Bystander killing of tumour cells by antibody-targeted enzymatic activation of a glucuronide prodrug. (1/14)

RHI-betaG-PEG, formed by linking poly(ethylene glycol)-modified beta-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 microg ml(-1) RH1-betaG-PEG and 20 microM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-betaG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-betaG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo.  (+info)

A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells. (2/14)

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.  (+info)

Quantitation of bystander effects in nitroreductase suicide gene therapy using three-dimensional cell cultures. (3/14)

The efficacy of cancer gene therapy depends critically on "bystander effects" by which genetic modification of tumor cells results in killing of unmodified cells in the local microenvironment. In gene-dependent enzyme-prodrug therapy, expression of a prodrug-activating suicide gene is used to generate a cytotoxic metabolite that diffuses to nontransduced cells. 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). Bystander effects were measured in three-dimensional multilayer cocultures of NTR+ and NTR- cells by determining clonogenic survival curves for both cell types using V79, Skov3, or WiDr as parental cell lines. Bystander killing by CB 1954 was much more efficient in multilayers than monolayers at equivalent cell:medium ratios, whereas the chloromustard analogue of CB 1954 showed even greater efficiency. For a series of dinitrobenzamides, bystander killing in multilayers showed a positive correlation with prodrug lipophilicity and also correlated with the bystander effect in mixed tumor xenografts grown from the same NTR+ and NTR- WiDr cell lines (r(2) = 0.84; P < 0.001). The multilayer model identified a bromomustard prodrug (SN 24927) with superior therapeutic activity to CB 1954 that provided curative activity against WiDr tumors comprising 1:1 mixtures of NTR+ and NTR- cells. This study demonstrates the utility of the multilayer tissue culture model for quantifying and optimizing bystander effects in tumors and identifies a new lead prodrug for NTR gene-dependent enzyme-prodrug therapy.  (+info)

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. (4/14)

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.  (+info)

Design, synthesis, and evaluation of estradiol-linked genotoxicants as anti-cancer agents. (5/14)

A series of bifunctional compounds was prepared consisting of 17beta estradiol linked to a DNA damaging N,N-bis-(2-chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with DNA and binding of the resultant covalent adducts to the estrogen receptor. Linker characteristics were pivotal determinants underlying the ability of the compounds to kill selectively breast cancer cells that express the estrogen receptor.  (+info)

Specific activation of glucuronide prodrugs by antibody-targeted enzyme conjugates for cancer therapy. (6/14)

Cancer chemotherapy may be improved by increasing antineoplastic drug specificity for tumor cells. We have synthesized a glucuronide prodrug that can be enzymatically converted to an antineoplastic agent at tumor cells that are able to bind beta-glucuronidase-monoclonal antibody conjugates. The glucuronide prodrug BHAMG, the tetra-n-butyl ammonium salt of (p-di-2-chloroethylaminophenyl-beta-D-glucopyranoside) uronic acid, was 150 times less toxic than the parent drug, N,N-di-(2-chloroethyl)-4-hydroxyaniline, to HepG2 human hepatoma cells and over 1000-fold less toxic than the parent drug to AS-30D rat hepatoma cells in vitro. In the presence of beta-glucuronidase, BHAMG was activated and became as toxic as the parent drug N,N-di-(2-chloroethyl)4-hydroxyaniline. A conjugate (RH1-beta G) was formed by linking beta-glucuronidase to a monoclonal antibody which binds to an antigen expressed on the surface of AS-30D cells. The concentration of BHAMG causing 50% inhibition of AS-30D cellular protein synthesis was reduced over 1000-fold, from greater than 770 microM to less than 0.74 microM after these cells were preincubated with RH1-beta G. Specificity of BHAMG activation at antigen-positive cells was shown by monoclonal antibody RH1 blocking of RH1-beta G conversion of BHAMG to toxic drug and by the inability of BHAMG to be converted to active drug when antigen-negative control cells were preincubated with RH1-beta G. Our results show that the targeted-beta-glucuronidase activation of BHAMG can increase the specificity of chemotherapy for rat hepatoma in vitro and suggest that the targeted activation of glucuronide prodrugs may be useful for cancer therapy.  (+info)

A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug. (7/14)

PURPOSE: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti-carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination. EXPERIMENTAL DESIGN: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized. RESULTS: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease. CONCLUSIONS: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.  (+info)

Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target. (8/14)

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