RESUMEN
Due to its antiangiogenic and anti-immunomodulatory activity, thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. In this study, a cohort of 27 chemically diverse thalidomide analogs was evaluated for antiangiogenic activity in an ex vivo rat aorta ring assay. The protein cereblon has been identified as the target for thalidomide, and in silico pharmacophore analysis and molecular docking with a crystal structure of human cereblon were used to investigate the cereblon binding abilities of the thalidomide analogs. The results suggest that not all antiangiogenic thalidomide analogs can bind cereblon, and multiple targets and mechanisms of action may be involved.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inhibidores de la Angiogénesis/farmacología , Aorta/efectos de los fármacos , Simulación del Acoplamiento Molecular , Neovascularización Fisiológica/efectos de los fármacos , Talidomida/análogos & derivados , Talidomida/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Inhibidores de la Angiogénesis/química , Animales , Simulación por Computador , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cereblon (CRBN) is a substrate recruiter element of the E3 cullin 4-RING ubiquitin ligase complex, and a binding target of immunomodulatory agents (IMiDs). CRBN is responsible for the pleiotropic effects of IMiDs, yet its function in angiogenesis and in mediating the antiangiogenic effects of IMiDs remains unclear. We investigated the role of CRBN in the angiogenic process and in propagating the antiangiogenic effects of IMiDs in vitro. siRNA-mediated CRBN knock down in human endothelial cells (HUVEC and HMVEC-L), did not affect endothelial cell proliferation, migration, or tube formation. Using CRBN-deficient mice, we further demonstrated that microvessal formation can occur independently of cereblon in the ex vivo mouse aortic ring model. The cereblon E3 ubiquitin ligase complex can recruit endothelial cell-specific factors, AGO2 (associated with angiogenesis), and SALL4 (associated with embryogenesis/angiogenesis), for ubiquitin-mediated degradation. Knockdown of CRBN caused a corresponding increase in AGO2 and SALL4 protein expression and IMiD treatment was able to rescue the siCRBN effect to increase the CRBN expression. These findings suggest one potential mechanism of action that likely involves a tightly coordinated regulation of CRBN with endothelial cell targets and highlight the need to further elucidate the mechanism(s), which could include cereblon-independent pathways, through which IMiDs exert their antiangiogenic effects.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Lenalidomida/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Interferencia de ARN , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Carfilzomib-lenalidomide-dexamethasone (KRd) therapy has yielded promising results in patients with newly diagnosed multiple myeloma (NDMM). Cereblon (CRBN) is the direct molecular target of lenalidomide and genetic polymorphisms in CRBN have been associated with lenalidomide efficacy. In this study, we assessed the correlation of five single nucleotide variants (SNVs) in the CRBN gene with clinical response and outcomes in patients with NDMM administered KRd therapy with lenalidomide maintenance, achieving favorable trial endpoints in a prospective Phase II study (NCT01402284). Of the observed SNVs, no associations with KRd therapy response were found in this patient cohort, although strong trends in hypoalbuminemia grade and hyperbilirubinemia grade emerged across the CRBN rs1672753 genotype (P = 0.0008) and the rs1714327 genotype (P = 0.0010), respectively. Our results do not provide conclusive support for the predictive utility of CRBN gene polymorphisms as potential biomarkers of clinical response to lenalidomide-based therapy in our patient population. However, these findings remain to be validated in prospective studies using larger patient populations.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Hiperbilirrubinemia/diagnóstico , Hipoalbuminemia/diagnóstico , Lenalidomida/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Polimorfismo de Nucleótido Simple , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Dexametasona/administración & dosificación , Dexametasona/efectos adversos , Esquema de Medicación , Femenino , Expresión Génica , Genotipo , Humanos , Hiperbilirrubinemia/inducido químicamente , Hiperbilirrubinemia/fisiopatología , Hipoalbuminemia/inducido químicamente , Hipoalbuminemia/fisiopatología , Lenalidomida/efectos adversos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Oligopéptidos/administración & dosificación , Oligopéptidos/efectos adversos , Estudios Prospectivos , Resultado del Tratamiento , Ubiquitina-Proteína LigasasRESUMEN
The introduction of fluorine into bioactive molecules is a matter of importance in medicinal chemistry. In this study, representatives of various chemical entities of fluoroaromatic compounds were synthesized. Depending on the reaction conditions, either tetrafluorophthalimides or ammonium tetrafluorophthalamates are accessible from tetrafluorophthalic anhydride and primary amines. Tetrafluorophthalamic acids undergo thermal decarboxylation to yield tetrafluorobenzamides. These could be successfully converted upon treatment with primary amines, in the course of an aromatic nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective amino substituents at the 4-position. The five structure types were characterized by means of spectroscopic and crystallographic methods. The synthesized compounds were evaluated as inhibitors of angiogenesis by measuring microvessel outgrowth in a rat aortic ring assay. The biological activity was maintained throughout these different polyfluorinated chemotypes.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Benzamidas/farmacología , Fluorocarburos/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/toxicidad , Animales , Aorta/efectos de los fármacos , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/toxicidad , Fluorocarburos/síntesis química , Fluorocarburos/química , Fluorocarburos/toxicidad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Microvasos/efectos de los fármacos , Estructura Molecular , Ftalimidas/síntesis química , Ftalimidas/química , Ftalimidas/farmacología , Ftalimidas/toxicidad , Ratas Sprague-Dawley , para-Aminobenzoatos/síntesis química , para-Aminobenzoatos/química , para-Aminobenzoatos/farmacología , para-Aminobenzoatos/toxicidadRESUMEN
Elements of the hypoxia inducible factor (HIF) transcriptional system, a key regulator of the cellular hypoxic response, are up-regulated in a range of cancer cells. HIF is fundamentally involved in tumor angiogenesis, invasion, and energy metabolism. Inhibition of the transcriptional activity of HIF may be of therapeutic benefit to cancer patients. We recently described the identification of two marine pyrroloiminoquinone alkaloids with potent activity in inhibiting the interaction between the oncogenic transcription factor HIF-1α and the coactivator protein p300. Herein, we present further characterization data for these two screening hits: discorhabdin H (1) and discorhabdin L (2), with a specific focus on their anti-angiogenic and anti-tumor effects. We demonstrated that only discorhabdin L (2) possesses excellent anti-angiogenic activity in inhibiting endothelial cell proliferation and tube formation, as well as decreasing microvessel outgrowth in the ex vivo rat aortic ring assay. We further showed that discorhabdin L (2) significantly inhibits in vivo prostate tumor growth in a LNCaP xenograft model. In conclusion, our findings suggest that discorhabdin L (2) represents a promising HIF-1α inhibitor worthy of further drug development.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Neovascularización Patológica/tratamiento farmacológico , Quinonas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Proteína p300 Asociada a E1A/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones SCID , Neovascularización Patológica/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
A library of 15 novel and heretofore uncharacterized adamantyl and noradamantyl phthalimidines was synthesized and evaluated for neuroprotective and anti-angiogenic properties. Phthalimidine treatment in LPS-challenged cells effected reductions in levels of secreted TNF-α and nitrite relative to basal amounts. The primary SAR suggests nitration of adamantyl phthalimidines has marginal effect on TNF-α activity but promotes anti-nitrite activity; thioamide congeners retain anti-nitrite activity but are less effective reducing TNF-α. Site-specific nitration and thioamidation provided phthalimidine 24, effecting an 88.5% drop in nitrite concurrent with only a 4% drop in TNF-α. Notable anti-angiogenesis activity was observed for 20, 21 and 22.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Diseño de Fármacos , Fármacos Neuroprotectores/farmacología , Nitritos/antagonistas & inhibidores , Ftalimidas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Nitritos/metabolismo , Ftalimidas/síntesis química , Ftalimidas/química , Células RAW 264.7 , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The development of new angiogenic inhibitors highlights a need for robust screening assays that adequately capture the complexity of vessel formation, and allow for the quantitative evaluation of the teratogenicity of new anti-angiogenic agents. This review discusses the use of screening assays in vertebrate embryos, specifically focusing upon chicken and zebrafish embryos, for the detection of anti-angiogenic agents.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Evaluación Preclínica de Medicamentos/métodos , Embrión de Mamíferos/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Animales , Embrión de Mamíferos/irrigación sanguínea , Embrión no Mamífero/irrigación sanguínea , Humanos , Modelos AnimalesRESUMEN
The development of novel thalidomide derivatives as immunomodulatory and anti-angiogenic agents has revived over the last two decades. Herein we report the design and synthesis of three chemotypes of barbituric acids derived from the thalidomide structure: phthalimido-, tetrafluorophthalimido-, and tetrafluorobenzamidobarbituric acids. The latter were obtained by a new tandem reaction, including a ring opening and a decarboxylation of the fluorine-activated phthalamic acid intermediates. Thirty compounds of the three chemotypes were evaluated for their anti-angiogenic properties in an exâ vivo assay by measuring the decrease in microvessel outgrowth in rat aortic ring explants. Tetrafluorination of the phthalimide moiety in tetrafluorophthalimidobarbituric acids was essential, as all of the nonfluorinated counterparts lost anti-angiogenic activity. An opening of the five-membered ring and the accompanying increased conformational freedom, in case of the corresponding tetrafluorobenzamidobarbituric acids, was well tolerated. Their activity was retained, although their molecular structures differ in torsional flexibility and possible hydrogen-bond networking, as revealed by comparative X-ray crystallographic analyses.
Asunto(s)
Inhibidores de la Angiogénesis/química , Barbitúricos/química , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Barbitúricos/síntesis química , Barbitúricos/farmacología , Cristalografía por Rayos X , Conformación Molecular , Ftalimidas/química , Ratas , Relación Estructura-Actividad , Talidomida/químicaRESUMEN
Angiogenesis, the formation of new blood vessels, is essential for tumor growth, stabilization and progression. Angiogenesis inhibitors are now widely used in the clinic; however, there are relatively few published studies on the mechanism of their presumed teratogenic effects. To address this issue, we screened a variety of angiogenesis inhibitors in developing zebrafish and chicken embryo models to assess for developmental defects and potential teratogenic effects. We confirmed previous reports that sunitinib, sorafenib and TNP-470 are teratogenic and demonstrate that axitinib, pazopanib, vandetanib, and everolimus are also teratogens in these models. A dose response study identified the drugs inhibit HUVEC cell proliferation in vitro, and also target the developing blood vessels of embryos in vivo. This provides further evidence for the potential risk of fetal toxicity when using these drugs in a clinical setting, and emphasizes the importance of the development and maintenance of the vasculature in the embryo. We conclude that angiogenesis inhibitors, regardless of the molecular target, are teratogenic when exposed to chicken embryos.
Asunto(s)
Inhibidores de la Angiogénesis/efectos adversos , Teratogénesis , Teratógenos/farmacología , Animales , Células Cultivadas , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Humanos , Pez Cebra/embriologíaRESUMEN
Thalidomide, a drug known for its teratogenic side-effects, is used successfully to treat a variety of clinical conditions including leprosy and multiple myeloma. Intense efforts are underway to synthesize and identify safer, clinically relevant analogs. Here, we conduct a preliminary in vivo screen of a library of new thalidomide analogs to determine which agents demonstrate activity, and describe a cohort of compounds with anti-angiogenic properties, anti-inflammatory properties and some compounds which exhibited both. The combination of the in vivo zebrafish and chicken embryo model systems allows for the accelerated discovery of new, potential therapies for cancerous and inflammatory conditions.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Embrión de Pollo/efectos de los fármacos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Talidomida/farmacología , Pez Cebra/embriología , Anomalías Inducidas por Medicamentos/etiología , Inhibidores de la Angiogénesis/toxicidad , Animales , Animales Modificados Genéticamente , Antiinflamatorios/toxicidad , Relación Dosis-Respuesta a Droga , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Neovascularización Fisiológica/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Medición de Riesgo , Talidomida/análogos & derivados , Talidomida/toxicidad , Flujo de Trabajo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
OBJECTIVE: To determine the safety and clinical efficacy of two anti-angiogenic agents, bevacizumab and lenalidomide, with docetaxel and prednisone. PATIENTS AND METHODS: Eligible patients with metastatic castration-resistant prostate cancer enrolled in this open-label, phase II study of lenalidomide with bevacizumab (15 mg/kg), docetaxel (75 mg/m(2) ) and prednisone (10 mg daily). Docetaxel and bevacizumab were administered on day 1 of a 3-week treatment cycle. To establish safety, lenalidomide dosing in this combination was escalated in a conventional 3 + 3 design (15, 20 and 25 mg daily for 2 weeks followed by 1 week off). Patients received supportive measures including prophylactic pegfilgrastim and enoxaparin. The primary endpoints were safety and clinical efficacy. RESULTS: A total of 63 patients enrolled in this trial. Toxicities were manageable with most common adverse events (AEs) being haematological, and were ascertained by weekly blood counts. Twenty-nine patients (46%) had grade 4 neutropenia, 20 (32%) had grade 3 anaemia and seven (11%) had grade 3 thrombocytopenia. Despite frequent neutropenia, serious infections were rare. Other common non-haematological grade 3 AEs included fatigue (10%) and diarrhoea (10%). Grade 2 AEs in >10% of patients included anorexia, weight loss, constipation, osteonecrosis of the jaw, rash and dyspnoea. Of 61 evaluable patients, 57 (93%), 55 (90%) and 33 (54%) had PSA declines of >30, >50 and >90%, respectively. Of the 29 evaluable patients, 24 (86%) had a confirmed radiographic partial response. The median times to progression and overall survival were 18.2 and 24.6 months, respectively. CONCLUSIONS: With appropriate supportive measures, combination angiogenesis inhibition can be safely administered and potentially provide clinical benefit. These hypothesis-generating data would require randomized trials to confirm the findings.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos Hormonales/uso terapéutico , Bevacizumab/uso terapéutico , Prednisona/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Talidomida/análogos & derivados , Anciano , Anciano de 80 o más Años , Humanos , Lenalidomida , Masculino , Persona de Mediana Edad , Talidomida/uso terapéuticoRESUMEN
Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenic pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort. We recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogues (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). In this study, we demonstrate the antiangiogenic properties of these newly synthesized compounds. We examined the specific antiangiogenic characteristics in vitro using rat aortic rings with carboxyamidotriazole as a positive control. In addition, further in vitro efficacy was evaluated using human umbilical vein endothelial cells (HUVEC) and PC3 cells treated with 5 and 10 µmol/L doses of each compound. All compounds were seen to reduce microvessel outgrowth in rat aortic rings as well as to inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The antiangiogenic properties of the compounds were also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos, whereas Gu998 and Gu992 showed no difference. The compounds' antitumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogues as a promising immunomodulatory class with anticancer effects that warrant further development to characterize their mechanisms of action.