RESUMEN
Oxidative stress is a key concept in basic, translational, and clinical research to understand the pathophysiology of various disorders, including cardiovascular and renal diseases. Although attempts to directly reduce oxidative stress with redox-active substances have until now largely failed to prove clinical benefit, indirect approaches to combat oxidative stress enzymatically have gained further attention as potential therapeutic strategies. The pantetheinase Vanin-1 is expressed on kidney proximal tubular cells, and its reaction product cysteamine is described to negatively affect redox homeostasis by inhibiting the replenishment of cellular antioxidative glutathione stores. Vanin-1-deficient mice were shown to be protected against oxidative stress damage. The aim of this study was to elucidate whether pharmacological inhibition of Vanin-1 protects mice from oxidative stress-related acute or chronic kidney injury as well. By studying renal ischemia-reperfusion injury in Col4α3-/- (Alport syndrome) mice and in vitro hypoxia-reoxygenation in human proximal tubular cells we found that treatment with a selective and potent Vanin-1 inhibitor resulted in ample inhibition of enzymatic activity in vitro and in vivo. However, surrogate parameters of metabolic and redox homeostasis were only partially and insufficiently affected. Consequently, apoptosis and reactive oxygen species level in tubular cells as well as overall kidney function and fibrotic processes were not improved by Vanin-1 inhibition. We thus conclude that Vanin-1 functionality in the context of cardiovascular diseases needs further investigation and the biological relevance of pharmacological Vanin-1 inhibition for the treatment of kidney diseases remains to be proven.
Asunto(s)
Lesión Renal Aguda/prevención & control , Amidohidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Nefritis Hereditaria/prevención & control , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Renal Crónica/prevención & control , Daño por Reperfusión/prevención & control , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacocinética , Fibrosis , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis Hereditaria/enzimología , Nefritis Hereditaria/genética , Nefritis Hereditaria/patología , Insuficiencia Renal Crónica/enzimología , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Daño por Reperfusión/enzimología , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
PURPOSE: We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity. EXPERIMENTAL DESIGN: Four PET tracers [(18)F-FDG, (18)F-Fpp(RGD)2, (18)F-FLT, and (18)F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes. RESULTS: Significant changes in uptake of (18)F-FAZA, (18)F-FLT, and (18)F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with (18)F-FAZA being the most prominent. (18)F-FDG uptake was unaffected. (18)F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. (18)F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting (18)F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced (18)F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed. CONCLUSIONS: Our studies suggest suitability of (18)F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243).
Asunto(s)
Antineoplásicos/uso terapéutico , Nitroimidazoles/farmacocinética , Oxadiazoles/uso terapéutico , Pirazoles/uso terapéutico , Radiofármacos/farmacocinética , Animales , Antineoplásicos/farmacología , Hipoxia de la Célula , Línea Celular Tumoral , Didesoxinucleósidos/farmacocinética , Femenino , Fluorodesoxiglucosa F18/farmacocinética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones Desnudos , Oxadiazoles/farmacología , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Pirazoles/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The transcription factor hypoxia-inducible factor-1 (HIF-1) pathway plays an important role in tumor response to cytotoxic treatments. We investigated the effects of a novel small molecule inhibitor of mitochondrial complex I and hypoxia-induced HIF-1 activity BAY-87-2243, on tumor microenvironment and response of human squamous cell carcinoma (hSCC) to clinically relevant fractionated radiotherapy (RT) with and without concomitant chemotherapy. METHODS: When UT-SCC-5 hSCC xenografts in nude mice reached 6 mm in diameter BAY-87-2243 or carrier was administered before and/or during RT or radiochemotherapy with concomitant cisplatin (RCT). Local tumor control was evaluated 150 days after irradiation and the doses to control 50% of tumors (TCD50) were compared between treatment arms. Tumors were excised at different time points during BAY-87-2243 or carrier treatment for western blot and immunohistological investigations. RESULTS: BAY-87-2243 markedly decreased nuclear HIF-1α expression and pimonidazole hypoxic fraction already after 3 days of drug treatment. BAY-87-2243 prior to RT significantly reduced TCD50 from 123 to 100 Gy (p=0.037). Additional BAY-87-2243 application during RT did not decrease TCD50. BAY-87-2243 before and during radiochemotherapy did not improve local tumor control. CONCLUSIONS: Pronounced reduction of tumor hypoxia by application of BAY-87-2243 prior to RT improved local tumor control. The results demonstrate that radiosensitizing effect importantly depends on treatment schedule. The data support further investigations of HIF-1 pathway inhibitors for radiotherapy and of predictive tests to select patients who will benefit from this combined treatment.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Oxadiazoles/farmacología , Pirazoles/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Antineoplásicos/farmacología , Western Blotting , Hipoxia de la Célula/efectos de los fármacos , Quimioradioterapia/métodos , Fraccionamiento de la Dosis de Radiación , Humanos , Ratones , Ratones Desnudos , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody-drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC(50) of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Proteínas Ligadas a GPI/inmunología , Inmunoconjugados/administración & dosificación , Maitansina/análogos & derivados , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Efecto Espectador , Línea Celular Tumoral , Proteínas Ligadas a GPI/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Maitansina/administración & dosificación , Mesotelina , Neoplasias/inmunología , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To study the effects of BAY-84-7296, a novel orally bioavailable inhibitor of mitochondrial complex I and hypoxia-inducible factor 1 (HIF-1) activity, on hypoxia, microenvironment, and radiation response of tumors. METHODS AND MATERIALS: UT-SCC-5 and UT-SCC-14 human squamous cell carcinomas were transplanted subcutaneously in nude mice. When tumors reached 4 mm in diameter BAY-84-7296 (Bayer Pharma AG) or carrier was daily administered to the animals. At 7 mm tumors were either excised for Western blot and immunohistologic investigations or were irradiated with single doses. After irradiation animals were randomized to receive BAY-84-7296 maintenance or carrier. Local tumor control was evaluated 150 days after irradiation, and the dose to control 50% of tumors (TCD50) was calculated. RESULTS: BAY-84-7296 decreased nuclear HIF-1α expression. Daily administration of inhibitor for approximately 2 weeks resulted in a marked decrease of pimonidazole hypoxic fraction in UT-SCC-5 (0.5% vs 21%, P<.0001) and in UT-SCC-14 (0.3% vs 19%, P<.0001). This decrease was accompanied by a significant increase in fraction of perfused vessels in UT-SCC-14 but not in UT-SCC-5. Bromodeoxyuridine and Ki67 labeling indices were significantly reduced only in UT-SCC-5. No significant changes were observed in vascular area or necrosis. BAY-84-7296 before single-dose irradiation significantly decreased TCD50, with an enhancement ratio of 1.37 (95% confidence interval [CI] 1.13-1.72) in UT-SCC-5 and of 1.55 (95% CI 1.26-1.94) in UT-SCC-14. BAY-84-7296 maintenance after irradiation did not further decrease TCD50. CONCLUSIONS: BAY-84-7296 resulted in a marked decrease in tumor hypoxia and substantially reduced radioresistance of tumor cells with the capacity to cause a local recurrence after irradiation. The data suggest that reduction of cellular hypoxia tolerance by BAY-84-7296 may represent the primary biological mechanism underlying the observed enhancement of radiation response. Whether this mechanism contributes to the improved outcome of fractionated chemoradiation therapy warrants further investigation.
Asunto(s)
Aminas/farmacología , Hipoxia de la Célula/efectos de los fármacos , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Animales , Bromodesoxiuridina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Hipoxia de la Célula/fisiología , Núcleo Celular/metabolismo , Fraccionamiento de la Dosis de Radiación , Femenino , Xenoinjertos , Humanos , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias/métodos , Nitroimidazoles/farmacología , Tolerancia a Radiación/fisiología , Fármacos Sensibilizantes a Radiaciones/farmacología , Distribución Aleatoria , Microambiente TumoralRESUMEN
The transcription factors hypoxia-inducible factor-1 and -2 (HIF-1 and HIF-2) orchestrate a multitude of processes that allow tumor cells to survive under conditions of low oxygen and nutrients, and that lead to resistance to some apoptotic pathways and facilitate invasion and metastasis. Therefore, inhibition of transactivation by HIF has become an attractive target in cancer research. Herein we present the results of a cell-based screening approach that led to the discovery of substituted 1H-pyrazole-3-carboxamides. Chemical optimization of the hit class with respect to potency and metabolic stability is described; it resulted in novel 5-(1H-pyrazol-3-yl)-1,2,4-oxadiazoles that inhibit the hypoxia-induced accumulation of HIF-1α and HIF-2α. The HIF inhibitory potency in the screening cell system was improved from IC50 190 to 0.7â nM, and significant parts of the SAR are disclosed. For a key compound, the ability to suppress the hypoxia-induced expression of HIF target genes was studied in A549 human lung adenocarcinoma cells. The same compound shows a favorable pharmacokinetic profile in rats after i.v. and p.o. administration.
Asunto(s)
Amidas/química , Hipoxia de la Célula , Oxadiazoles/química , Pirazoles/química , Administración Oral , Amidas/farmacocinética , Amidas/toxicidad , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Semivida , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inyecciones Intravenosas , Ratas , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacosRESUMEN
The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.