RESUMEN
Metallo-ß-lactamases (MBLs), such as New Delhi metallo-ß-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of ß-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine ß-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of ß-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Monobactamas/farmacología , Resistencia betalactámica/efectos de los fármacos , beta-Lactamasas/metabolismo , Animales , Antibacterianos/efectos adversos , Antibacterianos/química , Antibacterianos/metabolismo , Aztreonam/farmacología , Células CHO , Cricetulus , Estabilidad de Medicamentos , Escherichia coli/efectos de los fármacos , Femenino , Humanos , Meropenem , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Monobactamas/efectos adversos , Monobactamas/química , Monobactamas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Receptores de GABA-A/metabolismo , Convulsiones/inducido químicamente , Relación Estructura-Actividad , Tienamicinas/farmacologíaRESUMEN
This paper reports an LC/MS/MS method for analysis of salemeterol and fluticasone propionate in human plasma based on combined SPE-based extraction and separate LC/MS/MS conditions. Previously reported interaction between analytes was confirmed and eliminated by their separation in the sample preparation step to ensure no negative impact on their quantitation. The method was validated per FDA guidelines in the range of 2.5-500 pg/mL for salmeterol and 5-500 pg/mL for fluticasone propionate. The method is suitable for plasma analysis of combined salmeterol/fluticasone formulation without adverse effects of inter-analyte interactions on quantitation.
Asunto(s)
Albuterol/análogos & derivados , Androstadienos/sangre , Androstadienos/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Albuterol/sangre , Albuterol/metabolismo , Interacciones Farmacológicas , Estabilidad de Medicamentos , Fluticasona , Humanos , Reproducibilidad de los Resultados , Xinafoato de Salmeterol , Extracción en Fase Sólida/métodos , Solventes/químicaRESUMEN
Salmeterol is an inhaled bronchodilator drug used for treatment of asthma. Its concentrations in plasma are very low or undetectable by previously developed methods. The present paper describes a method for analysis of salmeterol in human plasma with 2.5 pg/mL lower limit of quantitation. Despite the basic character of the drug the method uses mixed mode anion-exchange solid phase extraction for sample preparation combined with a column switching approach to minimize matrix effects. Samples are separated and detected by LC/MS/MS. The method is easy to use, only requires 0.5 mL of plasma and was validated for use in bioanalytical applications. The method does not suffer from interference from co-administered fluticasone propionate.
Asunto(s)
Agonistas Adrenérgicos beta/sangre , Albuterol/análogos & derivados , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Albuterol/sangre , Humanos , Xinafoato de Salmeterol , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Neoplasias/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Sitio Alostérico , Cristalografía por Rayos X , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genéticaRESUMEN
Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.
Asunto(s)
Antineoplásicos/uso terapéutico , Cadherinas/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Macaca fascicularis , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Desoxiadenosinas/metabolismo , Eliminación de Gen , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferasas/genética , Purina-Nucleósido Fosforilasa/genética , ARN Interferente Pequeño/genética , Tionucleósidos/metabolismoRESUMEN
Cancer cells rely on aerobic glycolysis to maintain cell growth and proliferation via the Warburg effect. Phosphoglycerate dehydrogenase (PHDGH) catalyzes the first step of the serine biosynthetic pathway downstream of glycolysis, which is a metabolic gatekeeper both for macromolecular biosynthesis and serine-dependent DNA synthesis. Here, we report that PHDGH is overexpressed in many ER-negative human breast cancer cell lines. PHGDH knockdown in these cells leads to a reduction of serine synthesis and impairment of cancer cell proliferation. However, PHGDH knockdown does not affect tumor maintenance and growth in established breast cancer xenograft models, suggesting that PHGDH-dependent cancer cell growth may be context-dependent. Our findings suggest that other mechanisms or pathways may bypass exclusive dependence on PHGDH in established human breast cancer xenografts, indicating that PHGDH is dispensable for the growth and maintenance and of tumors in vivo.
Asunto(s)
Neoplasias de la Mama/enzimología , Fosfoglicerato-Deshidrogenasa/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Células MCF-7 , Ratones , Fosfoglicerato-Deshidrogenasa/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genéticaRESUMEN
Traditional chiral chromatographic separation method development is time consuming even for an experienced chromatographer. This paper describes the application of computer software ACD Lab to facilitate the development of chiral separation for the quantitation of eszopiclone using LC-MS/MS technology. Assisted by ACD/Chrom Manager and LC Simulator software, the optimal chiral chromatographic development was completed within hours. The baseline chiral separation was achieved with a total cycle time of 3 min. For sample extraction method development, a Waters Oasis Sorbent Selection Plate containing four different sorbents was utilized. Optimal conditions were determined using a single plate under various load, wash and elution conditions. This was followed by a GLP validation which demonstrated excellent intra- and inter-day accuracy and precision for the quantitation of eszopiclone in human plasma at 1.00-100 ng/mL range using LC/MS/MS technology. This method was utilized to support multiple clinic bioequivalence studies.
Asunto(s)
Compuestos de Azabiciclo/sangre , Cromatografía Liquida/métodos , Hipnóticos y Sedantes/sangre , Piperazinas/sangre , Espectrometría de Masas en Tándem/métodos , Compuestos de Azabiciclo/farmacocinética , Eszopiclona , Humanos , Piperazinas/farmacocinética , Control de Calidad , Extracción en Fase Sólida , Estereoisomerismo , ComprimidosRESUMEN
There is increasing scientific interest to understand the environmental fate of fluorotelomer alcohols (FTOHs) and fluorotelomer-based products which may break down to FTOHs. Both are expected to enter aqueous waste streams, which would be processed in a wastewater treatment plant and therein subject to microbial biodegradation. We investigated the biodegradation of 3-14C, 1H,1H,2H,2H-perfluorodecanol [CF3(CF2)6(14)CF2CH2CH2OH, 14C-8-2 FTOH] in mixed bacterial culture and activated sludge. 14CO2 and 14C-organic volatiles in the headspace of the sealed bottles and bottles with continuous air flow were analyzed up to 4 months. After sample extraction with acetonitrile, 14C-labeled biotransformation products (metabolites) were quantified by LC/ARC (on-line liquid chromatography/ accurate radioisotope counting) and identified by quadrupole time-of-flight (Q-TOF) mass spectrometry and GC/MSD (mass selective detector). Three metabolites not yet reported in the literature have been identified as CF3(CF2)6(14)CHOHCH3 (7-2 sFTOH), CF3(CF2)6(14)CH=CHCOOH (7-3 unsaturated acid or 7-3 u acid), and CF3(CF2)6(14)CH=CHCONH2 (7-3 u amide) along with five previously reported metabolites [CF3(CF2)6(14)CF2CH2CHO (8-2 FTAL), CF3(CF2)6 (14)CF2CH2COOH (8-2 acid), CF3(CF2)6(14)CF=CHCOOH (8-2 u acid), CF3(CF2)6(14)CH2CH2COOH (7-3 acid), and CF3(CF2)6(14)COOH (PFOA)]. No CF3(CF2)6(14)CF2COOH (14C-PFNA) was observed, indicating that alpha-oxidation does not take place. It was found that strong adsorption to the activated sludge and subsequent transformation, even under continuous air flow, greatly reduced partitioning of 8-2 FTOH or any transformation products to air. CF3(CF2)4COOH (PFHA; perfluorohexanoic acid) was observed and increased in mixed bacterial culture over 28 days and accounted for about 1% of the initial 14C-8-2 FTOH concentration from day 28 to day 90. 14CO2 accounted for 1% of initial 14C in activated sludge with continuous air flow at day 1 and increased over time. In closed bottles, 14CO2 in the headspace of activated sludge medium increased to 12% of the available 14C over 135 days with periodic addition of ethanol, as compared to 3% when no additional ethanol was added. These results show that replenishment of organic carbon enhanced microbial mineralization of multiple--CF2--groups in the fluorocarbon chain of 14C-8-2 FTOH. At day 90 the net increase of fluoride ion in the mixed bacterial culture was 93 microg L(-1), equivalent to 12% of total mineralization (destruction) of the 14C-8-2 FTOH. These results demonstrate that perfluorinated carbon bonds of 14C-8-2 FTOH are defluorinated and mineralized by microorganisms under conditions which may occur in a wastewater treatment plant, forming shorter fluorinated carbon metabolites.
Asunto(s)
Alcoholes/metabolismo , Bacterias/metabolismo , Fluorocarburos/metabolismo , Aguas del Alcantarillado/análisis , Adsorción , Biodegradación Ambiental , Dióxido de Carbono/análisis , Radioisótopos de Carbono/metabolismo , Cromatografía Liquida , Cinética , Espectrometría de Masas , Aguas del Alcantarillado/microbiologíaRESUMEN
This study investigated the biodegradation potential of 3-(14)C,1H,1H,2H,2H-perfluorodecanol [CF3(CF2)6(14)CF2CH2CH2OH, 14C-labeled 8-2 telomer B alcohol or 14C-labeled 8-2 TBA] by diluted activated sludge from a domestic wastewater treatment plant under aerobic conditions. After sample extraction with acetonitrile, biotransformation products were separated and quantified by LC/ARC (on-line liquid chromatography/accurate radioisotope counting) with a limit of quantification about 0.5% of the 14C counts applied to the test systems. Identification of biotransformation products was performed by quadrupole time-of-flight mass spectrometry. Three transformation products have been identified: CF3(CF2)6(14)CF2CH2COOH (8-2 saturated acid); CF3(CF2)6(14)CF=CHCOOH (8-2 unsaturated acid); and CF3(CF2)6(14)COOH (perfluorooctanoic acid, PFOA), representing 27, 6.0, and 2.1% of the initial 14C mass (14C counts applied) after 28 days, respectively. A transformation product, not yet reported in the literature, has also been observed and tentatively identified as CF3(CF2)6(14)CH2CH2COOH (2H,2H,3H,3H-perfluorodecanoic acid); it accounted for 2.3% of the mass balance after 28 days. The 2H,2H,3H,3H-perfluorodecanoic acid is likely a substrate for beta-oxidation, which represents one of the possible pathways for 8-2 telomer B alcohol degradation. The 8-2 saturated acid and 8-2 unsaturated acid cannot be directly used as substrates for beta-oxidation due to the proton deficiency in their beta-carbon (C3 carbon) and their further catabolism may be catalyzed by some other still unknown mechanisms. The 2H,2H,3H,3H-perfluorodecanoic acid may originate either from the major transformation product CF3(CF2)6(14)CF2CH2COOH or from other unidentified transformation products via multiple steps. Approximately 57% of the starting material remained unchanged after 28 days, likely due to its strong adsorption to the PTFE (poly(tetrafluoroethylene)) septa of the test vessels. No CF3(CF2)6(14)CF2COOH (perfluorononanoic acid) was observed, indicating that alpha-oxidation of CF3(CF2)6(14)CF2CH2COOH did not occur under the study conditions. Several 14C-labeled transformation products that have not yet been identified (each less than 1% of the mass balance) were also observed and together accounted for 7% of the total 14C mass balance after 28 days. It is not clear whether these unidentified transformation products were resulting from further metabolism of 8-2 saturated acid or 8-2 unsaturated acid. The results suggest that perfluorinated acid metabolites such as perfluorooctanoic acid account for only a very small portion of the transformation products observed. Also, the observed volatility and bioavailability of 14C-labeled 8-2 TBA for microbial degradation was markedly decreased as a result of the presence of a strongly adsorbing matrix such as PTFE in the experimental systems. It is apparent that the biological fate of 8-2 telomer B alcohol is determined by multiple degradation pathways, with neither beta-oxidation nor any other enzyme-catalyzed reactions as a single dominant (principal) mechanism under the study conditions.
Asunto(s)
Bacterias Aerobias , Fluorocarburos/metabolismo , Aguas del Alcantarillado/química , Adsorción , Biotransformación , Radioisótopos de Carbono/análisis , Catálisis , Oxidación-Reducción , Eliminación de Residuos LíquidosRESUMEN
The Wickbold decomposition method in combination with differential potentiometric detection via fluoride ion-selective electrode has been applied to analysis of total fluorine in biological matrices. The performance of the method has been evaluated for determination of total fluorine in rat blood. Total mineralization of the biological sample is achieved by combustion of the sample in oxygen/hydrogen flame and subsequent absorption of the resulting fluoride in aqueous absorption medium. The fluoride is then quantified by highly selective automated differential static potentiometry with fluoride ion-selective electrode. Total fluorine determination has been evaluated in terms of sample carryover, reproducibility, precision, as well as feasibility to routine analysis of alternative biological matrices. Our results indicate that, up to 100ppm fluorine in blood, the method does not suffer from sample carryover. Limits of quantitation of 0.5ppm and limits of detection of 0.24ppm fluorine in 0.5g blood samples were achieved by elimination of inherent limitations of fluoride ion-selective electrode detection via automated differential static potentiometric measurements. The Wickbold decomposition method was found to be suitable for routine total fluorine determination in blood samples despite its relatively low throughput and high operator skill requirements.