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1.
J Pharmacol Exp Ther ; 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30361237

RESUMO

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of OAT1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1,000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 h after receiving PROB (40 mg and 1,000 mg orally) on Days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased AUC of PDA by 3.1-fold (dosed alone; p < 0.05), and 3.2-fold (coadministered with FSM; p < 0.01), as compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold (p > 0.05) and 2.1-fold (p < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC whereas those of HVA are smaller (3.1-3.2 and 1.8-2.1 versus 3.3, respectively). PDA and HVA CLR were decreased by PROB to smaller extents compared to FSM (0.35-0.37 and 0.67-0.73 versus 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and its plasma exposure respond in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CLR between subjects is more favourable relative to HVA.

2.
Eur J Pharmacol ; 812: 104-112, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28690193

RESUMO

Alzheimer's disease is associated with the accumulation of amyloid-ß (Aß) in the brain. In particular, the 42-amino acid form, Aß1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aß1-42 production without inhibiting the production of other forms of Aß. Here we describe the novel discovery of synergistic inhibition of Aß by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aß peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aß occurred only when one GSM was of the "acid GSM" structural class and the other was of the "non-acid GSM" class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aß production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aß. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Fragmentos de Peptídeos/biossíntese , Inibidores de Proteases/farmacologia , Acetatos/farmacologia , Animais , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Camundongos , Piperidinas/farmacologia
3.
Drug Metab Dispos ; 45(8): 908-919, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28576766

RESUMO

Multiple endogenous compounds have been proposed as candidate biomarkers to monitor organic anion transporting polypeptide (OATP) function in preclinical species or humans. Previously, we demonstrated that coproporphyrins (CPs) I and III are appropriate clinical markers to evaluate OATP inhibition and recapitulate clinical drug-drug interactions (DDIs). In the present study, we investigated bile acids (BAs) dehydroepiandrosterone sulfate (DHEAS), hexadecanedioate (HDA), and tetradecanedioate (TDA) in plasma as endogenous probes for OATP inhibition and compared these candidate probes to CPs. All probes were determined in samples from a single study that examined their behavior and their association with rosuvastatin (RSV) pharmacokinetics after administration of an OATP inhibitor rifampin (RIF) in healthy subjects. Among endogenous probes examined, RIF significantly increased maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC)(0-24h) of fatty acids HDA and TDA by 2.2- to 3.2-fold. For the 13 bile acids in plasma examined, no statistically significant changes were detected between treatments. Changes in plasma DHEAS did not correlate with OATP1B inhibition by RIF. On the basis of the magnitude of effects for the endogenous compounds that demonstrated significant changes from baseline over interindividual variations, the overall rank order for the AUC change was found to be CP I > CP III > HDA ≈ TDA ≈ RSV > > BAs. Collectively, these results reconfirmed that CPs are novel biomarkers suitable for clinical use. In addition, HDA and TDA are useful for OATP functional assessment. Since these endogenous markers can be monitored in conjunction with pharmacokinetics analysis, the CPs and fatty acid dicarboxylates, either alone or in combination, offer promise of earlier diagnosis and risk stratification for OATP-mediated DDIs.


Assuntos
Ácidos e Sais Biliares/sangue , Biomarcadores/sangue , Coproporfirinas/sangue , Sulfato de Desidroepiandrosterona/sangue , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Ácidos Palmíticos/sangue , Adolescente , Adulto , Área Sob a Curva , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Interações de Medicamentos/fisiologia , Células HEK293 , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Rifampina/farmacologia , Rosuvastatina Cálcica/farmacologia , Adulto Jovem
4.
J Pharm Biomed Anal ; 138: 166-174, 2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28213177

RESUMO

Synthetic macrocyclic peptides with natural and unnatural amino acids have gained considerable attention from a number of pharmaceutical/biopharmaceutical companies in recent years as a promising approach to drug discovery, particularly for targets involving protein-protein or protein-peptide interactions. Analytical scientists charged with characterizing these leads face multiple challenges including dealing with a class of complex molecules with the potential for multiple isomers and variable charge states and no established standards for acceptable analytical characterization of materials used in drug discovery. In addition, due to the lack of intermediate purification during solid phase peptide synthesis, the final products usually contain a complex profile of impurities. In this paper, practical analytical strategies and methodologies were developed to address these challenges, including a tiered approach to assessing the purity of macrocyclic peptides at different stages of drug discovery. Our results also showed that successful progression and characterization of a new drug discovery modality benefited from active analytical engagement, focusing on fit-for-purpose analyses and leveraging a broad palette of analytical technologies and resources.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Descoberta de Drogas/métodos , Imagem por Ressonância Magnética/métodos , Peptídeos/química , Aminoácidos/química
5.
J Pharmacol Exp Ther ; 358(1): 138-50, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27189973

RESUMO

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aß39, Aß40, and Aß42 while increasing Aß37 and Aß38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aß40 and Aß42 were observed with no change in total Aß; in CSF, modest decreases in total Aß were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aß42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aß lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides , Compostos de Anilina/farmacologia , Compostos de Anilina/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Adolescente , Adulto , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Compostos de Anilina/efeitos adversos , Compostos de Anilina/química , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/química , Área Sob a Curva , Cromatografia Líquida , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Voluntários Saudáveis , Humanos , Limite de Detecção , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Pirimidinas/efeitos adversos , Pirimidinas/química , Adulto Jovem
6.
J Pharmacol Exp Ther ; 358(1): 125-37, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27189974

RESUMO

The amyloid-ß peptide (Aß)-in particular, the 42-amino acid form, Aß1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aß synthesis or increase the clearance of Aß have entered clinical trials, including γ-secretase inhibitors, anti-Aß antibodies, and amyloid-ß precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aß1-42 production, and may also decrease Aß1-40 while simultaneously increasing one or more shorter Aß peptides, such as Aß1-38 and Aß1-37. GSMs are particularly attractive because they do not alter the total amount of Aß peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, ß-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aß1-42 and Aß1-40 levels while increasing Aß1-38 and Aß1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Compostos de Anilina/farmacologia , Compostos de Anilina/farmacocinética , Encéfalo/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/genética , Compostos de Anilina/química , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Hidrocarbonetos Aromáticos com Pontes/química , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Macaca fascicularis , Pirimidinas/química , Ratos Sprague-Dawley , Receptores Notch/metabolismo , Especificidade da Espécie , Distribuição Tecidual
7.
Anal Biochem ; 503: 71-8, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27033006

RESUMO

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.


Assuntos
Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Marcação por Isótopo , Metabolômica , Animais , Ácidos e Sais Biliares/química , Cromatografia Líquida , Humanos , Espectrometria de Massas , Estrutura Molecular , Ratos
8.
Drug Metab Dispos ; 44(8): 1332-1340, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26961241

RESUMO

During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), a bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of bile samples revealed, in addition to multiple products of bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1 demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B inhibitors was assumed to result from two sequential reactions: hydroxylation of the bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of the hydroxylation product in vitro. The observation of this unexpected pathway prompted concerns about the possibility that 1 and 2 might interfere with routine phospholipid synthesis. These results demonstrate the participation in xenobiotic metabolism of a process whose function is ordinarily limited to the synthesis of endogenous compounds.

9.
J Pharm Sci ; 105(3): 1036-42, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26886323

RESUMO

Membrane-based devices typically used for serum protein binding determination are not fully applicable to highly lipophilic compounds because of nonspecific binding to the device membrane. Ultracentrifugation, however, completely eliminates the issue by using a membrane-free approach, although its wide application has been limited. This lack of utilization is mainly attributed to 2 factors: the high cost in acquiring and handling of radiolabeled compounds and low assay throughput owing to the difficulties in process automation. To overcome these challenges, we report a high-throughput workflow by cassette ultracentrifugation of nonradiolabeled compounds followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Twenty compounds with diverse physicochemical and protein binding properties were selected for the evaluation of the workflow. To streamline the working process, approaches of matrix balancing for all the samples for LC-MS/MS analysis and determining free fraction without analytical calibration curves were adopted. Both the discrete ultracentrifugation of individual compounds and cassette ultracentrifugation of all the test compounds followed by simultaneous LC-MS/MS analysis exhibited a linear correlation with literature values, demonstrating respectively the validity of the ultracentrifugation process and the cassette approach. The cassette ultracentrifugation using nonradiolabeled compounds followed by LC-MS/MS analysis has greatly facilitated its application for high-throughput protein binding screening in drug discovery.


Assuntos
Proteínas Sanguíneas/química , Cromatografia Líquida de Alta Pressão/métodos , Preparações Farmacêuticas/química , Ligação Proteica , Espectrometria de Massas em Tandem/métodos , Ultracentrifugação/métodos , Descoberta de Drogas/métodos , Marcação por Isótopo/métodos , Soro/química , Coloração e Rotulagem/métodos , Fluxo de Trabalho
10.
J Biomol Screen ; 21(2): 165-75, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26420787

RESUMO

The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)-based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)-tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1'-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4'-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)-based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays.


Assuntos
Inibidores das Enzimas do Citocromo P-450/química , Descoberta de Drogas/métodos , Bioensaio/métodos , Calibragem , Ensaios de Triagem em Larga Escala/métodos , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos
11.
J Lab Autom ; 21(1): 204-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26203056

RESUMO

In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample.


Assuntos
Tecnologia Biomédica/métodos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acústica , Controle de Qualidade , Soluções
12.
Bioanalysis ; 7(19): 2501-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26466806

RESUMO

The analysis of endogenous and exogenous analytes in biological matrices presents several challenges to the bioanalyst. These analytes are often present at low concentrations, typically in complex matrices, and may have physicochemical properties that are not amenable to LC-MS analysis. The bioanalyst thus relies heavily on the formation of analyte derivatives for the efficient quantification of these compounds. These derivatives are also critically employed to derive information on the biology of living systems, potential drug or disease targets, and biomarkers of drug efficacy, safety, or disease progression. In this perspective, we demonstrate how analyte derivatives are applied in modern bioanalytical workflows and we discuss the potential use of these derivatives in the future.


Assuntos
Biomarcadores/análise , Preparações Farmacêuticas/análise , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Humanos , Marcação por Isótopo , Preparações Farmacêuticas/química
13.
Bioanalysis ; 7(20): 2629-37, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26511148

RESUMO

Drug absorption, distribution, metabolism, excretion and toxicology study is one important step in drug discovery and development. MS imaging has become one of the popular methods in this field. Here, selected ionization methods such as matrix-assisted laser desorption/ionization, secondary ion MS and desorption electrospray ionization have been briefly discussed. To differentiate drug and drug metabolites from endogenous compounds present in the biological system, exact mass and/or tandem MS is necessary. As a result, mass analyzers such as time-of-flight, Fourier transform ion cyclotron resonance or Orbitrap are often the method of choice and are briefly introduced.


Assuntos
Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Análise de Fourier , Íons/química , Lasers , Peso Molecular , Preparações Farmacêuticas/metabolismo
14.
Biomed Chromatogr ; 29(9): 1375-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25712252

RESUMO

Acetyl-L-carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a high-throughput scale, a new and simple HILIC-MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via 'dilute and shoot' directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC-MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high-throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies.


Assuntos
Acetilcarnitina/líquido cefalorraquidiano , Espectrometria de Massas em Tandem/métodos , Acetilcarnitina/química , Animais , Cães , Humanos , Interações Hidrofóbicas e Hidrofílicas , Macaca fascicularis , Camundongos , Ratos , Espectrometria de Massas em Tandem/instrumentação
15.
Anal Chem ; 86(23): 11523-7, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25371986

RESUMO

Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.


Assuntos
Dissulfetos/química , Peptídeos Cíclicos/sangue , Peptídeos Cíclicos/química , Cromatografia Líquida , Humanos , Íons/análise , Íons/química , Espectrometria de Massas em Tandem
16.
J Biomol Screen ; 19(10): 1409-14, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25296658

RESUMO

Plastic labware is used in all processes of modern pharmaceutical research, including compound storage and biological assays. The use of these plastics has created vast increases in productivity and cost savings as experiments moved from glass test tubes and capillary pipettes to plastic microplates and multichannel liquid handlers. One consequence of the use of plastic labware, however, is the potential release of contaminants and their resultant effects on biological assays. We report herein the identification of biologically active substances released from a commonly used plastic microplate. The active contaminants were identified by gas chromatography-mass spectroscopy as dodecan-1-ol, dodecyl 3-(3-dodecoxy-3-oxopropyl)sulfanylpropanoate, and dodecanoic acid, and they were found to be selective monoamine oxidase-B inhibitors.


Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Inibidores da Monoaminoxidase/farmacologia , Plásticos/química , Ácido 3-Mercaptopropiônico/análogos & derivados , Ácido 3-Mercaptopropiônico/farmacologia , Dodecanol/química , Dodecanol/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Láuricos/farmacologia , Monoaminoxidase/metabolismo , Plásticos/farmacologia , Razão Sinal-Ruído , Sulfetos/farmacologia
17.
Anal Biochem ; 466: 65-71, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25175011

RESUMO

Microtubules (MTs) are highly dynamic polymers composed of α- and ß-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography-mass spectrometry (LC-MS) method to measure the fraction of [(13)C6]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [(13)C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.


Assuntos
Bioensaio/métodos , Marcação por Isótopo/métodos , Microtúbulos/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/citologia , Técnicas de Cultura de Células , Cromatografia Líquida , Espectrometria de Massas , Neurônios/citologia , Peptídeos/química , Ratos , Reprodutibilidade dos Testes
18.
Bioanalysis ; 6(14): 1901-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25158962

RESUMO

BACKGROUND: (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid, also known as LY379268, a group II metabotropic glutamate receptor agonist, has been widely used in neuroscience as a model compound in studies evaluating antipsychotic drugs for the treatment of schizophrenia. MATERIALS & METHODS: So far, no reports describing methods of the bioanalysis of LY379268 have been published. Here, a novel method is presented for determining LY379268 in rat plasma employing precolumn derivatization with pentafluorobenzoyl chloride reagent coupled to liquid chromatography/mass spectrometry. CONCLUSION: Chemical derivatization of a low-molecular-weight and highly polar molecule yields a derivative that is retained on a reversed-phase liquid chromatography column with improved tandem mass spectrometric response.


Assuntos
Aminoácidos/sangue , Compostos Bicíclicos Heterocíclicos com Pontes/sangue , Cromatografia Líquida/métodos , Receptores de Glutamato Metabotrópico/agonistas , Espectrometria de Massas em Tandem/métodos , Aminoácidos/química , Animais , Benzoatos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Ratos
19.
Int J Alzheimers Dis ; 2014: 431858, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25097793

RESUMO

Alzheimer's disease is the most prevalent cause of dementia and is associated with accumulation of amyloid-ß peptide (Aß), particularly the 42-amino acid Aß1-42, in the brain. Aß1-42 levels can be decreased by γ-secretase modulators (GSM), which are small molecules that modulate γ-secretase, an enzyme essential for Aß production. BMS-869780 is a potent GSM that decreased Aß1-42 and Aß1-40 and increased Aß1-37 and Aß1-38, without inhibiting overall levels of Aß peptides or other APP processing intermediates. BMS-869780 also did not inhibit Notch processing by γ-secretase and lowered brain Aß1-42 without evidence of Notch-related side effects in rats. Human pharmacokinetic (PK) parameters were predicted through allometric scaling of PK in rat, dog, and monkey and were combined with the rat pharmacodynamic (PD) parameters to predict the relationship between BMS-869780 dose, exposure and Aß1-42 levels in human. Off-target and safety margins were then based on comparisons to the predicted exposure required for robust Aß1-42 lowering. Because of insufficient safety predictions and the relatively high predicted human daily dose of 700 mg, further evaluation of BMS-869780 as a potential clinical candidate was discontinued. Nevertheless, BMS-869780 demonstrates the potential of the GSM approach for robust lowering of brain Aß1-42 without Notch-related side effects.

20.
Anal Biochem ; 452: 10-2, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24534252

RESUMO

L-serine-O-phosphate (L-SOP), the precursor of L-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of L-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine L-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization-LC/MS, d-LC/MS).


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Fosfosserina/líquido cefalorraquidiano , Fosfosserina/química , Compostos Organofosforados/química , Succinimidas/química
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