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1.
Metabolites ; 13(3)2023 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-36984817

RESUMO

With increased use of mass spectrometry imaging (MSI) in support of pharmaceutical research and development, there are opportunities to develop analytical pipelines that incorporate exploratory high-performance analysis with higher capacity and faster targeted MSI. Therefore, to enable faster MSI data acquisition we present analyte-targeted desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) utilizing a triple-quadrupole (TQ) mass analyzer. The evaluated platform configuration provided superior sensitivity compared to a conventional time-of-flight (TOF) mass analyzer and thus holds the potential to generate data applicable to pharmaceutical research and development. The platform was successfully operated with sampling rates up to 10 scans/s, comparing positively to the 1 scan/s commonly used on comparable DESI-TOF setups. The higher scan rate enabled investigation of the desorption/ionization processes of endogenous lipid species such as phosphatidylcholines and a co-administered cassette of four orally dosed drugs-erlotininb, moxifloxacin, olanzapine, and terfenadine. This was used to enable understanding of the impact of the desorption/ionization processes in order to optimize the operational parameters, resulting in improved compound coverage for olanzapine and the main olanzapine metabolite, hydroxy-olanzapine, in brain tissue sections compared to DESI-TOF analysis or matrix-assisted laser desorption/ionization (MALDI) platforms. The approach allowed reducing the amount of recorded information, thus reducing the size of datasets from up to 150 GB per experiment down to several hundred MB. The improved performance was demonstrated in case studies investigating the suitability of this approach for mapping drug distribution, spatially resolved profiling of drug-induced nephrotoxicity, and molecular-histological tissue classification of ovarian tumors specimens.

2.
Pharmaceuticals (Basel) ; 15(11)2022 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-36355479

RESUMO

Fixation of samples is broadly used prior to the histological evaluation of tissue samples. Though recent reports demonstrated the ability to use fixed tissues for mass spectrometry imaging (MSI) based proteomics, glycomics and tumor classification studies, to date comprehensive evaluation of fixation-related effects for spatially resolved metabolomics and drug disposition studies is still missing. In this study we used matrix assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) MSI to investigate the effect of formalin-fixation and formalin-fixation combined with paraffin embedding on the detectable metabolome including xenobiotics. Formalin fixation was found to cause significant washout of polar molecular species, including inorganic salts, amino acids, organic acids and carnitine species, oxidation of endogenous lipids and formation of reaction products between lipids and fixative ingredients. The slow fixation kinetics under ambient conditions resulted in increased lipid hydrolysis in the tissue core, correlating with the time-dependent progression of the fixation. Paraffin embedding resulted in subsequent partial removal of structural lipids resulting in the distortion of the elucidated biodistributions.

4.
Metabolites ; 12(3)2022 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-35323705

RESUMO

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a standard tool used for absolute quantification of drugs in pharmacokinetic (PK) studies. However, all spatial information is lost during the extraction and elucidation of a drugs biodistribution within the tissue is impossible. In the study presented here we used a sample embedding protocol optimized for mass spectrometry imaging (MSI) to prepare up to 15 rat intestine specimens at once. Desorption electrospray ionization (DESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) were employed to determine the distributions and relative abundances of four benchmarking compounds in the intestinal segments. High resolution MALDI-MSI experiments performed at 10 µm spatial resolution allowed to determine the drug distribution in the different intestinal histological compartments to determine the absorbed and tissue bound fractions of the drugs. The low tissue bound drug fractions, which were determined to account for 56-66% of the total drug, highlight the importance to understand the spatial distribution of drugs within the histological compartments of a given tissue to rationalize concentration differences found in PK studies. The mean drug abundances of four benchmark compounds determined by MSI were correlated with the absolute drug concentrations. Linear regression resulted in coefficients of determination (R2) ranging from 0.532 to 0.926 for MALDI-MSI and R2 values ranging from 0.585 to 0.945 for DESI-MSI, validating a quantitative relation of the imaging data. The good correlation of the absolute tissue concentrations of the benchmark compounds and the MSI data provides a bases for relative quantification of compounds within and between tissues, without normalization to an isotopically labelled standard, provided that the compared tissues have inherently similar ion suppression effects.

5.
Cancers (Basel) ; 13(23)2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34885051

RESUMO

BACKGROUND: Previous data on glycogen synthase kinase 3 (GSK-3) inhibition in cancer models support a cytotoxic effect with selectivity for tumor cells compared to normal tissue but the effect of these inhibitors in glioma has not been widely studied. Here, we investigate their potential as cytotoxics in glioma. METHODS: We assessed the effect of pharmacologic GSK-3 inhibition on established (U87, U251) and patient-derived (GBM1, GBM4) glioblastoma (GBM) cell lines using cytotoxicity assays as well as undertaking a detailed investigation of the effect on cell cycle, mitosis, and centrosome biology. We also assessed drug uptake and efficacy of GSK-3 inhibition alone and in combination with radiation in xenograft models. RESULTS: Using the selective GSK-3 inhibitor AZD2858, we demonstrated single agent cytotoxicity in two patient-derived glioma cell lines (GBM1, GBM4) and two established cell lines (U251 and U87) with IC50 in the low micromolar range promoting centrosome disruption, failed mitosis, and S-phase arrest. Glioma xenografts exposed to AZD2858 also showed growth delay compared to untreated controls. Combined treatment with radiation increased the cytotoxic effect of clinical radiation doses in vitro and in orthotopic glioma xenografts. CONCLUSIONS: These data suggest that GSK-3 inhibition promotes cell death in glioma through disrupting centrosome function and promoting mitotic failure and that AZD2858 is an effective adjuvant to radiation at clinical doses.

6.
Anal Chem ; 91(22): 14198-14202, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31660728

RESUMO

Absolute quantification of proteins in tissue is important for numerous fields of study. Liquid chromatography-mass spectrometry (LC-MS) methods are the norm but typically involve lengthy sample preparation including tissue homogenization, which results in the loss of information relating to spatial distribution. Here, we propose liquid extraction surface analysis (LESA) mass spectrometry (MS) of stable isotope labeled mimetic tissue models for the spatially resolved quantification of intact ubiquitin in rat and mouse brain tissue. Measured ubiquitin concentrations are in agreement with values found in the literature. Images of rat and mouse brain tissue demonstrate spatial variation in the concentration of ubiquitin and demonstrate the utility of spatially resolved quantitative measurement of proteins in tissue. Although we have focused on ubiquitin, the method has the potential for broader application to the absolute quantitation of any endogenous protein or protein-based drug in tissue.


Assuntos
Química Encefálica , Extração Líquido-Líquido/métodos , Espectrometria de Massas/métodos , Ubiquitina/análise , Animais , Cromatografia Líquida , Camundongos , Ratos
7.
Anal Chem ; 90(22): 13306-13314, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30350618

RESUMO

We have previously demonstrated liquid extraction surface analysis (LESA) high field asymmetric waveform ion mobility spectrometry (FAIMS) mass spectrometry imaging of proteins in thin tissue sections of brain and liver. Here, we present an improved approach that makes use of multiple static FAIMS parameters at each sampled location and allows a significant improvement in the number of proteins, lipids, and drugs that can be imaged simultaneously. The approach is applied to the mass spectrometry imaging of control and cassette-dosed rat kidneys. Mass spectrometry imaging of kidneys typically requires washing to remove excess hemoglobin; however, that is not necessary with this approach. Multistep static FAIMS mass spectrometry resulted in a 6- to 16-fold increase in the number of proteins detected in the absence of FAIMS, in addition to smaller increases over single step static FAIMS (chosen for optimum transmission of total protein ions). The benefits of multistep static FAIMS mass spectrometry for protein detection are also shown for sections of testes. The numbers of proteins detected following multistep FAIMS increased between 2- and 3-fold over single step FAIMS and between 2- and 14-fold over LESA alone. Finally, to date, LESA mass spectrometry of proteins in tissue has been undertaken solely on fresh frozen samples. In this work, we demonstrate that heat-preserved tissues are also suitable for these analyses. Heat preservation of tissue improved the number of proteins detected by LESA MS for both kidney and testes tissue (by between 2- and 4-fold). For both tissue types, the majority of the proteins additionally detected in the heat-treated samples were subsequently detected in the frozen samples when FAIMS was incorporated. Improvements in the numbers of proteins detected were observed for LESA FAIMS MS for the kidney tissue; for testes tissue, fewer total proteins were detected following heat preservation; however, approximately one-third were unique to the heat-preserved samples.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Extração Líquido-Líquido/métodos , Proteínas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Química Encefálica , Temperatura Baixa , Temperatura Alta , Rim/química , Masculino , Ratos Wistar , Propriedades de Superfície , Testículo/química
8.
Anal Chem ; 90(10): 6051-6058, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29668267

RESUMO

Described is a quantitative-mass-spectrometry-imaging (qMSI) methodology for the analysis of lactate and glutamate distributions in order to delineate heterogeneity among mouse tumor models used to support drug-discovery efficacy testing. We evaluate and report on preanalysis-stabilization methods aimed at improving the reproducibility and efficiency of quantitative assessments of endogenous molecules in tissues. Stability experiments demonstrate that optimum stabilization protocols consist of frozen-tissue embedding, post-tissue-sectioning desiccation, and storage at -80 °C of tissue sections sealed in vacuum-tight containers. Optimized stabilization protocols are used in combination with qMSI methodology for the absolute quantitation of lactate and glutamate in tumors, incorporating the use of two different stable-isotope-labeled versions of each analyte and spectral-clustering performed on each tissue section using k-means clustering to allow region-specific, pixel-by-pixel quantitation. Region-specific qMSI was used to screen different tumor models and identify a phenotype that has low lactate heterogeneity, which will enable accurate measurements of lactate modulation in future drug-discovery studies. We conclude that using optimized qMSI protocols, it is possible to quantify endogenous metabolites within tumors, and region-specific quantitation can provide valuable insight into tissue heterogeneity and the tumor microenvironment.


Assuntos
Ácido Glutâmico/análise , Ácido Láctico/análise , Espectrometria de Massas , Animais , Feminino , Ácido Glutâmico/metabolismo , Ácido Láctico/metabolismo , Camundongos , Camundongos Nus , Neoplasias Experimentais/química , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo
9.
Sci Rep ; 6: 37648, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27883030

RESUMO

Liquid extraction surface analysis mass spectrometry imaging (LESA-MSI) has been shown to be an effective tissue profiling and imaging technique, producing robust and reliable qualitative distribution images of an analyte or analytes in tissue sections. Here, we expand the use of LESA-MSI beyond qualitative analysis to a quantitative analytical technique by employing a mimetic tissue model previously shown to be applicable for MALDI-MSI quantitation. Liver homogenate was used to generate a viable and molecularly relevant control matrix for spiked drug standards which can be frozen, sectioned and subsequently analyzed for the generation of calibration curves to quantify unknown tissue section samples. The effects of extraction solvent composition, tissue thickness and solvent/tissue contact time were explored prior to any quantitative studies in order to optimize the LESA-MSI method across several different chemical entities. The use of a internal standard to normalize regional differences in ionization response across tissue sections was also investigated. Data are presented comparing quantitative results generated by LESA-MSI to LC-MS/MS. Subsequent analysis of adjacent tissue sections using DESI-MSI is also reported.


Assuntos
Cromatografia Líquida/métodos , Imageamento Tridimensional , Especificidade de Órgãos , Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Calibragem , Limite de Detecção , Masculino , Ratos Wistar , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
10.
Sci Transl Med ; 8(325): 325ra17, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26865565

RESUMO

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.


Assuntos
Aurora Quinases/antagonistas & inibidores , Nanopartículas/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Aurora Quinases/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Feminino , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos SCID , Organofosfatos/química , Organofosfatos/farmacocinética , Organofosfatos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Ratos Nus , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
11.
J Biomol Screen ; 21(2): 187-93, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26701101

RESUMO

Mass spectrometry imaging (MSI) provides pharmaceutical researchers with a suite of technologies to screen and assess compound distributions and relative abundances directly from tissue sections and offer insight into drug discovery-applicable queries such as blood-brain barrier access, tumor penetration/retention, and compound toxicity related to drug retention in specific organs/cell types. Label-free MSI offers advantages over label-based assays, such as quantitative whole-body autoradiography (QWBA), in the ability to simultaneously differentiate and monitor both drug and drug metabolites. Such discrimination is not possible by label-based assays if a drug metabolite still contains the radiolabel. Here, we present data exemplifying the advantages of MSI analysis. Data of the distribution of AZD2820, a therapeutic cyclic peptide, are related to corresponding QWBA data. Distribution of AZD2820 and two metabolites is achieved by MSI, which [(14)C]AZD2820 QWBA fails to differentiate. Furthermore, the high mass-resolving power of Fourier transform ion cyclotron resonance MS is used to separate closely associated ions.


Assuntos
Diagnóstico por Imagem/métodos , Preparações Farmacêuticas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Autorradiografia/métodos , Barreira Hematoencefálica/metabolismo , Descoberta de Drogas/métodos , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Peptídeos/metabolismo
12.
Anal Chem ; 87(19): 10146-52, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26350423

RESUMO

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a surface sampling technique that incorporates liquid extraction from the surface of tissue sections with nanoelectrospray mass spectrometry. Traditional tissue analysis techniques usually require homogenization of the sample prior to analysis via high-performance liquid chromatography mass spectrometry (HPLC-MS), but an intrinsic weakness of this is a loss of all spatial information and the inability of the technique to distinguish between actual tissue penetration and response caused by residual blood contamination. LESA-MS, in contrast, has the ability to spatially resolve drug distributions and has historically been used to profile discrete spots on the surface of tissue sections. Here, we use the technique as a mass spectrometry imaging (MSI) tool, extracting points at 1 mm spatial resolution across tissue sections to build an image of xenobiotic and endogenous compound distribution to assess drug blood-brain barrier penetration into brain tissue. A selection of penetrant and "nonpenetrant" drugs were dosed to rats via oral and intravenous administration. Whole brains were snap-frozen at necropsy and were subsequently sectioned prior to analysis by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and LESA-MSI. MALDI-MSI, as expected, was shown to effectively map the distribution of brain penetrative compounds but lacked sufficient sensitivity when compounds were marginally penetrative. LESA-MSI was used to effectively map the distribution of these poorly penetrative compounds, highlighting its value as a complementary technique to MALDI-MSI. The technique also showed benefits when compared to traditional homogenization, particularly for drugs that were considered nonpenetrant by homogenization but were shown to have a measurable penetration using LESA-MSI.


Assuntos
Encéfalo/metabolismo , Preparações Farmacêuticas/análise , Farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Masculino , Ratos , Ratos Wistar , Distribuição Tecidual
13.
Chem Res Toxicol ; 28(9): 1823-30, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26293472

RESUMO

Colistin and polymyxin B are effective treatment options for Gram-negative resistant bacteria but are used as last-line therapy due to their dose-limiting nephrotoxicity. A critical factor in developing safer polymyxin analogues is understanding accumulation of the drugs and their metabolites, which is currently limited due to the lack of effective techniques for analysis of these challenging molecules. Mass spectrometry imaging (MSI) allows direct detection of targets (drugs, metabolites, and endogenous compounds) from tissue sections. The presented study exemplifies the utility of MSI by measuring the distribution of polymyxin B1, colistin, and polymyxin B nonapeptide (PMBN) within dosed rat kidney tissue sections. The label-free MSI analysis revealed that the nephrotoxic compounds (polymyxin B1 and colistin) preferentially accumulated in the renal cortical region. The less nephrotoxic analogue, polymyxin B nonapeptide, was more uniformly distributed throughout the kidney. In addition, metabolites of the dosed compounds were detected by MSI. Kidney homogenates were analyzed using LC/MS/MS to determine total drug exposure and for metabolite identification. To our knowledge, this is the first time such techniques have been utilized to measure the distribution of polymyxin drugs and their metabolites. By simultaneously detecting the distribution of drug and drug metabolites, MSI offers a powerful alternative to tissue homogenization analysis and label or antibody-based imaging.


Assuntos
Rim/efeitos dos fármacos , Polimixinas/toxicidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Cromatografia Líquida , Masculino , Polimixinas/farmacocinética , Ratos , Ratos Wistar
14.
Anal Chem ; 86(16): 8473-80, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25084360

RESUMO

Cassette dosing of compounds for preclinical drug plasma pharmacokinetic analysis has been shown to be a powerful strategy within the pharmaceutical industry for increasing throughput while decreasing the number of animals used. Presented here for the first time is data on the application of a cassette dosing strategy for label-free tissue distribution studies. The aim of the study was to image the spatial distribution of eight nonproprietary drugs (haloperidol, bufuralol, midazolam, clozapine, terfenadine, erlotinib, olanzapine, and moxifloxacin) in multiple tissues after oral and intravenous cassette dosing (four compounds per dose route). An array of mass spectrometry imaging technologies, including matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI), liquid extraction surface analysis tandem mass spectrometry (LESA-MS/MS), and desorption electrospray ionization mass spectrometry (DESI-MS) was used. Tissue analysis following intravenous and oral administration of discretely and cassette-dosed compounds demonstrated similar relative abundances across a range of tissues indicating that a cassette dosing approach was applicable. MALDI MSI was unsuccessful in detecting all of the target compounds; therefore, DESI MSI, a complementary mass spectrometry imaging technique, was used to detect additional target compounds. In addition, by adapting technology used for tissue profiling (LESA-MS/MS) low spatial resolution mass spectrometry imaging (∼1 mm) was possible for all targets across all tissues. This study exemplifies the power of multiplatform MSI analysis within a pharmaceutical research and development (R&D) environment. Furthermore, we have illustrated that the cassette dosing approach can be readily applied to provide combined, label-free pharmacokinetic and drug distribution data at an early stage of the drug discovery/development process while minimizing animal usage.


Assuntos
Preparações Farmacêuticas/administração & dosagem , Farmacocinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Administração Intravenosa , Administração Oral , Animais , Descoberta de Drogas , Masculino , Camundongos , Ratos Wistar , Espectrometria de Massas em Tandem/métodos , Distribuição Tecidual
15.
J Med Chem ; 57(3): 970-86, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24422550

RESUMO

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) has been widely considered by the pharmaceutical industry as a target to treat metabolic syndrome in type II diabetics. We hypothesized that central nervous system (CNS) penetration might be required to see efficacy. Starting from a previously reported pyrimidine compound, we removed hydrogen-bond donors to yield 3, which had modest CNS penetration. More significant progress was achieved by changing the core to give 40, which combines good potency and CNS penetration. Compound 40 was dosed to diet-induced obese (DIO) mice and gave excellent target engagement in the liver and high free exposures of drug, both peripherally and in the CNS. However, no body weight reduction or effects on glucose or insulin were observed in this model. Similar data were obtained with a structurally diverse thiazole compound 51. This work casts doubt on the hypothesis that localized tissue modulation of 11ß-HSD1 activity alleviates metabolic syndrome.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Adamantano/análogos & derivados , Adamantano/síntese química , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/síntese química , Síndrome Metabólica/tratamento farmacológico , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Adamantano/farmacocinética , Adamantano/farmacologia , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Cristalografia por Raios X , Ciclopropanos/síntese química , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Gorduras na Dieta/administração & dosagem , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Insulina/sangue , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Modelos Moleculares , Pirazóis/síntese química , Pirazóis/farmacocinética , Pirazóis/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacocinética , Tiazóis/farmacologia
16.
Endocrinology ; 154(12): 4580-93, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24169553

RESUMO

The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11ß-HSD1 inhibitor (compound C) inhibited liver 11ß-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)-fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11ß-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11ß-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting "off-target" mechanisms. Based on the patent literature, we synthesized another 11ß-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11ß-HSD1 inhibitors occurs via 11ß-HSD1-independent pathways, and only limited efficacy is achievable from selective 11ß-HSD1 inhibition. These data challenge the concept that inhibition of 11ß-HSD1 is likely to produce a "step-change" treatment for diabetes and/or obesity.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Metabolismo Energético/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Triazóis/farmacologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Tecido Adiposo/metabolismo , Animais , Glicemia , Peso Corporal , Encéfalo/metabolismo , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Genótipo , Glucose/metabolismo , Hipoglicemiantes/química , Insulina/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Estrutura Molecular , Pirazóis/química , Pirimidinas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triazóis/química
17.
J Med Chem ; 55(22): 10136-47, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-23088558

RESUMO

Inhibition of 11ß-HSD1 is viewed as a potential target for the treatment of obesity and other elements of the metabolic syndrome. We report here the optimization of a carboxylic acid class of inhibitors from AZD4017 (1) to the development candidate AZD8329 (27). A structural change from pyridine to pyrazole together with structural optimization led to an improved technical profile in terms of both solubility and pharmacokinetics. The extent of acyl glucuronidation was reduced through structural optimization of both the carboxylic acid and amide substituents, coupled with a reduction in lipophilicity leading to an overall increase in metabolic stability.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Benzoatos/farmacologia , Inibidores Enzimáticos/farmacologia , Glucuronídeos/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/enzimologia , Animais , Benzoatos/síntese química , Benzoatos/farmacocinética , Cães , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Glucuronídeos/química , Cobaias , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Macaca fascicularis , Camundongos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Pirazóis/síntese química , Pirazóis/farmacocinética , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Bioorg Med Chem Lett ; 22(21): 6756-61, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23013933

RESUMO

11ß-HSD1 is increasingly seen as an attractive target for the treatment of type II diabetes and other elements of the metabolic syndrome. In this program of work we describe how a series of neutral 2-thioalkyl-pyridine 11ß-HSD1 inhibitors were optimized in terms of their pharmacokinetic properties to give compounds with excellent bioavailability in both rat and dog through a core change to pyrimidine. A potential reactive metabolite issue with 4-thioalkyl-pyrimidines was circumvented by a switch from sulfur to carbon substitution.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacocinética , Piridinas/química , Compostos de Sulfidrila/química , Animais , Cães , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Estrutura Molecular , Piridinas/farmacocinética , Ratos , Compostos de Sulfidrila/farmacocinética
19.
J Med Chem ; 55(12): 5951-64, 2012 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-22691057

RESUMO

Inhibition of 11ß-HSD1 is an attractive mechanism for the treatment of obesity and other elements of the metabolic syndrome. We report here the discovery of a nicotinic amide derived carboxylic acid class of inhibitors that has good potency, selectivity, and pharmacokinetic characteristics. Compound 11i (AZD4017) is an effective inhibitor of 11ß-HSD1 in human adipocytes and exhibits good druglike properties and as a consequence was selected for clinical development.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Niacinamida/análogos & derivados , Piperidinas/farmacologia , Piperidinas/farmacocinética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Modelos Moleculares , Niacinamida/administração & dosagem , Niacinamida/metabolismo , Niacinamida/farmacocinética , Niacinamida/farmacologia , Piperidinas/administração & dosagem , Piperidinas/metabolismo , Conformação Proteica , Ratos , Especificidade por Substrato
20.
Bioanalysis ; 4(11): 1327-35, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22720651

RESUMO

BACKGROUND: Laser diode thermal desorption (LDTD) is a relatively new sample introduction interface for MS. Analysis times are short as the technique does not require time-consuming separation steps, such as conventional HPLC, thus saving on the use of organic solvents, modifiers and cost, relating to their subsequent disposal. This paper compares the merits of LDTD-APCI-MS/MS and LC-MS/MS for the analysis of paracetamol (acetaminophen) in plasma from different species. RESULTS: LDTD-APCI-MS/MS compared favorably with our existing high-throughput generic LC-MS/MS method giving improved data quality. LDTD-APCI-MS/MS assay performance in terms of accuracy and precision in mouse, rat and dog plasma were within our local acceptance criteria (±30%). Run times were reduced approximately tenfold, while saving approximately 200 ml of solvent per 96-well plate. CONCLUSION: A rapid, sensitive and robust assay is reported. The method was successfully used for the analysis of spiked mouse, rat and dog plasma samples and the determination of oral pharmacokinetics. Reductions in electrical power and reagent consumption position LDTD as an environmentally 'green' bioanalytical method.


Assuntos
Acetaminofen/sangue , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Administração Oral , Animais , Cães , Lasers , Masculino , Camundongos , Ratos , Ratos Wistar , Temperatura
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