RESUMEN
AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2-M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent. SIGNIFICANCE: This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.
Asunto(s)
Antineoplásicos , Neoplasias de la Mama Triple Negativas , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carboplatino , Humanos , Indoles , Irinotecán , Morfolinas/farmacología , Ftalazinas , Piperazinas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Sulfonamidas/farmacología , Sulfóxidos/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 µg/mL). Lack of activity against E. coli was maintained (IC50 > 20 µM and MIC > 128 µg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/química , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/efectos de los fármacos , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum ß-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > CT ) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 ß-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 µg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log10 bioburden reduction of 8% and 44% fT > 2 µg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.
Asunto(s)
Cefalosporinas , Muslo , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Compuestos de Azabiciclo , Cefepima , Humanos , Klebsiella pneumoniae , Ratones , Pruebas de Sensibilidad Microbiana , Triazoles , beta-Lactamasas/genéticaRESUMEN
DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and CDKN2A/B deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G1 cells leading to G1 arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis. In vivo xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need. SIGNIFICANCE: ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas/farmacología , Sulfóxidos/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Indoles , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Morfolinas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genética , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Pirimidinonas/administración & dosificación , Sulfonamidas , Sulfóxidos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: AZD0156 and AZD6738 are potent and selective inhibitors of ataxia-telangiectasia-kinase (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR), respectively, important sensors/signallers of DNA damage. METHODS: We used multiplexed targeted-mass-spectrometry to select pRAD50(Ser635) as a pharmacodynamic biomarker for AZD0156-mediated ATM inhibition from a panel of 45 peptides, then developed and tested a clinically applicable immunohistochemistry assay for pRAD50(Ser635) detection in FFPE tissue. RESULTS: We found moderate pRAD50 baseline levels across cancer indications. pRAD50 was detectable in 100% gastric cancers (n = 23), 99% colorectal cancers (n = 102), 95% triple-negative-breast cancers (TNBC) (n = 40) and 87.5% glioblastoma-multiformes (n = 16). We demonstrated AZD0156 target inhibition in TNBC patient-derived xenograft models; where AZD0156 monotherapy or post olaparib treatment, resulted in a 34-72% reduction in pRAD50. Similar inhibition of pRAD50 (68%) was observed following ATM inhibitor treatment post irinotecan in a colorectal cancer xenograft model. ATR inhibition, using AZD6738, increased pRAD50 in the ATM-proficient models whilst in ATM-deficient models the opposite was observed, suggesting pRAD50 pharmacodynamics post ATR inhibition may be ATM-dependent and could be useful to determine ATM functionality in patients treated with ATR inhibitors. CONCLUSION: Together these data support clinical utilisation of pRAD50 as a biomarker of AZD0156 and AZD6738 pharmacology to elucidate clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Espectrometría de Masas/métodos , Animales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Biomarcadores/metabolismo , Línea Celular , Daño del ADN , Humanos , Inmunohistoquímica , Indoles , Irinotecán/farmacología , Ratones , Ratones Desnudos , Morfolinas , Ftalazinas/farmacología , Piperazinas/farmacología , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Transducción de Señal , Sulfonamidas , Sulfóxidos/farmacología , Sulfóxidos/uso terapéutico , Neoplasias de la Mama Triple Negativas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the DNA-damage response and the apical kinase which orchestrates the cellular processes that repair stalled replication forks (replication stress) and associated DNA double-strand breaks. Inhibition of repair pathways mediated by ATR in a context where alternative pathways are less active is expected to aid clinical response by increasing replication stress. Here we describe the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine morpholinopyrimidine ATR inhibitor with excellent preclinical physicochemical and pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for once or twice daily dosing and achieves biologically effective exposure at moderate doses. Compound 2 is currently being tested in multiple phase I/II trials as an anticancer agent.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Sulfóxidos/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Fenómenos Químicos , Ensayos Clínicos como Asunto , Femenino , Humanos , Indoles , Ratones , Modelos Moleculares , Conformación Molecular , Morfolinas , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/química , Pirimidinas/farmacocinética , Sulfonamidas , Sulfóxidos/química , Sulfóxidos/farmacocinética , Distribución TisularRESUMEN
Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro AZ32, with enhanced blood-brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637-47. ©2018 AACR.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Glioma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Administración Oral , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
Barasertib (AZD1152), a highly potent and selective aurora kinase B inhibitor, gave promising clinical activity in elderly acute myeloid leukemia (AML) patients. However, clinical utility was limited by the requirement for a 7-day infusion. Here we assessed the potential of a nanoparticle formulation of the selective Aurora kinase B inhibitor AZD2811 (formerly known as AZD1152-hQPA) in preclinical models of AML. When administered to HL-60 tumor xenografts at a single dose between 25 and 98.7 mg/kg, AZD2811 nanoparticle treatment delivered profound inhibition of tumor growth, exceeding the activity of AZD1152. The improved antitumor activity was associated with increased phospho-histone H3 inhibition, polyploidy, and tumor cell apoptosis. Moreover, AZD2811 nanoparticles increased antitumor activity when combined with cytosine arabinoside. By modifying dose of AZD2811 nanoparticle, therapeutic benefit in a range of preclinical models was further optimized. At high-dose, antitumor activity was seen in a range of models including the MOLM-13 disseminated model. At these higher doses, a transient reduction in bone marrow cellularity was observed demonstrating the potential for the formulation to target residual disease in the bone marrow, a key consideration when treating AML. Collectively, these data establish that AZD2811 nanoparticles have activity in preclinical models of AML. Targeting Aurora B kinase with AZD2811 nanoparticles is a novel approach to deliver a cell-cycle inhibitor in AML, and have potential to improve on the clinical activity seen with cell-cycle agents in this disease. Mol Cancer Ther; 16(6); 1031-40. ©2017 AACR.
Asunto(s)
Antineoplásicos/administración & dosificación , Aurora Quinasa B/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Nanopartículas , Organofosfatos/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular Tumoral , Citarabina/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Ratones , Organofosfatos/farmacocinética , Poliploidía , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/farmacocinética , Ratas , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084-94. ©2016 AACR.
Asunto(s)
Neoplasias Experimentales/tratamiento farmacológico , Ftalazinas/farmacología , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Animales , Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Descubrimiento de Drogas , Genes BRCA1 , Humanos , Ratones , Ftalazinas/administración & dosificación , Ftalazinas/toxicidad , Piperazinas/administración & dosificación , Piperidinas/toxicidad , Poli(ADP-Ribosa) Polimerasas/química , Ratas , Temozolomida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.
Asunto(s)
Aurora Quinasas/antagonistas & inhibidores , Nanopartículas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Aurora Quinasas/metabolismo , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Línea Celular Tumoral , Liberación de Fármacos , Femenino , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones SCID , Organofosfatos/química , Organofosfatos/farmacocinética , Organofosfatos/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/farmacología , Ratas Desnudas , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The mTOR kinase inhibitor AZD8055 inhibits both mTORC1 and mTORC2 leading to disruption of glucose metabolism and proliferation pathways. This study assessed the impact of single and multiple doses of AZD8055 on the uptake of the glucose metabolism marker 2-deoxy-2-[(18) F]fluoro-D-glucose ([(18) F]FDG) and the proliferation marker 3'-deoxy-3'-[(18) F]fluorothymidine ([(18) F]FLT) in U87-MG glioma xenografts. PROCEDURES: Mice bearing U87-MG tumours received either vehicle or AZD8055 (20 mg/kg) once daily p.o. Mice were imaged with either [(18) F]FDG or [(18) F]FLT PET to assess treatment response. Comparisons were made between in vivo imaging and ex vivo histopathology data. RESULTS: Tumour uptake of [(18) F]FDG was reduced by 33 % 1 h after a single dose of AZD8055 and by 49 % following 4 days of dosing. These changes coincided with suppression of the mTOR pathway biomarkers pS6 and pAKT. In contrast, the effect of AZD8055 on [(18) F]FLT uptake was inconsistent. CONCLUSIONS: The very rapid change in [(18) F]FDG uptake following acute AZD8055 treatment suggests that this could be used as an early mechanistic biomarker of metabolic changes resulting from mTOR inhibition. The utility of [(18) F]FLT for measuring the anti-proliferative effect of AZD8055 remains unclear.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Fluorodesoxiglucosa F18/farmacocinética , Glioma/metabolismo , Morfolinas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/farmacocinética , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Femenino , Glioma/tratamiento farmacológico , Humanos , Ratones , Ratones DesnudosRESUMEN
Reversed-phase gradient LC-MS was used to perform untargeted metabonomic analysis on extracts of human colorectal cancer (CRC) cell lines (COLO 205, HT-29, HCT 116 and SW620) subcutaneously implanted into age-matched athymic nude male mice to study small molecule metabolic profiles and examine possible correlations with human cancer biopsies. Following high mass accuracy data analysis using MS and MS/MS, metabolites were identified by searching against major metabolite databases including METLIN, MASSBANK, The Human Metabolome Database, PubChem, Biospider, LipidMaps and KEGG. HT-29 and COLO 205 tumor xenografts showed a distribution of metabolites that differed from SW620 and HCT 116 xenografts (predominantly on the basis of relative differences in the amounts of amino acids and lipids detected). This finding is consistent with NMR-based analysis of human colorectal tissue, where the metabolite profiles of HT-29 tumors exhibit the greatest similarity to human rectal cancer tissue with respect to changes in the relative amounts of lipids and choline-containing compounds. As the metabolic signatures of cancer cells result from oncogene-directed metabolic reprogramming, the HT-29 xenografts in mice may prove to be a useful model to further study the tumor microenvironment and cancer biology.
Asunto(s)
Biomarcadores de Tumor/aislamiento & purificación , Neoplasias Colorrectales/metabolismo , Metaboloma , Aminoácidos/aislamiento & purificación , Animales , Biopsia , Línea Celular Tumoral , Colina/aislamiento & purificación , Cromatografía Liquida , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Bases de Datos Factuales , Humanos , Lípidos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Especificidad de Órganos , Análisis de Componente Principal , Espectrometría de Masas en Tándem , Trasplante Heterólogo , Microambiente TumoralRESUMEN
ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Morfolinas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Animales , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada , Cristalografía por Rayos X , Descubrimiento de Drogas , Femenino , Células HeLa , Humanos , Ratones , Modelos Moleculares , Morfolinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Cediranib (AZD2171) is a highly potent inhibitor of all three vascular endothelial growth factor receptors. The aim of this preclinical study was to examine the effect of combining cediranib with mechanistically distinct anti-tumour therapies. METHODS: Cediranib (1.5 or 3 mg/kg/day) was evaluated alone and in combination with either gefitinib, imatinib, ZD6126, saracatinib, selumetinib, bevacizumab, 5-fluorouracil (5-FU), docetaxel, oxaliplatin, gemcitabine, pemetrexed, irinotecan or cisplatin in human tumour xenograft models. Anti-tumour activity was measured by assessing the change in tumour volume following treatment compared with vehicle-treated time-matched controls. RESULTS: In all cases, the combination regimens, at tolerated doses and schedules, inhibited tumour growth to a greater extent than the corresponding monotherapy treatments. Compared with cediranib alone, statistically significant enhancements in anti-tumour activity were observed with all combination regimens. Notably, after 14 days of treatment, the combination of cediranib with ZD6126 induced substantial tumour regression (60 % compared with pre-treatment volume), whilst treatment with each agent alone led only to partial growth inhibition. A combination of cediranib with gefitinib also induced tumour regressions, and cediranib combined with either gemcitabine or irinotecan was found to inhibit tumour growth profoundly (by 99 and 98 %, respectively). CONCLUSIONS: Combining cediranib with selected cytotoxic or targeted agents proved efficacious in a range of human tumour xenograft models.
Asunto(s)
Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Quinazolinas/uso terapéutico , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Humanos , Ratones , Quinazolinas/administración & dosificación , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Aurora family of kinases, play a fundamental role in cell division and are overexpressed in several cancers including colon. The activity of barasertib-hQPA, a selective inhibitor of Aurora-B kinase (ABK) was investigated in a range of preclinical models of gastrointestinal cancer. Treatment with barasertib-hQPA produced anti-proliferative and cytotoxic effects across a panel of human colorectal cancer (CRC) cell lines in vitro. Prodrug, barasertib [48-h subcutaneous (s.c.) infusion; 150 mg/kg/day] inhibited the growth of SW620, Colo205, HCT116 human colorectal tumor xenografts in nude mice significantly (Student's t-test, P<0.05, n=10-12 per group). Flow cytometric analysis of single cells from disaggregated barasertib-treated SW620 tumors revealed a decrease in phosphorylated histone H3 (phH3) and an increase in tumor cells with ≥4N DNA content P<0.05). The activity of barasertib was then examined in ApcMin/+ mice, a spontaneous model of early intestinal neoplasia. Macroscopic evaluation of the small intestine revealed that barasertib treatment [25 mg/kg intra-peritoneal (i.p.) Q1Dx4 each week for 3 weeks] of 8-week old ApcMin/+ mice produced a 39% reduction in macroadenoma number (P=0.02) and a 43% reduction in overall adenoma burden (P=0.02) compared with vehicle-treated controls. Quantification of microscopic adenomas revealed a >64% reduction in the number of adenomas spanning more than one villus. Histological analysis of these adenomas revealed a number of distinct changes in barasertib-treated ApcMin/+ mice, including a 94% reduction in the proportion of phospho-histone H3-positive cells (P<0.001) and a 53% reduction in the number of cells per adenoma (P=0.001). These results provide a scientific rationale for investigating ABK inhibitors as a treatment for intestinal cancer.
Asunto(s)
Aurora Quinasa B/biosíntesis , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Gastrointestinales/genética , Animales , Aurora Quinasa B/antagonistas & inhibidores , Aurora Quinasa B/genética , Neoplasias Colorrectales/patología , Neoplasias Gastrointestinales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Histonas/genética , Humanos , Ratones , Fosforilación , Quinazolinas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Thin films of ceria (CeO(2)) have many applications, and their synthesis by liquid-injection MOCVD (metal-organic chemical vapor deposition) or ALD (atomic layer deposition) requires volatile precursor compounds. Here we report the synthesis of a series of homoleptic and heteroleptic Ce(IV) complexes with donor-functionalized alkoxide ligands mmp (1-methoxy-2-methylpropan-2-olate), dmap (1-(dimethylamino)propan-2-olate), and dmop (2-(4,4-dimethyl-4,5-dihydrooxazol-2-yl)propan-2-olate) and their potential as precursors for MOCVD and ALD of CeO(2). New complexes were synthesized by alcohol exchange reactions with [Ce(OBu(t))(4)]. [Ce(mmp)(4)] and [Ce(dmap)(4)] were both found to be excellent precursors for liquid-injection MOCVD of CeO(2), depositing high purity thin films with very low carbon contamination, and both have a large temperature window for diffusion controlled growth (350-600 °C for [Ce(mmp)(4)]; 300-600 °C for [Ce(dmap)(4)]). [Ce(mmp)(4)] is also an excellent precursor for liquid-injection ALD of CeO(2) using H(2)O as oxygen source and demonstrates self-limiting growth from 150 to 350 °C. [Ce(dmap)(4)] has lower thermal stability than [Ce(mmp)(4)] and does not show self-limiting growth in ALD. Heteroleptic complexes show a tendency to undergo ligand redistribution reactions to form mixtures in solution and are unsuitable as precursors for liquid-injection CVD.
RESUMEN
Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Piperidinas/farmacología , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Humanos , Ratones , Ratones SCID , Neoplasias/fisiopatología , Fenotipo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of rat or mouse plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were precipitated with three volumes of either methanol or acetonitrile. Chromatographic separations were performed on a C18-bonded stationary phase using 3.5 and 5 mum particles packed into 2.1 and 4.6 mm i.d. formats, respectively, and on a C8 phase using 3.5 mum particles and a 2.1 mm i.d. column. Three reversed-phase gradient solvent systems, based on acidified water-acetonitrile, acidified water-methanol and acidified water-methanol-acetonitrile mixtures, were investigated. The column eluent was analysed with both positive and negative electrospray ionisation using a quadrupole-linear ion trap mass spectrometer. These studies revealed that while accurate classification of sample type can be made, there are a number of methodological problems associated with the analysis of plasma with respect to factors such as repeatability and column longevity. In particular, special care has to be taken to ensure that the analytical system is properly "conditioned" by the repeated injection of matrix samples. The use of biological quality control (QC) samples provided an important means of monitoring method performance. Finally, the source of the plasma (Zucker wild-type or (fa/fa) rat or mouse tumour model) also appeared to have an effect on the repeatability of the methodology.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Metabolómica/métodos , Plasma/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Ratones , Ratas , Reproducibilidad de los ResultadosRESUMEN
Aurora kinases play a critical role in regulating mitosis and cell division, and their overexpression has been implicated in the survival and proliferation of human cancer. In this study, we report the in vitro and in vivo activities of AZD1152, a compound that has selectivity for aurora B kinase, in acute myeloid leukemia (AML) cell lines, primary AML samples, and cord blood cells. AZD1152 exerted antiproliferative or cytotoxic effects in all cell lines studied, inhibited the phosphorylation of histone H3 (pHis H3) on Ser10 in a dose-dependent manner, and resulted in cells with >4N DNA content. THP-1 cells treated with AZD1152 accumulated in a state of polyploidy and showed a senescent response to the drug, in contrast to the apoptotic response seen in other cell lines. Accordingly, AZD1152 profoundly affected the growth of AML cell lines and primary AML in an in vivo xenotransplantation model. However, concentration-dependent effects on cell growth, apoptosis, and cell cycle progression were also observed when human cord blood and primary lineage-negative stem and progenitor cells were analyzed in vitro and in vivo. These data suggest that the inhibition of aurora B kinase may be a useful therapeutic strategy in the treatment of AML and that further exploration of dosing and treatment schedules is warranted in clinical trials.