RESUMO
The optimal evaluation of molecularly targeted anticancer agents requires the integration of pharmacodynamic assays into early clinical investigations. Phase '0' trials conducted under the new Exploratory Investigational New Drug Guidance from the US Food and Drug Administration can provide a platform to establish the feasibility of assays for target modulation in human samples, evaluate biomarkers for drug effects and provide pharmacokinetic data. Phase 0 trials could facilitate rational drug selection, identify therapeutic failures early, and might compress timelines for anticancer drug development. We expect that such trials will become a routine part of early-phase oncological drug development in the future.
Assuntos
Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Desenho de Fármacos , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Ética Médica , HumanosRESUMO
Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8±8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R(2)=0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5µg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.
Assuntos
Ensaio de Imunoadsorção Enzimática , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/patologia , Manejo de Espécimes/métodos , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Humanos , CamundongosRESUMO
The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a critical role in the maintenance of DNA integrity as part of the base excision pathway of DNA repair. PARP1 is overexpressed in a variety of cancers, and its expression has been associated with overall prognosis in cancer, especially breast cancer. A series of new therapeutic agents that are potent inhibitors of the PARP1 and PARP2 isoforms have demonstrated important clinical activity in patients with breast or ovarian cancers that are caused by mutations in either the BRCA1 or 2 genes. Results from such studies may define a new therapeutic paradigm, wherein simultaneous loss of the capacity to repair DNA damage may have antitumor activity in itself, as well as enhance the antineoplastic potential of cytotoxic chemotherapeutic agents.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Ensaios Clínicos como Assunto , Humanos , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
ST-20 (sodium 2,2-dimethylbutyrate) is a potential therapeutic agent for treatment of ß-thalassemia and sickle cell disease. A subchronic oral toxicity study was conducted in Sprague-Dawley rats (10/sex/dose) at gavage dosages of 0 (vehicle control), 200, 600, or 1,000 mg/kg, once daily for up to 15 days followed by a 14-day recovery. Ataxia (females), rough coat/thin appearance (males), and decreased body weights were observed at 1,000 mg/kg. Functional observational battery (FOB) deficits were observed more frequently in females and included decreased body tone, rectal temperature, emotional reactivity, neuromotor-neuromuscular activity (as exhibited by a deficit in visual/tactile placing accuracy, ataxia, hind limb dragging, and decreased grip strength), and rearing. ST-20 caused a decrease in WBC/RBC counts and RBC parameters; increase in reticulocytes and red cell inclusion bodies; decrease in total protein, globulin, and glucose; and increase in AG ratio. Micronucleated polychromatic erythrocytes of the bone marrow increased significantly in males at 1,000 mg/kg. Mean liver and kidney weights increased, and hepatocellular hypertrophy was observed in males at 1,000 mg/kg. Toxicologic findings were fully recovered during the 14-day recovery period. In conclusion, the no-observed adverse effect level for FOB and general toxicity was 200 mg/kg following gavage administration of ST-20 for up to 15 consecutive days.
Assuntos
Butiratos/toxicidade , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Reticulócitos/efeitos dos fármacos , Testes de Toxicidade Aguda/métodos , Administração Oral , Animais , Peso Corporal , Butiratos/farmacologia , Relação Dose-Resposta a Droga , Contagem de Eritrócitos , Feminino , Contagem de Leucócitos , Modelos Lineares , Masculino , Testes para Micronúcleos , Nível de Efeito Adverso não Observado , Ratos , Ratos Sprague-DawleyRESUMO
The PD-driven phase 0 trial is a new form, designed to be a first-in-man study, often of a new agent, conducted to assess drug effect on a molecular target, by means of a pharmacodynamic (PD) assay, in a very small number (10-15) of patients. Such a study is meant to be a proof of principle trial to determine whether the agent yields the PD effect predicted by pre-clinical studies. The dosage is meant to be pharmacologically active, but is neither toxic nor likely to yield clinical benefit. Such a trial may be used to serve as a very early test of an agent's biologic effect, allowing for early weeding out of ineffective agents, or as an early means of determining the most promising of competing analogue agents. This manuscript will present designs for such PD-driven studies that are statistically efficient and rigorous, focusing on non-comparative trials. The phase 0 trial promises to become an increasingly important tool for facilitating and speeding the development of new therapeutic agents, particularly in oncology.
Assuntos
Ensaios Clínicos como Assunto/métodos , Interpretação Estatística de Dados , Drogas em Investigação/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Drogas em Investigação/farmacocinética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , FarmacocinéticaRESUMO
BACKGROUND: Reovirus type 3 Dearing strain (ReoT3D) has an inherent propensity to preferentially infect and destroy cancer cells. The oncolytic activity of ReoT3D as a single agent has been demonstrated in vitro and in vivo against various cancers, including colon, pancreatic, ovarian and breast cancers. Its human safety and potential efficacy are currently being investigated in early clinical trials. In this study, we investigated the in vitro combination effects of ReoT3D and chemotherapeutic agents against human non-small cell lung cancer (NSCLC). RESULTS: ReoT3D alone exerted significant cytolytic activity in 7 of 9 NSCLC cell lines examined, with the 50% effective dose, defined as the initial virus dose to achieve 50% cell killing after 48 hours of infection, ranging from 1.46 +/- 0.12 approximately 2.68 +/- 0.25 (mean +/- SD) log10 pfu/cell. Chou-Talalay analysis of the combination of ReoT3D with cisplatin, gemcitabine, or vinblastine demonstrated strong synergistic effects on cell killing, but only in cell lines that were sensitive to these compounds. In contrast, the combination of ReoT3D and paclitaxel was invariably synergistic in all cell lines tested, regardless of their levels of sensitivity to either agent. Treatment of NSCLC cell lines with the ReoT3D-paclitaxel combination resulted in increased poly (ADP-ribose) polymerase cleavage and caspase activity compared to single therapy, indicating enhanced apoptosis induction in dually treated NSCLC cells. NSCLC cells treated with the ReoT3D-paclitaxel combination showed increased proportions of mitotic and apoptotic cells, and a more pronounced level of caspase-3 activation was demonstrated in mitotically arrested cells. CONCLUSION: These data suggest that the oncolytic activity of ReoT3D can be potentiated by taxanes and other chemotherapeutic agents, and that the ReoT3D-taxane combination most effectively achieves synergy through accelerated apoptosis triggered by prolonged mitotic arrest.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Orthoreovirus Mamífero 3/fisiologia , Terapia Viral Oncolítica/métodos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/virologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/virologia , Paclitaxel/administração & dosagem , Poli(ADP-Ribose) Polimerases/metabolismo , Vimblastina/administração & dosagem , Vírion/fisiologia , Proteínas ras/metabolismo , GencitabinaRESUMO
PURPOSE: The National Cancer Institute has completed a first-in-human clinical pharmacodynamic trial of the targeted agent ABT-888, a poly (ADP-ribose) polymerase (PARP) inhibitor, under the auspices of the U.S. Food and Drug Administration's Exploratory Investigational New Drug Application. Performance of the study design, needle biopsy procedure, and validated pharmacodynamic assay were evaluated in human tumor xenograft models. EXPERIMENTAL DESIGN: A validated ELISA was used to quantify PAR, a product of the PARP 1/2 enzyme activity. Sampling variability from tumor heterogeneity was determined by comparing PAR content in multiple tumors, and in different areas of the same tumor in a particular animal, collected under anesthesia by needle biopsy or resection before and after administration of nontoxic doses of ABT-888. The degree of PARP inhibition following single-dose treatment was evaluated in the time frame anticipated for biopsy in humans. RESULTS: Sampling variability around the mean (approximately 50%) for untreated and vehicle-treated animals was random and due to specimen heterogeneity. PAR levels in initial and repeat tumor biopsies, separated by 1 week, were not altered by the stress induced by daily handling of the animals. A single ABT-888 dose (3 or 12.5 mg/kg) reduced intratumor PAR levels by >95%. ABT-888 (1.56-25 mg/kg) significantly decreased PAR levels at 2 h post-dosing. CONCLUSION: The detailed methodologies developed for this study facilitated the design of a phase 0, first-in-human clinical trial of ABT-888 and could serve as a model for developing proof-of-principle clinical trials of molecularly targeted anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Ensaios Clínicos como Assunto , Melanoma Experimental/química , Modelos Biológicos , Poli(ADP-Ribose) Polimerases/análise , Animais , Benzimidazóis/uso terapêutico , Biópsia por Agulha , Sistemas de Liberação de Medicamentos , Humanos , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Nus , Inibidores de Poli(ADP-Ribose) Polimerases , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Phase 0 trials are designed primarily to evaluate the pharmacodynamic and/or pharmacokinetic properties of selected investigational agents before initiating more traditional phase I testing. One of the major objectives of phase 0 trials is to interrogate and refine a target or biomarker assay for drug effect in human samples implementing procedures developed and validated in preclinical models. Thus, close collaboration between laboratory scientists and clinical investigators is essential to the design and conduct of phase 0 trials. Given the relatively small number of patients and tissue samples, showing a significant drug effect in phase 0 trials requires precise and reproducible assay procedures and innovative statistical methodology. Furthermore, phase 0 trials involving limited exposure of a study agent administered at low doses and/or for a short period allow them to be initiated under the Food and Drug Administration exploratory investigational new drug guidance with less preclinical toxicity data than usually required for traditional first-in-human studies. Because of the very limited drug exposure, phase 0 trials offer no chance of therapeutic benefit, which can impede patient enrollment, particularly if invasive tumor biopsies are required. The challenges to accrual are not insurmountable, however, and well-designed and executed phase 0 trials are feasible and have great potential for improving the efficiency and success of subsequent trials, particularly those evaluating molecularly targeted agents.
Assuntos
Ensaios Clínicos como Assunto/métodos , Projetos de Pesquisa , Algoritmos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Modelos BiológicosRESUMO
The past several years have seen unprecedented advances in the application of various therapeutic strategies for the treatment of patients with renal cancer. The availability of active immunotherapy, antiangiogenic therapy, and targeted therapy for this disease has brought front and center issues related to choosing the appropriate treatment for particular patient populations. It is increasingly evident that the most promising treatment selection strategies will incorporate identifying specific features of the tumor itself. To facilitate this move toward personalized medicine, it is critically important to establish some standard principles for renal cancer tissue collection, preparation, and analysis for translational research studies. In this article, we identify and discuss some critical issues related to tissue-based kidney cancer research. We focus on five major areas as follows: (a) surgical and image-guided techniques for tissue collection; (b) quality control of specimen collection, processing, storage, and review; (c) issues related to analysis of paraffin embedded tissues; (d) genomic studies; and (e) assessment of reproducibility of assays across institutions. In addition, some practical implementation strategies are proposed. Although many of the topics discussed are specific for renal cancer, several are also relevant to tissue based biomarker investigations in a broad array of malignancies.
Assuntos
Carcinoma de Células Renais/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/tendências , Neoplasias Renais/patologia , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/tendências , Algoritmos , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , Genômica/métodos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Modelos Biológicos , Inclusão em Parafina/métodos , Controle de Qualidade , Projetos de Pesquisa , Cirurgia Assistida por Computador/métodos , Bancos de TecidosRESUMO
Non-clinical studies were conducted to evaluate the toxicity of Antalarmin, a corticotropin-releasing hormone type 1 receptor antagonist being developed for therapy of stress-related pathologies. Antalarmin was not genotoxic in bacterial mutagenesis assays, mammalian cell mutagenesis assays, or in vivo DNA damage assays. In a 14-day range-finding study in rats, Antalarmin doses >or=500 mg/kg/day (3,000 mg/m(2)/day) induced mortality. In a 90-day toxicity study in rats, no gross toxicity was seen at doses of 30, 100, or 300 mg/kg/day (180, 600, or 1,800 mg/m(2)/day, respectively). Antalarmin (300 mg/kg/day) induced mild anemia, increases in serum gamma-glutamyl transferase activity, and microscopic hepatic pathology (bile duct hyperplasia and epithelial necrosis, periportal inflammation). Microscopic renal changes (cortical necrosis, inflammation, hypertrophy, nephropathy) were observed in rats at all Antalarmin doses. In a 14-day range-finding study in dogs, Antalarmin doses >or=50mg/kg/day (1,000 mg/m(2)/day) induced repeated emesis and bone marrow suppression. In a 90-day toxicity study in dogs, Antalarmin (4, 8, or 16 mg/kg/day (80, 160, or 320 mg/m(2)/day, respectively)) induced bone marrow and lymphoid depletion, but no gross toxicity. Comparative in vitro studies using rat, dog, and human neutrophil progenitors demonstrated that canine bone marrow cells are highly sensitive to Antalarmin cytotoxicity, while rat and human bone marrow cells are relatively insensitive. As such, the bone marrow toxicity observed in dogs is considered likely to over-predict Antalarmin toxicity in humans. The hepatic and renal toxicities seen in rats exposed to Antalarmin identify those tissues as the most likely targets for Antalarmin toxicity in humans.
Assuntos
Pirimidinas/toxicidade , Pirróis/toxicidade , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Ensaio de Unidades Formadoras de Colônias , Cães , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Testes de Mutagenicidade , Nível de Efeito Adverso não Observado , Ratos , Ratos Endogâmicos F344 , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Vômito/induzido quimicamenteRESUMO
The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/efeitos dos fármacos , Antígeno AC133/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Introdução de Genes , Fator de Crescimento de Hepatócito/genética , Humanos , Indicadores e Reagentes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Circulating tumor cells (CTCs) are increasingly employed for research and clinical monitoring of cancer, though most current methods do not permit the isolation of non-epithelial tumor cells. Furthermore, CTCs isolated with antibody-dependent methods are not suitable for downstream experimental uses, including in vitro culturing and implantation in vivo. In the present study, we describe the development, validation, and transfer across laboratories of a new antibody-independent device for the enrichment of CTCs from blood samples of patients with various cancer diagnoses. The ApoStream® device uses dielectrophoresis (DEP) field-flow assist to separate non-hematopoietic cells from the peripheral blood mononuclear fraction by exposing cells in a laminar flow stream to a critical alternating current frequency. The ApoStream® device was calibrated and validated in a formal cross-laboratory protocol using 3 different cancer cell lines spanning a range of distinct phenotypes (A549, MDA-MB-231, and ASPS-1). In spike-recovery experiments, cancer cell recovery efficiencies appeared independent of spiking level and averaged between 68% and 55%, depending on the cell line. No inter-run carryover was detected in control samples. Moreover, the clinical-readiness of the device in the context of non-epithelial cancers was evaluated with blood specimens from fifteen patients with metastatic sarcoma. The ApoStream® device successfully isolated CTCs from all patients with sarcomas examined, and the phenotypic heterogeneity of the enriched cells was demonstrated by fluorescence in situ hybridization or with multiplex immunophenotyping panels. Therefore, the ApoStream® technology expands the clinical utility of CTC evaluation to mesenchymal cancers.
Assuntos
Separação Celular/instrumentação , Células Neoplásicas Circulantes/metabolismo , Sarcoma Alveolar de Partes Moles/sangue , Células A549 , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Separação Celular/métodos , Proteínas Correpressoras , Humanos , Proteínas Repressoras/metabolismo , Sarcoma Alveolar de Partes Moles/patologiaRESUMO
Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.
Assuntos
Bioensaio/métodos , Dano ao DNA/genética , Histonas/metabolismo , Fosforilação/fisiologia , Animais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Histonas/genética , Humanos , Camundongos , Camundongos Nus , MutaçãoRESUMO
This study aimed at characterizing the interspecies absorption, distribution, metabolism and elimination (ADME) profile of N-geranyl-N'-(2-adamantyl)ethane-1,2-diamine (SQ109), a new diamine-based antitubercular drug. Single doses of SQ109 were administered (intravenously (i.v.) and per os (p.o.)) to rodents and dogs and blood samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Based on i.v. equivalent body surface area dose, the terminal half-life (t1/2) of SQ109 in dogs was longer than that in rodents, reflected by a larger volume of distribution (Vss) and a higher clearance rate of SQ109 in dogs, compared to that in rodents. The oral bioavailability of SQ109 in dogs, rats and mice were 2.4-5, 12 and 3.8%, respectively. After oral administration of [14C]SQ109 to rats, the highest level of radioactivity was in the liver, followed by the lung, spleen and kidney. Tissue-to-blood ratios of [14C]SQ109 were greater than 1. Fecal elimination of [14C]SQ109 accounted for 22.2% of the total dose of [14C]SQ109, while urinary excretion accounted for only 5.6%. The binding of [14C]SQ109 (0.1-2.5 microg ml-1) to plasma proteins varied from 6 to 23% depending on the species (human, mouse, rat and dog). SQ109 was metabolized by rat, mouse, dog and human liver microsomes, resulting in 22.8, 48.4, 50.8 or 58.3%, respectively, of SQ109 remaining after a 10-min incubation at 37 degrees C. The predominant metabolites in the human liver microsomes gave intense ion signals at 195, 347 and 363m/z, suggesting the oxidation, epoxidation and N-dealkylation of SQ109. P450 reaction phenotyping using recombinant cDNA-expressed human CYPs in conjunction with specific CYP inhibitors indicated that CYP2D6 and CYP2C19 were the predominant CYPs involved in SQ109 metabolism.
Assuntos
Adamantano/análogos & derivados , Antituberculosos/farmacocinética , Etilenodiaminas/farmacocinética , Adamantano/farmacocinética , Animais , Hidrocarboneto de Aril Hidroxilases/fisiologia , Proteínas Sanguíneas/metabolismo , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/fisiologia , Cães , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Taxa de Depuração Metabólica , Camundongos , Oxigenases de Função Mista/fisiologia , Ligação Proteica , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie , Distribuição TecidualRESUMO
SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.
Assuntos
Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA/efeitos dos fármacos , Pirróis/farmacologia , Animais , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Ensaio Cometa , DNA/metabolismo , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor suppressor p53 plays a critical role in suppressing cancer growth and progression and is an attractive target for the development of new targeted therapies. We synthesized several indolo-pyrido-isoquinolin based alkaloids to activate p53 function and examined their therapeutic efficacy using NCI-60 screening. Here, we provide molecular evidence that one of these compounds, 11-methoxy-2,3,4,13-tetrahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ylium-bromide (termed P18 or NSC-768219) inhibits growth and clonogenic potential of cancer cells. P18 treatment results in downregulation of mesenchymal markers and concurrent upregulation of epithelial markers as well as inhibition of migration and invasion. Experimental epithelial-mesenchymal-transition (EMT) induced by exposure to TGFß/TNFα is also completely reversed by P18. Importantly, P18 also inhibits mammosphere-formation along with a reduction in the expression of stemness factors, Oct4, Nanog and Sox2. We show that P18 induces expression, phosphorylation and accumulation of p53 in cancer cells. P18-mediated induction of p53 leads to increased nuclear localization and elevated expression of p53 target genes. Using isogenic cancer cells differing only in p53 status, we show that p53 plays an important role in P18-mediated alteration of mesenchymal and epithelial genes, inhibition of migration and invasion of cancer cells. Furthermore, P18 increases miR-34a expression in p53-dependent manner and miR-34a is integral for P18-mediated inhibition of growth, invasion and mammosphere-formation. miR-34a mimics potentiate P18 efficacy while miR-34a antagomirs antagonize P18. Collectively, these data provide evidence that P18 may represent a promising therapeutic strategy for the inhibition of growth and progression of breast cancer and p53-miR-34a axis is important for P18 function.
Assuntos
Alcaloides/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Isoquinolinas/farmacologia , MicroRNAs/metabolismo , Piridinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Alcaloides/química , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Clonais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Isoquinolinas/química , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação/efeitos dos fármacos , Piridinas/química , Alcaloides de Triptamina e Secologanina/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. EXPERIMENTAL DESIGN: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. RESULTS: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. CONCLUSIONS: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Dipeptídeos/farmacologia , Indóis/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunoensaio , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos Nus , Proteínas Mitocondriais/química , Mimetismo Molecular , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect. EXPERIMENTAL DESIGN: Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models. RESULTS: By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation. CONCLUSIONS: These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field. Clin Cancer Res; 22(14); 3683-94. ©2016 AACR.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células A549 , Animais , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Crizotinibe , Células HEK293 , Células HT29 , Humanos , Imunoensaio/métodos , Indazóis , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Camundongos , Camundongos Nus , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
SQ109 is a novel [1,2]-diamine-based ethambutol (EMB) analog developed from high-throughput combinatorial screening. The present study aimed at characterizing its pharmacodynamics and pharmacokinetics. The antimicrobial activity of SQ109 was confirmed in vitro (Mycobacterium tuberculosis-infected murine macrophages) and in vivo (M. tuberculosis-infected C57BL/6 mice) and compared to isoniazid (INH) and EMB. SQ109 showed potency and efficacy in inhibiting intracellular M. tuberculosis that was similar to INH, but superior to EMB. In vivo oral administration of SQ109 (0.1-25 mg kg(-1) day(-1)) to the mice for 28 days resulted in dose-dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg(-1) day(-1), but was less potent than INH at 25 mg kg(-1) day(-1). Monitoring of SQ109 levels in mouse tissues on days 1, 14 and 28 following 28-day oral administration (10 mg kg(-1) day(-1)) revealed that lungs and spleen contained the highest concentration of SQ109, at least 10 times above its MIC. Pharmacokinetic profiles of SQ109 in mice following a single administration showed its C(max) as 1038 (intravenous (i.v.)) and 135 ng ml(-1) (p.o.), with an oral T(max) of 0.31 h. The elimination t(1/2) of SQ109 was 3.5 (i.v.) and 5.2 h (p.o.). The oral bioavailability was 4%. However, SQ109 displayed a large volume of distribution into various tissues. The highest concentration of SQ109 was present in lung (>MIC), which was at least 120-fold (p.o.) and 180-fold (i.v.) higher than that in plasma. The next ranked tissues were spleen and kidney. SQ109 levels in most tissues after a single administration were significantly higher than that in blood. High tissue concentrations of SQ109 persisted for the observation period (10 h). This study demonstrated that SQ109 displays promising in vitro and in vivo antitubercular activity with favorable targeted tissue distribution properties.
Assuntos
Antituberculosos/farmacocinética , Diaminas/farmacocinética , Etambutol/análogos & derivados , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Administração Oral , Animais , Antituberculosos/administração & dosagem , Antituberculosos/sangue , Antituberculosos/química , Antituberculosos/uso terapêutico , Disponibilidade Biológica , Linhagem Celular , Diaminas/sangue , Diaminas/uso terapêutico , Relação Dose-Resposta a Droga , Esquema de Medicação , Etambutol/administração & dosagem , Etambutol/química , Etambutol/uso terapêutico , Feminino , Injeções Intravenosas , Isoniazida/administração & dosagem , Isoniazida/uso terapêutico , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Distribuição Tecidual , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
PURPOSE: 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs. METHODS: Doses of 0, 2.4, 12 and 24 mg/m2 per day were administered to rats, while dogs received doses of 0, 8 or 16 mg/m2 per day. In both species, 17-DMAG was administered i.v. (slow bolus for rats; 1-h infusion for dogs) daily for 5 days. An additional cohort of dogs received 16 mg/m2 per day orally for 5 days. Clinical observations were noted, and standard hematology and clinical chemistry parameters were monitored. Selected tissues were evaluated microscopically for drug-related lesions. Tissue and plasma 17-DMAG concentrations were measured by HPLC/MS at selected time-points on days 1 and 5. RESULTS: Daily i.v. administration of 17-DMAG at doses of 24 mg/m2 per day in rats or 16 mg/m2 per day in dogs produced lethality on day 6, approximately 24 h following the last dose. Body weight loss was common in rats and dogs. Drug-related gastrointestinal, bone marrow and hepatic toxicities were also common in rats and dogs. Dogs also exhibited signs of renal and gallbladder toxicity. Plasma concentrations of 17-DMAG increased proportionately with dose in rats and disproportionately with dose in dogs. In rat tissues, however, only fourfold to sixfold increases in 17-DMAG concentrations were observed with a tenfold increase in dose. The highest concentrations of 17-DMAG were found in the liver of rats, with progressively lower concentrations in the spleen, lung, kidney and plasma. Regardless of the route of administration, higher drug concentrations were present in plasma (rat and dog) and tissue (rat) samples obtained on day 5 compared to those obtained on day 1. Although plasma concentrations decreased with time, 17-DMAG was still detected in dog plasma for at least 24 h after drug administration. CONCLUSIONS: With the recent approval of 17-DMAG for clinical use, the data generated from these preclinical studies will provide guidance to clinicians as they administer this drug to their patients. The MTD of 17-DMAG was 12 mg/m2 per day in rats and 8 mg/m2 per day in dogs; therefore, the recommended starting dose for phase I trial is 1.3 mg/m2 per day for 5 days. Gastrointestinal and bone marrow toxicity were dose-limiting in rats, and gastrointestinal, renal, gallbladder and bone marrow toxicity were dose-limiting in dogs. All adverse effects were fully reversible in surviving animals after treatment was complete.