RESUMO
The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol). The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity. Conjugate levels in tumors decreased to 36% of peak levels at this time. Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72. 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors. Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72. 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/uso terapêutico , Glucuronidase/farmacocinética , Polietilenoglicóis/farmacocinética , Pró-Fármacos/farmacocinética , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/química , Mostarda de Anilina/uso terapêutico , Mostarda de Anilina/toxicidade , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/química , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Escherichia coli/enzimologia , Glucuronidase/química , Glucuronidase/imunologia , Humanos , Técnicas Imunoenzimáticas/métodos , Fragmentos Fab das Imunoglobulinas/química , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Polietilenoglicóis/química , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Distribuição Tecidual , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
We have investigated the sequence selectivity, DNA binding site characteristics, interstrand cross-linking ability and cytotoxicity of four 4-anilinoquinoline aniline mustards related to the AT-selective minor groove-binding bisquaternary ammonium heterocycles. The compounds studied include two full mustards that differ in alkylating power, a half mustard and a quaternary anilinoquinolinium bismustard. We have also compared their cytotoxicity with their precursor diols and their toxicity and cross-linking ability with the classical alkylating agents melphalan and chlorambucil. We find that the anilinoquinoline aniline mustards weakly and non-specifically alkylate guanines in the major groove and that they bind strongly to AT-rich sequences in the minor groove, where they alkylate both adenines and guanines at the N3 position. The most preferred sites are classical minor groove binder AT-tracts to which all four ligands bind equally well. The remaining sites are AT-rich, but include GC base pairs, to which the ligands bind with preferences depending on their structure. The full mustards alkylate at the 3' ends of the binding site in an orientation that depends on the spatial disposition of the purines within the two strands. Generally speaking guanines are found to be much less reactive than adenines. The anilinoquinoline aniline mustards are interstrand cross-linking agents that are 60- to 100-fold more effective than melphalan, with the quaternary compound being the most efficacious. However, the type of binding site at which the cross-links occur is not clear, since distamycin challenge fails to antagonize them fully. The full mustards are 20- to 50-fold more cytotoxic than their diol precursors, are more cytotoxic than the half mustard and are 20- to 30-fold more active than melphalan and chlorambucil. The quaternary ligand is the most potent. Given the evidence to hand, it appears that antitumour activity correlates with capacity to cause interstrand cross-links at classical or near-classical AT-minor groove binder sites, rather than with ability to discriminate between the subsets of potential anilinoquinoline aniline mustard binding sites.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/toxicidade , DNA/metabolismo , Alquilação , Mostarda de Anilina/metabolismo , Mostarda de Anilina/toxicidade , Antivirais/metabolismo , Antivirais/toxicidade , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Reagentes de Ligações Cruzadas/metabolismo , Reagentes de Ligações Cruzadas/toxicidade , DNA/antagonistas & inibidores , Distamicinas/metabolismo , Distamicinas/toxicidade , Células HT29/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/genética , Quinolinas/metabolismo , Quinolinas/toxicidade , Especificidade por SubstratoRESUMO
We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos/uso terapêutico , Ascite/terapia , Carcinoma Hepatocelular/terapia , Glucuronidase/metabolismo , Imunotoxinas/uso terapêutico , Neoplasias Hepáticas/terapia , Pró-Fármacos/uso terapêutico , Mostarda de Anilina/metabolismo , Mostarda de Anilina/uso terapêutico , Mostarda de Anilina/toxicidade , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Ascite/metabolismo , Carcinoma Hepatocelular/metabolismo , Imunotoxinas/metabolismo , Leucócitos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidade , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
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.
Assuntos
Antineoplásicos/farmacocinética , Glucuronidase/farmacocinética , Imunoconjugados/farmacocinética , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Polietilenoglicóis/farmacocinética , Pró-Fármacos/farmacocinética , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/toxicidade , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos/toxicidade , Estabilidade Enzimática , Glucuronatos/farmacocinética , Glucuronatos/farmacologia , Glucuronidase/sangue , Glucuronidase/farmacologia , Imunoconjugados/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Polietilenoglicóis/farmacologia , Ratos , Transplante HeterólogoRESUMO
It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4'-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(-) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard-phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard-DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.
Assuntos
Mostarda de Anilina/síntese química , Mostarda de Anilina/toxicidade , Antineoplásicos Alquilantes/síntese química , Dano ao DNA , Compostos de Mostarda Nitrogenada/síntese química , Compostos de Mostarda Nitrogenada/toxicidade , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Mostarda de Anilina/química , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/toxicidade , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Estrutura Molecular , Compostos de Mostarda Nitrogenada/química , Oligodesoxirribonucleotídeos , Neoplasias Ovarianas/metabolismo , Receptores de Estrogênio/química , Fatores de Transcrição/químicaRESUMO
A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study.
Assuntos
Alquilantes/toxicidade , Mostarda de Anilina/toxicidade , Bisbenzimidazol/análogos & derivados , Bisbenzimidazol/toxicidade , DNA/efeitos dos fármacos , Alquilantes/metabolismo , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Sítios de Ligação , Bisbenzimidazol/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Troca Genética , DNA/metabolismo , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/metabolismo , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Eletroforese em Gel de Ágar , Mutação da Fase de Leitura , Ligantes , Testes de Mutagenicidade , Conformação de Ácido Nucleico , Mutação Puntual , Saccharomyces cerevisiae/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
During the past 30 years, thousands of quantitative structure-activity relationships (QSAR) have been published for all sorts of chemicals acting on many forms of life or parts thereof (DNA, enzymes, organelles, etc.). Very little effort has been made to show the relationship among these equations. In this report, we discuss two examples, the toxicity of phenols to rats and the effect of aniline mustards on a variety of living systems, where the electronic effects in the QSAR can be correlated to QSAR from physical organic chemistry. This enables one to make better mechanistic deductions about the biological structure-activity relationships. From this, it is concluded that radicals formed from the phenols cause birth defects.
Assuntos
Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/farmacologia , Mostarda de Anilina/toxicidade , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Teratogênicos/toxicidade , Animais , Humanos , Relação Estrutura-AtividadeRESUMO
The ortho, meta and para isomers of N,N-bis(2-chloroethyl)aminocinnamic acid were tested for their ability to mutate Salmonella typhimurium strains in the Salmonella/microsome mutagenicity test. The aim of the work was to establish a structure-activity relationship between these three isomers. The drugs were found to induce base-pair substitutions, causing dose-dependent increases in his+ revertants, in strains TA100 and TA1535. The study showed that the position of the substituent groups influenced the mutagenic activity of the compounds. The ortho isomer exhibited a poorer mutagenic effect than meta and this was found to be a weaker mutagen than para. The presence of metabolic activation enzymes in the test system induced a further increase in his+ revertants, in strains TA100 and TA1535, which is consistent with the findings for melphalan, a cancer chemotherapeutic agent with a chemical structure similar to that of the isomers tested.
Assuntos
Mostarda de Anilina/toxicidade , Testes de Mutagenicidade , Mutagênicos/toxicidade , Mutação Puntual , Salmonella typhimurium/efeitos dos fármacos , Mostarda de Anilina/química , Mostarda de Anilina/metabolismo , Distribuição de Qui-Quadrado , Cinamatos/química , Cinamatos/metabolismo , Cinamatos/toxicidade , Relação Dose-Resposta a Droga , Extratos Hepáticos , Microssomos Hepáticos/enzimologia , Reprodutibilidade dos Testes , Salmonella typhimurium/genética , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The prodrug p-[N,N-bis(2-chloroethyl)amino]phenyl phosphate (phenol mustard phosphate, POMP) was prepared from p-[N,N-bis(2-chloroethyl)amino]phenol (phenol mustard, POM) by phosphorylation with phosphoryl chloride, followed by aqueous hydrolysis. It was found that POMP was much less cytotoxic than POM when tested against H2981 human lung and H3396 human breast carcinoma cells in vitro. Pretreatment of the H2981 cells with L6-alkaline phosphatase (L6-AP), a monoclonal antibody conjugate that could bind to cell surface antigens, greatly enhanced the cytotoxic effects of POMP in an immunologically specific manner. Owing to its reduced toxicity in nude mice, larger amounts of POMP compared to POM could be administered. Neither agent exhibited significant in vivo antitumor activity when tested against subcutaneous H2981 tumors in nude mice. However, antitumor activity was observed in animals receiving L6-AP 48 h prior to POMP administration. This level of activity was greater than with the drugs alone, or a combination of 1F5-AP (nonbinding control) with POMP.
Assuntos
Fosfatase Alcalina/química , Mostarda de Anilina/análogos & derivados , Anticorpos Monoclonais/química , Antineoplásicos/química , Imunotoxinas/uso terapêutico , Pró-Fármacos/química , Mostarda de Anilina/química , Mostarda de Anilina/uso terapêutico , Mostarda de Anilina/toxicidade , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Pró-Fármacos/uso terapêutico , Pró-Fármacos/toxicidade , Células Tumorais CultivadasRESUMO
A series of even numbered normal alkyl ethers (C2-C14) of p-N,N-bis(2-chloroethyl)aminophenol were synthesized and evaluated as to acute toxicity in mice and effects on survival in L-1210 leukemic mice. All of the ether derivatives demonstrated significantly lower acute toxicity than the parent phenol mustard. Significant survival times (greater than or equal to 125%) were obtained with all compounds except the hexyl derivative. The decyl ether produced the greatest significant increase and the ethyl ether the lowest significant increase in mean survival time. Significant survival times were produced at four dosage levels for the butyl, decyl, and dodecyl derivatives, three dosage levels for the octyl and tetradecyl derivatives, and one dosage level for the ethyl derivative.
Assuntos
Mostarda de Anilina/síntese química , Antineoplásicos/síntese química , Compostos de Mostarda Nitrogenada/síntese química , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/farmacologia , Mostarda de Anilina/toxicidade , Animais , Fenômenos Químicos , Química , Dose Letal Mediana , Leucemia L1210/tratamento farmacológico , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICRRESUMO
A set of 15 derivatives of aniline mustard (I) was tested to give a quantitative measure of mutagenicity in Salmonella typhimurium TA-1535 and TA-100 and also carcinogenicity as lung tumors in strain-A mice. The structural variation in the set was chosen to minimize collinearity between hydrophobic, electronic, and molar refractive properties. By these measures, there was not a direct relationship between mutagenicity and carcinogenicity; in fact, since the 4-OPh analogue ranked highest in mutagenicity and among the lowest in carcinogenicity, while the reverse was noted for the 3,5-(NHCONH2)2 analogue, an inverse relationship was marginally significant. S-9 activation was required in the Ames test using TA-100, and the dose-response curve, prior to toxicity, appeared biphasic.
Assuntos
Mostarda de Anilina/toxicidade , Carcinógenos , Mutagênicos , Compostos de Mostarda Nitrogenada/toxicidade , Adenoma/tratamento farmacológico , Mostarda de Anilina/análogos & derivados , Animais , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Neoplasias Experimentais/tratamento farmacológicoRESUMO
A more rapid cell-counting technique using an electronic cell counter was developed as an improvement over the slower hemocytometer method. The electronic counting method produced cell counts that had a smaller standard deviation but were not significantly different from the hemocytometer method. The acetate, hexanoate, and decanoate esters of p-N,N-bis(2-chloroethyl)aminophenol were investigated for acute toxicity in mice, effects on survival times, and effects on L-1210 cell populations in L-1210 leukemic mice. The decanoate ester was the least toxic compound and was most effective in lengthening the lifespan of the mice. The acetate and hexanoate esters were more effective in reducing L-1210 cell populations.