CB 1954 revisited

Summary Although it has been the subject of considerable interest for 15 years, originally as a cytotoxic agent and more recently as a radiosensitizer, there is very little pharmacokinetic information on CB 1954 (2,4-dinitro-5-aziridinylbenzamide). We have developed a rapid high-performance liquid chromatography assay for the drug and its metabolites and applied it to detailed examination of the pharmacokinetics of CB 1954 in mice and dogs. With IV administration a dose of 50 mg/kg gave peak blood concentrations of 100 g/ml in mice, while 25 mg/kg gave peak palsma concentrations of 27 g/ml in dogs. Peak concentrations were 3 to 5-fold lower for the IP route in mice and the oral route in dogs, and the bioavailabilities were 85% and 40%, respectively. Elimination t_1/2 values were 1.4–2 h in mice and 2.5–4 h in dogs and were independent of route of administration. Plasma protein binding was 57% but tissue penetration in mice was generally good. Tumour: plasma ratios were 50%–90%, while brain: plasma ratios were lower, at 37%–50%. The parent drug and several metabolites were identified and quantified in mouse urine, the total recovery being 24%–29%, of which 16%–25% was parent drug. The metabolites were also found in the circulation and in tissues. No changes in pharmacokinetics were seen with repeated dosing in mice or with administration of the protective agent phenyl AIC. Phenobarbitone pretreatment produced a small reduction in elimination t_1/2, mainly by accelerating aziridine ring removal. Allopurinol increased the blood levels of the 5-amino nitroreduction product. These studies provide a pharmacokinetic basis for interpreting the antitumour activity and toxicity of CB 1954, as well as for the development of new mixed-function sensitizers.     

Pharmacology of the mixed-function radio- and chemosensitizers CB 1954 and RSU 1069

We have studied the pharmacokinetics and metabolism in mice of CB 1954 and RSU 1069. Containing both nitro and alkylating (aziridine) substituents, these are lead compounds in the mixed-function analogue series, which show particular promise for sensitizer development. Both compounds are degraded extensively, via pathways including nitro reduction, aziridine ring hydrolysis and aziridine ring removal RSU 1069 (plasma t1/2 22 min) was eliminated more rapidly than CB 1954 (blood t1/2 84 min), and the AUC was three times less. Tissue/plasma ratios tended to be rather lower than those for simple nitroimidazoles of intermediate lipophilicity, which are usually close to 100%. With CB 1954, for example, tumor/plasma and brain/plasma ratios were 58 and 37% respectively, whereas with RSU 1069 the values were 29 and 26%. Nevertheless, tumor concentrations were consistent with potent sensitization. There is, however, scope for pharmacokinetic fine-tuning to modify tissue penetration as appropriate.     

Comparative DNA damage and repair induced by misonidazole, CB 1954 and RSU 1069

We have studied the ability of CB 1954, misonidazole, and RSU 1069 to induce biologically relevant DNA damage in single- and double-stranded phi X174 DNA under oxic, anoxic, and anoxic reductive conditions using a double transfection technique. In addition, the ability of the three drugs to induce the SOS repair response in E. coli under the same conditions was measured. Whereas the relative order of DNA damage was RSU 1069 greater than CB 1954 greater than misonidazole the order in inducing SOS repair was RSU 1069 greater than misonidazole greater than CB 1954. Drug-induced damage by RSU 1069 involves enhanced damage by endonuclease III suggesting drug-induced pyrimidine damage. There appears to be no correlation between drug-induced damage and the degree of SOS repair induction. Thus it appears that enzymes other than, or in addition to, those of the SOS repair system are involved in the repair of DNA damage induced by these drugs.     

硝基还原酶/CB1954自杀基因系统对宫颈癌Hela细胞杀伤效应的实验研究

目的:体外观察大肠杆菌硝基还原酶/[5-(1-氮丙啶)-2,4-二硝基苯甲酰胺](以下简称NTR/CB1954)自杀基因系统对宫颈癌Hela细胞的杀伤效应,探索一种新的宫颈癌基因治疗方法.方法: 利用PCR技术从Escherichia coli K12的基因组中扩增出编码NTR的基因nfsB,酶切后,连接到真核表达载体pcDNA3上,获得重组载体pcDNA3-nfsB,lipofectamineTM2000脂质体转染法将pcDNA3-nfsB转染Hela细胞,筛选稳定表达细胞株,应用RT-PCR以及SDS-PAGE检测NTR在Hela细胞中的表达,MTT法检测NTR/CB1954对Hela细胞活力的影响,流式细胞术检测亚二倍体细胞率改变,PI/Hoechest33258双染荧光显微镜下观察Hela细胞凋亡率.结果: 成功构建了真核表达载体pcDNA3-nfsB,获得稳定表达NTR的Hela细胞株,在mRNA水平以及蛋白水平检测到NTR在Hela细胞中的表达,NTR/CB1954自杀基因系统明显影响Hela细胞的活力,增加了Hela细胞的凋亡率.结论: NTR/CB1954自杀基因系统对Hela细胞在体外通过凋亡产生明显的杀伤效应.     

硝基还原酶／CB1954自杀基因系统对宫颈癌Hela细胞杀伤效应的实验研究

目的：体外观察大肠杆菌硝基还原酶／[5-（1-氮丙啶）-2,4-二硝基苯甲酰胺]（以下简称NTR／CB1954）自杀基因系统对宫颈癌Hela细胞的杀伤效应,探索一种新的宫颈癌基因治疗方法。方法：利用PCR技术从Escherichia coli K12的基因组中扩增出编码NTR的基因nfsB,酶切后,连接到真核表达载体pcDNA3上,获得重组载体pcDNA3-nfsB,lipofectamine^TM2000脂质体转染法将pcDNA3-nfsB转染Hela细胞,筛选稳定表达细胞株,应用RT—PCR以及SDS—PAGE检测NTR在Hela细胞中的表达,Mrrr法检测NTR／CB1954对Hela细胞活力的影响,流式细胞术检测亚二倍体细胞率改变,PI／Hoechest33258双染荧光显微镜下观察Hela细胞凋亡率。结果：成功构建了真核表达载体pcDNA3-nfsB,获得稳定表达NTR的Hela细胞株,在mRNA水平以及蛋白水平检测到NTR在Hela细胞中的表达,NTR／CB1954自杀基因系统明显影响Hela细胞的活力,增加了Hela细胞的凋亡率。结论：NTR／CB1954自杀基因系统对Hela细胞在体外通过凋亡产生明显的杀伤效应。     

Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954

Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. 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. 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. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy.     

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.     

E. coli nitroreductase/CB1954 gene-directed enzyme prodrug therapy: role of arylamine N-acetlytransferase 2

Gene-directed enzyme prodrug therapy is a promising approach to the local management of cancer and a number of gene prodrug combinations have entered clinical trials. The antitumor activity of Escherichia coli nitroreductase (NTR) in combination with the prodrug CB1954 relies on the reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells. We examined whether secondary metabolic activation of the N-hydroxylamines by sulfotransferases or acetyltransferases altered cell responsiveness to the drug. We evaluated the coexpression of NTR with the human cytosolic sulfotransferases SULT1A1, 1A2, 1A3, 1E1 and 2A1, or the human arylamine N-acetyltransferases NAT1 and NAT2 on SKOV3 cell survival. Only NAT2 significantly altered the toxicity of CB1954, decreasing the IC(50) 16-fold from 0.61 to 0.04 microM. These results suggest that one or more of the N-hydroxyl metabolites are a substrate for O-acetylation by NAT2. We also examined the bystander effect of SKOV3 cells expressing NTR or NTR plus NAT2. Addition of the acetyltransferase resulted in a significant decreased bystander effect (P>0.01), possibly due to a lower concentration of reactive metabolites in the culture medium. These results suggest that a combination of bacterial NTR and NAT2 may provide a greater clinical response at therapeutic concentrations of CB1954 provided the reduction in bystander effect is not clinically significant. Moreover, endogenous NAT2, which is localized predominantly in the liver and gut, may be involved in the dose-limiting hepatic toxicity and gastrointestinal side effects seen in patients treated with the higher doses of CB1954.     

Chemopotentiation by CB 1954: the importance of postincubations and the possible involvement of poly(ADP-ribosylation)

CB 1954 potentiates the cytotoxic action of the bifunctional alkylating agent melphalan (L-PAM). In vitro, this potentiation does not require the preincubation in hypoxia normally needed for other nitroaromatic compounds such as misonidazole. Chemopotentiation is observed when cells are held in CB 1954 in air after treatment with L-PAM. This may reflect an inhibition of DNA repair process(es). Structural considerations suggested that CB 1954 might be acting as an inhibitor of poly(ADP-ribosylation). However, an inhibition of the drop in NAD levels consequent on exposure to melphalan was not obtained. Furthermore, unlike the known poly(ADP-ribose) inhibitor, 3-aminobenzamide, CB 1954 does not potentiate the cytotoxicity of the monofunctional alkylator N-methyl-N nitro N-nitrosoguanidine, or inhibit NAD depletion caused by this agent. Therefore the evidence suggests that CB 1954 is not an inhibitor of poly(ADP ribosylation).     

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO₂ or 4-NO₂ positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO₂ group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.Gene-directed enzyme prodrug therapy (GDEPT) is a developing strategy for cancer treatment, involving delivery to tumour cells of an exogenous gene, encoding an enzyme that can convert a non-toxic prodrug into cytotoxic products. In principle, local generation of highly reactive cytotoxins within the cancer cells allows optimal therapeutic effect, whereas systemic toxicity remains lower than with conventional chemotherapy (McNeish et al, 1997; Niculescu-Duvaz and Springer, 2005; Russell and Khatri, 2006). As specific gene delivery to all tumour cells seems unattainable using current methods, the ability of an activated prodrug to spread locally from cell to cell is considered an essential feature for a successful GDEPT system. It allows tumour cells that have escaped gene transfer to be killed as a result of prodrug activation in nearby cells that express the activating enzyme. This is known as the ‘bystander effect'' (Freeman et al, 1993).CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) is a prodrug, which is converted from a weak, monofunctional alkylating agent to a potent bifunctional alkylating agent upon nitroreduction (Knox et al, 1988, 1991; Friedlos et al, 1992). Anlezark et al (1992) reported that the nitroreductase encoded by the nfsB gene of Escherichia coli could activate CB1954, leading to the initial adoption of this enzyme for use with CB1954 in GDEPT (Anlezark et al, 1992; Bridgewater et al, 1995; Grove et al, 1999; Searle et al, 2004). The expression of NfsB in cancer cells using replication-defective retrovirus or adenovirus vectors was shown to confer greatly increased sensitivity to CB1954, and anti-tumour activity was shown in vivo (McNeish et al, 1998; Djeha et al, 2000, 2001; Weedon et al, 2000). After the initial phase I clinical trials of CB1954 alone (Chung-Faye et al, 2001), and of a replication-defective adenovirus expressing NfsB (CTL102) (Palmer et al, 2004), the combination has been tested in a phase I/II trial in patients with prostate cancer. A decline in serum levels of prostate specific antigen in some patients suggests anti-tumour activity (Patel et al, 2009); however, greater efficacy is desirable.NfsB is a homodimeric flavoenzyme which can use either NADH or NADPH to reduce the tightly bound FMN; after dissociation of the NAD(P)⁺; a variety of nitroaromatic or quinone substrates can be reduced in a second reaction step (Anlezark et al, 1992; Lovering et al, 2001; Race et al, 2005). The published K_m for NfsB nitroreductase with CB1954 is 862 μM (Anlezark et al, 1992), which is considerably higher than its peak serum concentration achievable in humans (5–10 μM) (Chung-Faye et al, 2001). Thus, the activation of CB1954 by NfsB in vivo will be very inefficient, justifying the consideration of alternative enzymes (Anlezark et al, 2002). NfsB was originally identified through its role in bacterial sensitivity to nitrofuran antibiotics (Sastry and Jayaraman, 1984). Selection of E. coli for nitrofurazone resistance leads first to mutations in the major nitroreductase gene, nfsA (Whiteway et al, 1998), and only subsequently in nfsB. Alignment of the amino acid sequences shows only 28 identical and 32 conserved residues out of the 242 or 217 residues of NfsA and NfsB, respectively, and antibodies specific for NfsB do not cross-react with NfsA (unpublished results). The two enzymes share many structural features, including a 5-stranded anti-parallel β-sheet core and surrounding α-helices, with the two active sites occupying clefts at the dimer interface and presenting the re-face of the FMN isoalloxazine ring towards the substrate pocket (Kobori et al, 2001; Lovering et al, 2001). Although some residues around the active site are conserved, the active site of NfsA is more open than that of NfsB, an observation that contributed to our decision to investigate its activity with CB1954 and other prodrugs. Kinetic studies of the purified enzymes showed that NfsA has a two- to three-fold greater activity with nitrofurazone and several other nitroaromatic substrates, compared with NfsB (Zenno et al, 1996). The report that NfsA has a marked preference for the cofactor NADPH (Zenno et al, 1996) suggested that this enzyme may have been overlooked in the initial isolation of NfsB for the reduction of CB1954, which used NADH as a cofactor (Anlezark et al, 1992). One study has shown that NfsA is capable of CB1954 activation (Barak et al, 2006); however, to our knowledge, no earlier study has compared the activities of NfsA and NfsB with CB1954. In this study, we compare the ability of these two E. coli nitroreductases to sensitise cells to CB1954 and a selection of other prodrugs. We also compare their kinetics of CB1954 activation in vitro, and show that NfsA preferentially reduces the 2-NO₂ group of CB1954, resulting in an improved bystander cell killing. Overall, the results suggest that NfsA could have advantages over NfsB for use in GDEPT with CB1954 or several other nitroaromatic prodrugs.     

The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT)

Walker cellsin vivo orin vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of k_cat= 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated fromE. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.     

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001).     

Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954

A recombinant retrovirus encoding E. coli nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTR/CB1954 cell killing was effective in non-cycling cells. Co-operative killing was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.     

Studies on the reversal of the selective antitumour effect of the aziridinyl derivative CB 1954 by 4-amino-5-imidazolecarboxamide

The selective toxicity of CB 1954 (5-aziridinyl-2,4-dinitrobenzamide) for Walker tumour cells, previously shown in the whole animal and in an in vitro/in vivo system, has been confirmed in an in vitro system In each case its actions on the Walker tumour were reversed by 4-amino-5-imidazolecarboxamide, the ribotide of which is a purine precursor, provided it was given before or soon after the CB 1954. Other purines also produced this reversal, while pyrimidines and several other compounds did not. This evidence suggested that CB 1954, in addition to its alkylating properties, had features of a purine antimetabolite. However, anthranilamide was also very effective in protecting against CB 1954, suggesting that reversal could be achieved by compounds structurally related to CB 1954 which competed with it for some receptor site.     

The nitroreductase/CB1954 combination in Epstein-Barr virus-positive B-cell lines: induction of bystander killing in vitro and in vivo

Epstein-Barr virus (EBV)-based gene delivery vectors that preferentially express toxic genes in EBV-infected cells could be used to target EBV-positive tumors for destruction. We have shown previously that the cytosine deaminase (CD) enzyme, which converts the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil efficiently kills EBV-positive cells in the presence of 5-FC, with a substantial bystander killing effect in vitro and in vivo. To identify the optimal enzyme/prodrug combination for treating EBV-positive lymphomas, we have compared the effectiveness of the CD/5-FC combination with the nitroreductase (NTR)/CB1954 combination for killing EBV-positive B-cell lines. NTR metabolizes CB1954 into an alkylating agent that cross-links DNA. When the CD gene or the NTR gene were transfected into two different EBV-positive B-cell lines in vitro, approximately 90% of cells were killed in a prodrug-dependent manner, although the transfection efficiency was <5%. However, severe combined immunodeficient mouse tumors containing either 30% or 100% of NTR-expressing Burkitt lymphoma (Jijoye) cells were growth inhibited, but not cured, by treatment with intraperitoneal CB1954 (20 mg/kg/day) for 10 days. These results suggest that the NTR/CB1954 combination induces efficient bystander killing of EBV-positive B-cell lines in vitro but may not be as effective as the CD/5-FC combination for treating B-cell lymphomas in vivo.     

Studies on the mechanism of action of 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), a selective inhibitor of the Walker tumour

CB 1954, a monofunctional alkylating agent, has exceptional activity against the Walker carcinoma yet is inactive against a large number of other animal tumours. A line of the Walker tumour with acquired resistance to CB 1954 was cross-resistant to Melphalan, implying a common mechanism of action, although other tumours highly sensitive to Melphalan were not affected by CB 1954. CB 1954 was also similar to Melphalan in its effects on DNA and RNA synthesis. No correlation was observed between binding of CB 1954 to the DNA of tumour cells and their sensitivity to it. Differences in response of the Walker tumour when incubated with Melphalan and CB 1954 suggest that the latter agent may have some features of an anti-metabolite. In preliminary experiments, its selective anti-tumour effect against the Walker carcinoma has been completely reversed by an intermediate in purine biosynthesis.     

Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70

Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach to local management of cancer through targeted chemotherapy. Killing localized tumors by GDEPT in a manner that induces strong antitumor cellular immune responses might improve local management and allow benefit in disseminated cancer. Here we evaluated the combination of nitroreductase (NTR)/CB1954 GDEPT with high-level expression of heat shock protein 70 (HSP70, a stress protein that can shuttle cytosolic peptides into antigen-presenting cells) for induction of antitumor immunity using adenovirus gene delivery in an aggressive and nonimmunogenic BALB/c syngeneic 4T1 breast cancer model. The mechanism of cell death and spectrum of stress proteins induced are likely to be important determinants of the resulting immune responses. We showed that NTR/CB1954 treatment of 4T1 cells gave both apoptotic and nonapoptotic killing. In vivo killing of 4T1 cells expressing NTR gave weak antitumor immunity and very limited induction of stress proteins including HSP70. High-level coexpression of HSP70 during NTR/CB1954-mediated killing of 4T1 cells in vivo gave much greater protection from tumor challenge (67% long-term survivors compared to 17%) and induced 4T1-specific cytotoxic T-cell responses. The enhancement of antitumor responses resulting from HSP70 coexpression was similar to that conferred by coexpression of GM-CSF.     

Virus-directed, enzyme prodrug therapy with nitroimidazole reductase: a phase I and pharmacokinetic study of its prodrug, CB1954.

CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is converted by the bacterial enzyme nitroimidazole reductase (NTR) into a potent cytotoxic bifunctional alkylating agent, which can be delivered to tumors in adenoviral vectors as virus-directed, enzyme prodrug therapy. This report summarizes a Phase I and pharmacokinetic study of the prodrug, CB1954. Thirty patients, ages 23-78 years (median 62 years), with predominantly gastrointestinal malignancies were treated. CB1954 was administered by i.v. injection every 3 weeks or i.p. followed by 3-weekly i.v. injections, toward a maximum of six cycles. The dose was escalated from 3 to 37.5 mg/m2. No significant toxicity was seen until 24 mg/m2 (recommended i.v. dose). Dose-limiting toxicities (DLT) were diarrhea and hepatic toxicity, seen at 37.5 mg/m2. DLT has not been observed at the current i.p. dose of 24 mg/m2. There was no alopecia, marrow suppression, or nephrotoxicity. Clearance data suggest hepatic metabolism, and <5% of CB1954 was renally excreted. There was a nonlinear relationship between i.v. dose and area under the curve (AUC). At the recommended i.v. dose of 24 mg/m2, the AUC was 5.8 microM/h. Intraperitoneal administration (24 mg/m2) achieved an AUC of 387 microM/h, giving a considerable regional advantage. 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. We are now conducting a Phase I trial combining adenovirus-mediated NTR and i.v. CB1954 (24 mg/m2) in patients with primary and secondary liver tumors.