RESUMO
Described is a quantitative-mass-spectrometry-imaging (qMSI) methodology for the analysis of lactate and glutamate distributions in order to delineate heterogeneity among mouse tumor models used to support drug-discovery efficacy testing. We evaluate and report on preanalysis-stabilization methods aimed at improving the reproducibility and efficiency of quantitative assessments of endogenous molecules in tissues. Stability experiments demonstrate that optimum stabilization protocols consist of frozen-tissue embedding, post-tissue-sectioning desiccation, and storage at -80 °C of tissue sections sealed in vacuum-tight containers. Optimized stabilization protocols are used in combination with qMSI methodology for the absolute quantitation of lactate and glutamate in tumors, incorporating the use of two different stable-isotope-labeled versions of each analyte and spectral-clustering performed on each tissue section using k-means clustering to allow region-specific, pixel-by-pixel quantitation. Region-specific qMSI was used to screen different tumor models and identify a phenotype that has low lactate heterogeneity, which will enable accurate measurements of lactate modulation in future drug-discovery studies. We conclude that using optimized qMSI protocols, it is possible to quantify endogenous metabolites within tumors, and region-specific quantitation can provide valuable insight into tissue heterogeneity and the tumor microenvironment.
Assuntos
Ácido Glutâmico/análise , Ácido Láctico/análise , Espectrometria de Massas , Animais , Feminino , Ácido Glutâmico/metabolismo , Ácido Láctico/metabolismo , Camundongos , Camundongos Nus , Neoplasias Experimentais/química , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismoRESUMO
Osimertinib (AZD9291) is a potent, selective, irreversible inhibitor of EGFR-sensitizing (exon 19 and L858R) and T790M-resistant mutation. In vivo, in the mouse, it is metabolized to an active des-methyl metabolite, AZ5104. To understand the therapeutic potential in patients, this study aimed to assess the relationship between osimertinib pharmacokinetics, the pharmacokinetics of the active metabolite, the pharmacodynamics of phosphorylated EGFR reduction, and efficacy in mouse xenograft models of EGFR-driven cancers, including two NSCLC lines. Osimertinib was dosed in xenografted models of EGFR-driven cancers. In one set of experiments, changes in phosphorylated EGFR were measured to confirm target engagement. In a second set of efficacy studies, the resulting changes in tumor volume over time after repeat dosing of osimertinib were observed. To account for the contributions of both molecules, a mathematical modeling approach was taken to integrate the resulting datasets. The model was able to describe the pharmacokinetics, pharmacodynamics, and efficacy in A431, PC9, and NCI-H1975 xenografts, with the differences in sensitivity described by the varying potency against wild-type, sensitizing, and T790M-mutant EGFR and the phosphorylated EGFR reduction required to reduce tumor volume. It was inferred that recovery of pEGFR is slower after chronic dosing due to reduced resynthesis. It was predicted and further demonstrated that although inhibition is irreversible, the resynthesis of EGFR is such that infrequent intermittent dosing is not as efficacious as once daily dosing. Mol Cancer Ther; 15(10); 2378-87. ©2016 AACR.
Assuntos
Antineoplásicos/farmacocinética , Receptores ErbB/antagonistas & inibidores , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Acrilamidas , Algoritmos , Compostos de Anilina , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Receptores ErbB/química , Humanos , Camundongos , Modelos Biológicos , Piperazinas/química , Inibidores de Proteínas Quinases/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.
Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/genética , Técnicas de Química Sintética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Ratos Endogâmicos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle. SIGNIFICANCE: We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented.
Assuntos
Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Acrilamidas/química , Acrilamidas/farmacologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Receptores ErbB/química , Feminino , Genes erbB-2 , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Conformação Molecular , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Several small-molecule FGF receptor (FGFR) kinase inhibitors are currently in clinical development; however, the predominant activity of the most advanced of these agents is against the kinase insert domain receptor (KDR), which compromises the FGFR selectivity. Here, we report the pharmacologic profile of AZD4547, a novel and selective inhibitor of the FGFR1, 2, and 3 tyrosine kinases. AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines with deregulated FGFR expression. In a representative FGFR-driven human tumor xenograft model, oral administration of AZD4547 was well tolerated and resulted in potent dose-dependent antitumor activity, consistent with plasma exposure and pharmacodynamic modulation of tumor FGFR. Importantly, at efficacious doses, no evidence of anti-KDR-related effects were observed, confirming the in vivo FGFR selectivity of AZD4547. Taken together, our findings show that AZD4547 is a novel selective small-molecule inhibitor of FGFR with potent antitumor activity against FGFR-deregulated tumors in preclinical models. AZD4547 is under clinical investigation for the treatment of FGFR-dependent tumors.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Piperazinas/farmacologia , Pirazóis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Benzamidas/farmacocinética , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacocinéticaRESUMO
The SAR and improvement in potency against Tie2 of novel thienopyrimidine and thiazolopyrimidine kinase inhibitors are reported. The crystal structure of one of these compounds bound to the Tie-2 kinase domain is consistent with the SAR. These compounds have moderate potency in cellular assays of Tie-2 inhibition, good physical properties, DMPK, and show evidence of in vivo inhibition of Tie-2.