Disease relevance of CCRIS 1631

• Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954 [1].
• Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be approximately 5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 micro M than was the WT enzyme [1].
• The high level of nitroreductase expression observed at 1 to 5 x 10(11) virus particles mandates further studies in patients with inoperable tumors who will receive CTL102 and CB1954 [2].
• Virus-directed enzyme prodrug therapy utilizing the bacterial enzyme nitroreductase delivered by a replication-defective adenovirus vector to activate the prodrug CB1954 is a promising strategy currently undergoing clinical trials in patients with a range of cancers [3].
• Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954 [4].

High impact information on CCRIS 1631

• Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy [1].
• Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement [1].
• The objective of this study was to develop a physiologically relevant tissue culture model for quantifying bystander effects and to validate the model using as an example the activation of dinitrobenzamide prodrugs (e.g., CB 1954) by Escherichia coli aerobic nitroreductase (NTR) [5].
• In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954 [6].
• Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy [6].

Chemical compound and disease context of CCRIS 1631

• Importantly for clinical application in ovarian cancer, a cisplatin-resistant ovarian tumour cell line remains as susceptible to the NTR-dependent cytotoxicity of CB1954 as parental cells [4].
• The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells [7].
• It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced) [8].
• 5. Alternative prodrugs may eventually prove superior to CB1954; a nitroaryl phosphoramide mustard prodrug activated by NTR shows a greater therapeutic index than CB1954 in a human ovarian carcinoma [9].
• Using multiparameter regression analysis, we have found that the toxicity of CB-1954 derivatives as well as that of several non-alkylating nitroaromatics, increased upon the increase in their single-electron reduction potential (E(1)7) and octanol/water partition coefficient (P), and almost did not depend on their reactivity towards DT-diaphorase [10].

Biological context of CCRIS 1631

• Placing the nitroreductase gene under the control of the telomerase gene promoters sensitized cancer cells in tissue culture to the pro-drug CB1954 and promoter activity was predictive of sensitization to the pro-drug (2-20-fold sensitization), with cell death restricted to lines exhibiting high levels of promoter activity [11].
• Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128 [12].
• In vitro, the AUC required to achieve the IC50 for CB1954, in NTR-expressing cancer cells, ranges from 10-50 microM/h. Thus, CB1954 is well tolerated at a dose of 24 mg/m2, and sufficient serum/peritoneal levels are achieved for an enzyme-prodrug approach to be feasible [13].
• Gene-directed enzyme prodrug therapy: quantitative bystander cytotoxicity and DNA damage induced by CB1954 in cells expressing bacterial nitroreductase [14].
• Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector [15].

Anatomical context of CCRIS 1631

• There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH [6].
• Infection with adenoviral telomerase-NTR constructs in a panel of seven cancer cell lines resulted in up to 18-fold sensitization to the prodrug CB1954, an effect that was retained in two drug-resistant ovarian lines [16].
• Unlike the Tcf-NTR virus, the CMV-NTR virus expresses NTR in human lung fibroblasts and sensitizes these normal cells to CB1954 [17].
• Upon CB1954 treatment, transgenic mice show extensive cell depletion in thymus and spleen (14-16% of normal cell numbers), whereas all other tissues are unaffected by prodrug administration [18].
• The astrocytes expressing NTR were selectively ablated after administration of the prodrug CB1954, resulting in motor discoordination [19].

Associations of CCRIS 1631 with other chemical compounds

• One example of suicide gene therapy is the bacterial nitroreductase (NTR) gene, which bioactivates the prodrug CB1954 into an active cytotoxic alkylating agent [16].
• The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach [20].
• While CB 1954 gave both 2- and 4-hydroxylamine metabolites in NTR+ve cells, related analogues with substituted carboxamides gave only a single hydroxylamine metabolite possibly because the steric bulk in the side chain constrains binding within the active site [21].
• Compound 20 is a much less efficient substrate than CB 1954 for the major aerobic nitroreductase from rat Walker tumor cells, NAD(P)H:quinone oxidoreductase (DT diaphorase) [22].
• Irrespective of the differences between the various reduced pyridinium derivatives in their ability to act as co-factors for the reduction of menadione by DT diaphorase, all the compounds that showed activity in this assay were equally effective co-factors for the reduction of the nitrobenzamide, CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) [23].

Gene context of CCRIS 1631

• The human form of NQO1 metabolizes CB 1954 much less efficiently than rat NQO1 [24].
• The PSA-based nitroreductase virus produced comparable amounts of nitroreductase and sensitization to CB1954 approaching that of the CMV-driven virus [25].
• Moreover, injection of SW480 xenografts (mutated p53) with Ad.p53R resulted in a clear inhibition of growth in response to the prodrug CB1954 [26].
• These results agree well with those of the in vitro enzyme assays that show that Q104Y is significantly more active than the wild-type DT-diaphorase in the activation of CB 1954 [27].
• Our site-directed mutagenesis study has revealed that residue 104 (Tyr in the rat enzyme and Gln in the human enzyme) is an important residue responsible for the catalytic differences between the rat and the human enzymes in the activation of CB 1954 (S. Chen et al., 1997, J. Biol. Chem. 272, 1437-1439) [27].

Analytical, diagnostic and therapeutic context of CCRIS 1631

• The use of the bacterial enzyme nitroreductase to activate CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a short lived, highly toxic DNA cross-linking agent has been demonstrated in tumor xenografts [2].
• The in vivo activity of the viruses was tested in SW620 xenografts in nude mice by intravenous injection of 1,011 particles of virus followed 1 week later by intraperitoneal injections of CB1954 [17].
• Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects [28].
• Towards gene therapy in prosthesis loosening: efficient killing of interface cells by gene-directed enzyme prodrug therapy with nitroreductase and the prodrug CB1954 [29].
• Chemopotentiation is observed when cells are held in CB 1954 in air after treatment with L-PAM [30].

References