RESUMEN
A design strategy for macromolecular prodrugs is described, that are expected to exhibit robust activity against most solid tumor types while resulting in minimal toxicities to normal tissues. This approach exploits the enhanced permeability, and retention (EPR) effect, and utilizes carefully engineered rate constants to selectively target tumor tissue with short-lived cytotoxic moieties. EPR based tumor accumulation (half-life ~ 15 h) is dependent upon the ubiquitous abnormal solid tumor capillary morphology and is expected to be independent of individual tumor cell genetic variability that leads to resistance to molecularly targeted agents. The macromolecular sulfonylhydrazine-based prodrugs hydrolyze spontaneously with long half-life values (~10 h to >300 h dependent upon their structure) resulting in the majority of the 1,2-bis(sulfonyl)-1-alkylhydrazines (BSHs) cytotoxic warhead being released only after tumor sequestration. The very short half-life (seconds) of the finally liberated BSHs localizes the cytotoxic stress to the tumor target site by allowing insufficient time for escape. Thus, short lifespan anticancer species are liberated, and exhibit their activity largely within the tumor target. The abnormal tumor cell membrane pH gradients favor the uptake of BSHs compared to that of normal cells, further enhancing their selectivity. The reliance on physicochemical/chemical kinetic parameters and the EPR effect is expected to reduce response variability, and the acquisition of resistance.
Asunto(s)
Antineoplásicos , Sistemas de Liberación de Medicamentos , Hidrazinas , Neoplasias/tratamiento farmacológico , Profármacos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Humanos , Hidrazinas/síntesis química , Hidrazinas/química , Neoplasias/metabolismo , Profármacos/síntesis química , Profármacos/químicaRESUMEN
Hypoxic tumor cell sub-populations are highly resistant to radiotherapy and their presence frequently causes disease recurrence and death. Here, we described the physicochemical properties required to develop superior tumor-targeted hypoxia-activated modular prodrugs that liberate extremely short-lived bis(sulfonyl)hydrazines (BSHs) as reactive cytotoxins, thereby precisely focusing cytotoxic stress on these radio-resistant hypoxic sub-populations. Therefore, cytotoxic stress will be focused on radiation resistant areas and thus strongly synergizing with radiotherapy.
Asunto(s)
Antineoplásicos/química , Hidrazinas/química , Profármacos/química , Animales , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Humanos , Hidrazinas/administración & dosificación , Hipoxia , Neoplasias/tratamiento farmacológico , Profármacos/administración & dosificaciónRESUMEN
Laromustine (also known as cloretazine, onrigin, VNP40101M, 101M) is a prodrug of 90CE, a short-lived chloroethylating agent with anticancer activity. The short half-life of 90CE necessitates the use of latentiated prodrug forms for in vivo treatments. Alkylaminocarbonyl-based prodrugs such as laromustine exhibit significantly superior in vivo activity in several murine tumor models compared to analogs utilizing acyl, and alkoxycarbonyl latentiating groups. The alkylaminocarbonyl prodrugs possess two exclusive characteristics: (i) They are primarily unmasked by spontaneous base catalyzed elimination; and (ii) they liberate a reactive carbamoylating species. Previous speculations as to the therapeutic superiority of laromustine have focused upon the inhibition of enzymes by carbamoylation. We have investigated the therapeutic interactions of analogs with segregated chloroethylating and carbamoylating activities (singly and in combination) in the in vivo murine L1210 leukemia model. The combined treatment with chloroethylating and carbamoylating prodrugs failed to result in any synergism and produced a reduction in the therapeutic efficacy compared to the chloroethylating prodrug alone. Evidence supporting an alternative explanation for the superior tumor selectivity of laromustine is presented that is centered upon the high pH sensitivity of its base catalyzed activation, and the more alkaline intracellular pH values commonly found within tumor cells.
Asunto(s)
Antineoplásicos/química , Hidrazinas/química , Isocianatos/metabolismo , Profármacos/química , Sulfonamidas/química , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Catálisis , Línea Celular Tumoral , ADN/química , ADN/metabolismo , Diseño de Fármacos , Femenino , Semivida , Humanos , Hidrazinas/farmacocinética , Hidrazinas/farmacología , Hidrazinas/uso terapéutico , Concentración de Iones de Hidrógeno , Leucemia/tratamiento farmacológico , Leucemia/patología , Ratones , Profármacos/farmacología , Profármacos/uso terapéutico , Carbamilación de Proteína , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Trasplante HomólogoRESUMEN
Alkylating agents are a significant class of environmental carcinogens as well as commonly used anticancer therapeutics. Traditional alkylating activity assays have utilized the colorimetric reagent 4-(4-nitrobenzyl)pyridine (4NBP). However, 4NBP based assays have a relatively low sensitivity towards harder, more oxophilic alkylating species and are not well suited for the identification of the trapped alkyl moiety due to adduct instability. Herein we describe a method using water as the trapping agent which permits the trapping of simple alkylating electrophiles with a comparatively wide range of softness/hardness and permits the identification of donated simple alkyl moieties.
Asunto(s)
Alcoholes/química , Alquilantes/análisis , Alquilantes/aislamiento & purificación , Carcinógenos Ambientales/análisis , Carcinógenos Ambientales/aislamiento & purificación , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Agua Dulce/químicaRESUMEN
Although epithelial ovarian cancers (EOCs) are initially treated with platinum-based chemotherapy, EOCs vary in platinum responsiveness. Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive EOC cell lines is valuable for development of therapeutic strategies to avoid platinum inefficacy and to exploit platinum sensitivity. TOV-21G devoid of FANCF expression, OV-90 and SKOV-3 were employed as examples of platinum-sensitive, platinum-intermediate and platinum-resistant cell lines, respectively. Antineoplastic agents examined included mitomycin C, doxorubicin, etoposide, gemcitabine, chlorambucil, paclitaxel, triapine and X-rays. Their effectiveness against cell lines was analyzed by clonogenic assays. Cytotoxic profiles of mitomycin C and carboplatin were similar, with mitomycin C exhibiting greater potency and selectivity against TOV-21G than carboplatin. Cytotoxic profiles of doxorubicin, etoposide and X-rays overlapped with that of carboplatin, while OV-90 overexpressing Rad51 was more resistant to chlorambucil than SKOV-3. The efficacy of paclitaxel and triapine was independent of platinum sensitivity or resistance. Consistent with these cytotoxic profiles, cisplatin/mitomycin C, triapine, and paclitaxel differed in the capacity to induce phosphorylation of H2AX, and produced unique inhibitory patterns of DNA/RNA syntheses in HL-60 human leukemia cells. Paclitaxel and triapine in combination produced additive antitumor effects in M109 murine lung carcinoma. In conclusion, mitomycin C is potentially more effective against Fanconi anemia pathway-deficient EOCs than carboplatin. Doxorubicin and etoposide, because of their overlapping cytotoxic properties with carboplatin, are unlikely to be efficacious against platinum-refractory EOCs. Paclitaxel and triapine are effective regardless of platinum sensitivity status, and promising in combination for both platinum-sensitive and platinum-refractory EOCs.
RESUMEN
1,2-Bis(sulfonyl)-1-alkylhydrazines (BSHs) were conceived as more specific DNA guanine O-6 methylating and chloroethylating agents lacking many of the undesirable toxicophores contained in antitumor nitrosoureas. O(6)-Alkylguanine-DNA alkyltransferase (MGMT) is the sole repair protein for O(6)-alkylguanine lesions in DNA and has been reported to be absent in 5-20% of most tumor types. Many BSHs exhibit highly selective cytotoxicity toward cells deficient in MGMT activity. The development of clinically useful MGMT assays should permit the identification of tumors with this vulnerability and allow for the preselection of patient subpopulations with a high probability of responding. The BSH system is highly versatile, permitting the synthesis of many prodrug types with the ability to incorporate an additional level of tumor-targeting due to preferential activation by tumor cells. Furthermore, it may be possible to expand the spectrum of activity of these agents to include tumors with MGMT activity by combining them with tumor-targeted MGMT inhibitors.
Asunto(s)
Antineoplásicos/química , Resistencia a Antineoplásicos , Hidrazinas/química , Sulfonas/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Hipoxia de la Célula , Quimioterapia del Cáncer por Perfusión Regional , Terapia Combinada , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Diseño de Fármacos , Humanos , Hidrazinas/farmacología , Hidrazinas/uso terapéutico , Medicina de Precisión , Relación Estructura-Actividad , Sulfonas/farmacología , Sulfonas/uso terapéutico , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu(2+) inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 µM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.
Asunto(s)
Antineoplásicos/farmacología , Cobre/farmacología , Piridinas/farmacología , Tiosemicarbazonas/farmacología , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Enzimas/metabolismo , Células HL-60/efectos de los fármacos , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Factores de TiempoRESUMEN
Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) are promising anticancer agents. The 90CE moiety is a readily latentiated, short-lived (t1/2 â¼ 30 s) chloroethylating agent that can generate high yields of oxophilic electrophiles responsible for the chloroethylation of the O-6 position of guanine in DNA. These guanine O-6 alkylations are believed to be responsible for the therapeutic effects of 90CE and its prodrugs. Thus, 90CE demonstrates high selectivity toward tumors with diminished levels of O(6)-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O(6)-alkylguanine repair. The formation of O(6)-(2-chloroethyl)guanine lesions ultimately leads to the generation of highly cytotoxic 1-(N(3)-cytosinyl),-2-(N(1)-guaninyl)ethane DNA interstrand cross-links via N(1),O(6)-ethanoguanine intermediates. The anticancer activity arising from this sequence of reactions is thus identical to this component of the anticancer activity of the clinically used chloroethylnitrosoureas. Herein, we evaluate the ability of glutathione (GSH) and other low molecular weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields of cross-links generated by 90CE when added prior to or immediately following the initial chloroethylation step to determine the major point(s) of interaction. In contrast to studies utilizing BCNU as a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably quench DNA interstrand cross-link precursors. While thiols alone offered little protection at either alkylation step, the GSH/GST couple was able to diminish the initial yields of cross-link precursors. 90CE exhibited a very different GST isoenzyme susceptibility to that reported for BCNU, this could have important implications in the relative resistance of tumor cells to these agents. The protection afforded by GSH/GST was compared to that produced by MGMT.
Asunto(s)
Antineoplásicos/química , ADN/química , Glutatión Transferasa/metabolismo , Glutatión/química , Hidrazinas/química , Sulfonamidas/química , Alquilación , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , ADN/metabolismo , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/genética , Guanina/química , Guanina/metabolismo , Hidrazinas/metabolismo , Ratones , Profármacos/química , Profármacos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Sulfonamidas/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Prodrugs of the short-lived chloroethylating agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine (KS90) are potentially useful anticancer agents. This class of agents frequently yields higher ratios of therapeutically active oxophilic electrophiles responsible for DNA O(6)-guanine alkylations to other electrophiles with lower therapeutic relevance than the nitrosoureas. This results in improved selectivity toward tumors with diminished levels of O(6)-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O(6)-alkylguanine repair. The formation of O(6)-(2-chloroethyl)guanine, which leads to the formation of a DNA-DNA interstrand cross-link, accounts for the bulk of the anticancer activity of 90CE prodrugs. Herein, we describe a new decomposition pathway that is available to 90CE but not to its methylating counterpart. This pathway appears to be subject to general/acid base catalysis with phosphate (Pi), phosphomonoesters, and phosphodiesters, being particularly effective. This pathway does not yield a chloroethylating species and results in a major change in nucleophile preference since thiophilic rather than oxophilic electrophiles are produced. Thus, a Pi concentration dependent decrease in DNA-DNA interstand cross-link formation was observed. Changes in 90CE decomposition products but not alkylation kinetics occurred in the presence of Pi since the prebranch point elimination of the N-1 methanesulfinate moiety remained the rate-limiting step. The Pi catalyzed route is expected to dominate at Pi and phosphoester concentrations totaling >25-35 mM. In view of the abundance of Pi and phosphoesters in cells, this pathway may have important effects on agent toxicity, tumor selectivity, and resistance to prodrugs of 90CE. Furthermore, it may be possible to design analogues that diminish this thiophile-generating pathway, which is likely superfluous at best and potentially detrimental to the targeting of hypoxic regions where Pi concentrations can be significantly elevated.
Asunto(s)
Antineoplásicos/metabolismo , Hidrazinas/metabolismo , Sustancias Intercalantes/metabolismo , Profármacos/metabolismo , Sulfonamidas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , ADN/química , ADN/metabolismo , Hidrazinas/química , Hidrazinas/farmacología , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fosfatos/metabolismo , Profármacos/química , Profármacos/farmacología , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included (a) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, (b) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and (c) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor-selective reduction in MGMT activity exist in human tissue.
RESUMEN
Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.
Asunto(s)
Antineoplásicos Alquilantes/química , Metilnitrosourea/análogos & derivados , Sulfonamidas/química , Animales , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/química , ADN/metabolismo , Aductos de ADN/química , Metilación de ADN , Semivida , Humanos , Hidrazinas/química , Hidrazinas/farmacocinética , Hidrazinas/toxicidad , Metilnitrosourea/síntesis química , Metilnitrosourea/química , Metilnitrosourea/toxicidad , Ratones , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Sulfonamidas/síntesis química , Sulfonamidas/toxicidadRESUMEN
O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein which removes alkyl groups from the O-6 position of guanine, thereby providing strong resistance to anticancer agents which alkylate this position. The clinical usefulness of these anticancer agents would be substantially augmented if AGT could be selectively inhibited in tumor tissue, without a corresponding depletion in normal tissue. We report the synthesis of a new AGT inhibitor (5c) which selectively depletes AGT in hypoxic tumor cells.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Profármacos/farmacología , Línea Celular Tumoral , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Profármacos/síntesis química , Profármacos/químicaRESUMEN
The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O(6)-alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves a protective function. A newly synthesized prodrug of the AGT inhibitor O(6)-benzylguanine (O(6)-BG) with an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demonstrated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery. However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT inhibitors with improved formulatability such as O(6)-[(3-aminomethyl)benzylguanine (1) have been developed. However, acetylamino (N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)acetamide) (2) exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility, we synthesized 6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-amine (3) in which the 3-aminomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-yl)carbamate (7), a hypoxia targeted prodrug of 3 utilizing an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitization of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved solubility.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Hipoxia , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Profármacos/síntesis química , Profármacos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HL-60 , Humanos , Ratones , Estructura Molecular , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Profármacos/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Solubilidad , Relación Estructura-ActividadRESUMEN
Agents with selective toxicity to hypoxic cells have shown promise as adjuncts to radiotherapy. Our previous studies showed that the bioreductive alkylating agent KS119 had an extremely large differential toxicity to severely hypoxic and aerobic cells in cell culture, and was effective in killing the hypoxic cells of EMT6 mouse mammary tumors in vivo. However, the limited solubility of that compound precluded its development as an anticancer drug. Here we report our initial studies with KS119W, a water-soluble analog of KS119. The cytotoxicity of KS119W to EMT6 cells in vitro was similar to that of KS119, with both agents producing only minimal cytotoxicity to aerobic cells even after intensive treatments, while producing pronounced cytotoxicity to oxygen-deficient cells. This resulted in large differentials in the toxicities to hypoxic and aerobic cells (>1,000-fold at 10 µM). Low pH had only minimal effects on the cytotoxicity of KS119W. Under hypoxic conditions, EMT6 cells transfected to express high levels of either human or mouse versions of the repair protein O(6)-alkylguanine-DNA alkyltransferase, which is also known as O(6)-methylguanine DNA-methyltransferase, were much more resistant to KS119W than parental EMT6 cells lacking O(6)-alkylguanine-DNA alkyltransferase, confirming the importance of DNA O-6-alkylation to the cytotoxicity of this agent. Studies with EMT6 tumors in BALB/c Rw mice using both tumor cell survival and tumor growth delay assays showed that KS119W was effective as an adjunct to irradiation for the treatment of solid tumors in vivo, producing additive or supra-additive effects in most combination regimens for which the interactions could be evaluated. Our findings encourage additional preclinical studies to examine further the antineoplastic effects of KS119W alone and in combination with radiation, and to examine the pharmacology and toxicology of this new bioreductive alkylating agent so that its potential for clinical use as an adjuvant to radiotherapy can be evaluated.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Hidrazinas/química , Hidrazinas/farmacología , Agua/química , Aerobiosis , Animales , Antineoplásicos/uso terapéutico , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Humanos , Hidrazinas/uso terapéutico , Concentración de Iones de Hidrógeno , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/radioterapia , Ratones , SolubilidadRESUMEN
Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC(50) = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.
Asunto(s)
Antineoplásicos/farmacología , Oxadiazoles/farmacología , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Peróxido de Hidrógeno/metabolismo , Dosificación Letal Mediana , Peroxidación de Lípido/efectos de los fármacos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Oxadiazoles/administración & dosificación , Oxidación-Reducción/efectos de los fármacos , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Cellular resistance to chemotherapeutics that alkylate the O-6 position of guanine residues in DNA correlates with their O(6)-alkylguanine-DNA alkyltransferase activity. In normal cells high [O(6)-alkylguanine-DNA alkyltransferase] is beneficial, sparing the host from toxicity, whereas in tumor cells high [O(6)-alkylguanine-DNA alkyltransferase] prevents chemotherapeutic response. Therefore, it is necessary to selectively inactivate O(6)-alkylguanine-DNA alkyltransferase in tumors. The oxygen-deficient compartment unique to solid tumors is conducive to reduction, and could be utilized to provide this selectivity. Therefore, we synthesized 2-nitro-6-benzyloxypurine, an analog of O(6)-benzylguanine in which the essential 2-amino group is replaced by a nitro moiety, and 2-nitro-6-benzyloxypurine is >2000-fold weaker than O(6)-benzylguanine as an O(6)-alkylguanine-DNA alkyltransferase inhibitor. We demonstrate oxygen concentration sensitive net reduction of 2-nitro-6-benzyloxypurine by cytochrome P450 reductase, xanthine oxidase, and EMT6, DU145, and HL-60 cells to yield O(6)-benzylguanine. We show that 2-nitro-6-benzyloxypurine treatment depletes O(6)-alkylguanine-DNA alkyltransferase in intact cells under oxygen-deficient conditions and selectively sensitizes cells to laromustine (an agent that chloroethylates the O-6 position of guanine) under oxygen-deficient but not normoxic conditions. 2-Nitro-6-benzyloxypurine represents a proof of concept lead compound; however, its facile reduction (E(1/2) - 177 mV versus Ag/AgCl) may result in excessive oxidative stress and/or the generation of O(6)-alkylguanine-DNA alkyltransferase inhibitors in normoxic regions in vivo.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Oxígeno/metabolismo , Antineoplásicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Inhibidores Enzimáticos/síntesis química , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Células HL-60 , Humanos , Hidrazinas/farmacología , Peróxido de Hidrógeno/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Oxidación-Reducción , Purinas/síntesis química , Purinas/química , Purinas/farmacología , Sulfonamidas/farmacología , Superóxidos/metabolismoRESUMEN
PURPOSE: These studies explored questions related to the potential use of Laromustine in the treatment of solid tumors and in combination with radiotherapy. MATERIALS AND METHODS: The studies used mouse EMT6 cells (both parental and transfected with genes for O(6)-alkylguanine-DNA transferase [AGT]), repair-deficient human Fanconi Anemia C and Chinese hamster VC8 (BRCA2(-/-)) cells and corresponding control cells, and EMT6 tumors in mice assayed using cell survival and tumor growth assays. RESULTS: Hypoxia during Laromustine treatment did not protect EMT6 cells or human fibroblasts from this agent. Rapidly proliferating EMT6 cells were more sensitive than quiescent cultures. EMT6 cells expressing mouse or human AGT, which removes O(6)-alkyl groups from DNA guanine, thereby protecting against G-C crosslink formation, increased resistance to Laromustine. Crosslink-repair-deficient Fanconi Anemia C and VC8 cells were hypersensitive to Laromustine, confirming the importance of crosslinks as lethal lesions. In vitro, Laromustine and radiation produced additive toxicities to EMT6 cells. Studies using tumor cell survival and tumor growth assays showed effects of regimens combining Laromustine and radiation that were compatible with additive or subadditive interactions. CONCLUSIONS: The effects of Laromustine on solid tumors and with radiation are complex and are influenced by microenvironmental and proliferative heterogeneity within these malignancies.
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Antineoplásicos/farmacología , Hidrazinas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Sulfonamidas/farmacología , Animales , Antineoplásicos/uso terapéutico , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Terapia Combinada , Cricetinae , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Humanos , Hidrazinas/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/radioterapia , Ratones , Neoplasias/metabolismo , Neoplasias/patología , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Sulfonamidas/uso terapéutico , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A series of 4-nitrobenzyloxycarbonyl prodrug derivatives of O(6)-benzylguanine (O(6)-BG), conceived as prodrugs of O(6)-BG, an inhibitor of the resistance protein O(6)-alkylguanine-DNA alkyltransferase (AGT), were synthesized and evaluated for their ability to undergo bioreductive activation by reductase enzymes under oxygen deficiency. Three agents of this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1) and its monomethyl (2) and gem-dimethyl analogues (3), were tested for activation by reductase enzyme systems under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O(6)-BG following reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency, providing evidence for the preferential release of the AGT inhibitor O(6)-BG under hypoxia.
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Antineoplásicos Alquilantes/síntesis química , ADN/metabolismo , Guanina/análogos & derivados , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Profármacos/síntesis química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Hipoxia de la Célula , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Humanos , Hidrazinas/farmacología , Ratones , NADPH-Ferrihemoproteína Reductasa/química , Profármacos/química , Profármacos/farmacología , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/farmacología , Xantina Oxidasa/químicaRESUMEN
The anticancer prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively releases a short-lived cytotoxin following enzymatic reduction in hypoxic environments found in solid tumors. KS119, in addition to two enantiomers, has two stable atropisomers (conformers differing in structure owing to hindered bond rotation) that interconvert at 37 °C in aqueous solution by first-order kinetics with t(1/2) values of â¼50 and â¼64 h. The atropisomers differ in physical properties such as partition coefficients that allow their chromatographic separation on non-chiral columns. A striking difference in the rate of metabolism of the two atropisomers occurs in intact EMT6 murine mammary carcinoma cells under oxygen-deficient conditions. A structurally related molecule, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-hydroxy-4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119WOH), was also found to exist in similar stable atropisomers. The ratio of the atropisomers of KS119 and structurally related agents has the potential to impact the bioavailability, activation, and therapeutic activity. Thus, thermally stable atropisomers/conformers in small molecules can result in chemically and enantiomerically pure compounds having differences in biological activities.
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Antineoplásicos/química , Antineoplásicos/farmacología , Hidrazinas/química , Hidrazinas/farmacología , Neoplasias Mamarias Animales/tratamiento farmacológico , Profármacos/química , Profármacos/farmacología , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Hidrazinas/metabolismo , Isomerismo , Neoplasias Mamarias Animales/metabolismo , Ratones , Modelos Moleculares , Conformación Molecular , Profármacos/metabolismoRESUMEN
To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O(6)-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC(50) value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O(6)-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.