RESUMO
KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Quinazolinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Mutação , Piperazinas/química , Piperazinas/uso terapêutico , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinazolinas/química , Quinazolinas/uso terapêuticoRESUMO
Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor ß (TGF-ß) type I receptor, and endoglin, a TGF-ß co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-ß to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.
Assuntos
Receptores de Activinas Tipo II/imunologia , Endotélio Vascular/metabolismo , Fator 2 de Diferenciação de Crescimento/fisiologia , Transdução de Sinais/fisiologia , Receptores de Activinas Tipo II/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Citometria de Fluxo , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Reação em Cadeia da Polimerase , Ligação ProteicaRESUMO
In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (k(on) and k(off)), internalization of the drug-target complex (k(int)), target degradation rate (k(deg)), and target abundance (R(0)), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD.
Assuntos
Anticorpos Monoclonais/farmacocinética , Simulação por Computador , Modelos Biológicos , Receptores de Ativinas Tipo I/imunologia , Animais , Anticorpos Monoclonais Humanizados , Ensaios Clínicos Fase I como Assunto , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Haplorrinos , Humanos , Imunoglobulina G/imunologia , Camundongos , Ressonância de Plasmônio de Superfície , Veias UmbilicaisAssuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Indóis/uso terapêutico , Morfolinas/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , HumanosRESUMO
The anti-angiogenic class of drugs is one of the few where representatives have gained international approval for clinical use in oncology during the past decade. Most of the biological and clinical activity of the currently available generation of anti-angiogenic drugs targets vascular endothelial growth factor (VEGF) and its related pathways. However, the clinical benefits associated with the use of these drugs have, so far, been limited. There is, therefore, an unmet need for biomarkers that can be used to identify patients who are most likely to benefit therapeutically and also to predict the best schedule and dosage for these drugs. Here, we discuss some of the emerging new combination strategies involving the approved anti-angiogenic drugs, some of the emerging targets associated with neoplastic angiogenesis and some novel agents used as a paradigm of the next generation of anti-angiogenic drugs.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We report a comparison between tumor perfusion estimates acquired using contrast-enhanced MRI and motion-corrected contrast-enhanced ultrasound before and after treatment with AG-028262, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor. Antiangiogenic activity was determined by assessing weekly ultrasound and MRI images of rats with bilateral hind flank mammary adenocarcinomas before and after treatment with AG-028262. Images were acquired with a spoiled gradient, 1.5 T magnetic resonance sequence and a destruction-replenishment ultrasound protocol. For ultrasound, a time to 80% contrast replenishment was calculated for each tumor voxel; for MR imaging, a measure of local flow rate was estimated from a linear fit of minimum to maximum intensities. AG-028262 significantly decreased tumor growth and increased the time required to replenish tumor voxels with an ultrasound contrast agent from 2.66 to 4.54 s and to fill with an MR contrast agent from 29.5 to 50.8 s. Measures of flow rate derived from MRI and ultrasound demonstrated a positive linear correlation of r2 = 0.86.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Imagem de Difusão por Ressonância Magnética/métodos , Neoplasias Mamárias Experimentais/diagnóstico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neovascularização Patológica/diagnóstico , Neovascularização Patológica/tratamento farmacológico , Ultrassonografia/métodos , Adenocarcinoma/complicações , Adenocarcinoma/diagnóstico , Adenocarcinoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Feminino , Masculino , Neoplasias Mamárias Experimentais/complicações , Neovascularização Patológica/etiologia , Ratos , Ratos Endogâmicos F344 , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Resultado do TratamentoRESUMO
Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/uso terapêutico , Animais , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Humanos , Camundongos , Camundongos SCID , Terapia de Alvo Molecular/métodos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Integrin α5ß1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5ß1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Integrina alfa5beta1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Bevacizumab , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células HEK293 , Haplorrinos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Indóis/farmacologia , Integrina alfa5beta1/biossíntese , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos Transgênicos , Células NIH 3T3 , Fagocitose/imunologia , Pirróis/farmacologia , Receptores de IgG/imunologia , SunitinibeRESUMO
Molecular and cellular mechanisms underlying resistance/low responsiveness to antiangiogenic compounds are under extensive investigations. Both populations of tumor and stroma (nontumor compartment) seem to contribute in inherent/acquired resistance to antiangiogenic therapy. Here, investigating in vivo efficacy of sunitinib in experimental models resulted in the identification of tumors that were resistant/sensitive to the therapy. Analysis of tumor protein lysates indicated a greater concentration of hepatocyte growth factor (HGF) in resistant tumors than in sensitive ones. In addition, using flow cytometry, c-Met expression was found to be significantly higher in endothelial cells than in tumor cells, suggesting that HGF might target the vascular endothelial cells in resistant tumors. Combination of sunitinib and a selective c-Met inhibitor significantly inhibited tumor growth compared with sunitinib or c-Met inhibitor alone in resistant tumors. Histology and in vitro analyses suggested that combination treatment mainly targeted the vasculature in the resistant tumors. Conversely, systemic injection of HGF in the sensitive tumor models conferred resistance to sunitinib through maintenance of tumor angiogenesis. In conclusion, our study indicates a role for HGF/c-Met pathway in development of resistance to antiangiogenic therapy and suggests a potential strategy to circumvent resistance to vascular endothelial growth factor receptor tyrosine kinase inhibitor in the clinic.
Assuntos
Inibidores da Angiogênese/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Indóis/farmacologia , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirróis/farmacologia , Animais , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/genética , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Sunitinibe , TransfecçãoRESUMO
Ricinus communis agglutinin I (RCA I), a galactose-binding lectin from castor beans, binds to endothelial cells at sites of plasma leakage, but little is known about the amount and functional consequences of binding to tumor endothelial cells. We addressed this issue by examining the effects of RCA I on blood vessels of spontaneous pancreatic islet-cell tumors in RIP-Tag2 transgenic mice. After intravenous injection, RCA I bound strongly to tumor vessels but not to normal blood vessels. At 6 minutes, RCA I fluorescence of tumor vessels was largely diffuse, but over the next hour, brightly fluorescent dots appeared as the lectin was internalized by endothelial cells. RCA I injection led to a dose- and time-dependent decrease in vascular endothelial growth factor receptor-2 (VEGFR-2) immunoreactivity in tumor endothelial cells, with 95% loss over 6 hours. By comparison, VEGFR-3, CD31, and CD105 had decreases in the range of 21% to 33%. Loss of VEGFR-2 was followed by increased activated caspase-3 in tumor vessels. Prior inhibition of VEGF signaling by AG-028262 decreased RCA I binding and internalization into tumor vessels. These findings indicate RCA I preferentially binds to and is internalized by tumor endothelial cells, which leads to VEGFR-2 down-regulation, endothelial cell apoptosis, and tumor vessel regression. Together, the results illustrate the selective impact of RCA I on VEGF signaling in tumor blood vessels.
Assuntos
Apoptose , Regulação para Baixo , Células Endoteliais/citologia , Regulação Neoplásica da Expressão Gênica , Lectinas de Plantas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Relação Dose-Resposta a Droga , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica , Fatores de TempoRESUMO
Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their receptors are important targets in cancer therapy based on angiogenesis inhibition. However, it is unclear whether inhibition of VEGF and PDGF together is more effective than inhibition of either one alone. Here, we used two contrasting tumor models to compare the effects of inhibiting VEGF or PDGF alone, by adenovirally generated soluble receptors, to the effects of inhibiting both together. In RIP-Tag2 tumors, VEGF and PDGF inhibition together reduced tumor vascularity and abundance of pericytes. However, VEGF inhibition reduced tumor vascularity without decreasing pericyte density, and PDGF inhibition reduced pericytes without reducing tumor vascularity. By contrast, in Lewis lung carcinomas (LLC), inhibition of VEGF or PDGF reduced blood vessels and pericytes to the same extent as did inhibition of both together. Similar results were obtained using tyrosine kinase inhibitors AG-013736 and imatinib. In LLC, VEGF expression was largely restricted to pericytes and PDGF was largely restricted to endothelial cells, but, in RIP-Tag2 tumors, expression of both growth factors was more widespread and significantly greater than in LLC. These findings suggest that inhibition of PDGF in LLC reduced pericytes, and then tumor vessels regressed because pericytes were the main source of VEGF. The vasculature of RIP-Tag2 tumors, in which most VEGF is from tumor cells, was more resistant to PDGF inhibition. The findings emphasize the interdependence of pericytes and endothelial cells in tumors and the importance of tumor phenotype in determining the cellular effects of VEGF and PDGF inhibitors on tumor vessels.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Antineoplásicos/uso terapêutico , Axitinibe , Benzamidas , Humanos , Mesilato de Imatinib , Imidazóis/uso terapêutico , Fragmentos Fc das Imunoglobulinas/genética , Indazóis/uso terapêutico , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Piperazinas/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Pirimidinas/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
PURPOSE: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data. EXPERIMENTAL DESIGN: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models. RESULTS: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC(50) values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure. CONCLUSIONS: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.
Assuntos
Inibidores da Angiogênese/farmacologia , Imidazóis/farmacologia , Indazóis/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Axitinibe , Western Blotting , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dynamic contrast-enhanced MRI (DCE-MRI) was used to noninvasively evaluate the effects of AG-03736, a novel inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, on tumor microvasculature in a breast cancer model. First, a dose response study was undertaken to determine the responsiveness of the BT474 human breast cancer xenograft to AG-013736. Then, DCE-MRI was used to study the effects of a 7-day treatment regimen on tumor growth and microvasculature. Two DCE-MRI protocols were evaluated: (1) a high molecular weight (MW) contrast agent (albumin-(GdDTPA)(30)) with pharmacokinetic analysis of the contrast uptake curve and (2) a low MW contrast agent (GdDTPA) with a clinically utilized empirical parametric analysis of the contrast uptake curve, the signal enhancement ratio (SER). AG-013736 significantly inhibited growth of breast tumors in vivo at all doses studied (10-100 mg/kg) and disrupted tumor microvasculature as assessed by DCE-MRI. Tumor endothelial transfer constant (K(ps)) measured with albumin-(GdDTPA)(30) decreased from 0.034+/-0.005 to 0.003+/-0.001 ml min(-1) 100 ml(-1) tissue (P<.0022) posttreatment. No treatment-related change in tumor fractional plasma volume (fPV) was detected. Similarly, in the group of mice studied with GdDTPA DCE-MRI, AG-013736-induced decreases in tumor SER measures were observed. Additionally, our data suggest that 3D MRI-based volume measurements are more sensitive than caliper measurements for detecting small changes in tumor volume. Histological staining revealed decreases in tumor cellularity and microvessel density with treatment. These data demonstrate that both high and low MW DCE-MRI protocols can detect AG-013736-induced changes in tumor microvasculature. Furthermore, the correlative relationship between microvasculature changes and tumor growth inhibition supports DCE-MRI methods as a biomarker of VEGF receptor target inhibition with potential clinical utility.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Permeabilidade Capilar/efeitos dos fármacos , Imidazóis/uso terapêutico , Indazóis/uso terapêutico , Imageamento por Ressonância Magnética/métodos , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Animais , Antineoplásicos/administração & dosagem , Axitinibe , Neoplasias da Mama/irrigação sanguínea , Proliferação de Células/efeitos dos fármacos , Meios de Contraste , Feminino , Camundongos , Camundongos Nus , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Resultado do Tratamento , Quinases da Família src/antagonistas & inibidoresRESUMO
Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Insulinoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Actinas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Axitinibe , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Membrana Basal/patologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/patologia , Colágeno Tipo IV/imunologia , Colágeno Tipo IV/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indazóis/farmacologia , Indazóis/uso terapêutico , Insulinoma/irrigação sanguínea , Insulinoma/patologia , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Compostos Orgânicos/farmacologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Pericitos/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With the exception of a few malignancies, seemingly driven by a single genetic mutation in a signaling protein, most tumors are the product of multiple mutations in multiple aberrant signaling pathways. Consequently, simultaneous targeted inhibition of multiple signaling pathways could be more effective than inhibiting a single pathway in cancer therapies. Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using RTK inhibitors with broad target selectivity. SU14813, a small molecule identified from the same chemical library used to isolate sunitinib, has broad-spectrum RTK inhibitory activity through binding to and inhibition of VEGFR, PDGFR, KIT, and FLT3. In cellular assays, SU14813 inhibited ligand-dependent and ligand-independent proliferation, migration, and survival of endothelial cells and/or tumor cells expressing these targets. SU14813 inhibited VEGFR-2, PDGFR-beta, and FLT3 phosphorylation in xenograft tumors in a dose- and time-dependent fashion. The plasma concentration required for in vivo target inhibition was estimated to be 100 to 200 ng/mL. Used as monotherapy, SU14813 exhibited broad and potent antitumor activity resulting in regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. Treatment in combination with docetaxel significantly enhanced both the inhibition of primary tumor growth and the survival of the tumor-bearing mice compared with administration of either agent alone. In summary, SU14813 inhibited target RTK activity in vivo in association with reduction in angiogenesis, target RTK-mediated proliferation, and survival of tumor cells, leading to broad and potent antitumor efficacy. These data support the ongoing phase I clinical evaluation of SU14813 in advanced malignancies.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Indóis/uso terapêutico , Morfolinas/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células , Humanos , Indóis/química , Indóis/farmacologia , Camundongos , Morfolinas/química , Morfolinas/farmacologia , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Células Tumorais Cultivadas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibodies and other macromolecular therapeutics can gain access to tumor cells via leaky tumor vessels. Inhibition of vascular endothelial growth factor (VEGF) signaling can reduce the vascularity of tumors and leakiness of surviving vessels, but little is known about how these changes affect the distribution of antibodies within tumors. We addressed this issue by examining the distribution of extravasated antibodies in islet cell tumors of RIP-Tag2 transgenic mice and implanted Lewis lung carcinomas using fluorescence and confocal microscopic imaging. Extravasated nonspecific immunoglobulin G (IgG) and antibodies to fibrin or E-cadherin accumulated in irregular patchy regions of stroma. Fibrin also accumulated in these regions. Anti-E-cadherin antibody, which targets epitopes on tumor cells of RIP-Tag2 adenomas, was the only antibody to achieve detectable levels within tumor cell clusters at 6 hours after i.v. injection. Treatment for 7 days with AG-013736, a potent inhibitor of VEGF signaling, reduced the tumor vascularity by 86%. The overall area density of extravasated IgG/antibodies decreased after treatment but the change was less than the reduction in vascularity and actually increased when expressed per surviving tumor vessel. Accumulation of anti-E-cadherin antibody in tumor cell clusters was similarly affected. The patchy pattern of antibodies in stroma after treatment qualitatively resembled untreated tumors and surprisingly coincided with sleeves of basement membrane left behind after pruning of tumor vessels. Together, the findings suggest that antibody transport increases from surviving tumor vessels after normalization by inhibition of VEGF signaling. Basement membrane sleeves may facilitate this transport. Antibodies preferentially distribute to tumor stroma but also accumulate on tumor cells if binding sites are accessible.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/irrigação sanguínea , Adenoma de Células das Ilhotas Pancreáticas/imunologia , Anticorpos Antineoplásicos/metabolismo , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/imunologia , Imunoglobulina G/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adenoma de Células das Ilhotas Pancreáticas/sangue , Animais , Anticorpos Antineoplásicos/sangue , Anticorpos Antineoplásicos/imunologia , Axitinibe , Caderinas/imunologia , Carcinoma Pulmonar de Lewis/sangue , Fibrina/imunologia , Fibrina/metabolismo , Imidazóis/farmacologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Indazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microesferas , Neovascularização Patológica/sangue , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth.
Assuntos
Capilares/citologia , Capilares/fisiologia , Indazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Axitinibe , Membrana Basal/metabolismo , Capilares/crescimento & desenvolvimento , Capilares/metabolismo , Colágeno Tipo IV/metabolismo , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Imidazóis , Camundongos , Camundongos Endogâmicos , Pericitos/citologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Traqueia/irrigação sanguínea , Grau de Desobstrução Vascular/efeitos dos fármacosRESUMO
Antiangiogenesis is emerging as efficient strategy for targeting and potentially eliminating neoplastic tumor vessels. The main goal of this study was to establish whether absolute tumor blood volume (V(b)) change could be used as an early predictor of antiangiogenesis in ectopic and orthotopic colon carcinomas. To assess therapy-induced changes of V(b), we did comparative analysis of signal intensities in tumors and muscle using steady-state magnetic resonance imaging (MRI) assisted with an intravascular paramagnetic contrast agent [gadolinium-labeled protected graft copolymer (PGC-Gd)]. Athymic mice with implanted human MV522 tumors were treated with vascular endothelial growth factor type 2 receptor tyrosine kinase inhibitor (VEGFR2-TKI) that has been shown to inhibit VEGFR2 phosphorylation and tumor growth in vivo. Animals were imaged either after a single day or after a 1-week course of treatments. The measured V(b) in ectopic tumors was 2.5 +/- 1.5% of total tissue volume 1 week after the implantation (n = 8). Two doses of VEGFR2-TKI (25 mg/kg, p.o., b.i.d.) resulted in a decrease of V(b) to 1.3 +/- 0.3%. In orthotopic tumors, the measured V(b) was initially higher (11.9 +/- 2.0%); however, VEGFR2-TKI treatment also resulted in a statistically significant decrease of V(b). The absolute V(b) was not affected in the muscle as a result of treatments. MRI measurements were corroborated by using isotope and correlative histology experiments. Our results show that steady-state MRI is highly sensitive to early antiangiogenic effects caused by small molecule drugs.
Assuntos
Adenocarcinoma/irrigação sanguínea , Neoplasias do Colo/irrigação sanguínea , Inibidores de Proteínas Quinases/farmacologia , Adenocarcinoma/sangue , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/sangue , Volume de Eritrócitos , Humanos , Indazóis/farmacologia , Angiografia por Ressonância Magnética , Camundongos , Camundongos Nus , Neovascularização Patológica/sangue , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/enzimologia , Fosforilação , Cintilografia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small molecule vascular endothelial growth factor (VEGF) receptor tyrosinase kinase inhibitors (VEGFR-TKIs) show great promise in inducing antiangiogenic responses in tumors. We investigated whether antiangiogenic tumor responses induced by an experimental VEGFR-TKI (AG013925; Pfizer Global Research and Development) could be reported by magnetic resonance imaging (MRI) during the initial phase of treatment. We used MRI and superparamagnetic nanoparticles for measuring relative vascular volume fraction (rVVF) in a drug-resistant colon carcinoma model. Athymic mice harboring MV522 xenografts were treated with VEGFR-TKI (25 mg/kg, p.o., with a 12-hour interval in between treatments) and were imaged after three consecutive treatments. Relative tumor blood volume fractions were calculated using deltaR2* maps that were scaled by the known VVF value of an in-plane skeletal muscle (1.9%). There was a pronounced and statistically significant (P < .001) decrease of tumor rVVF in treated animals (0.95 +/- 0.24%; mean +/- SEM, n = 66 slices, eight mice) compared to mice that received a placebo (2.91 +/- 0.24%; mean +/- SEM, n = 66 slices, nine mice). Tumor histology confirmed a three-fold decrease of vascular density and a concomitant increase of apoptotic cell index. Hence, we demonstrated that: 1) the VEGFR-TKI resulted in antiangiogenic effects that were manifested by a decrease or rVVF; and 2) iron oxide nanoparticles and steady-state MRI enable an early detection of tumor response to antiangiogenic therapies.
Assuntos
Adenocarcinoma/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Indazóis/uso terapêutico , Imageamento por Ressonância Magnética/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Volume Sanguíneo/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Compostos Férricos , Humanos , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To investigate the heterogeneity in the angiogenic response of a human breast cancer xenograft to a novel vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor, AG-013736, using dynamic contrast-enhanced MR imaging (DCE-MRI). MATERIALS AND METHODS: Changes in pharmacokinetic parameters in a seven-day interval were compared between AG-treated and control groups, using Gd-DTPA and albumin-(Gd-DTPA)30. A voxel-by-voxel analysis was performed to produce parametric spatial pharmacokinetic parametric maps and histograms. Histogram segmentation was used to quantify the heterogeneity in tumor response to therapy, and compared with conventional descriptive measures of distribution in terms of their capacity to separate control from AG-treated tumors. RESULTS: The albumin-(Gd-DTPA)30 endothelial transfer constant, Kps, showed changes with AG-013736 treatment and tumor growth. The changes were highly heterogeneous for individual segments of the histogram with different Kps values, and the overall patterns in which the frequency distribution changed differed significantly between the two groups. A change in the number of voxels with Kps ranging from 0.03 to 0.14 mL/min/(100 mL tissue) was the most sensitive variable for separating control from AG-treated tumors (P = 0.0008). Parametric maps of the kinetic parameters also showed spatial heterogeneity in tumor response to treatment. The Kps maps depicted rapid development of central necrosis as a result of AG-013736 treatment. Maps of v(p) demonstrated a marked increase at peripheral regions of necrotic areas. Similar trends were noted in the Gd-DTPA rate constant Ktrans distribution. CONCLUSION: This study demonstrates the value of histogram analysis of maps of pharmacokinetic parameters for assessing heterogeneity in tumor response to antiangiogenic therapy. Changes in the number of voxels within certain segments of the Kps histogram were the most sensitive variable for separating control from AG-treated tumors.