RESUMEN
Clinical trials in autism spectrum disorder (ASD) often rely on clinician rating scales and parent surveys to measure autism-related features and social behaviors. To aid in the selection of these assessments for future clinical trials, the Autism Biomarkers Consortium for Clinical Trials (ABC-CT) directly compared eight common instruments with respect to acquisition rates, sensitivity to group differences, equivalence across demographic sub-groups, convergent validity, and stability over a 6-week period. The sample included 280 children diagnosed with ASD (65 girls) and 119 neurotypical children (36 girls) aged from 6 to 11 years. Full scale IQ for ASD ranged from 60 to 150 and for neurotypical ranged from 86 to 150. Instruments measured clinician global assessment and autism-related behaviors, social communication abilities, adaptive function, and social withdrawal behavior. For each instrument, we examined only the scales that measured social or communication functioning. Data acquisition rates were at least 97.5% at T1 and 95.7% at T2. All scales distinguished diagnostic groups. Some scales significantly differed by participant and/or family demographic characteristics. Within the ASD group, most clinical instruments exhibited weak (≥ |0.1|) to moderate (≥ |0.4|) intercorrelations. Short-term stability was moderate (ICC: 0.5-0.75) to excellent (ICC: >0.9) within the ASD group. Variations in the degree of stability may inform viability for different contexts of use, such as identifying clinical subgroups for trials versus serving as a modifiable clinical outcome. All instruments were evaluated in terms of their advantages and potential concerns for use in clinical trials.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Niño , Femenino , Humanos , Habilidades Sociales , Trastorno del Espectro Autista/diagnóstico , Comunicación , BiomarcadoresRESUMEN
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
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.
Asunto(s)
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
The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to prodrugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.
Asunto(s)
Antineoplásicos/metabolismo , Catalasa/metabolismo , Hipoxia de la Célula , Glucosa Oxidasa/metabolismo , Hidrazinas/metabolismo , Neoplasias/metabolismo , Animales , Células CHO , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Glucosa/análisis , Humanos , Ratones , Profármacos/metabolismo , Células Tumorales CultivadasRESUMEN
Cloretazine [1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia (AML) patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The numbers of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produces a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line.
Asunto(s)
Antineoplásicos/toxicidad , Reactivos de Enlaces Cruzados/toxicidad , ADN/química , Hidrazinas/toxicidad , Leucemia/tratamiento farmacológico , Sulfonamidas/toxicidad , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/química , Citosina/química , Etano/química , Guanina/química , Células HL-60 , Humanos , Hidrazinas/química , Hidrazinas/uso terapéutico , Ratones , Sulfonamidas/química , Sulfonamidas/uso terapéutico , Células U937RESUMEN
Cloretazine is an antitumor sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species. The cytotoxic potency of these species was analyzed in L1210 leukemia cells using analogues with chloroethylating or carbamoylating function only. Clonogenic assays showed that the chloroethylating-only agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) produced marked differential cytotoxicity against wild-type and O6-alkylguanine-DNA alkyltransferase-transfected L1210 cells (LC10, 1.4 versus 31 micromol/L), indicating that a large portion of the cytotoxicity was due to alkylation of DNA at the O-6 position of guanine. Consistent with the concept that O-6 chloroethylation of DNA guanine progresses to interstrand cross-links, the comet assay, in which DNA cross-links were measured by a reduction in DNA migration induced by strand breaks, showed that cloretazine and 90CE, but not the carbamoylating-only agent 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), produced DNA cross-links and that cloretazine caused more DNA cross-links than 90CE at equimolar concentrations. Cell cycle analyses showed that 90CE and 101MDCE at concentrations of 5 and 80 micromol/L, respectively, produced similar degrees of G2-M arrest. 90CE produced selective inhibition of DNA synthesis after overnight incubation, whereas 101MDCE caused rapid and nonselective inhibition of RNA, DNA, and protein syntheses. Both 90CE and 101MDCE induced phosphorylation of histone H2AX, albeit with distinct kinetics. These results indicate that (a) differential expression of O6-alkylguanine-DNA alkyltransferase in tumor and host cells seems to be responsible for tumor selectivity exerted by cloretazine; (b) 101MDCE enhances DNA cross-linking activity; and (c) 90CE induces cell death at concentrations lower than those causing alterations in the cell cycle and macromolecular syntheses.
Asunto(s)
Antineoplásicos/toxicidad , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Hidrazinas/química , Hidrazinas/toxicidad , Sulfonamidas/química , Sulfonamidas/toxicidad , Animales , Antineoplásicos/química , Ensayo Cometa , Histonas/metabolismo , Leucemia L1210 , Ratones , O(6)-Metilguanina-ADN Metiltransferasa/efectos de los fármacos , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Fosforilación , Profármacos , TransfecciónRESUMEN
To target malignant cells residing in hypoxic regions of solid tumors, we have designed and synthesized prodrugs generating the cytotoxic alkylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) after bioreductive activation. We postulate that one of these agents, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), requires enzymatic nitro reduction to produce 90CE, whereas another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine (PNBC), can also be activated by nucleophilic attack by thiols such as glutathione (GSH)/GST. We demonstrated that these agents selectively kill hypoxic EMT6 mouse mammary carcinoma and CHO cells. In hypoxia, 50 microM KS119 produced 5 logs of kill of EMT6 cells without discernable cytotoxicity in air; similar effects were observed with CHO cells. PNBC was less efficacious against hypoxic tumor cells and also had some toxicity to aerobic cells, presumably because of GST/thiol activation, making PNBC less interesting as a selective hypoxic-cell cytotoxin. BALB/c mice with established EMT6 solid tumors were used to demonstrate that KS119 could reach and kill hypoxic cells in solid tumors. To gain information on bioreductive enzymes involved in the activation of KS119, cytotoxicity was measured in CHO cell lines overexpressing NADH:cytochrome b5 reductase (NBR), NADPH:cytochrome P450 reductase (NPR), or NADPH: quinone oxidoreductase 1 (NQO1). Increased cytotoxicity occurred in cells overexpressing NBR and NPR, whereas overexpressed NQO1 had no effect. These findings were supported by enzymatic studies using purified NPR and xanthine oxidase to activate KS119. KS119 has significant potential as a hypoxia-selective tumor-cell cytotoxin and is unlikely to cause major toxicity to well oxygenated normal tissues.
Asunto(s)
Antineoplásicos/farmacología , Hidrazinas/farmacología , Hipoxia , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Sulfonamidas/farmacología , Animales , Células CHO , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Cricetinae , Reactivos de Enlaces Cruzados/farmacología , Citocromo-B(5) Reductasa/metabolismo , ADN/química , Relación Dosis-Respuesta a Droga , Ácido Edético/química , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hipoxia/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Químicos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Trasplante de Neoplasias , Nitrógeno/metabolismo , Oxígeno/metabolismo , Compuestos de Sulfhidrilo , Factores de Tiempo , Resultado del Tratamiento , Xantina Oxidasa/metabolismo , Zinc/químicaRESUMEN
Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence. Immunofluorescent imaging of transfected cell lines expressing the fusion protein confirmed the nuclear location of the enzyme. Regardless of the oxygenation state of the cell, mitomycin C (MC) cytotoxicity was enhanced in cells with overexpressed NPR localized to the nuclear compartment compared with cells overexpressing an endoplasmic reticulum localized enzyme. Enhanced cytotoxicity in cells treated under hypoxic conditions correlated with increases in genomic DNA alkylations, with more MC-DNA adducts being formed when the enzyme was expressed closer to its DNA target. No change was observed in the hypoxic/aerobic differential toxicity as a function of enzyme localization. These findings indicate that drug efficacy is increased when the subcellular site of drug activation corresponds to its site of action.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Mitomicina/toxicidad , NADPH-Ferrihemoproteína Reductasa/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Células CHO , Cricetinae , Sinergismo Farmacológico , NADPH-Ferrihemoproteína Reductasa/toxicidadRESUMEN
Cloretazine {1,2-bis(methylsulfonyl)-1-[(2-chloroethyl)-2-(methylamino)carbonyl]hydrazine; VNP40101M; 101M} is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy against transplanted murine and human tumor models and has shown activity in clinical trials against relapsed or refractory acute myeloid leukemia. Base catalyzed activation of this prodrug generates two different reactive intermediates: chloroethylating species that covalently interact with DNA at the O6-position of guanine residues that progress to a G-C interstrand cross-link, and a carbamoylating agent, methyl isocyanate. Previous findings from this laboratory have provided initial evidence that methyl isocyanate can contribute to the efficacy of Cloretazine by enhancing the cytotoxicity of the generated chloroethylating species. This action may be due in part to inhibition of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT); however, activity in cells devoid of AGT indicates that other actions are involved in the synergistic cytotoxicity. Herein we demonstrate that O6-benzylguanine can also produce synergistic cell kill with the alkylating component of Cloretazine but differs from methyl isocyanate in that the enhancement occurs in AGT-containing cells, but not in cells devoid of AGT. Methyl isocyanate generated by the decomposition of 1,2-bis(methylsulfonyl)-1-[methylaminocarbonyl]hydrazine also acts to enhance the activity of a variety of DNA cross-linking agents, while only producing additive cytotoxicity with methylating agents. Flow cytometric studies using annexin as a marker for apoptosis indicate that in Chinese hamster ovary cells and in human leukemia cells Cloretazine-induced apoptosis is primarily caused by the generated methyl isocyanate. Comet assays designed to detect DNA cross-links in intact cells indicate that the chloroethylating species generated by the activation of Cloretazine produce DNA cross-links, with the co-generated methyl isocyanate increasing the degree of cross-linking produced by the reactive chloroethylating species. These findings provide further evidence that the methyl isocyanate produced by the activation of Cloretazine can be a major contributor to the cytotoxicity produced by this antineoplastic agent.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Hidrazinas/farmacología , Profármacos/farmacología , Sulfonamidas/farmacología , Animales , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Biotransformación , Células CHO , Línea Celular , Células Cultivadas , Cricetinae , Sinergismo Farmacológico , Guanina/análogos & derivados , Guanina/farmacología , Células HL-60 , Humanos , Hidrazinas/farmacocinética , Isocianatos/farmacocinética , Isocianatos/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Profármacos/farmacocinética , Sulfonamidas/farmacocinética , TransfecciónRESUMEN
The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.