RESUMO
A pretargeted imaging strategy based on the HaloTag dehalogenase enzyme is described. Here, a HaloTag-Trastuzumab conjugate has been used as the primary agent targeting HER2 expression, and three new radiolabelled HaloTag ligands have been used as secondary agents, two of which offer dual-modality (SPECT/optical) imaging capability.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Halogênios/metabolismo , Hidrolases/metabolismo , Imagem Óptica/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Linhagem Celular Tumoral , Humanos , Ligantes , TrastuzumabRESUMO
Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/fisiologia , Glucose/genética , Glucose/metabolismo , Células HeLa , Humanos , ATPases Mitocondriais Próton-Translocadoras , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA , Ativação Transcricional/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
This study describes a system for quantifying paclitaxel activity using the C-terminus of α-tubulin as a biomarker. Following stabilization of microtubules with paclitaxel, a specific detyrosination reaction occurs at the C-terminus of α-tubulin which could be used to assess efficacy. A fluorescence resonance energy transfer (FRET) based biosensor was synthesized comprising a short peptide that corresponded to the C-terminus of α-tubulin, a fluorophore (Abz), and a quencher (Dnp). The fluorophore added to the end of the peptide can be released upon enzymatic detyrosination. In addition, a single fluorophore-tagged peptide was also conjugated to mesoporous silica nanoparticles to examine the feasibility of combining the drug with the peptide biomarker. As a proof of concept, we found that the degree of peptide cleavage, and therefore enzymatic activity, was directly correlated with exogenous bovine carboxypeptidase (CPA) an enzyme that mimics endogenous detyrosination. In addition, we show that cell lysates obtained from paclitaxel-treated cancer cells competed with exogenous CPA for biosensor cleavage in a paclitaxel dose-dependent manner. Our work provides strong evidence for the feasibility of combining paclitaxel with a novel biosensor in a multi-load nanoparticle.
Assuntos
Monitoramento de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Nanocápsulas/química , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Tubulina (Proteína)/farmacocinética , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/análise , Antineoplásicos Fitogênicos/farmacologia , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Nanocápsulas/administração & dosagem , Nanocápsulas/ultraestrutura , Nanoconjugados/administração & dosagem , Nanoconjugados/química , Nanoconjugados/ultraestrutura , Paclitaxel/análise , Tamanho da Partícula , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício/química , Tubulina (Proteína)/análise , Tubulina (Proteína)/químicaRESUMO
BACKGROUND: The vascular disrupting agent combretastatin A4 phosphate (CA4P) causes major regression of animal tumours when given as combination therapy. METHODS: Patients with advanced cancer refractory to standard therapy were treated with CA4P as a 10-min infusion, 20 h before carboplatin, paclitaxel, or paclitaxel, followed by carboplatin. RESULTS: Combretastatin A4 phosphate was escalated from 36 to 54 mg m(-2) with the carboplatin area under the concentration curve (AUC) 4-5, from 27 to 54 mg m(-2) with paclitaxel 135-175 mg m(-2), and from 54 to 72 mg m(-2) with carboplatin AUC 5 and paclitaxel 175 mg m(-2). Grade 3 or 4 neutropenia was seen in 17%, and thrombocytopenia only in 4% of 46 patients. Grade 1-3 hypertension (26% of patients) and grade 1-3 tumour pain (65% of patients) were the most typical non-haematological toxicities. Dose-limiting toxicity of grade 3 hypertension or grade 3 ataxia was seen in two patients at 72 mg m(-2). Responses were seen in 10 of 46 (22%) patients with ovarian, oesophageal, small-cell lung cancer, and melanoma. CONCLUSION: The combination of CA4P with carboplatin and paclitaxel was well tolerated in the majority of patients with adequate premedication and had antitumour activity in patients who were heavily pretreated.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/uso terapêutico , Neoplasias/tratamento farmacológico , Paclitaxel/uso terapêutico , Estilbenos/uso terapêutico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Ataxia/induzido quimicamente , Carboplatina/toxicidade , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Relação Dose-Resposta a Droga , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Feminino , Humanos , Infusões Intravenosas , Expectativa de Vida , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Melanoma/tratamento farmacológico , Melanoma/patologia , Pessoa de Meia-Idade , Neoplasias/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Paclitaxel/toxicidade , Seleção de Pacientes , Estilbenos/administração & dosagem , Estilbenos/toxicidadeRESUMO
Gene-directed enzyme prodrug therapy is a form of targeted cancer therapy, in which an enzyme is used to convert a non-toxic prodrug to a cytotoxin within the tumor. Horseradish peroxidase (HRP) is able to convert the indole prodrugs indole-3-acetic acid (IAA) and the halogenated derivative 5-bromo-IAA (5Br-IAA) to toxic agents able to induce cell kill in vitro. This study characterized HRP-directed gene therapy in vivo. Human nasopharyngeal squamous cell carcinoma cells, FaDu, stably expressing HRP were grown as xenografts in SCID mice. Pharmacokinetic analysis of IAA and 5Br-IAA showed satisfactory drug profiles, and millimolar concentrations could be achieved in tumor tissue at non-toxic doses. HRP-expressing tumors showed a modest growth delay when treated with IAA compared with drug-vehicle controls. Treatment response could not be improved using different drug scheduling or drug vehicle, nor by combining HRP-directed gene therapy with fractionated radiotherapy.
Assuntos
Terapia Genética/métodos , Peroxidase do Rábano Silvestre/metabolismo , Ácidos Indolacéticos/metabolismo , Neoplasias/terapia , Animais , Área Sob a Curva , Bromo/química , Linhagem Celular Tumoral , Terapia Combinada , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peroxidase do Rábano Silvestre/genética , Humanos , Ácidos Indolacéticos/química , Ácidos Indolacéticos/farmacocinética , Camundongos , Camundongos SCID , Neoplasias/genética , Pró-Fármacos/administração & dosagem , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Radioterapia/métodos , Transgenes/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To determine whether tumour radiosensitization and the therapeutic benefit of administering carbogen with nicotinamide depend upon irradiating at the time of peak drug concentration. MATERIALS AND METHODS: Local tumour control of CaNT tumours in CBA mice and acute skin reactions in albino WHT mice were assessed after treatment with 10 X-ray fractions in air, carbogen alone or combined with 0.1, 0.2 or 0.5 mg g(-1) nicotinamide, injected 15, 30 or 60 min before irradiation. Plasma and tumour drug pharmacokinetics were performed. RESULTS: Nicotinamide was rapidly taken up into tumours; a six- and threefold higher concentration was obtained with 0.5 mg g(-1) compared with 0.1 and 0.2 mg g(-1), respectively. Tumour, but not skin, radiosensitization increased as the dose of nicotinamide increased (p = 0.03), but at each dose level there was no significant difference in radiosensitivity when irradiations were done at or after the time of peak concentration. An almost eightfold increase in plasma levels increased tumour enhancement ratios from 1.74 to 1.92 (p < 0.0001). In tumours all schedules gave significant enhancement relative to carbogen alone (p < or = 0.04). CONCLUSIONS: Tumour and skin radiosensitivity was independent of time of nicotinamide administration. Higher drug concentrations were not mirrored by proportionally higher enhancement ratios. Lower plasma levels than previously suggested significantly enhanced tumour radiosensitivity relative to carbogen alone. The clinical implications of these findings are discussed.
Assuntos
Dióxido de Carbono/farmacologia , Neoplasias Mamárias Experimentais/radioterapia , Niacinamida/farmacologia , Oxigênio/farmacologia , Radiossensibilizantes/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos CBA , Niacinamida/farmacocinética , Pele/efeitos da radiaçãoRESUMO
BACKGROUND: Serotonin (5HT), a naturally occurring vasoactive substance, is released from platelets into plasma under various pathological conditions. Recently, anticancer drugs that act by selectively disrupting tumour blood flow have been found to increase plasma 5HT concentrations in mice. Two such antivascular agents, flavone acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have completed Phase I clinical trial and raise the important question of whether suitable surrogate markers for antivascular effects can be identified. METHODS: 5HT is unstable to storage, precluding routine clinical assay, but the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) accumulates in plasma following 5HT release and is a more suitable marker because of its greater stability. We have developed an automated procedure for the assay of the low concentrations of 5HIAA found in humans by combining solid-phase extraction with high-performance liquid chromatography (HPLC). RESULTS: Efficient separation of 5HIAA from possible interfering substances in human plasma, including a variety of pharmaceutical agents, was achieved on C18 columns using cetyltrimethylammonium bromide (CETAB) as an organic modifier. Adequate precision, accuracy and sensitivity were achieved by electrochemical detection (ECD) at +400 mV. Analysis of plasma from two patients treated with DMXAA in a Phase I trial demonstrated DMXAA-induced elevation of plasma 5HIAA with a time course similar to that previously described in mice. CONCLUSIONS: Measurement of changes in plasma 5HIAA provides a new approach to the monitoring of therapies with an antivascular effect. The assay is sensitive to dietary sources of 5HT, which should be minimised.
Assuntos
Inibidores da Angiogênese/farmacologia , Ácido Hidroxi-Indolacético/sangue , Xantonas , Inibidores da Angiogênese/uso terapêutico , Biomarcadores , Calibragem , Cromatografia Líquida de Alta Pressão , Ensaios Clínicos Fase I como Assunto , Eletroquímica , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Controle de Qualidade , Padrões de Referência , Fluxo Sanguíneo Regional/efeitos dos fármacos , Reprodutibilidade dos Testes , Soluções , Xantenos/farmacologia , Xantenos/uso terapêuticoRESUMO
PURPOSE: To test the hypothesis that there is a link between plasma glutathione (GSH) or other antioxidants (uric acid, ascorbate) and the severity of radiation mucositis following radiation treatment of tumors of the head and neck. PATIENTS AND METHODS: Patients with carcinomas of the head-and-neck region were treated with the continuous hyperfractionated accelerated radiotherapy (CHART) regimen (54 Gy in 36 fractions over 12 days). Samples of blood plasma were analyzed for GSH, cysteine, urate, and ascorbate by high-pressure liquid chromatography. Patients were graded for dysphagia and requirement for analgesics. The areas under the curves of scores over 2-6 weeks following treatment were computed, and Spearman's rank-correlation coefficient was used to test for an association between plasma GSH levels (or those of other antioxidants) and mucositis. RESULTS: The pretreatment plasma GSH level in 18 patients scored in the study was 1.0 +/- 0.7 M. Analysis of these and the dysphagia scores produced a correlation coefficient of 0.22 (confidence interval -0.28, 0.61; p = 0.39). No correlation was seen between mucositis severity and other measures of plasma antioxidants: cysteine (7.6 +/- 1.7 M), cysteine + GSH (8.6 +/- 1.9 M), uric acid (317 +/- 86 M), ascorbate (29 +/- 20 M), or whole-blood GSH concentrations (1,010 +/- 239 M). CONCLUSION: The measurements of approximately micromolar levels of plasma GSH, or about 10 M cysteine + GSH (almost all of the total nonprotein thiols), are consistent with most other published data for either healthy adults or cancer patients; however, the values reported in an earlier study, suggesting a link between GSH and mucositis, are much higher. The hypothesis of a possible link between radiation mucositis and plasma-free (nonprotein) thiols was not supported.
Assuntos
Carcinoma de Células Escamosas/radioterapia , Glutationa/sangue , Mucosa Bucal/efeitos da radiação , Neoplasias Bucais/radioterapia , Estomatite/sangue , Ácido Ascórbico/sangue , Cisteína/sangue , Fracionamento da Dose de Radiação , Humanos , Modelos Lineares , Sensibilidade e Especificidade , Estomatite/etiologia , Ácido Úrico/sangueRESUMO
BACKGROUND: Combretastatin A4 Phosphate (CA4P) is a tubulin binding agent which causes rapid tumour vascular shutdown. It has anti-proliferative and apoptotic effects on dividing endothelial cells after prolonged exposure, but these effects occur on a much longer time scale than the reduction in tumour blood flow. This study compared the time course of CA4P effects on endothelial cell shape and reduction in red cell velocity. METHODS: Endothelial cell area and form factor (1-4 pi x area x perimeter-2) were measured for proliferating and confluent HUVECs after CA4P treatment. Recovery of shape after CA4P and colchicine was compared. Window chamber studies of tumours were used to measure red cell velocity. Results 70% reduction in red cell velocity and 44% reduction in HUVEC form factor occurred by 10 minutes. Proliferating HUVECs underwent greater cell shape change after CA4P, which occurred at lower doses than for confluent cells. Cell shape recovered 24 hours after 30 minutes exposure to CA4P, but not after colchicine. CONCLUSIONS: The similar time course of cell shape change and red cell velocity reduction suggests endothelial cell shape change may be involved early in the in vivo events leading to vascular shutdown. Differences in the recovery from the shape changes induced by CA4P and colchicine could underlie the different toxicity profiles of these drugs.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Neoplasias Experimentais/irrigação sanguínea , Estilbenos/farmacologia , Animais , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Colchicina/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Humanos , Cinética , Masculino , Microtúbulos/efeitos dos fármacos , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacosRESUMO
The role of nitric oxide (NO) in the response to Photofrin-based photodynamic therapy (PDT) was investigated using mouse tumour models characterized by either relatively high or low endogenous NO production (RIF and SCCVII vs EMT6 and FsaR, respectively). The NO synthase inhibitors Nomega-nitro-L-arginine (L-NNA) or Nomega-nitro-L-arginine methyl ester (L-NAME), administered to mice immediately after PDT light treatment of subcutaneously growing tumours, markedly enhanced the cure rate of RIF and SCCVII models, but produced no obvious benefit with the EMT6 and FsaR models. Laser Doppler flowmetry measurement revealed that both L-NNA and L-NAME strongly inhibit blood flow in RIF and SCCVII tumours, but not in EMT6 and FsaR tumours. When injected intravenously immediately after PDT light treatment, L-NAME dramatically augmented the decrease in blood flow in SCCVII tumours induced by PDT. The pattern of blood flow alterations in tumours following PDT indicates that, even with curative doses, regular circulation may be restored in some vessels after episodes of partial or complete obstruction. Such conditions are conducive to the induction of ischaemia-reperfusion injury, which is instigated by the formation of superoxide radical. The administration of superoxide dismutase immediately after PDT resulted in a decrease in tumour cure rates, thus confirming the involvement of superoxide in the anti-tumour effect. The results of this study demonstrate that NO participates in the events associated with PDT-mediated tumour destruction, particularly in the vascular response that is of critical importance for the curative outcome of this therapy. The level of endogenous production of NO in tumours appears to be one of the determinants of sensitivity to PDT.
Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Óxido Nítrico/biossíntese , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , NG-Nitroarginina Metil Éster/farmacologia , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , FotoquimioterapiaRESUMO
Urinary markers of bone resorption, pyridinoline and deoxypyridinoline were measured before and at 4 weeks after radiotherapy for metastatic bone pain. An association was shown between relief of metastatic skeletal pain by radiotherapy and low marker concentrations before and after treatment, lending support to the hypothesis that relief of metastatic bone pain by radiotherapy relates to an effect on bone, rather than tumour physiology.
Assuntos
Aminoácidos/urina , Biomarcadores/urina , Neoplasias Ósseas/complicações , Neoplasias Ósseas/secundário , Reabsorção Óssea/etiologia , Reabsorção Óssea/urina , Dor/etiologia , Dor/radioterapia , Neoplasias da Mama/patologia , Feminino , Humanos , Masculino , Dor/diagnóstico , Medição da Dor , Estudos Prospectivos , Neoplasias da Próstata/patologia , Resultado do TratamentoRESUMO
2-Nitroimidazoles were synthesised substituted with aspirin or salicylic acid, as leaving groups linked through the (imidazol-5-yl)methyl position. Activation of aqueous solutions by CO2*- (a model one-electron reductant) resulted in release of aspirin or salicylate, probably via the 2-hydroxyaminoimidazole. The analogous 2-nitroimidazole with bromide as leaving group eliminated bromide in < 1 ms via the radical-anion.
Assuntos
Aspirina/química , Hipóxia Celular/efeitos dos fármacos , Nitroimidazóis/síntese química , Pró-Fármacos/química , Animais , Brometos/química , Humanos , Salicilatos/química , Fatores de TempoRESUMO
The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Estilbenos/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Neoplasias Experimentais/irrigação sanguínea , Fosforilação , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Estilbenos/metabolismo , Resistência Vascular/efeitos dos fármacosRESUMO
A series of regioisomeric derivatives of a 1-methylindole-4,7-dione were synthesised, substituted with a 2-acetoxybenzoate leaving group linked through the (indol-2-yl)methyl or (indol-3-yl)methyl (or propenyl) positions. Reductive elimination of the leaving group occurred from the (indol-3-yl)methyl derivatives but not the 2-substituted regioisomers, indicating that only the C-3 position may be utilised in bioreductively-activated drug delivery, which was demonstrated with an aspirin prodrug.
Assuntos
Aspirina/química , Indóis/química , Pró-Fármacos/química , Quinonas/química , Animais , Anti-Inflamatórios não Esteroides , Artrite/metabolismo , Aspirina/metabolismo , Radicais Livres/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Indóis/metabolismo , Neoplasias/metabolismo , Oxirredução , Pró-Fármacos/metabolismo , Quinonas/metabolismo , Relação Estrutura-AtividadeRESUMO
High-performance liquid chromatography with both absorbance and fluorescence detection has been applied to the determination of the potential anti-tumour agent combretastatin A-4 and its phosphate ester in murine and human plasma. The presence of different interfering peaks in the two species makes absorbance detection at 295 nm the method of choice for the mouse, and fluorescence detection (295 nm/390 nm) for human plasma. The calibration was linear over the range studied (0.01-50 microM for combretastatin A-4, 0.02-200 microM for combretastatin A-4 phosphate), with quantitation limits of 0.05 microM for both drugs in the mouse, and 0.05 microM and 0.0125 microM for the phosphate ester and free drug, respectively, in human plasma. The method should be useful for pharmacokinetic studies in the forthcoming Phase I clinical trial of combretastatin A-4 phosphate.
Assuntos
Antineoplásicos Fitogênicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Pró-Fármacos/análise , Estilbenos/sangue , Animais , Antineoplásicos Fitogênicos/química , Calibragem , Ésteres , Humanos , Isomerismo , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Estilbenos/químicaRESUMO
This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.
Assuntos
DNA Super-Helicoidal/efeitos dos fármacos , Peroxidase do Rábano Silvestre/farmacologia , Ácidos Indolacéticos/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Cricetinae , Cricetulus , Adutos de DNA/biossíntese , Radicais Livres , Ácidos Indolacéticos/metabolismoRESUMO
BACKGROUND: The EORTC has initiated studies to combine nicotinamide with carbogen in accelerated fractionation schedules (ARCON), since for some tumour types, acute and chronic hypoxia as well as treatment protraction may prejudice the outcome of radiotherapy. The tolerable dose of nicotinamide and the optimal interval for administration need to be ascertained. AIM: Full pharmacokinetic profiles of nicotinamide concentrations in plasma were analyzed repeatedly in 15 patients to determine the inter- and intra-patient variability in peak plasma concentrations and the optimum times for administering nicotinamide as a radiosensitizer. METHODS: Nicotinamide (Nicobion) was administered in tablet form to patients with advanced head and neck and non-small cell lung carcinomas. A standard 6 g dose was given regardless of body weight after an overnight fast and at least 30 min before breakfast. In 15 patients, blood samples were taken prior to and 1, 2, 4, 6, 8, 12 and 24 h after administration of the drug. This full profile was determined on two to four occasions for the head and neck cancer patients and on two occasions for the lung cancer patients. For each profile, the maximum concentration of nicotinamide (Cmax), time to peak plasma concentration (Tmax), elimination half-lives (t1/2) and area under the curve (AUC) were determined. Compliance was recorded and nausea and vomiting were graded on a 0-3 scale. Complete profiles of the five major metabolites were also obtained. RESULTS: In the 48 complete sets of blood samples, peak plasma concentrations ranged from 787 to 2312 nmol/ml with a median value of 1166 nmol/ml. The peak plasma concentration was achieved at 1 h in only 54% of the pharmacokinetic profiles, but at this time 92% of the profiles had already exceeded the target concentration of 700 nmol/ml, the level required in the mouse for tumour radiosensitization. The median t1/2 for all 15 cases was 9.3 h, with minimum and maximum values of 4.2 and 26.8 h. The highest concentrations of nicotinamide metabolites were found to be the N-oxide, 2-pyridone and 1-methylnicotinamide. The toxicity (nausea and vomiting) was scored and found not to be correlated with any of the pharmacokinetic parameters. CONCLUSIONS: The plasma concentrations considered necessary to radiosensitize can easily be exceeded with a dose of 6 g taken as 12 x 500 mg in tablet form; 700 nmol/ml was achieved in all patients and apparently would have been achieved in most even with a considerable reduction in dose. An adequate time between administration and radiotherapy appeared to be 1 h with this drug formulation for 92% of the profiles.
Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias Pulmonares/radioterapia , Niacinamida/farmacocinética , Radiossensibilizantes/farmacocinética , Administração Oral , Área Sob a Curva , Dióxido de Carbono/farmacocinética , Dióxido de Carbono/uso terapêutico , Carcinoma/radioterapia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Hipóxia Celular/efeitos dos fármacos , Fracionamento da Dose de Radiação , Seguimentos , Meia-Vida , Humanos , Náusea/induzido quimicamente , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Niacinamida/sangue , Niacinamida/uso terapêutico , Oxigênio/farmacocinética , Oxigênio/uso terapêutico , Piridonas/sangue , Radiossensibilizantes/administração & dosagem , Radiossensibilizantes/análise , Radiossensibilizantes/uso terapêutico , Comprimidos , Fatores de Tempo , Resultado do Tratamento , Vômito/induzido quimicamenteRESUMO
Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO.). In vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH.) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO.) and normal skin. Significantly greater OH. generation followed I/R of murine adenocarcinoma CaNT tumours (low NO. production) compared to round cell sarcoma SaS tumours (high NO. production) and normal skin. These data suggest that tumour production of NO. confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH. generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO.. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.
Assuntos
Adenocarcinoma/metabolismo , Radical Hidroxila/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Óxido Nítrico/fisiologia , Traumatismo por Reperfusão/metabolismo , Sarcoma de Células Pequenas/metabolismo , Adenocarcinoma/irrigação sanguínea , Animais , Catalase/farmacologia , Desferroxamina/farmacologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Hidroxilação , L-Lactato Desidrogenase/sangue , Neoplasias Mamárias Experimentais/irrigação sanguínea , Camundongos , Camundongos Endogâmicos CBA , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Estresse Oxidativo , Salicilatos/farmacologia , Ácido Salicílico , Sarcoma de Células Pequenas/irrigação sanguíneaRESUMO
A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol-was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.