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Cancer Immunology, Immunotherapy : CII Aug 1997Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of...
Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.
Topics: Aniline Mustard; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Enzyme Stability; Glucuronates; Glucuronidase; Immunoconjugates; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Polyethylene Glycols; Prodrugs; Rats; Transplantation, Heterologous
PubMed: 9298932
DOI: 10.1007/s002620050387 -
Journal of the American Chemical Society Mar 2002We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming...
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.
Topics: Aniline Mustard; Antineoplastic Agents, Alkylating; Breast Neoplasms; DNA Adducts; Drug Design; Estradiol; Humans; Kinetics; Receptors, Estrogen; Substrate Specificity; Tumor Cells, Cultured
PubMed: 11866593
DOI: 10.1021/ja017344p -
International Journal of Cancer Dec 2001Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is...
Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is generated during ADEPT by employing an immunoenzyme composed of the monoclonal antibody (MAb) RH1 conjugated to beta-glucuronidase to target rat AS-30D hepatocellular carcinoma tumors. A glucuronide prodrug of p-hydroxyaniline mustard was used to treat malignant ascites after immunoenzyme localization at the cancer cells. ADEPT cured more than 96% of Sprague-Dawley rats bearing advanced malignant ascites, and all cured rats were protected from a lethal challenge of AS-30D cells. Immunization with radiation-killed AS-30D cells or AS-30D cells coated with immunoenzyme did not provide tumor protection. Likewise, ex vivo treatment of tumor cells by ADEPT before injection into rats did not protect against a tumor challenge. AS-30D and N1-S1 hepatocellular carcinoma cells but not unrelated syngeneic tumor cells were lysed by peritoneal exudate cells isolated from ADEPT-cured rats. Depletion of CD8(+) but not CD4(+) T cells or natural killer (NK) cells reduced the cytolytic activity of peritoneal lymphocytes. ADEPT did not cure tumor-bearing rats depleted of CD4(+) and CD8(+) T cells even though it was curative when given 7 days after tumor transplantation in rats with an intact immune system, indicating that ADEPT can synergize with host immunity to increase therapeutic efficacy. These results have important implications for the clinical application of ADEPT.
Topics: Aniline Mustard; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cytosine Deaminase; Glucuronidase; Liver Neoplasms, Experimental; Nucleoside Deaminases; Prodrugs; Rats; Rats, Sprague-Dawley; T-Lymphocytes, Cytotoxic
PubMed: 11745488
DOI: 10.1002/ijc.1550 -
Chemico-biological Interactions Jun 1997The rates of the non-enzymatic conjugation of the substituted aniline mustards, melphalan, chlorambucil and p-(N,N-bis(2-chloroethyl))toluidine with glutathione and...
The rates of the non-enzymatic conjugation of the substituted aniline mustards, melphalan, chlorambucil and p-(N,N-bis(2-chloroethyl))toluidine with glutathione and thiosulfate were determined using nuclear magnetic resonance spectroscopy. Using this method, the disappearance of drug and the formation of both the mono-thioether and bis-thioether conjugates can be monitored directly. For glutathione conjugation, the rate constants for the formation of the first and second aziridinium intermediates were similar. With thiosulfate conjugation, the rate constant for the formation of the first aziridinium intermediate is greater than the rate constant for the formation of the second aziridinium. This demonstrates that the type of nucleophile has a significant influence on the overall alkylating activity of these bifunctional mustards. The bisthioether adduct formed from the reaction between p-(N,N-bis([2-13C]-2-chloroethyl))toluidine and glutathione and thiosulfate can be identified and scrambling of the 13C label in the product provides strong evidence that the alkylation must occur through an aziridinium intermediate.
Topics: Aniline Mustard; Aziridines; Carbon Radioisotopes; Glutathione; Kinetics; Magnetic Resonance Spectroscopy; Melphalan; Thiosulfates
PubMed: 9233374
DOI: 10.1016/s0009-2797(97)00036-7 -
Proceedings of the National Academy of... Jan 1996Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many...
Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used chemotherapeutic agents. Most ADEPT systems incorporate a bacterial enzyme, and thus their potential is reduced because of the immunogenicity of that component of the conjugate. This limitation can be circumvented by the use of a catalytic antibody, which can be "humanized," in place of the bacterial enzyme catalyst. We have explored the scope of such antibody-directed "abzyme" prodrug therapy, ADAPT, to evaluate the potential for a repeatable targeted cancer chemotherapy. We report the production of a catalytic antibody that can hydrolyze the carbamate prodrug 4-[N,N-bis(2-chloroethyl)]aminophenyl-N-[(1S)-(1,3- dicarboxy)propyl]carbamate (1) to generate the corresponding cytotoxic nitrogen mustard (Km = 201 microM, kcat = 1.88 min-1). In vitro studies with this abzyme, EA11-D7, and prodrug 1 lead to a marked reduction in viability of cultured human colonic carcinoma (LoVo) cells relative to appropriate controls. In addition, we have found a good correlation between antibody catalysis as determined by this cytotoxicity assay in vitro and competitive binding studies of candidate abzymes to the truncated transition-state analogue ethyl 4-nitrophenylmethylphosphonate. This cell-kill assay heralds a general approach to direct and rapid screening of antibody libraries for catalysts.
Topics: Aniline Mustard; Antibodies, Catalytic; Antibody Affinity; Antineoplastic Agents, Alkylating; Carcinoma; Colonic Neoplasms; Haptens; Humans; Hydrolysis; Nitrogen Mustard Compounds; Prodrugs; Tumor Cells, Cultured
PubMed: 8570638
DOI: 10.1073/pnas.93.2.799 -
British Journal of Cancer Dec 1971The rat Walker 256 tumour was grown in mice that had previously been thymectomized and treated with anti-lymphocyte serum. These rat tumour-bearing mice were used to... (Comparative Study)
Comparative Study
The rat Walker 256 tumour was grown in mice that had previously been thymectomized and treated with anti-lymphocyte serum. These rat tumour-bearing mice were used to determine the therapeutic indices of 4 anti-tumour drugs.The agent with the highest index of the four examined was 5-aziridino 2,4-dinitrobenzamide (CB1954), followed by melphalan, aniline mustard and methotrexate, in that order. This rank order is the same as that found when therapeutic indices are determined on the Walker tumour growing in the rat. In this system, therefore, drugs have been ranked correctly in effectiveness against a rat tumour by measuring their effects on the tumour when growing in an immunosuppressed xenogeneic species. The implications for testing the drug sensitivity of individual human tumours before treating the patient are discussed.
Topics: Animals; Antilymphocyte Serum; Antineoplastic Agents; Azirines; Benzoates; Carcinoma 256, Walker; Immunosuppression Therapy; Lethal Dose 50; Melphalan; Methotrexate; Mice; Neoplasm Transplantation; Nitro Compounds; Nitrogen Mustard Compounds; Rats; Thymectomy; Transplantation, Heterologous
PubMed: 5144544
DOI: 10.1038/bjc.1971.97 -
British Journal of Cancer Nov 1995ADEPT is an antibody-based targeting strategy for the treatment of cancer. We have developed two new prodrugs, 4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl-L-...
ADEPT is an antibody-based targeting strategy for the treatment of cancer. We have developed two new prodrugs, 4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl-L- glutamic acid (PGP) and (S)-2-[N-[4-[N,N-bis(2-chloroethyl)amino]- phenoxycarbonyl]amino]-4-(5-tetrazoyl)butyric acid (PTP), which are cleaved by the bacterial enzyme CPG2 to release the 4-[N,N-bis(2-chloroethyl)amino] phenol drug. In vitro, both prodrugs are approximately 100- to 200-fold less potent than the parent drug (1 h IC50 = 1.4 microM) in LoVo colorectal tumour cells. These prodrugs have been evaluated for utility in ADEPT when used in combination with a conjugate of CPG2 and the F(ab')2 fragment of the anti-CEA monoclonal antibody, A5B7. The conjugate was shown to localise specifically to established LoVo tumour xenografts growing in nude mice and optimal tumour-normal tissue ratios were achieved after 72 h. Administration of either prodrug, at doses which cause 6-8% body weight loss, 72 h after administration of the A5B7-CPG2 conjugate to the LoVo tumour-bearing mice resulted in tumour regressions and growth delays of 14-28 days. The PTP prodrug in combination with a high dose of conjugate (10 mg kg-1) gave the best anti-tumour activity despite being a 10-fold worse substrate for CPG2 than PGP. Prodrug alone, active drug alone or prodrug in combination with a non-specific conjugate had minimal anti-tumour activity in this tumour model.
Topics: Aniline Mustard; Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antineoplastic Agents, Alkylating; Bacterial Proteins; Biotransformation; Carcinoembryonic Antigen; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Female; Humans; Immunoconjugates; Immunoglobulin Fab Fragments; Mice; Mice, Nude; Neoplasm Transplantation; Prodrugs; Pseudomonas; Substrate Specificity; Tumor Cells, Cultured; gamma-Glutamyl Hydrolase
PubMed: 7577451
DOI: 10.1038/bjc.1995.469 -
British Journal of Cancer Jan 1982The HT29R colonic adenocarcinoma xenograft has been shown to be rich in the enzyme beta-glucuronidase. Experiments in rodent systems have demonstrated a marked...
The HT29R colonic adenocarcinoma xenograft has been shown to be rich in the enzyme beta-glucuronidase. Experiments in rodent systems have demonstrated a marked anti-tumour effect of the drug aniline mustard (AM) on tumours with high levels of this enzyme (e.g. the plasmacytomas PC5 and PC6). We have found that AM is no more effective than its analogue paramethyl aniline mustard (PMAM) or other alkylating agents against the HT29R xenograft. Amongst the possible explanations for this may be: (1) The wide shoulder on the cell-survival curve shown for exposure to alkylating agents of HT29R in vivo. (2) Lack of correlation between physiological availability of beta-glucuronidase and the high levels measured by the standard assay. (3) Increased beta-glucuronidase levels in host mouse marrow, making the latter potentially more susceptible to AM damage.
Topics: Adenocarcinoma; Aniline Mustard; Animals; Colonic Neoplasms; Glucuronidase; Humans; Male; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Neoplasms, Experimental; Nitrogen Mustard Compounds; Transplantation, Heterologous
PubMed: 7059462
DOI: 10.1038/bjc.1982.4 -
Bioorganic & Medicinal Chemistry Letters Jul 2004A 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...
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.
Topics: Aniline Compounds; Aniline Mustard; Antineoplastic Agents, Alkylating; Binding Sites; Breast Neoplasms; Cell Survival; DNA Adducts; Dose-Response Relationship, Drug; Drug Design; Estradiol; Evaluation Studies as Topic; Female; Humans; Receptors, Estrogen; Tumor Cells, Cultured
PubMed: 15203171
DOI: 10.1016/j.bmcl.2004.04.064 -
British Journal of Cancer Aug 1984The response of clonal subpopulations isolated from the RIF-1 mouse sarcoma to melphalan treatment is independent of cell ploidy, whereas a clear relationship exists...
The response of clonal subpopulations isolated from the RIF-1 mouse sarcoma to melphalan treatment is independent of cell ploidy, whereas a clear relationship exists between ploidy and cell sensitivity to CCNU treatment. In the present study RIF-1 clones have been exposed to nitrogen mustard, aniline mustard and chlorambucil, and to nitrosoureas BCNU, MeCCNU and chlorozotocin, in order to evaluate whether or not the different physiochemical and biological activities of these agents would affect the patterns of drug sensitivity obtained for melphalan and CCNU. Irrespective of the different lipophilicities, transport properties and chemical reactivities of the nitrogen mustards, RIF-1 clones showed the same pattern of sensitivity as previously observed for melphalan. Similarly, RIF-1 clones when exposed to nitrosoureas BCNU, MeCCNU and chlorozotocin, showed the same pattern of sensitivity as that obtained for CCNU exposure. These data suggest (a) that the variation in the sensitivity of RIF-1 clones to treatment by the nitrogen mustards is unlikely to reflect differences in either membrane permeability or in drug transport and (b) that the ploidy dependent nitrosourea responses shown by RIF-1 clones similarly do not reflect differences in drug uptake.
Topics: Animals; Cell Survival; Clone Cells; Mice; Nitrogen Mustard Compounds; Nitrosourea Compounds; Ploidies; Sarcoma, Experimental
PubMed: 6466534
DOI: 10.1038/bjc.1984.157