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During the production process, the author order of Zhandong Don Zhong and Lynn L. Jiang were inadvertently placed. Lynn L. Jiang is the first author of this manuscript; Zhandong Don Zhong is the last author.
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Biologics can potentially induce unwanted immune responses, leading to formation of antidrug antibodies (ADA) of various affinity, isotypes, and subclasses. Among them, antigen and drug-specific immunoglobulin E (IgE) antibodies have been reported to have potential correlation with hypersensitivity and anaphylaxis in particular. Recent regulatory guidance on immunogenicity testing has recommended the measurement of antigen-specific IgE antibodies for biologics with a reported high risk of anaphylaxis using assays with sensitivities in the high pg/mL to low ng/mL range. Nevertheless, IgE ADA remains challenging to detect due to their being the least abundant isotype in blood serum samples and the potential for interference in the bioanalytical methods due to high levels of endogenous immunoglobulin G (IgG) and immunoglobulin M (IgM) ADA, not to mention the nonspecific total serum IgE antibodies. Another challenge in developing IgE ADA assays is the need to create a surrogate drug-specific IgE antibody positive control to monitor the performance of the assay for the intended use. In this case study, utilizing a human IgE antidrug antibody positive control and a human IgE receptor as capture, an enzyme-linked immunosorbent assay (ELISA) method was developed for the measurement of IgE ADA, meeting the regulatory expectations, with excellent assay sensitivity, selectivity, specificity, and tolerance towards potential interference in serum samples. This assay format could be readily adapted and implemented to assess drug-specific IgE antibodies in the event of drug-related anaphylaxis in clinical and in nonclinical development programs.
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Produtos Biológicos/imunologia , Hipersensibilidade a Drogas/diagnóstico , Ensaio de Imunoadsorção Enzimática , Imunoglobulina E/sangue , Produtos Biológicos/efeitos adversos , Hipersensibilidade a Drogas/sangue , Hipersensibilidade a Drogas/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
To compare pharmacokinetics, metabolism and excretion of levodopa and a triply deuterated form, which is being developed as an improved treatment for Parkinson's disease, methods were needed for quantification of the deuterated and nondeuterated forms of levodopa and five metabolites in human plasma and urine. Results: The natural heavy isotopes in the nondeuterated compounds caused an absolute contribution of up to 100% in the response of the deuterated compounds. Similarly, heavy isotopes in the deuterated analytes contributed to the response of the internal standards, but this did not affect the reliability of the results. Conclusion: Deuterated and nondeuterated analytes can be quantified together by LC-MS/MS, but overestimation of the concentrations of the deuterated molecules may be unavoidable and a careful interpretation of the concentration data is essential.
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Sensitive LC-MS/MS methods were developed to measure lidocaine and its metabolite 2,6-dimethylaniline (2,6-DMA) with application to transdermal studies. The methods for lidocaine in minipig plasma, tissue biopsies, and dermal tapes utilized mixed mode/SCX solid phase extraction, with lower quantitation limits of 25â¯pg/mL in plasma, 15â¯ng/g tissue, and 5â¯ng/tape. 2,6-DMA was measured in plasma and skin tissue homogenates by ultrafiltration and (for tissue) by further derivatization with 4-methoxybenzoyl chloride to form the corresponding benzamide derivative, which extended the lower limit of quantitation to 200â¯pg/mL. The methods allowed local measurement of lidocaine in stratum corneum, punch biopsies, and plasma and of 2,6-DMA in plasma and biopsies obtained from minipigs dosed with experimental transdermal formulations. Quantitation limits were approximately 7-fold lower than previously reported for lidocaine and 3-fold lower for 2,6-DMA.
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Compostos de Anilina/sangue , Cromatografia Líquida de Alta Pressão/métodos , Lidocaína/sangue , Pele/química , Espectrometria de Massas em Tandem/métodos , Adesivos , Administração Cutânea , Compostos de Anilina/administração & dosagem , Compostos de Anilina/análise , Compostos de Anilina/farmacocinética , Animais , Feminino , Lidocaína/administração & dosagem , Lidocaína/análise , Lidocaína/farmacocinética , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pele/metabolismo , SuínosRESUMO
MDT-637 is a new chemical entity that shows potent and selective antiviral activity against respiratory syncytial virus. As part of the new drug development program, a method for quantitating MDT-637 in nasal wash collected from human subjects was required. This article describes the method development and validation and application of a bioanalytical assay for MDT-637 in human nasal wash using liquid chromatography-tandem mass spectrometry. Sample pretreatment utilized supported liquid extraction and final extracts were injected onto an Agilent Poroshell 120 EC-C18 column and eluted under isocratic conditions of 65:35 (v/v) 10 mm ammonium formate in water and acetonitrile Detection was performed on an API 5000 tandem mass spectrometer operating in negative electrospray ionization mode applying multiple reaction monitoring. The assay was validated in accordance with the US Food and Drug Administration guidance on bioanalytical method validation and was applied in support of a Phase IIa human safety and tolerability study.
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A sensitive, selective, and reliable LC-MS/MS method was developed and validated for simultaneous quantification of venlafaxine (VEN) and its 5 metabolites (ODV, NDV, NNDDV, OHV and NODDV) in rat plasma. The calibration ranges are 15.0 to 6000â¯ng/mL for VEN, 1.00 to 400â¯ng/mL for ODV, 5.00 to 2000â¯ng/mL for NDV, 1.00 to 400â¯ng/mL for NNDDV, 10.0 to 4000â¯ng/mL for OHV, and 0.200 to 20.0â¯ng/mL for NODDV. Briefly, 50⯵L of rat plasma was extracted using liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE). The analytes were separated on an Agilent SB-Phenyl (50â¯mmâ¯×â¯4.6â¯mm, 3.5⯵m) column using a binary gradient of 0.1% formic acid in water versus 0.1% formic acid in acetonitrile at a flow rate of 0.8â¯mL/min. The method was validated following FDA guidance for bioanalytical method validation. Validated method was successfully applied to a pharmacokinetic study of VEN orally administered to rats.
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Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Cloridrato de Venlafaxina/sangue , Cloridrato de Venlafaxina/farmacocinética , Animais , Estabilidade de Medicamentos , Feminino , Modelos Lineares , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Cloridrato de Venlafaxina/químicaRESUMO
The reliable quantification of carbidopa in biological samples at low concentrations is challenging because of the polar and highly unstable nature of the compound. In this paper, LC-MS/MS methods are described for the determination of carbidopa in 50µL of human plasma and 25µL of human urine in the concentration ranges 1-1,000ng/mL and 100-50,000ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40°C. The product is a relatively non-polar molecule that is suitable for reversed-phase liquid chromatography (3.5min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalization. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5mg/mL sodium metabisulfite and 1mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5mg oral dose.
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Carbidopa/sangue , Carbidopa/urina , Cromatografia Líquida/métodos , Pentanonas/química , Espectrometria de Massas em Tandem/métodos , Carbidopa/farmacocinética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Antibody drug conjugates (ADCs) have emerged as a viable option in targeted delivery of highly potent cytotoxic drugs in treatment of solid tumors. At the time of writing, only two ADCs have received regulatory approval with >40 others in clinical development. The first generation ADCs suffered from a lack of specificity in amino acid site-conjugations, yielding statistically heterogeneous stoichiometric ratios of drug molecules per antibody molecule. For the second generation ADCs, however, site-specific amino acid conjugation using enzymatic ligation, introduction of unnatural amino acids, and site-specific protein engineering hold promise to alleviate some of the current technical limitations. The rapid progress in technology platforms and antibody engineering has introduced novel linkers, site-specific conjugation chemistry, and new payload candidates that could possibly be exploited in the context of ADCs. A search using the Clinical Trial Database registry ( www.clinicaltrials.gov ), using the keyword 'antibody drug conjugate', yielded ~270 hits. The main focus of this article is to present a brief overview of the recent developments and current challenges related to ADC development.
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Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Antineoplásicos/química , Sítios de Ligação de Anticorpos , Ensaios Clínicos como Assunto , Humanos , Imunoconjugados/química , Neoplasias/imunologia , Engenharia de ProteínasRESUMO
After more than 40 years, immunoassays are still the backbone of protein biomarker analysis in clinical diagnostics and drug development. They have come a long way since their inception, incorporating technical developments including monoclonal antibodies, novel labels and lately microfluidics. A number of microfluidic platforms have been tested, such as centrifugational compact disc assays, lab-on-a-chip, arrays and digital electrochemical assays. This review focuses on commercial applications of microfluidic immunoassays with reference to some applied academic examples of interest. Advantages and disadvantages of the platform technologies are discussed in general.
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Imunoensaio/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Miniaturização/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Miniaturização/métodosRESUMO
BACKGROUND: Dalotuzumab (MK 0646), an anti-IGF1R antibody intended for cancer therapy, has progressed to Phase III clinical trials. To evaluate pharmacokinetic properties, we developed and compared two ELISAs to measure dalotuzumab in human serum and validated the second method following regulatory guidelines for ligand-binding assays. RESULTS: After an IGF1R-mediated capture step, dalotuzumab was detected by either an antihuman IgGFc- or by an antihuman IgG1-specific antibody. The assay range was 20 to 2000 ng/ml with mean inter-day accuracy of controls ranging from 97 to 108% (method A) and 83 to 97% (method B), respectively. Mean assay precision was ≤20% CV both intra- and inter-day. Other parameters that were validated included dilution linearity, stability, interferences and incurred sample reanalysis. In addition, application of both assay formats to clinical sample analysis was demonstrated establishing time-concentration curves. CONCLUSION: As the methods rely on commercial reagents, they may be applicable to other anti-IGF1R antibodies and facilitate the development of new therapeutics.
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Anticorpos Monoclonais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Receptor IGF Tipo 1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Biotina/química , Biotina/metabolismo , Estabilidade de Medicamentos , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Estreptavidina/química , Estreptavidina/metabolismoRESUMO
Pharmacokinetic data derived from assays that accurately and precisely quantitate a therapeutic drug in circulation are critical to appropriately designing suitable dosing schedules. This manuscript describes the validation and implementation of methods to quantitate a therapeutic anti-human PCSK9 monoclonal antibody in rat and monkey sera as well as immunogenicity methods to screen the possible presence of rat and monkey antibodies directed against the antibody. As soluble, endogenous PCSK9 can interfere with a PCSK9-mediated capture step in ELISA, an indirect target-capture assay was used that potentially could capture free and target-engaged therapeutic mAb. Immunogenicity assays were based on a standard bridge ELISA using the therapeutic antibody for capture and detection. Both pharmacokinetic and immunogenicity assays were implemented in preclinical studies of the therapeutic antibody. The methods presented here may enable further pharmacokinetic studies.
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Anticorpos Monoclonais/análise , Serina Endopeptidases/análise , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Ensaio de Imunoadsorção Enzimática/métodos , Haplorrinos , Humanos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Serina Endopeptidases/imunologia , Serina Endopeptidases/farmacocinética , SolubilidadeRESUMO
INTRODUCTION: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays. METHODS: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support. RESULTS: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10µL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less. DISCUSSION: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.
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Biofarmácia/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Imunoensaio/métodos , Farmacocinética , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/farmacocinética , Biofarmácia/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio/instrumentação , Macaca fascicularis , Macaca mulatta , Camundongos , Camundongos Transgênicos , Miniaturização , Ratos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/farmacocinéticaRESUMO
INTRODUCTION: Rapid lateral flow immunogenicity assays for the detection of anti-drug antibodies (ADAs) to two biotherapeutic antibodies, an anti-HER2 antibody and an anti-TNF-α antibody, were developed using ANP Technologies, Inc.'s proprietary Nano-Intelligent Detection System (NIDS®) and compared to their ELISA counterparts. METHODS: Biotin and hapten-labeled drugs are incubated with the patient serum sample to allow ADA to form a bridge complex with each drug conjugate. The reaction mixture is then added to a test strip with an anti-hapten capture zone which captures the mixed bridge complex. The bridge-complexed biotinylated drug then reacts with streptavidin-labeled gold particles in situ. The signal developed at the capture zone, which is directly proportional to ADA in the sample, is then quantitatively measured with a handheld reader. The counterpart ELISAs were run using the same reagents. Dose-response, specificity/free drug depletion, and screening cut-point assays were performed using both methods. RESULTS: The rapid assays' performance compare very closely to their ELISA counterparts'. Both types of assays identified the same positive samples in screening a limited population of 50 normal serum samples for the anti-HER2 antibody. In the case of anti-TNF-α, both assays identified the same positive samples out of 50 normal and 20 rheumatoid arthritis patient serum samples but differed in the assessment of two others. The rapid assay correctly identified as negative an ELISA false positive sample, and correctly tested as positive an ELISA false negative sample. Positive results were verified with a specificity/free drug depletion assay. DISCUSSION: The NIDS® rapid immunogenicity assay offers distinct advantages over current methods in simplicity, low cost, and short time to result. More importantly, the method requires no sample dilution and no washing steps which can perturb fragile complexes formed by low-affinity ADAs. Thus, the assay can potentially detect ADAs with various affinities.
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Ensaio de Imunoadsorção Enzimática/métodos , Imunogenética/métodos , Anticorpos/imunologia , Formação de Anticorpos/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Relação Dose-Resposta Imunológica , Humanos , Receptor ErbB-2/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.
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Anticorpos/imunologia , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/sangue , Polietilenoglicóis/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia de Afinidade/métodos , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Tripsina/metabolismoRESUMO
Biomarkers have shown promising utilities at various stages of the pharmaceutical R & D. With the recent technological advancements and the introduction of protein and gene arrays, high performance instrumentation (e.g., high-field nuclear magnetic resonance and high-resolution mass spectrometers), and bioinformatics, decisions on safety and efficacy criteria can be made with a higher degree of confidence. However, there is a scarcity of robust and valid biomarkers to accelerate the drug development process from pre-clinical through all stages of clinical studies. In this article, a brief overview of current definitions, biomarker categories, challenges in biological and analytical validation, along with several clinical examples will be presented.
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Biomarcadores/análise , Desenho de Fármacos , Indústria Farmacêutica/métodos , Animais , Diagnóstico por Imagem/métodos , Humanos , Oncologia/métodos , Neurociências/métodos , Toxicologia/métodosRESUMO
During the last decade, quantification of low molecular weight molecules using liquid chromatography-tandem mass spectrometry in biological fluids has become a common procedure in many preclinical and clinical laboratories. This overview highlights a number of issues involving "small molecule drugs", bioanalytical liquid chromatography-tandem mass spectrometry, which are frequently encountered during assay development. In addition, possible solutions to these issues are proposed with examples in some of the case studies. Topics such as chromatographic peak shape, carry-over, cross-talk, standard curve non-linearity, internal standard selection, matrix effect, and metabolite interference are presented. Since plasma is one of the most widely adopted biological fluid in drug discovery and development, the focus of this discussion will be limited to plasma analysis. This article is not intended to be a comprehensive overview and readers are encouraged to refer to the citations herein.
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Bioensaio/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Preparações Farmacêuticas/análise , Peso Molecular , Preparações Farmacêuticas/metabolismo , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Electron capture dissociation (ECD) represents one of the most recent and significant advancements in tandem mass spectrometry (MS/MS) for the identification and characterization of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally activated dissociation (CAD), ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation--an important attribute for characterizing post-translational modifications. Herein, we present a brief overview of the ECD technique as well as selected applications in characterization of peptides and proteins. Case studies including characterization and localization of amino acid glycosylation, methionine oxidation, acylation, and "top-down" protein mass spectrometry using ECD will be presented. A recent technique, coined as electron transfer dissociation (ETD), will be also discussed briefly.
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Mapeamento de Peptídeos/instrumentação , Mapeamento de Peptídeos/métodos , Peptídeos/química , Proteínas/química , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Mapeamento de Peptídeos/tendências , Espectrometria de Massas em Tandem/tendênciasRESUMO
Electron capture dissociation (ECD) represents a significant advance in tandem mass spectrometry for the identification and characterization of post-translational modifications (PTMs) of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally induced dissociation and infrared multi-photon dissociation, ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation. This unique attribute offers ECD as an attractive alternative for detection and localization of PTMs. The success and rapid adoption of ECD recently led to the culmination of The 1st International Uppsala Symposium on Electron Capture Dissociation of Biomolecules and Related Phenomena (October 19-22, 2003, Stockholm, Sweden). Herein, we present a general overview of the ECD technique as well as selected applications in characterization of post-translationally modified polypeptides.
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Perfilação da Expressão Gênica/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , ElétronsRESUMO
A high throughput method with ultra-low level quantification limit (10 pg/ml) was developed and validated for the quantitative determination of LAG078, a lipid modulator, in human plasma to support clinical studies employing low doses of the compound. The method consisted of reverse phase chromatographic separation of the analyte from plasma extract followed by electrospray ionization (ESI) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring mode (MRM). Extraction was performed using a combination of protein precipitation and liquid-liquid extraction in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short and high-resolution column (50 mm x 2.0 mm i.d., 3 microm particle size) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r=0.996) within the concentration range of 0.01 (lower limit of quantification) to 50 ng/ml using 0.5 ml of human plasma. The accuracy of the method varied from 95-101% with a precision (CV) of 5.29-13.2% over the concentration range. The method was simple and rapid.
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Hidrocarbonetos Fluorados/administração & dosagem , Hidrocarbonetos Fluorados/sangue , Lipídeos/sangue , Compostos de Enxofre/administração & dosagem , Compostos de Enxofre/sangue , Cromatografia Líquida de Alta Pressão/métodos , HumanosRESUMO
MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione-containing dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK-0767 contains a chiral center at the C-5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)-(R) to (-)-(S) enantiomers of MK-0767 were determined in plasma from humans (in vitro only) and nonclinical species used in the toxicological evaluation of rac-MK-0767, namely CD-1 mice, Sprague-Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (approximately 1.5-1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo. In mice, the ratio was higher in vitro (approximately 1) than in vivo (approximately 0.6), while in rabbits it was higher in vivo (approximately 1) than in vitro (approximately 0.5). These results suggested that differential binding of the MK-0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo, since in protein-free systems MK-0767 exists as the racemate.