgenic control female mice were treated with CB1954 in the same manner. [2]

- For the p53 study, female mice aged 10–12 weeks with various genotypes were mated, and CB1954 was administered i.p. three times at 30 mg/kg body weight on lactation days 4 to 6. Mice of the same genotypes were mock-injected as controls. The mice were killed 24 hours after the last injection, and mammary glands and liver tissues were collected for analysis. In some cases, female mice failed to feed their pups after only two injections of CB1954, indicating early failure of lactation; tissues were collected immediately to prevent involution. [2]

- CB1954 was suspended in arachis oil containing 10% acetone at a concentration of either 3 mg/ml or 5 mg/ml. The suspension was warmed to 37°C before injection. [2]

All mice injected with CB1954, regardless of genotype, showed temporary weight loss. At the higher dose of 40 mg/kg body weight (three injections), transgenic mice showed acute morbidity including hunching, hypothermia (body temperature drop to 32°C ± 0.5), and death of some mice. At the lower dose of 20 mg/kg body weight, no hypothermic response or morbidity signs were observed. Weight loss was also attributed partly to effects of CB1954 on NTR-expressing bacteria in the gut. No direct toxic effect (necrosis) was observed. [3]

[1]. Oncol Lett . 2012 Sep;4(3):566-570.

[2]. Gene Ther . 1999 May;6(5):764-70.

[3]. J Endocrinol . 2002 Nov;175(2):487-98.

Tretazicar is being investigated in the clinical trial NCT00746590 (Study on the antitumor activity and safety of Prolarix™ in hepatocellular carcinoma). Tretazicar is a prodrug of a bifunctional alkylated dinitrobenzamide derivative with antitumor activity. In the presence of the cosubstrate calicotermine (an analog of the natural cosubstrate dihydronicotinamide nucleoside (NRH), with NRH acting as an electron donor), tretazicar can be activated by human quinone oxidoreductase 2 (NQO2). The resulting active but short-lived metabolite, dinitrobenzamide, inhibits DNA replication and induces apoptosis in NQO2-expressing cancer cells. NQO2 expression is typically latent due to the lack of the natural cosubstrate NRH, but it is upregulated in certain types of tumor cells.

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CB1954 is a monofunctional alkylating agent. NOR1 (oxidored-nitro domain containing protein 1) enhances CB1954-induced cell cytotoxicity through upregulation of Grb2 expression and activation of MAPK signal transduction in HepG2 cells. CB1954-induced cell killing is dependent on tyrosine kinase activity, Grb2, and the MEK/MAPK pathway. The human liver is capable of aerobic reductive bioactivation of CB1954 to cytotoxic metabolites in vitro. CB1954 has entered clinical trials in combination with E. coli NTR for gene-directed enzyme prodrug therapy (GDEPT). [1]

C9H8N4O5

252.1836

252.049

C, 42.86; H, 3.20; N, 22.22; O, 31.72

21919-05-1

89105

White solid powder

1.7±0.1 g/cm3

427.2±45.0 °C at 760 mmHg

173 °C

212.2±28.7 °C

0.0±1.0 mmHg at 25°C

1.715

1.28

1

6

2

18

385

0

[O-][N+](C1C([H])=C(C(C(N([H])[H])=O)=C([H])C=1N1C([H])([H])C1([H])[H])[N+](=O)[O-])=O

WOCXQMCIOTUMJV-UHFFFAOYSA-N

InChI=1S/C9H8N4O5/c10-9(14)5-3-7(11-1-2-11)8(13(17)18)4-6(5)12(15)16/h3-4H,1-2H2,(H2,10,14)

5-(aziridin-1-yl)-2,4-dinitrobenzamide

CB-1954; CB1954; CB 1954

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)

DMSO: 50~125 mg/mL (198.3~495.7 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.25 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 (8.25 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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 (Please use freshly prepared in vivo formulations for optimal results.)