Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.002
Filtrar
1.
Biochem Pharmacol ; 173: 113639, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31536727

RESUMO

The side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in the cardiovascular system mainly result from its inhibitory effect on cyclooxygenase-2 (COX-2). Since NSAIDs are one of the most commonly used anti-inflammatory drugs in the clinic, it is necessary to identify new anti-inflammatory drugs that are safer than NSAIDs. Nardosinanone N (NAN), a compound isolated from the roots and rhizomes of Nardostachys chinensis, was evaluated for its anti-inflammatory effects using the lipopolysaccharide (LPS)-stimulated RAW264.7 cell line and rat peritoneal macrophage models. First, we found that NAN down regulated the levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2), but not cyclooxygenase-2 (COX-2). Additionally, NAN reduced the M1 macrophage phenotype and increased the M2 macrophage phenotype. Furthermore, mechanistic studies showed that NAN activated the nuclear factor-erythroid 2 -related factor 2 (Nrf2) signaling pathway, which, in turn, increased the expression of antioxidant protein heme oxygenase-1 (HO-1) to achieve its anti-inflammatory effect. Finally, Nrf2 siRNA and the HO-1 inhibitor significantly attenuated the anti-inflammatory effect of NAN. More interestingly, we found that NAN did not affect COX-2 expression and activity but reduced the PGE2 concentration by selective inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). In conclusion, NAN may be a new anti-inflammatory drug that has fewer side effects than NSAIDs and can be a new potential Nrf2 activator and mPGES-1 inhibitor.


Assuntos
Compostos de Epóxi/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Nardostachys/química , Preparações de Plantas/farmacologia , Prostaglandina-E Sintases/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Compostos de Epóxi/química , Expressão Gênica/efeitos dos fármacos , Macrófagos/classificação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Estrutura Molecular , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Preparações de Plantas/química , Prostaglandina-E Sintases/genética , Células RAW 264.7 , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
2.
Drug Metab Dispos ; 48(1): 8-17, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31685482

RESUMO

The liver and small intestine restrict oral bioavailability of drugs and constitute the main sites of pharmacokinetic drug-drug interactions. Hence, detailed data on hepatic and intestinal activities of drug metabolizing enzymes is important for modeling drug disposition and optimizing pharmacotherapy in different patient populations. The aim of this study was to determine the activities of seven cytochrome P450 (P450) enzymes in paired liver and small intestinal samples from patients with obesity. Biopsies were obtained from 20 patients who underwent Roux-en-Y gastric bypass surgery following a 3-week low-energy diet. Individual hepatic and intestinal microsomes were prepared and specific probe substrates in combined incubations were used for determination of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A activities. The activities of CYP2C8, CYP2C9, CYP2D6, and CYP3A were quantified in both human liver microsomes (HLM) and human intestinal microsomes (HIM), while the activities of CYP1A2, CYP2B6, and CYP2C19 were only quantifiable in HLM. Considerable interindividual variability was present in both HLM (9- to 23-fold) and HIM (5- to 55-fold). The median metabolic HLM/HIM ratios varied from 1.5 for CYP3A to 252 for CYP2C8. The activities of CYP2C9 in paired HLM and HIM were positively correlated (r = 0.74, P < 0.001), while no interorgan correlations were found for activities of CYP2C8, CYP2D6, and CYP3A (P > 0.05). Small intestinal CYP3A activities were higher in females compared with males (P < 0.05). Hepatic CYP2B6 activity correlated negatively with body mass index (r = -0.72, P < 0.001). These data may be useful for further in vitro-in vivo predictions of drug disposition in patients with obesity. SIGNIFICANCE STATEMENT: Hepatic and intestinal drug metabolism is the key determinant of oral drug bioavailability. In this study, paired liver and jejunum samples were obtained from 20 patients with obesity undergoing gastric bypass surgery following a 3-week low-energy diet. We determined the hepatic and small intestinal activities of clinically important P450 enzymes and provide detailed enzyme kinetic data relevant for predicting in vivo disposition of P450 substrates in this patient population.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Jejuno/enzimologia , Fígado/enzimologia , Microssomos/enzimologia , Obesidade/enzimologia , Índice de Massa Corporal , Sistema Enzimático do Citocromo P-450/genética , Ativação Enzimática , Feminino , Genótipo , Humanos , Técnicas In Vitro , Cinética , Masculino , Microssomos Hepáticos/enzimologia , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacologia , Especificidade de Órgãos , Caracteres Sexuais , Especificidade por Substrato
3.
Pestic Biochem Physiol ; 161: 68-76, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31685199

RESUMO

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.


Assuntos
Abelhas/metabolismo , Microssomos/metabolismo , Fosfolipases A2/isolamento & purificação , Abdome , Animais , Cumarínicos/metabolismo , Hidroxilação , Microssomos/enzimologia , Neonicotinoides/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Especificidade por Substrato , Xenobióticos/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-31669773

RESUMO

The pond wolf spider, Pardosa pseudoannulata, is one of the dominant natural enemies in farmlands and plays important roles in controlling a range of insect pests. The spider is less sensitive to many insecticides than the target pests such as the brown planthopper, Nilaparvata lugens. The different sensitivity to a certain insecticide between species is mostly attributed to the differences in both molecular targets and detoxification enzymes. As one of the most important detoxification enzymes, glutathione transferases (GSTs) play a key role as phase II enzyme in the enzymic detoxification in organisms. Until now, there are few studies on spiders' GSTs, limiting the understanding of insecticide selectivity between insect pests and natural enemy spiders. In this study, based on the transcriptome and genome sequencing of P. pseudoannulata, thirteen full-length transcripts encoding GSTs were identified and analyzed. Interestingly, Delta family, which is thought to be specific to the Insecta, was identified in P. pseudoannulata. Further, vertebrate/mammalian-specific Mu family was also identified in P. pseudoannulata. The mRNA expression levels of cytosolic GSTs in different tissues were determined, and most GST genes were abundant in the gut and the fat body. To investigate GST candidates involving in insecticide detoxification, the mRNA levels of cytosolic GSTs were tested after spiders' exposure to either imidacloprid or deltamethrin. The results showed that PpGSTD3 and PpGSTT1 responded to at least one of these two insecticides. The present study helped understand the function of GSTs in P. pseudoannulata and enriched the genetic information of natural enemy spiders.


Assuntos
Genoma de Inseto , Glutationa Transferase/genética , Aranhas/enzimologia , Sequência de Aminoácidos , Animais , Citosol/enzimologia , Éxons , Corpo Adiposo/enzimologia , Glutationa Transferase/química , Inseticidas/farmacologia , Íntrons , Microssomos/enzimologia , Mitocôndrias/enzimologia , Homologia de Sequência de Aminoácidos , Aranhas/genética
5.
Plant Physiol ; 181(4): 1468-1479, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31619508

RESUMO

In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.


Assuntos
Acil Coenzima A/metabolismo , Brassica napus/enzimologia , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Erúcicos/metabolismo , Proteínas de Plantas/metabolismo , Brassica napus/genética , Diacilglicerol O-Aciltransferase/genética , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos/enzimologia , Filogenia , Proteínas de Plantas/genética , Sementes/embriologia , Especificidade por Substrato/genética , Triglicerídeos
6.
Int J Mol Sci ; 20(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614497

RESUMO

Besides the liver, which has always been considered the major source of endogenous glucose production in all post-absorptive situations, kidneys and intestines can also produce glucose in blood, particularly during fasting and under protein feeding. However, observations gained in different experimental animals have given ambiguous results concerning the presence of the glucose-6-phosphatase system in the small intestine. The aim of this study was to better define the species-related differences of this putative gluconeogenic organ in glucose homeostasis. The components of the glucose-6-phosphatase system (i.e., glucose-6-phosphate transporter and glucose-6-phosphatase itself) were analyzed in homogenates or microsomal fractions prepared from the small intestine mucosae and liver of rats, guinea pigs, and humans. Protein and mRNA levels, as well as glucose-6-phosphatase activities, were detected. The results showed that the glucose-6-phosphatase system is poorly represented in the small intestine of rats; on the other hand, significant expressions of glucose-6-phosphate transporter and of the glucose-6-phosphatase were found in the small intestine of guinea pigs and homo sapiens. The activity of the recently described fructose-6-phosphate transporter-intraluminal hexose isomerase pathway was also present in intestinal microsomes from these two species. The results demonstrate that the gluconeogenic role of the small intestine is highly species-specific and presumably dependent on feeding behavior (e.g., fructose consumption) and the actual state of metabolism.


Assuntos
Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Intestino Delgado/enzimologia , Animais , Frutose/metabolismo , Cobaias , Humanos , Microssomos/enzimologia , Ratos , Especificidade da Espécie
7.
Anal Bioanal Chem ; 411(26): 7005-7013, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31440781

RESUMO

Estrogens are key factors in the development of the estrogen receptor-positive (ER+) breast cancer. Estrogens, estrone (E1), and estradiol (E2) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER+ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E1 (ratio E1/AD) using isotopic analogues as internal standards. The method showed good linearity (r2 = 0.9908 for AD and 0.9944 for E1), high accuracy (more than 74% for AD and more than 66% for E1), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC50 values of the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from the radiometric assay are also compared, and similar IC50 values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs. Graphical abstract.


Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Microssomos/enzimologia , Aromatase/análise , Ensaios Enzimáticos/métodos , Feminino , Humanos , Microextração em Fase Líquida/métodos , Placenta/enzimologia , Gravidez
8.
Toxicol Lett ; 314: 181-186, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31404594

RESUMO

The cytochrome P450 enzyme (CYP) family includes key enzymes for the metabolism of drugs and xenobiotics. Several animal models have been used to determine the metabolite profile of specific drugs. Among these are porcine microsomes prepared from Göttingen minipigs. However, CYP expression profile in microsomes from this pig breed is unknown. In the present study, we determined the mRNA and protein profiles of a comprehensive selection of CYPs in microsomes prepared from male and female Göttingen minipigs. Using RT-PCR, western blotting and mass spectroscopy, we found that the expression levels of CYP1A, CYP2A and CYP2E1 were significantly higher in females than males. Moreover, some of the transcription factors controlling CYP transcription also showed a sex-dependent expression pattern. Conversely, expression of CYP2B, CYP2D and CYP3A was comparable between sexes. The overall CYP expression distribution showed high similarity with what previously been reported in humans. In conclusion, our results suggest that Göttingen minipigs are a reliable model for studying CYPs.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Isoenzimas , Masculino , Microssomos/enzimologia , Modelos Animais , Fatores Sexuais , Especificidade por Substrato , Suínos , Porco Miniatura
10.
Sci Rep ; 9(1): 8607, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197208

RESUMO

Cell membrane phospholipids regulate various biological functions. We previously reported enzymatic fluorometric methods for quantifying phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, phosphatidylglycerol and cardiolipin. In the present report, a new enzymatic fluorometric assay was developed for quantifying phosphatidylinositol. These simple, sensitive and high-throughput methods enabled us to quantify all major phospholipid classes in cultured cells and intracellular organelles. By conducting comprehensive quantitative analyses of major phospholipid classes, we demonstrated that the contents of phospholipid classes in HEK293 cells changed with cell density and that overexpression of phosphatidylinositol synthase or CDP-diacylglycerol synthase significantly affected the phospholipid compositions of microsomal and mitochondrial membranes. These enzymatic fluorometric assays for measuring all major phospholipid classes may be applicable to tissues, fluids, lipoproteins, extracellular vesicles and intracellular organelles of many organisms and will further our understanding of cellular, physiological and pathological processes.


Assuntos
Ensaios Enzimáticos , Fluorometria/métodos , Espaço Intracelular/metabolismo , Organelas/enzimologia , Fosfolipídeos/metabolismo , Contagem de Células , Células HEK293 , Humanos , Microssomos/enzimologia , Fosfatidilinositóis/metabolismo , Fosfolipase D/metabolismo
11.
Biomed Res Int ; 2019: 5747436, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214617

RESUMO

The metabolic syndrome is defined by impaired carbohydrate metabolism and lipid disorders and often accompanied by hypertension, all of which will lead to obesity and insulin resistance. Glucocorticoids play a regulatory role in the metabolism of proteins, lipids, and carbohydrates. There is growing evidence for a role of glucocorticoids in the development of the metabolic syndrome. The most important factor that regulates the access of endogenous glucocorticoids to receptors after release of glucocorticoids and their diffusion into the cytoplasm of target cells is the steroid metabolism involving a microsomal enzyme, 11ß-hydroxysteroid dehydrogenase (11ß-HSD). The changes in intracellular glucocorticoid metabolism in the pathogenesis of obesity indicate the participation of modulation by 11ß-HSD1, which may represent a new therapeutic target for the treatment of diseases such as type 2 diabetes, visceral obesity, or atherosclerosis. The aim of our study was to determine the fast and effective method to assess inhibition activity of compounds in relation with 11ß-hydroxysteroid dehydrogenase. The material for this study was human liver and kidney microsomes. In this study we used ELISA technique using 96-well microplates coated with antibodies which were specific for analyzed enzymes. The method can quickly and efficiently measure the inhibition of both 11ß-HSD1 and 11ß-HSD2. This method can be used to search for and determine inhibitors of this enzyme. Cortisone and cortisol were used as the substrates for corresponding enzyme assays. Furthermore, 3-N-allyl-2-thiouracil derivatives were used by us for comparison purposes in developing the method, although, due to their structure, those derivatives have not previously been considered as potential inhibitors of 11ß-HSD1. 3-N-Allyl-2-thiouracil derivatives are a group worth considering, because by modifying their structure (e.g., by introducing other substituents into the pyrimidine ring) it will be possible to obtain an increase in the activity of compounds in this regard. In conclusion, this study shows an efficient and fast method of determining inhibition activity of compounds in relation with 11ß-hydroxysteroid dehydrogenase.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Inibidores Enzimáticos , Microssomos/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , Aterosclerose/tratamento farmacológico , Aterosclerose/enzimologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Obesidade Abdominal/tratamento farmacológico , Obesidade Abdominal/enzimologia
12.
Drug Metab Dispos ; 47(7): 732-742, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31043439

RESUMO

Nonspecific drug partitioning into microsomal membranes must be considered for in vitro-in vivo correlations. This work evaluated the effect of including lipid partitioning in the analysis of complex TDI kinetics with numerical methods. The covariance between lipid partitioning and multiple inhibitor binding was evaluated. Simulations were performed to test the impact of lipid partitioning on the interpretation of TDI kinetics, and experimental TDI datasets for paroxetine (PAR) and itraconazole (ITZ) were modeled. For most kinetic schemes, modeling lipid partitioning results in statistically better fits. For MM-IL simulations (KI,u = 0.1 µM, kinact = 0.1 minute-1), concurrent modeling of lipid partitioning for an fumic range (0.01, 0.1, and 0.5) resulted in better fits compared with post hoc correction (AICc: -526 vs. -496, -579 vs. -499, and -636 vs. -579, respectively). Similar results were obtained with EII-IL. Lipid partitioning may be misinterpreted as double binding, leading to incorrect parameter estimates. For the MM-IL datasets, when fumic = 0.02, MM-IL, and EII model fits were indistinguishable (δAICc = 3). For less partitioned datasets (fumic = 0.1 or 0.5), the inclusion of partitioning resulted in better models. The inclusion of lipid partitioning can lead to markedly different estimates of KI,u and kinact A reasonable alternate experimental design is nondilution TDI assays, with post hoc fumic incorporation. The best fit models for PAR (MIC-M-IL) and ITZ (MIC-EII-M-IL and MIC-EII-M-Seq-IL) were consistent with their reported mechanism and kinetics. Overall, experimental fumic values should be concurrently incorporated into TDI models with complex kinetics, when dilution protocols are used.


Assuntos
Metabolismo dos Lipídeos , Microssomos/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Itraconazol/farmacocinética , Microssomos/enzimologia , Modelos Biológicos , Paroxetina/farmacocinética
13.
Eur J Med Chem ; 171: 332-342, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928706

RESUMO

New pyrazole derivatives 2-5 were synthesized and evaluated for their COX-1 and COX-2 inhibitory activity in vitro. All compounds showed good inhibitory activity at a nanomolar level and most compounds exhibited selectivity towards COX-2 inhibition. Compounds 2a, 3b, 4a, 5b and 5e exhibited IC50 towards COX-2 enzyme of 19.87, 39.43, 61.24, 38.73 and 39.14 nM, respectively. Furthermore, compounds 3b, 4a, 5b and 5e exhibited a selectivity index of 22.21, 14.35, 17.47 and 13.10, respectively. The most active compounds were further subjected to in vivo anti-inflammatory assay. The tested compounds showed better or comparable activity to celecoxib as positive control. In order to explore their binding mode and selectivity behaviour, molecular docking in the active site of COX-2 was carried out for these derivatives. Analysis of the docked poses of the compounds showed that they adopt similar conformations to the highly selective COX-2 inhibitor, SC-558. The docking pose of compound 3b was confirmed by molecular dynamics. All the tested compounds exhibited potent inhibitory effect on the production of PGE2, in addition to their inhibition of COX-2 enzyme.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Microssomos/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Pirazóis/farmacologia , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/química , Relação Dose-Resposta a Droga , Humanos , Microssomos/enzimologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade
14.
Br J Clin Pharmacol ; 85(6): 1367-1377, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30817016

RESUMO

AIMS: To characterize mRNA expression levels (17 cytochromes P450) and activity (9 isoforms) of major cytochromes P450 expressed throughout the human small intestine. METHODS: Tissue samples were obtained from 9 deceased subjects and intestinal sections (n = 10) were isolated for each subject. Relative mRNA expression levels were determined using quantitative real-time PCR. Intestinal microsomes were prepared from 5 subsections: duodenum, jejunum (proximal and mid-jejunum) and ileum (proximal and mid-ileum) regions. In vitro incubations were performed with various cytochrome P450 probe substrates: bupropion (CYP2B6), repaglinide (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), chlorzoxazone (CYP2E1), ebastine (CYP2J2), midazolam (CYP3A4/5) and lauric acid (CYP4A11). Metabolite formation was assessed using validated liquid chromatography-tandem mass spectrometry assays. RESULTS: Cytochrome P450 mRNA levels ranked as follows: CYP3A4 > CYP2C9 > CYP2C19 > CYP2J2 > CYP4F2. Cytochrome P450 mRNA transcripts showed different patterns in their relative expression from 1 region to the other but CYP3A4, CYP2C9, CYP2C19 and CYP2J2 displayed the highest levels of mRNA expression (>5%) in all intestinal sections. Cytochrome P450 activities were greater in proximal part of the small intestine with the jejunum showing the greatest drug-metabolism activity. Spearman's correlation analyses indicated that cytochrome P450 mRNA expressions and corresponding cytochrome P450 activities in the human intestine were moderately associated for CYP2C19, CYP2D6 and CYP4A11 (rs  = 0.44-0.56). CONCLUSIONS: Our study provides new and additional information on the expression and activities of selected cytochromes P450 in various sections of the human small intestine.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Intestino Delgado/enzimologia , RNA Mensageiro/metabolismo , Adulto , Idoso , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas , Masculino , Microssomos/enzimologia , Pessoa de Meia-Idade , RNA Mensageiro/genética , Especificidade por Substrato
15.
Biomed Khim ; 65(1): 63-66, 2019 Jan.
Artigo em Russo | MEDLINE | ID: mdl-30816099

RESUMO

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredutases Intramoleculares/metabolismo , Humanos , Microssomos/enzimologia , Mitocôndrias/enzimologia , Prostaglandinas I/biossíntese , Tromboxano A2/biossíntese
16.
Eur J Pharm Sci ; 132: 125-131, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30878380

RESUMO

Two types of haloperidol prodrugs in which a chemical modification was carried out on the hydroxyl group or carbonyl group were synthesized, and their metabolic activation abilities were evaluated in a human liver microsome (HLM) solution, a human small intestine microsome (HIM) solution and solutions of human recombinant carboxylesterases (hCESs). The metabolic activation rates of alcohol ester prodrugs in HLM solution were similar to those in hCES2 solution, and haloperidol pentanoate and haloperidol hexanoate showed high metabolic activation rates in the synthesized alcohol ester prodrugs. In addition, haloperidol acetate and haloperidol 2-methylbutanoate were hydrolyzed as slowly as haloperidol decanoate. The results suggested that haloperidol prodrugs with a small chain or a branched chain are useful as prodrugs for sustained release. The metabolic activation rate of the enol ester prodrug in HLM solution was similar to that in hCES1 solution, and the enol ester prodrug was found to behave differently from alcohol ester prodrugs, which were metabolically activated by hCES2.


Assuntos
Carboxilesterase/metabolismo , Haloperidol/análogos & derivados , Haloperidol/síntese química , Microssomos/enzimologia , Pró-Fármacos/síntese química , Estabilidade de Medicamentos , Ésteres , Haloperidol/metabolismo , Humanos , Técnicas In Vitro , Inativação Metabólica , Intestino Delgado/enzimologia , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Pró-Fármacos/metabolismo
17.
J Lipid Res ; 60(5): 953-962, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30792183

RESUMO

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.


Assuntos
Serina C-Palmitoiltransferase/metabolismo , Serina/química , Serina/metabolismo , Sphingomonas/enzimologia , Humanos , Marcação por Isótopo , Cinética , Microssomos/enzimologia , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/isolamento & purificação , Especificidade por Substrato
18.
Biochimie ; 160: 28-33, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30763639

RESUMO

Osteoarthritis (OA) is one of the main locomotor disorders in horses. Although nonsteroidal anti-inflammatory drugs are the first-line treatment for OA, opioids could also be used. In previous studies, opioids showed promising anti-inflammatory and analgesic effects. In this study, we aimed to investigate the effects of two opioids (morphine and methadone) against inflammation in lipopolysaccharide (LPS)-stimulated synoviocytes by analyzing microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. Synoviocytes were obtained from the joints at the distal limbs of dead animals. The cytotoxic effects of LPS, morphine, and methadone were investigated by using a cell viability assay with crystal violet dye. Synoviocytes were treated with LPS, LPS plus morphine, or LPS plus methadone for 3, 6, and 12 h, and mPGES-1 and PTGS2 expression was measured using real-time polymerase chain reaction. LPS, and morphine did not affect the viability of synoviocytes, even at high concentrations. LPS treatment increased mPGES-1 and PTGS2 expression, whereas morphine inhibited the increase in mPGES-1 and PTGS2 expression in LPS-stimulated synoviocytes. Methadone did not inhibit mPGES-1 or PTGS2 expression. These results suggest that morphine may exhibit anti-inflammatory effect; therefore, it might be beneficial for the treatment of OA.


Assuntos
Ciclo-Oxigenase 2/química , Inflamação/tratamento farmacológico , Lipopolissacarídeos/toxicidade , Metadona/farmacologia , Microssomos/enzimologia , Morfina/farmacologia , Prostaglandina-E Sintases/antagonistas & inibidores , Sinoviócitos/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Animais , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cavalos , Inflamação/induzido quimicamente , Inflamação/enzimologia , Masculino , Sinoviócitos/imunologia , Sinoviócitos/metabolismo , Sinoviócitos/patologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-30267892

RESUMO

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ±â€¯4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ±â€¯0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ±â€¯5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ±â€¯0.42 mmol L-1), Mg2+ (VM = 130.6 ±â€¯6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ±â€¯0.042 mmol L-1), K+ (VM = 126.7 ±â€¯7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ±â€¯0.0079 mmol L-1) and NH4+ (VM = 134.5 ±â€¯8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ±â€¯0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ±â€¯0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.


Assuntos
Proteínas de Artrópodes/metabolismo , Microssomos/enzimologia , Palaemonidae/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Biocatálise , Brasil , Inibidores Enzimáticos/farmacologia , Brânquias/enzimologia , Brânquias/crescimento & desenvolvimento , Brânquias/fisiologia , Microssomos/efeitos dos fármacos , Ouabaína/farmacologia , Palaemonidae/citologia , Palaemonidae/crescimento & desenvolvimento , Palaemonidae/fisiologia , Rios , Tolerância ao Sal , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores
20.
Xenobiotica ; 49(10): 1133-1142, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30392427

RESUMO

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions. Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis-Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot. Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (∼50-fold) affinity but a higher (∼six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes. Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction.


Assuntos
Encéfalo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Microssomos/enzimologia , Mitocôndrias/enzimologia , Oxirredutases O-Desmetilantes/metabolismo , Animais , Química Encefálica , Sistema Enzimático do Citocromo P-450/química , Família 2 do Citocromo P450/química , Cinética , Masculino , Oxirredutases O-Desmetilantes/química , Ratos , Ratos Sprague-Dawley
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA