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1.
J Med Chem ; 62(20): 8973-8995, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31318208

RESUMO

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

2.
J Med Chem ; 62(20): 8953-8972, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31314518

RESUMO

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

3.
ACS Med Chem Lett ; 10(3): 383-388, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30891145

RESUMO

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

4.
Obesity (Silver Spring) ; 25(6): 1069-1076, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28452429

RESUMO

OBJECTIVE: Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). METHODS: A principal component analysis for all 91 metabolites measured indicated that metabolome perturbation is greater and less varied for humans than for rodents. RESULTS: Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g., asparagine, citrulline, gamma-aminobutyric acid, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). CONCLUSIONS: Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain end points of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.


Assuntos
Metabolômica/métodos , Obesidade/complicações , Animais , Dieta , Progressão da Doença , Humanos , Fígado/patologia , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ratos , Ratos Sprague-Dawley
5.
PLoS One ; 11(6): e0157111, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27310468

RESUMO

A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability.


Assuntos
Metabolômica , Estresse Oxidativo/genética , Ácidos Siálicos/metabolismo , Transcriptoma/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Perfilação da Expressão Gênica , Glucose/metabolismo , Glicólise/genética , Glicosilação , Manose/genética , Manose/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Oxigênio/metabolismo
6.
Regul Toxicol Pharmacol ; 73(1): 27-42, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26111605

RESUMO

This comparative study was conducted to assess background physiologic and pharmacologic parameters of cynomolgus macaques (Macaca fascicularis) from Cambodia, from a mixed Asian source (Cambodia, Vietnam and Indonesia), and from Mauritius. This evaluation provides a comprehensive assessment of several of these parameters in a single study. Ten male and 10 female captive-bred, age-matched macaques from each source were evaluated. Criteria for evaluation included weight gain, assessment of drug metabolizing enzyme activity, metabolomic analysis, immunologic assessments (lymphocyte subsets, TDAR, and serum Ig isotyping), clinical pathology evaluations, physical (respiratory, neurologic, cardiovascular, and ophthalmologic) examinations, pathogen screening, organ weights, and gross and microscopic pathology analyses. The results of this evaluation indicate that, compared to macaques of Asian origin, macaques from Mauritius had the lowest incidence and/or severity of spontaneous pathologic findings in several organs and tissues (lymphoid organs, stomach, kidney, urothelium, heart, arteries and lung) and better testicular maturity at a given age with minimal variability in organ weights. Although slight differences were observed in other parameters, none were considered detrimental to the use of macaques of Asian or Mauritius origin in pharmaceutical candidate safety studies with the use of a consistent source, concomitant controls, and appropriate background knowledge and screening.


Assuntos
Macaca fascicularis/fisiologia , Tamanho do Órgão/fisiologia , Animais , Grupo com Ancestrais do Continente Asiático , Feminino , Humanos , Masculino , Maurício
7.
Amino Acids ; 47(3): 603-15, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25534430

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.


Assuntos
Isoleucina/metabolismo , Leucina/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Valina/metabolismo , Carnitina/genética , Carnitina/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Isoleucina/genética , Leucina/genética , Masculino , Metabolômica , Hepatopatia Gordurosa não Alcoólica/genética , Transdução de Sinais/genética , Valina/genética
8.
J Lipid Res ; 55(7): 1366-74, 2014 07.
Artigo em Inglês | MEDLINE | ID: mdl-24755647

RESUMO

A method is described that allows noninvasive identification and quantitative assessment of lipid classes present in sebaceous excretions in rodents. The method relies on direct high-field proton NMR analysis of common group lipid protons in deuterated organic solvent extracts of fur. Extracts from as little as 15 mg of fur from rat, mouse, and hamster provided acceptable results on a 600 MHz NMR equipped with a cryogenically cooled proton-observe probe. In rats, sex- and age-related differences in lipid composition are larger than differences in fur collected from various body regions within an individual and much larger than interanimal differences in age- and sex-matched specimens. The utility of this method to noninvasively monitor drug-induced sebaceous gland atrophy in rodents is demonstrated in rats dosed with a stearoyl-CoA desaturase 1 (SCD1) inhibitor. In this model, a 35% reduction in sebum lipids, extracted from fur, was observed. Finally, structural elucidation of cholesta-7,24-dien-3ß-ol ester as the most prominent, previously unidentified sebum sterol ester in male Syrian hamsters is described. The utility of this method for drug and cosmetic safety and efficacy assessment is discussed.


Assuntos
Pelo Animal/metabolismo , Inibidores Enzimáticos/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Doenças das Glândulas Sebáceas/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Mesocricetus , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ratos Sprague-Dawley , Doenças das Glândulas Sebáceas/induzido quimicamente , Estearoil-CoA Dessaturase/metabolismo
9.
Methods Mol Biol ; 1104: 223-36, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24297419

RESUMO

Metabolomics has become an important tool for measuring pools of small molecules in mammalian cell cultures expressing therapeutic proteins. NMR spectroscopy has played an important role, largely because it requires minimal sample preparation, does not require chromatographic separation, and is quantitative. The concentrations of large numbers of small molecules in the extracellular media or within the cells themselves can be measured directly on the culture supernatant and on the supernatant of the lysed cells, respectively, and correlated with endpoints such as titer, cell viability, or glycosylation patterns. The observed changes can be used to generate hypotheses by which these parameters can be optimized. This chapter focuses on the sample preparation, data acquisition, and analysis to get the most out of NMR metabolomics data from CHO cell cultures but could easily be extended to other in vitro culture systems.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Espectroscopia de Ressonância Magnética/métodos , Mamíferos/metabolismo , Animais , Células CHO , Cricetulus , Metabolômica , Software
10.
Dig Dis Sci ; 59(2): 365-74, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24048683

RESUMO

BACKGROUND: The worldwide prevalences of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are estimated to range from 30 to 40 % and 5-17 %, respectively. Hepatocellular carcinoma (HCC) is primarily caused by hepatitis B infection, but retrospective data suggest that 4-29 % of NASH cases will progress to HCC. Currently the connection between NASH and HCC is unclear. AIMS: The purpose of this study was to identify changes in expression of HCC-related genes and metabolite profiles in NAFLD progression. METHODS: Transcriptomic and metabolomic datasets from human liver tissue representing NAFLD progression (normal, steatosis, NASH) were utilized and compared to published data for HCC. RESULTS: Genes involved in Wnt signaling were downregulated in NASH but have been reported to be upregulated in HCC. Extracellular matrix/angiogenesis genes were upregulated in NASH, similar to reports in HCC. Iron homeostasis is known to be perturbed in HCC and we observed downregulation of genes in this pathway. In the metabolomics analysis of hepatic NAFLD samples, several changes were opposite to what has been reported in plasma of HCC patients (lysine, phenylalanine, citrulline, creatine, creatinine, glycodeoxycholic acid, inosine, and alpha-ketoglutarate). In contrast, multiple acyl-lyso-phosphatidylcholine metabolites were downregulated in NASH livers, consistent with observations in HCC patient plasma. CONCLUSIONS: These data indicate an overlap in the pathogenesis of NAFLD and HCC where several classes of HCC related genes and metabolites are altered in NAFLD. Importantly, Wnt signaling and several metabolites are different, thus implicating these genes and metabolites as mediators in the transition from NASH to HCC.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Análise por Conglomerados , Bases de Dados Genéticas , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Metabolômica , Hepatopatia Gordurosa não Alcoólica , Transdução de Sinais/genética
11.
Toxicol Appl Pharmacol ; 268(2): 132-40, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23391614

RESUMO

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the 'classical' (neutral) and 'alternative' (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH.


Assuntos
Ácidos e Sais Biliares/metabolismo , Fígado Gorduroso/metabolismo , Fígado/efeitos dos fármacos , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/genética , Ácidos e Sais Biliares/toxicidade , Análise por Conglomerados , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Metabolômica , Hepatopatia Gordurosa não Alcoólica
12.
Toxicol Sci ; 129(2): 268-79, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22821849

RESUMO

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.


Assuntos
Adipatos/urina , Malonatos/urina , Músculo Esquelético/efeitos dos fármacos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Sequência de Bases , Western Blotting , Carnitina/sangue , Primers do DNA , Cães , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Metabolômica , Reação em Cadeia da Polimerase , Ensaio Radioligante , Receptor CB1 de Canabinoide/genética
13.
Toxicol Sci ; 122(2): 587-97, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21561886

RESUMO

Organic anion-transporting polypeptides (Oatp) 1a1 and 1a4 were deleted by homologous recombination, and mice were characterized for Oatp expression in liver and kidney, transport in isolated hepatocytes, in vivo disposition of substrates, and urinary metabolomic profiles. Oatp1a1 and Oatp1a4 proteins were undetected in liver, and both lines were viable and fertile. Hepatic constitutive messenger RNAs (mRNAs) for Oatp1a4, 1b2, or 2b1 were unchanged in Oatp1a1⁻/⁻ mice, whereas renal Oatp1a4 mRNA decreased approximately 50% (both sexes). In Oatp1a4⁻/⁻ mice, no changes in constitutive mRNAs for other Oatps were observed. Uptake of estradiol-17ß-D-glucuronide and estrone-3-sulfate in primary hepatocytes decreased 95 and 75%, respectively, in Oatp1a1⁻/⁻ mice and by 60 and 30%, respectively, in Oatp1a4⁻/⁻ mice. Taurocholate uptake decreased by 20 and 50% in Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, respectively, whereas digoxin was unaffected. Plasma area under the curve (AUC) for estradiol-17ß-D-glucuronide increased 35 and 55% in male and female Oatp1a1⁻/⁻ mice, respectively, with a concurrent 50% reduction in liver-to-plasma ratios. In contrast, plasma AUC or tissue concentrations of estradiol-17ß-D-glucuronide were unchanged in Oatp1a4⁻/⁻ mice. Plasma AUCs for dibromosulfophthalein increased nearly threefold in male Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, increased by 40% in female Oatp1a4⁻/⁻ mice, and were unchanged in female Oatp1a1⁻/⁻ mice. In both lines, no changes in serum ALT, bilirubin, and cholesterol were noted. NMR analyses showed no generalized increase in urinary excretion of organic anions. However, urinary excretion of taurine decreased by 30-40% and was accompanied by increased excretion of isethionic acid, a taurine metabolite generated by intestinal bacteria, suggesting some perturbations in intestinal bacteria distribution.


Assuntos
Deleção de Genes , Recombinação Homóloga , Metabolômica , Transportadores de Ânions Orgânicos/metabolismo , Animais , Área Sob a Curva , Transporte Biológico/genética , Western Blotting , Estradiol/análogos & derivados , Estradiol/sangue , Estradiol/farmacocinética , Estrona/análogos & derivados , Estrona/sangue , Estrona/farmacocinética , Feminino , Hepatócitos/efeitos dos fármacos , Ácido Isetiônico/urina , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Taurina/urina , Ácido Taurocólico/farmacocinética
14.
J Biomol NMR ; 49(3-4): 195-206, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21373840

RESUMO

NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Animais , Células CHO , Colina , Ciclo do Ácido Cítrico , Cricetinae , Cricetulus , Histidina , Fígado/metabolismo , Teoria Quântica , Proteínas Recombinantes/biossíntese
15.
Chem Res Toxicol ; 24(4): 481-7, 2011 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-21381695

RESUMO

The overnight (16-h) fast is one of the most common experimental manipulations performed in rodent studies. Despite its ubiquitous employment, a comprehensive evaluation of metabolomic and transcriptomic sequelae of fasting in conjunction with routine clinical pathology evaluation has not been undertaken. This study assessed the impact of a 16-h fast on urine and serum metabolic profiles, transcript profiles of liver, psoas muscle, and jejunum as well as on routine laboratory clinical pathology parameters. Fasting rats had an approximate 12% relative weight decrease compared to ad libitum fed animals, and urine volume was significantly increased. Fasting had no effect on hematology parameters, though several changes were evident in serum and urine clinical chemistry data. In general, metabolic changes in biofluids were modest in magnitude but broad in extent, with a majority of measured urinary metabolites and from 1/3 to 1/2 of monitored serum metabolites significantly affected. Increases in fatty acids and bile acids dominated the upregulated metabolites. Downregulated serum metabolites were dominated by diet-derived and/or gut-microflora derived metabolites. Major transcriptional changes included genes with roles in fatty acid, carbohydrate, cholesterol, and bile acid metabolism indicating decreased activity in glycolytic pathways and a shift toward increased utilization of fatty acids. Typically, several genes within these metabolic pathways, including key rate limiting genes, changed simultaneously, and those changes were frequently correlative to changes in clinical pathology parameters or metabolomic data. Importantly, up- or down-regulation of a variety of cytochrome P450s, transporters, and transferases was evident. Taken together, these data indicate profound consequences of fasting on systemic biochemistry and raise the potential for unanticipated interactions, particularly when metabolomic or transcriptomic data are primary end points.


Assuntos
Jejum , Perfilação da Expressão Gênica , Metaboloma , Animais , Feminino , Glucose/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
16.
Xenobiotica ; 41(2): 144-54, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21043805

RESUMO

2-Bromoethanamine (BEA) causes renal papillary necrosis (RPN) in rats after a single dose and has been widely used as a model compound for studying the lesion. Although the metabolism of BEA may be an important determinant of toxicity, the metabolic fate of the compound has not been fully elucidated. To date, the only identified BEA metabolites are aziridine, 2-oxazolidone and 5-hydroxy-2-oxazolidone. In this study, stable isotope labelling (SIL) of BEA analogs ((¹³C and ²H) were used to differentiate generated BEA metabolites from endogenous molecules which enabled the accurate liquid chromatography mass spectrometry detection of more than 180 novel metabolites. BEA metabolism was evaluated in rats after acute administration of a non-toxic dose (50 mg/kg) and a toxic dose (250 mg/kg) that caused frank RPN and polyuria. Newly identified metabolites include three carbamoylation products, two mercapturic acids and a group of amino acid conjugates. Overall, the results indicate that BEA metabolism is very complex, suggest the potential formation of reactive intermediates and establish that BEA is subject to conjugation with glutathione. The results also demonstrate the utility and sensitivity of the SIL approach for identification of metabolites from small, reactive compounds.


Assuntos
Carbamatos/metabolismo , Etilaminas/urina , Glutationa/metabolismo , Marcação por Isótopo/métodos , Aminoácidos/metabolismo , Animais , Etilaminas/química , Etilaminas/toxicidade , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
17.
Anal Biochem ; 410(1): 84-91, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21094120

RESUMO

Nuclear magnetic resonance (NMR)-based metabolomic profiling identified urinary 1- and 3-methylhistidine (1- and 3-MH) as potential biomarkers of skeletal muscle toxicity in Sprague-Dawley rats following 7 and 14 daily doses of 0.5 or 1mg/kg cerivastatin. These metabolites were highly correlated to sex-, dose- and time-dependent development of cerivastatin-induced myotoxicity. Subsequently, the distribution and concentration of 1- and 3-MH were quantified in 18 tissues by gas chromatography-mass spectrometry. The methylhistidine isomers were most abundant in skeletal muscle with no fiber or sex differences observed; however, 3-MH was also present in cardiac and smooth muscle. In a second study, rats receiving 14 daily doses of 1mg/kg cerivastatin (a myotoxic dose) had 6- and 2-fold elevations in 1- and 3-MH in urine and had 11- and 3-fold increases in 1- and 3-MH in serum, respectively. Selectivity of these potential biomarkers was tested by dosing rats with the cardiotoxicant isoproterenol (0.5mg/kg), and a 2-fold decrease in urinary 1- and 3-MH was observed and attributed to the anabolic effect on skeletal muscle. These findings indicate that 1- and 3-MH may be useful urine and serum biomarkers of drug-induced skeletal muscle toxicity and hypertrophy in the rat, and further investigation into their use and limitations is warranted.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Metilistidinas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismo , Biomarcadores/urina , Creatina/metabolismo , Creatina/urina , Relação Dose-Resposta a Droga , Feminino , Masculino , Metilistidinas/farmacocinética , Metilistidinas/urina , Doenças Musculares/induzido quimicamente , Doenças Musculares/metabolismo , Doenças Musculares/urina , Piridinas/toxicidade , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
18.
J Med Chem ; 53(9): 3814-30, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-20405922

RESUMO

Leukocyte function-associated antigen-1 (LFA-1), also known as CD11a/CD18 or alpha(L)beta(2), belongs to the beta(2) integrin subfamily and is constitutively expressed on all leukocytes. The major ligands of LFA-1 include three intercellular adhesion molecules 1, 2, and 3 (ICAM 1, 2, and 3). The interactions between LFA-1 and the ICAMs are critical for cell adhesion, and preclinical animal studies and clinical data from the humanized anti-LFA-1 antibody efalizumab have provided proof-of-concept for LFA-1 as an immunological target. This article will detail the structure-activity relationships (SAR) leading to a novel second generation series of highly potent spirocyclic hydantoin antagonists of LFA-1. With significantly enhanced in vitro and ex vivo potency relative to our first clinical compound (1), as well as demonstrated in vivo activity and an acceptable pharmacokinetic and safety profile, 6-((5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro-[4.4]nonan-7-yl)nicotinic acid (2e) was selected to advance into clinical trials.


Assuntos
Hidantoínas/farmacocinética , Fatores Imunológicos/química , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Ácidos Nicotínicos/farmacocinética , Humanos , Hidantoínas/farmacologia , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/imunologia , Ácidos Nicotínicos/toxicidade , Relação Estrutura-Atividade
19.
Magn Reson Chem ; 47 Suppl 1: S12-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19768707

RESUMO

In the present study, NMR-based urinary metabonomic profiles resulting from dosing with widely recognized microsomal enzyme inducers were evaluated in male rats. Wistar or Sprague-Dawley rats were dosed daily by oral gavage with phenobarbital (PB; 100 mg/kg), diallyl sulfide (DAS; 500 mg/kg), the investigational compound DMP-904 (150 mg/kg), or beta-naphthoflavone (BNF; 100 mg/kg) for 4 days, and urine was collected daily for analysis. Compounds known to increase cytochrome P450 2B enzymes, including PB, DAS and DMP-904, increased the urinary excretion of gulonic and ascorbic acid in a time-dependent manner, reaching a maximum following 3-4 days of dosing. In contrast, BNF, an agent that induces primarily Cyp1A enzymes, did not increase gulonic or ascorbic acid excretion, despite inducing Cyp1A1 more than 200-fold. Given the metabonomic results, hepatic transcriptional changes in the regulation of ascorbic acid biosynthesis were determined by RT-PCR. All Cyp2B inducers increased hepatic mRNA levels of aldo-keto reductase 1A1, an enzyme that catalyzes the formation of gulonic acid from glucuronate with concurrent decreased expression of both regucalcin (Rgn), the enzyme responsible for conversion of gulonic acid to gulono-1, 4-lactone and gulonolactone oxidase (Gulo), the rate-limiting enzyme in ascorbate biosynthesis. These effects would be expected to increase levels of gulonic acid. In addition, Cyp2B inducers also increased hepatic expression of enzymes regulating ascorbic acid reutilization including glutaredoxin reductase (Glrx2) and thioredoxin reductase (Txnrd1). In contrast, BNF did not effect hepatic expression of any enzyme regulating gulonic or ascorbic acid biosynthesis. Thus, some microsomal enzyme inducers alter transcriptional regulation of ascorbic acid biosynthesis, and these changes are detected by noninvasive metabonomic profiling. However, not all microsomal enzyme inducers appear to alter ascorbic acid metabolism. Finally, the work illustrates how metabonomic results can direct additional studies to determine the biochemical mechanisms underlying changes in urinary metabolite excretion.


Assuntos
Ácido Ascórbico/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Metabolômica , Açúcares Ácidos/metabolismo , Compostos Alílicos/farmacologia , Animais , Ácido Ascórbico/urina , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Fígado/enzimologia , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Fenobarbital/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Açúcares Ácidos/urina , Sulfetos/farmacologia , Fatores de Tempo , Ativação Transcricional
20.
Methods Mol Biol ; 358: 247-71, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17035690

RESUMO

Nuclear magnetic resonance (NMR)-based metabonomics is gaining popularity in drug discovery and development and in academia in a variety of settings, ranging from toxicology, preclinical, and clinical approaches to nutrition research, studies on microorganisms, and research on plants. This chapter focuses on the basic steps in a metabonomics study and emphasizes experience and lessons learned in our lab where we focused on metabonomic analyses of plant extracts, cell lines, and a variety of animal tissues and biofluids. We emphasize that a comprehensive and suitable study design is pivotal for a correct biological interpretation of the results, as well as highly controlled experimental conditions. Sample preparation and NMR protocols are detailed for a wide range of sample types. We discuss alternative data processing strategies and considerations for a general data analysis approach, paying particular attention to the statistical interpretation and validation of the results while also highlighting approaches to avoid possible pitfalls resulting from systematic and random errors. A tutorial written for the R statistical package and other small utilities are available from the authors upon request.


Assuntos
Desenho de Drogas , Espectroscopia de Ressonância Magnética/métodos , Extratos Vegetais/análise , Extratos de Tecidos/análise , Animais , Coleta de Amostras Sanguíneas/métodos , Interpretação Estatística de Dados , Células Epiteliais/metabolismo , Fezes/química , Hepatócitos/metabolismo , Humanos , Extratos Vegetais/isolamento & purificação , Folhas de Planta/metabolismo , Análise de Componente Principal/métodos , Ratos , Software , Extratos de Tecidos/isolamento & purificação , Urina/química , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
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