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1.
Artigo em Inglês | MEDLINE | ID: mdl-30553974

RESUMO

INTRODUCTION: Low intrinsic solubility leading to poor oral bioavailability is a common challenge in drug discovery that can often be overcome by formulation strategies, however, it remains a potential limitation that can pose challenges for early risk assessment and represent a significant obstacle to drug development. We identified a selective inhibitor (BMS-986126) of the IL-1 receptor-associated kinase 4 (IRAK4) with favorable properties as a lead candidate, but with unusually low intrinsic solubility of <1 µg/mL. METHODS: Conventional histopathology identified the issue of crystal formation in vivo. Subsequent investigative work included confocal Raman micro-spectroscopy, MALDI-MS, polarized light microscopy of fresh wet-mount tissue scrapings and transmission electron microscopy. RESULTS: BMS-986126 was advanced into a 2-week toxicology study in rats. The main finding in this study was minimal granulomatous inflammation in the duodenum, associated with the presence of birefringent crystals at the highest dosage of 100 mg/kg/day. Considering the safety margin, and the single location of the lesion, BMS-986126 was further progressed into IND-enabling toxicology studies where tolerability deteriorated with increasing dosing duration. Birefringent crystals and granulomatous inflammation were detected in multiple organs at dosages ≥20 mg/kg/day. Raman spectroscopy confirmed the identity of the crystals as BMS-986126. Therefore, follow up investigations were conducted to further characterize drug crystallization and to evaluate detection methods for their potential to reliably detect in vivo crystallization early. DISCUSSION: The purpose of our efforts was to identify critical factors influencing in vivo drug crystallization and to provide a preliminary assessment (based on one compound) which method would be best suited for identifying crystals. Results indicated a combination of methods was required to provide a complete assessment of drug crystallization and that a simple technique, scraping of freshly collected tissue followed by evaluation under polarizing light was suitable for detecting crystals. However, dosing for 2 weeks was required for crystals to grow to a clearly detectable size.


Assuntos
Cristalização , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Pirazóis/química , Piridinas/química , Animais , Disponibilidade Biológica , Descoberta de Drogas , Duodeno/patologia , Feminino , Quinases Associadas a Receptores de Interleucina-1/química , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Cultura Primária de Células , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Piridinas/administração & dosagem , Piridinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Risco , Solubilidade , Análise Espectral Raman
2.
Bioorg Med Chem Lett ; 27(4): 855-861, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28108251

RESUMO

As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model.


Assuntos
Aminas/química , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Aminas/metabolismo , Aminas/uso terapêutico , Animais , Doenças Autoimunes/tratamento farmacológico , Sítios de Ligação , Cristalografia por Raios X , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Piperazina , Piperazinas/química , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Relação Estrutura-Atividade , Triazinas/química
3.
J Immunol ; 198(3): 1308-1319, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28003376

RESUMO

The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Prednisolona/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia
4.
Bioorg Med Chem Lett ; 26(17): 4256-60, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27476421

RESUMO

Aberrant Class I PI3K signaling is a key factor contributing to many immunological disorders and cancers. We have identified 4-amino pyrrolotriazine as a novel chemotype that selectively inhibits PI3Kδ signaling despite not binding to the specificity pocket of PI3Kδ isoform. Structure activity relationship (SAR) led to the identification of compound 30 that demonstrated efficacy in mouse Keyhole Limpet Hemocyanin (KLH) and collagen induced arthritis (CIA) models.


Assuntos
Aminas/química , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Triazinas/química , Aminas/metabolismo , Aminas/uso terapêutico , Animais , Artrite/tratamento farmacológico , Artrite/metabolismo , Artrite/patologia , Sítios de Ligação , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Camundongos , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
5.
ACS Med Chem Lett ; 6(8): 850-5, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288683

RESUMO

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

6.
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
7.
Mol Cell Proteomics ; 5(10): 1876-86, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16705188

RESUMO

Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. MALDI imaging MS represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well known nephrotoxicant. Differential analysis of the mass spectrum profiles revealed a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (Ser(28)-Gln(146)) using an innovative combination of tissue microextraction and fractionation by reverse-phase liquid chromatography followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of imaging MS in the discovery of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms.


Assuntos
Gentamicinas/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Rim/efeitos dos fármacos , Pré-Albumina/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Biomarcadores/análise , Biomarcadores/química , Western Blotting , Rim/citologia , Rim/patologia , Nefropatias/patologia , Masculino , Dados de Sequência Molecular , Pré-Albumina/química , Análise Serial de Proteínas , Ratos , Ratos Wistar , Reprodutibilidade dos Testes
8.
Toxicol Appl Pharmacol ; 207(2 Suppl): 161-70, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15990132

RESUMO

Male Wistar rats were treated with various model compounds or the appropriate vehicle controls in order to create a reference database for toxicogenomics assessment of novel compounds. Hepatotoxic compounds in the database were either known hepatotoxicants or showed hepatotoxicity during preclinical testing. Histopathology and clinical chemistry data were used to anchor the transcript profiles to an established endpoint (steatosis, cholestasis, direct acting, peroxisomal proliferation or nontoxic/control). These reference data were analyzed using a supervised learning method (support vector machines, SVM) to generate classification rules. This predictive model was subsequently used to assess compounds with regard to a potential hepatotoxic liability. A steatotic and a non-hepatotoxic 5HT(6) receptor antagonist compound from the same series were successfully discriminated by this toxicogenomics model. Additionally, an example is shown where a hepatotoxic liability was correctly recognized in the absence of pathological findings. In vitro experiments and a dog study confirmed the correctness of the toxicogenomics alert. Another interesting observation was that transcript profiles indicate toxicologically relevant changes at an earlier timepoint than routinely used methods. Together, these results support the useful application of toxicogenomics in raising alerts for adverse effects and generating mechanistic hypotheses that can be followed up by confirmatory experiments.


Assuntos
Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , RNA Mensageiro/genética , Testes de Toxicidade , Animais , Cães , Masculino , Ratos , Ratos Wistar , Receptores de Serotonina/efeitos dos fármacos
9.
Mutat Res ; 575(1-2): 102-15, 2005 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-15924886

RESUMO

Almost 10 years ago, microarray technology was established as a new powerful tool for large-scale analysis of gene expression. Soon thereafter the new technology was discovered by toxicologists for the purpose of deciphering the molecular events underlying toxicity, and the term "Toxicogenomics" appeared in scientific literature. Ever since, the toxicology community was fascinated by the multiplicity of sophisticated possibilities toxicogenomics seems to offer: genome-wide analysis of toxicant-induced expression profiles may provide a means for prediction of toxicity prior to classical toxicological endpoints such as histopathology or clinical chemistry. Some researchers even speculated of the classical methods being superfluous before long. It was assumed that by using toxicogenomics it would be possible to classify compounds early in drug development and consequently save animals, time, and money in pre-clinical toxicity studies. Moreover, it seemed within reach to unravel the molecular mechanisms underlying toxicity. The feasibility of bridging data derived from in vitro and in vivo systems, identifying new biomarkers, and comparing toxicological responses "across-species" was also excessively praised. After several years of intensive application of microarray technology in the field of toxicology, not only by the pharmaceutical industry, it is now time to survey its achievements and to question how many of these wishes and promises have really come true.


Assuntos
Desenho de Drogas , Indústria Farmacêutica/tendências , Toxicogenética/tendências , Animais , Biomarcadores , Bases de Dados Factuais , Indústria Farmacêutica/economia , Interações de Medicamentos , Perfilação da Expressão Gênica , Genômica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da Espécie , Toxicogenética/economia
10.
Environ Health Perspect ; 112(12): 1236-48, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15345370

RESUMO

Male rats were treated with various model compounds or the appropriate vehicle controls. Most substances were either well-known hepatotoxicants or showed hepatotoxicity during preclinical testing. The aim of the present study was to determine if biological samples from rats treated with various compounds can be classified based on gene expression profiles. In addition to gene expression analysis using microarrays, a complete serum chemistry profile and liver and kidney histopathology were performed. We analyzed hepatic gene expression profiles using a supervised learning method (support vector machines; SVMs) to generate classification rules and combined this with recursive feature elimination to improve classification performance and to identify a compact subset of probe sets with potential use as biomarkers. Two different SVM algorithms were tested, and the models obtained were validated with a compound-based external cross-validation approach. Our predictive models were able to discriminate between hepatotoxic and nonhepatotoxic compounds. Furthermore, they predicted the correct class of hepatotoxicant in most cases. We provide an example showing that a predictive model built on transcript profiles from one rat strain can successfully classify profiles from another rat strain. In addition, we demonstrate that the predictive models identify nonresponders and are able to discriminate between gene changes related to pharmacology and toxicity. This work confirms the hypothesis that compound classification based on gene expression data is feasible.


Assuntos
Algoritmos , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Toxicogenética/métodos , Xenobióticos/classificação , Xenobióticos/toxicidade , Animais , Biomarcadores/análise , Masculino , Ratos , Ratos Wistar
11.
J Pharm Biomed Anal ; 35(1): 93-105, 2004 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15030884

RESUMO

Gene chip array (Affymetrix) data from liver tissue and high resolution 1H NMR spectra from intact liver tissue, tissue extracts and plasma have been analyzed to identify biochemical changes arising from hepatotoxicity in mice dosed with acetaminophen. These data sets have been co-interpreted in terms of common metabolic pathways. The principal metabolic changes comprised a decrease in hepatic glucose and glycogen in intact tissue, coupled with an increase in lipid content, with increases in the levels of glucose, pyruvate, acetate and lactate in plasma, and increases in alanine and lactate in the aqueous tissue extracts. Collectively these data provide evidence for an increased rate of hepatic glycolysis. The metabolic observations were consistent with the altered levels of gene expression relating to lipid and energy metabolism in liver which both preceded and were concurrent with the metabolic perturbations. The results show that these two technology platforms together offer a complementary view into cellular responses to toxic processes, providing new insight into the toxic consequences, even for well-studied therapeutic agents such as acetaminophen.


Assuntos
Acetaminofen/toxicidade , Analgésicos não Entorpecentes/toxicidade , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Testes de Toxicidade/métodos , Acetaminofen/sangue , Alanina Transaminase/metabolismo , Analgésicos não Entorpecentes/sangue , Animais , Aspartato Aminotransferases/metabolismo , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Metabolismo dos Lipídeos , Fígado/enzimologia , Fígado/patologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
12.
Toxicol Sci ; 65(1): 135-50, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11752693

RESUMO

Overdose of acetaminophen (APAP) causes severe centrilobular hepatic necrosis in humans and experimental animals. Here, to explore its mechanism, we administered APAP at subtoxic (150 mg/kg ip) and toxic (500 mg/kg ip) doses to overnight fasted mice. Animals were sacrificed at different time points from 15 min to 4 h postinjection. We assessed liver toxicity by plasma ALT activity and by electron microscopy. Using nylon filter arrays and RTQPCR, we performed genomics analysis in liver. We ran proteomics on liver mitochondrial subfractions using the newly developed quantitative fluorescent 2D-DIGE method (Amersham Pharmacia Biotech UK Limited). As soon as 15 min postinjection, centrilobular hepatocyte mitochondria were already slightly enlarged and GSH total content dropped by a third at top dose. GM-CSF mRNA, which is a granulocyte specific gene likely coming from resident Kupffer cells, was also induced to its maximum of 3-fold at both doses. Chaperone proteins Hsp10 and Hsp60 were readily decreased by half in mitochondria at both doses, most likely by leaking into cytoplasm. Although APAP is known as an apoptotic trigger, no apoptosis was observed at any time point. Most of the protein changes in mitochondria were present at 15 min postinjection, thus preceding most of the gene regulations. The decrease of ATP synthase subunits and beta-oxidation pathway proteins indicated a loss of energy production. As the morphology of mitochondria was also affected very early at top dose, we concluded that APAP toxicity was a direct action of its known reactive metabolite NAPQI, rather than a consequence of gene regulation. However, the latter will either worsen the toxicity or lead toward cell recovery depending on the cellular damage level.


Assuntos
Acetaminofen/toxicidade , Genômica , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Proteoma , Acetaminofen/sangue , Animais , Western Blotting , Adutos de DNA/metabolismo , Relação Dose-Resposta a Droga , Overdose de Drogas , Eletroforese em Gel de Poliacrilamida , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Macrófagos do Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Microscopia Eletrônica , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Estresse Oxidativo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo
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