RESUMEN
PURPOSE: Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK). EXPERIMENTAL DESIGN: Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo, antitumor activity, plasma, and tumor RAD001 levels were measured. Activity in several different angiogenic/vascular assays in vitro and in vivo was assessed and compared with PTK/ZK. RESULTS: RAD001 inhibited proliferation in vitro (IC50 values<1 nmol/L to >1 micromol/L), and in sensitive and insensitive tumor cells, pS6 kinase and 4E-BP1 were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC50 values>10-fold higher than tumor RAD001 concentrations. In vitro, RAD001 inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo, in tumor models derived from either sensitive or insensitive cells, RAD001 reduced Tie-2 levels, the amount of mature and immature vessels, total plasma, and tumor VEGF. RAD001 did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (Ktrans) as measured by dynamic contrast-enhanced magnetic resonance imaging. However, the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001. CONCLUSIONS: VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology, which has implications for their clinical activity alone or in combination.
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Inhibidores de la Angiogénesis/farmacología , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Ftalazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Piridinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Sirolimus/análogos & derivados , Inhibidores de la Angiogénesis/farmacocinética , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Everolimus , Femenino , Humanos , Técnicas para Inmunoenzimas , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Ftalazinas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Quinasas/metabolismo , Piridinas/farmacocinética , Ratas , Ratas Endogámicas BN , Ratas Endogámicas WF , Receptor TIE-2/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Sirolimus/farmacocinética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Distribución Tisular , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.
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Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/tratamiento farmacológico , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Animales , Calcio/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Córnea/irrigación sanguínea , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Femenino , Clorhidrato de Fingolimod , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Ftalazinas/farmacología , Glicoles de Propileno/farmacocinética , Piridinas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Despite bacteraemia is present in the majority of patients with pneumococcal, little is known about the influence of the systemic infection on the meningeal inflammatory response. METHODS: To explore the role of systemic infection on the meningeal inflammation, experimental meningitis was induced by intracisternal injection of approximately 1 x 10(6) CFU Streptococcus pneumoniae, type 3, and the 26 rabbits were either provided with approximately 1 x 10(6) CFU S. pneumoniae intravenously at 0 hour ("bacteraemic" rabbits, n = 9), immunized with paraformaldehyde-killed S. pneumoniae for 5 weeks prior to the experiment ("immunized" rabbits", n = 8), or not treated further ("control" rabbits, n = 9). WBC and bacterial concentrations were determined in CSF and blood every second hour during a 16 hours study period together with CSF IL-8 and protein levels. We also studied CSF and blood WBC levels in 153 pneumococcal meningitis patients with and without presence of bacteraemia. RESULTS: As designed, blood bacterial concentrations were significantly different among three experimental groups during the 16 hours study period (Kruskal Wallis test, P < 0.05), whereas no differences in CSF bacterial levels were observed (P > 0.05). Blood WBC decreased in bacteraemic rabbits between approximately 10-16 hours after the bacterial inoculation in contrast to an increase for both the immunized rabbits and controls (P < 0.05). The CSF pleocytosis was attenuated in bacteraemic rabbits as compared to the two other groups between 12-16 hours from time of infection (P < 0.017), despite accelerated CSF IL-8 levels in bacteraemic rabbits. In patients with pneumococcal meningitis, no significant difference in CSF WBC was observed between patients with or without bacteraemia at admission (n = 103, 1740 cells/microL (123-4032) vs. n = 50, 1961 cells/microL (673-5182), respectively, P = 0.18), but there was a significant correlation between CSF and blood WBC (n = 127, Spearman rho = 0.234, P = 0.008). CONCLUSION: Our results suggest that a decrease in peripheral WBC induced by enhanced bacteraemia in pneumococcal meningitis results in an attenuated CSF pleocytosis.
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Bacteriemia/microbiología , Meningitis Neumocócica/patología , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/patogenicidad , Animales , Bacteriemia/sangre , Bacteriemia/líquido cefalorraquídeo , Vacunas Bacterianas/administración & dosificación , Líquido Cefalorraquídeo/citología , Líquido Cefalorraquídeo/microbiología , Proteínas del Líquido Cefalorraquídeo/líquido cefalorraquídeo , Humanos , Inflamación/inmunología , Inflamación/microbiología , Interleucina-8/líquido cefalorraquídeo , Recuento de Leucocitos , Leucocitosis/líquido cefalorraquídeo , Meningitis Neumocócica/sangre , Meningitis Neumocócica/líquido cefalorraquídeo , Meningitis Neumocócica/inmunología , ConejosRESUMEN
PURPOSE: Receptor tyrosine kinases of the ErbB family play important roles in the control of tumor growth. Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation, enhances vascular permeability, and plays an important role in tumor vascularization. We evaluated the effects of selective VEGF receptor (VEGFR; PTK787/ZK222584) and ErbB (PKI166 and ZD1839) inhibitors on tumor growth and angiogenesis and asked whether additional therapeutic benefit was conferred by combination treatment. EXPERIMENTAL DESIGN: The antitumor activity of each inhibitor alone or in combination was assessed in human cancer models in immunocompromised mice. ErbB receptor expression and activation of downstream signaling pathway was evaluated in both tumor and endothelial cells. RESULTS: Both ErbB inhibitors significantly enhanced the antitumor activity of PTK787/ZK222584. In vitro, ErbB1 inhibition blocked VEGF release by tumor cells and proliferation of both tumor and endothelial cells. In an in vitro angiogenesis assay, epidermal growth factor (EGF) stimulated the release of VEGF by smooth muscle cells resulting in increased angiogenesis, a response blocked by administration of PTK787/ZK222584. Under basal condition, both ZD1839 and PTK787/ZK222584 blocked sprouting, likely via inhibition of an autocrine ErbB1 loop and VEGFR signaling, respectively, in endothelial cells. In conditions of limiting VEGF, EGF plays an important role in endothelial cell proliferation, survival, and sprouting. CONCLUSION: We have shown that activation of ErbB1 triggers a plethora of effects, including direct effects on tumor and endothelial cells and indirect effects mediated via induction of VEGF release. Simultaneous blockade of ErbB1 and VEGFR pathways results in a cooperative antitumor effect, indicating that this combination may represent a valid therapeutic strategy.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Ftalazinas/farmacología , Piridinas/farmacología , Quinazolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Sinergismo Farmacológico , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Técnica del Anticuerpo Fluorescente , Gefitinib , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Inhibidores de Proteínas Quinasas/farmacología , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models. EXPERIMENTAL DESIGN: The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001 and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses. RESULTS: Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented (P < 0.001, two-way ANOVA, synergy by isobologram analysis). Increased activity of the combination correlated with more profound effects on G1 progression and a significant decrease in cell viability (P < 0.01, two-way ANOVA) defined as apoptosis (P < 0.05, Friedman test). Increased cell death was particularly evident with optimal drug concentrations. CONCLUSION: mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormone-dependent breast cancers.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Carcinoma/patología , Inmunosupresores/farmacología , Nitrilos/farmacología , Proteínas Quinasas/fisiología , Receptores de Estrógenos/fisiología , Sirolimus/análogos & derivados , Triazoles/farmacología , Células Tumorales Cultivadas , Proliferación Celular , Supervivencia Celular , Interacciones Farmacológicas , Estrógenos/fisiología , Everolimus , Femenino , Humanos , Letrozol , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TORRESUMEN
PURPOSE: Evaluation of vascular disruptive activity in orthotopic models as potential surrogate biomarkers of tumor response to the microtubule-stabilizing agent patupilone. EXPERIMENTAL DESIGN: Mice bearing metastatic B16/BL6 melanoma and rats bearing mammary BN472 tumors received vehicle or efficacious patupilone doses (4 and 0.8-1.5 mg/kg i.v., respectively). Tumor vascularity assessment by dynamic contrast-enhanced or dynamic susceptibility contrast magnetic resonance imaging and interstitial fluid pressure (IFP) occurred at baseline, 2 days (mice and rats), and 6 days (rats) after treatment and were compared with histologic measurements and correlated with tumor response. RESULTS: In B16/BL6 metastases, patupilone (4 mg/kg) induced a 21 +/- 5% decrease (P < 0.001) in tumor blood volume and a 32 +/- 15% decrease (P = 0.02) in IFP after 2 days and reduced tumor growth and vessel density (>42%) after 2 weeks (P < or = 0.014). Patupilone dose-dependently inhibited BN472 tumor growth (day 6) and reduced IFP on days 2 and 6 (-21% to -70%), and the percentage change in IFP correlated (P < 0.01) with the change in tumor volume. In both models, histology and vascular casts confirmed decreases in tumor blood volume. One patupilone (0.8 mg/kg) administration decreased (P < 0.01) tumor IFP (54 +/- 4%), tumor blood volume (50 +/- 6%), and vessel diameter (40 +/- 11%) by day 6 but not the apparent diffusion coefficient, whereas histology showed that apoptosis was increased 2.4-fold and necrosis was unchanged. Apoptosis correlated negatively (P < 0.001) with IFP, tumor blood volume, and tumor volume, whereas tumor blood volume and IFP were correlated positively (P = 0.0005). CONCLUSIONS: Vascular disruptive effects of patupilone were detected in situ using dynamic contrast-enhanced or dynamic susceptibility contrast magnetic resonance imaging and IFP. Changes in IFP preceded and correlated with tumor response, suggesting that IFP may be a surrogate biomarker for patupilone efficacy.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Epotilonas/farmacología , Neovascularización Patológica , Animales , Apoptosis , Vasos Sanguíneos/patología , Caspasa 3 , Caspasas/metabolismo , Medios de Contraste/farmacología , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Femenino , Inmunohistoquímica , Metástasis Linfática , Imagen por Resonancia Magnética/métodos , Melanoma/patología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Presión , Ratas , Factores de TiempoRESUMEN
The orally bioavailable rapamycin derivative RAD001 (everolimus) targets the mammalian target of rapamycin pathway and possesses potent immunosuppressive and anticancer activities. Here, the antitumor activity of RAD001 was evaluated in the CA20948 syngeneic rat pancreatic tumor model. RAD001 demonstrated dose-dependent antitumor activity with daily and weekly administration schedules; statistically significant antitumor effects were observed with 2.5 and 0.5 mg/kg RAD001 administered daily [treated tumor versus control tumor size (T/C), 23% and 23-30%, respectively], with 3-5 mg/kg RAD001 administered once weekly (T/C, 14-36%), or with 5 mg/kg RAD001 administered twice weekly (T/C, 36%). These schedules were well tolerated and exhibited antitumor potency similar to that of the cytotoxic agent 5-fluorouracil (T/C, 23%). Moreover, the efficacy of intermittent treatment schedules suggests a therapeutic window allowing differentiation of antitumor activity from the immunosuppressive properties of this agent. Detailed biochemical profiling of mammalian target of rapamycin signaling in tumors, skin, and peripheral blood mononuclear cells (PBMCs), after a single administration of 5 mg/kg RAD001, indicated that RAD001 treatment blocked phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1 and inactivated the translational activator ribosomal protein S6 kinase 1 (S6K1). The efficacy of intermittent treatment schedules was associated with prolonged inactivation of S6K1 in tumors and surrogate tissues (> or =72 h). Furthermore, detailed analysis of the dose dependency of weekly treatment schedules demonstrated a correlation between antitumor efficacy and prolonged effects (> or =7 days) on PBMC-derived S6K1 activity. Analysis of human PBMCs revealed that S6K1 also underwent a concentration-dependent inactivation after RAD001 treatment ex vivo (>95% inactivation with 20 nM RAD001). In contrast, human PBMC-derived eukaryotic initiation factor 4E-binding protein 1 was present predominantly in the hypophosphorylated form and was unaffected by RAD001 treatment. Taken together, these results demonstrate a correlation between the antitumor efficacy of intermittent RAD001 treatment schedules and prolonged S6K1 inactivation in PBMCs and suggest that long-term monitoring of PBMC-derived S6K1 activity levels could be used for assessing RAD001 treatment schedules in cancer patients.
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Inmunosupresores/uso terapéutico , Leucocitos Mononucleares/enzimología , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Sirolimus/uso terapéutico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Monitoreo de Drogas/métodos , Everolimus , Humanos , Inmunosupresores/administración & dosificación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/inmunología , Ratas , Ratas Endogámicas Lew , Proteínas Quinasas S6 Ribosómicas 90-kDa/sangre , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Many components of mitogenic signaling pathways in normal and neoplastic cells have been identified, including the large family of protein kinases, which function as components of signal transduction pathways, playing a central role in diverse biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases caused by abnormalities in these signaling pathways is widely considered a promising approach for drug development. Because of their deregulation in human cancers, protein kinases, such as Bcr-Abl, those in the epidermal growth factor-receptor (HER) family, the cell cycle regulating kinases such as the cyclin-dependent kinases, as well as the vascular endothelial growth factor-receptor kinases involved in the neo-vascularization of tumors, are among the protein kinases considered as prime targets for the development of selective inhibitors. These drug-discovery efforts have generated inhibitors and low-molecular weight therapeutics directed against the ATP-binding site of various protein kinases that are in various stages of development (up to Phase II/III clinical trials). Three examples of inhibitors of protein kinases are reviewed, including low-molecular weight compounds targeting the cell cycle kinases; a potent and selective inhibitor of the HER1/HER2 receptor tyrosine kinase, the pyrollopyrimidine PKI166; and the 2-phenyl-aminopyrimidine STI571 (Glivec(R), Gleevec) a targeted drug therapy directed toward Bcr-Abl, the key player in chronic leukemia (CML). Some members of the HER family of receptor tyrosine kinases, in particular HER1 and HER2, have been found to be overexpressed in a variety of human tumors, suggesting that inhibition of HER signaling would be a viable antiproliferative strategy. The pyrrolo-pyrimidine PKI166 was developed as an HER1/HER2 inhibitor with potent in vitro antiproliferative and in vivo antitumor activity. Based upon its clear association with disease, the Bcr-Abl tyrosine kinase in CML represents the ideal target to validate the clinical utility of protein kinase inhibitors as therapeutic agents. In a preclinical model, STI571 (Glivec(R), Gleevec) showed potent in vitro and in vivo antitumor activity that was selective for Abl, c-Kit, and the platelet-derived growth factor-receptor. Phase I/II studies demonstrated that STI571 is well tolerated, and that it showed promising hematological and cytogenetic responses in CML and clinical responses in the c-Kit-driven gastrointestinal tumors.
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Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Quinasas , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Benzamidas , Ciclo Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Humanos , Mesilato de Imatinib , Ratones , Piperazinas , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Pirimidinas/uso terapéuticoRESUMEN
BACKGROUND: Resistance to microtubule-stabilizing agents is a major hurdle for successful cancer therapy. We investigated combined treatment of microtubule-stabilizing agents (MSAs) with inhibitors of angiogenesis to overcome MSA resistance. METHODS: Treatment regimens of clinically relevant MSAs (patupilone and paclitaxel) and antiangiogenic agents (everolimus and bevacizumab) were investigated in genetically defined MSA-resistant lung (A549EpoB40) and colon adenocarcinoma (SW480) tumor xenografts in nude mice (CD1-Foxn1
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Adenocarcinoma/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Resistencia a Antineoplásicos , Neoplasias Pulmonares/tratamiento farmacológico , Microtúbulos/efectos de los fármacos , Moduladores de Tubulina/uso terapéutico , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Bevacizumab , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Epotilonas/administración & dosificación , Everolimus , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Desnudos , Paclitaxel/administración & dosificación , ARN Neoplásico/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Deregulation of protein kinase activity has been shown to play a central role in the pathogenesis of human cancer. The molecular pathogenesis of chronic myelogenous leukemia (CML) in particular, depends on formation of the bcr-abl oncogene, leading to constitutive expression of the tyrosine kinase fusion protein, Bcr-Abl. Based on these observations, imatinib was developed as a specific inhibitor for the Bcr-Abl protein tyrosine kinase. The expanding understanding of the basis of imatinib-mediated tyrosine kinase inhibition has revealed a spectrum of potential new antitumor applications beyond the powerful activity already reported in the treatment of CML. Imatinib has shown activity in vivo against PDGF-driven tumor models including glioblastoma, dermatofibrosarcoma protuberans and chronic myelomonocytic leukemia. Antiangiogenic effects have been demonstrated by inhibition of PDGF-, VEGF (vascular endothelial growth factor)- and bFGF- (basic fibroblast growth factor) induced angiogenesis in vivo, and by inhibition of angiogenesis and tumor growth in an experimental bone metastasis model. Imatinib has been shown to reduce interstitial fluid pressure in an experimental colonic carcinoma model by blocking PDGF-mediated effects on tumor-associated blood vessels and stromal tissue. It is also a potent inhibitor of the Kit receptor tyrosine kinase, and has demonstrated activity clinically against the Kit-driven gastrointestinal stromal tumor (GIST) and experimentally in small-cell lung cancer cell lines. The pharmacology of imatinib and its activity in various tumor models is discussed.
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Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias Gastrointestinales/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Neoplasias de Tejido Conjuntivo/tratamiento farmacológico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Células del Estroma , Benzamidas , Humanos , Mesilato de Imatinib , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidoresRESUMEN
During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.
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Actinobacillus pleuropneumoniae/crecimiento & desarrollo , Haemophilus/crecimiento & desarrollo , NAD/metabolismo , Infecciones del Sistema Respiratorio/veterinaria , Enfermedades de los Porcinos/microbiología , Infecciones por Actinobacillus/microbiología , Infecciones por Actinobacillus/veterinaria , Actinobacillus pleuropneumoniae/aislamiento & purificación , Actinobacillus pleuropneumoniae/patogenicidad , Animales , Líquido Extracelular/química , Líquido Extracelular/microbiología , Haemophilus/aislamiento & purificación , Haemophilus/patogenicidad , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/veterinaria , Pulmón/microbiología , Fenotipo , Infecciones del Sistema Respiratorio/microbiología , PorcinosRESUMEN
Tumor biomarker studies are integral to oncology clinical trials but may yield artifactual results owing to variation in sample procurement and processing. Ethanol, 70% vol/vol, was validated as a sample transport medium using markers of the PI3K/Akt/mTOR pathway. BT474 tumor xenografts were excised and slices were immediately placed into formaldehyde and fixed for 24 hours. Fixed tissue slices were immediately processed into paraffin or transferred to 70% vol/vol ethanol and stored at room temperature for 1, 2, and 4 weeks before further processing. Freshly cut tissue sections were evaluated for pAKT(S473), HER2, pHER-2(Y1248), pS6(S235/236), and pS6(S240/244), Ki-67, and HER2 by fluorescence in situ hybridization and stained with H&E and Masson trichrome. No significant changes were observed when comparing samples stored in 70% ethanol for up to 4 weeks with immediately processed tissue. Ethanol, 70% vol/vol, provides a safe storage medium for formaldehyde-fixed tumor tissue, facilitating sample transport during multicenter clinical trials.
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Técnicas de Preparación Histocitológica/métodos , Hibridación Fluorescente in Situ , Patología Molecular/métodos , Manejo de Especímenes/métodos , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , ADN/análisis , ADN/química , Etanol/química , Femenino , Fijadores/química , Formaldehído/química , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Factores de TiempoRESUMEN
PURPOSE: Everolimus (RAD001, Afinitor) is an mTORC1 pathway inhibitor, and vatalanib (PTK/ZK) is a pan VEGF-R tyrosine kinase inhibitor (TKI). These two drugs have been shown to have overlapping but also distinct anti-angiogenic effects. Consequently, we investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of their combination in vivo. METHODS: Murine melanoma B16/BL6 cells were grown orthotopically in BL6/C57 mice by injection into the derma of both ears to create a primary tumour which metastasized rapidly to the cervical lymph nodes. Mice were treated daily p.o. with PTK/ZK (100 mg/kg) or everolimus (1 mg/kg) or their combination, and anti-tumour efficacy (PD) assessed. In the same model, plasma PK of everolimus was measured following single doses of the monotherapy or combination schedules. RESULTS: Two independent experiments showed that combination of everolimus and PTK/ZK caused at least additive increases in anti-tumour activity compared to either monotherapy, without increases in toxicity. Pooling the data to improve the statistical power demonstrated the interactions to be synergistic. PK modelling showed that although PTK/ZK increased everolimus plasma concentrations by about twofold, this PK drug-drug interaction could not account for the increased anti-tumour effect of the combination. Modelling of the PTK/ZK dose-response curve in this model suggested that any effect of everolimus on the PK of PTK/ZK was unlikely to affect efficacy. Measurement of changes in tumour and plasma VEGF levels at the endpoint of therapy confirmed earlier observations of differential effects of these two agents. CONCLUSIONS: The combination of everolimus and PTK/ZK hold promise for the treatment of human cancers.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Melanoma Experimental/tratamiento farmacológico , Modelos Biológicos , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Everolimus , Femenino , Diana Mecanicista del Complejo 1 de la Rapamicina , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos , Metástasis de la Neoplasia , Ftalazinas/administración & dosificación , Proteínas/antagonistas & inhibidores , Piridinas/administración & dosificación , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TORRESUMEN
PURPOSE: Reactivation of p53 tumor suppressor activity in diseases such as soft-tissue sarcoma is considered an attractive means of targeted therapy. By systematically assessing alterations affecting the p53 pathway, we aimed to (a) classify sarcoma subtypes, (b) define a potential role in malignancy, and (c) identify potential patient biomarkers in this heterogeneous disease. EXPERIMENTAL DESIGN: We have mapped mutational events in a panel of 192 benign or malignant bone and soft-tissue sarcomas. Analyses included TP53 and CDKN2A mutational and SNP status, MDM2 and MDM4 amplification and MDM2 SNP309 status. RESULTS: We found an inverse relationship between MDM2 amplification and TP53 mutations, with a predominantly wild-type CDKN2A background. A high rate of point mutations in TP53 was observed uniquely in leiomyosarcoma, osteosarcoma, and MFH. Both MDM2 and MDM4 were also amplified in a subtype-specific manner, which was frequently seen as a coamplification event. We have also analyzed the risk allele frequencies for MDM2 SNP309, and show that the G allele was strongly associated with both liposarcomas and MDM2 amplification. CONCLUSIONS: Our data emphasize the critical role of p53 inactivation in sarcomagenesis, whereby different pathway alterations may be related to the heterogeneity of the disease. Moreover, we observed a strong association of malignancy with TP53 mutation, or MDM2 amplification and the presence of a G allele in SNP309, especially in lipoma versus liposarcoma. We propose, therefore, that MDM2 markers along with TP53 sequencing should be considered as patient biomarkers in clinical trials of sarcomas using MDM2 antagonists.
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Neoplasias Óseas/genética , Amplificación de Genes , Genes p53 , Proteínas Proto-Oncogénicas c-mdm2/genética , Sarcoma/genética , Neoplasias de los Tejidos Blandos/genética , Biomarcadores de Tumor/análisis , Mapeo Cromosómico , Dosificación de Gen , Humanos , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
INTRODUCTION: The critical role of increased activity of mammalian target of rapamycin (mTOR) in the pathophysiology of multiple diseases is well established. Inhibition of the mTOR pathway may block disease progression and improve patient outcomes. Everolimus, an mTOR inhibitor, began in clinical development as part of a regimen (Certican, Zortress) for prevention of organ transplant rejection and is now an approved oncology agent. AREAS COVERED: The objective of this review is to discuss the history of key findings and innovative cancer research undertaken to successfully develop everolimus as an oncology therapy (Afinitor) now approved for patients with advanced renal cell carcinoma (RCC) and for subependymal giant cell astrocytomas (SEGAs) associated with tuberous sclerosis. In addition, data for the use of everolimus in the treatment of other cancers and rare diseases are also discussed. A PubMed search of English articles without time restrictions was conducted using the search terms 'everolimus or rapamycin' and 'cancer'. Bibliographies of retrieved articles were manually searched for additional relevant articles. Major cancer congresses were also searched. EXPERT OPINION: The clinical efficacy of everolimus alone and in combination with other agents has been observed in recently completed Phase II-III studies in a wide spectrum of tumors, including RCC, neuroendocrine tumors, tuberous sclerosis complex, SEGAs and angiomyolipomas, lymphoma and gastric, breast and hepatocellular cancers. These findings emphasize the importance of mTOR in diverse cancers and rare diseases and underscore the potential role for everolimus as an effective agent in multiple indications.
RESUMEN
The mTOR inhibitor everolimus (RAD001, Afinitor) is an orally active anticancer agent. Everolimus demonstrates growth-inhibitory activity against a broad range of tumor cell histotypes in vitro and has the capacity to retard tumor growth in preclinical tumor models in vivo through mechanisms directed against both the tumor cell and the solid tumor stroma components. These properties have rendered it to be a clinically active drug, with subsequent registration in renal cell carcinoma (Motzer et al. [2008]. Lancet372, 449-456) as well as showing strong potential as a combination partner (André F et al. [2008]. J Clin Oncol26. Abstract 1003). Although everolimus has a high specificity for its molecular target, the ubiquitous nature of mTOR and the multifactorial influence that mTOR signaling has on cell physiology have made studies difficult on the identification and validation of a biomarker set to predict and monitor drug sensitivity for clinical use. In this review, a summary of the preclinical and clinical data relevant to biomarker development for everolimus is presented, and the advantages and problems of current biomarkers are reviewed. In addition, alternative approaches to biomarker development are proposed on the basis of examples of a combination of markers and functional noninvasive imaging. In particular, we show how basal levels of pAKT and pS6 together could, in principle, be used to stratify patients for likely response to an mTOR inhibitor.
RESUMEN
PURPOSE: Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice. METHODS: Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity. RESULTS: The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t(1/2), oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a 2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a 7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04-0.35) were observed in both species after daily administration with similar C(max) and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t(1/2) compared to the systemic circulation. CONCLUSIONS: The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.
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Inmunosupresores/farmacocinética , Neoplasias Experimentales/metabolismo , Sirolimus/análogos & derivados , Animales , Área Bajo la Curva , Línea Celular Tumoral , Everolimus , Heces/química , Femenino , Humanos , Inmunosupresores/sangre , Inmunosupresores/orina , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/sangre , Neoplasias Experimentales/orina , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Sirolimus/farmacocinética , Especificidad de la Especie , Factores de Tiempo , Distribución Tisular , Trasplante HeterólogoRESUMEN
Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.
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Antineoplásicos/farmacología , Epotilonas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Epotilonas/síntesis química , Epotilonas/metabolismo , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismoRESUMEN
INTRODUCTION: Patupilone is a microtubule stabilizer (MTS) currently in clinical development. Here, we evaluate the anti-cancer activity in vitro and in vivo in comparison to paclitaxel and describe the pharmacokinetics (PK) of patupilone in tumor-bearing nude mice and rats. METHODS: The potency in vitro of patupilone and two other MTS, paclitaxel and ixabepilone, was determined using human colon carcinoma cell lines with low (HCT-116, HT-29, RKO) and high (HCT-15) P-glycoprotein expression (P-gp), as well as two multi-drug resistance (MDR) model cell pairs, MCF7/ADR and KB-8511 cells and their respective drug-sensitive parental counterparts. The PK of patupilone was investigated in nude mice bearing HCT-15 or HT-29 xenografts and in rats bearing s.c. pancreatic CA20498 tumors or A15 glioma tumors. Anti-cancer activity in vivo was compared to that of paclitaxel using three different human tumor colon models. The retention and efficacy of patupilone was compared in small and large HT-29 xenografts whose vascularity was determined by non-invasive magnetic resonance imaging. RESULTS: Patupilone was highly potent in vitro against four different colon carcinoma cell lines including those showing multi-drug-resistance. In contrast, paclitaxel and ixabepilone displayed significantly reduced activity with markedly increased resistance factors. In both rats and mice, a single i.v. bolus injection of patupilone (1.5-4 mg/kg) rapidly distributed from plasma to all tissues and was slowly eliminated from muscle, liver and small intestine, but showed longer retention in tumor and brain with no apparent elimination over 24 h. Patupilone showed significant activity against three human colon tumor models in vivo, unlike paclitaxel, which only had activity against low P-gp expressing tumors. In HT-29 tumors, patupilone activity and retention were independent of tumor size, blood volume and flow. CONCLUSIONS: The high potency of patupilone, which is not affected by P-gp expression either in vitro or in vivo, and favorable PK, independent of tumor vascularity, suggest that it should show significant activity in colorectal cancer and in other indications where high P-gp expression may compromise taxane activity.