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1.
Chem Biol Interact ; 352: 109775, 2022 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-34910929

RESUMO

Vicagrel, an antiplatelet drug candidate targeting platelet P2Y12 receptor and has finished its phase II clinical trial. The inhibition of six major cytochrome P450 enzymes (P450) (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six UDP-glucuronosyltransferases (UGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) by vicagrel was evaluated using pooled human liver microsomes and specific probe substrates. Physiology-based pharmacokinetic (PBPK) simulation was further applied to predict the in vivo drug-drug interaction (DDI) potential between vicagrel and bupropion as well as S-mephenytoin. The results suggested that vicagrel inhibited CYP2B6 and CYP2C19 potently with apparent IC50 values of 1.6 and 2.0 µM, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of CYP2B6-catalyzed bupropion hydroxylation and noncompetitive inhibition of CYP2C19-mediated S-mephenytoin 4'-hydroxylation with Ki values of 0.19 µM and 1.2 µM, respectively. Vicagrel displayed profound time-dependent inhibition towards CYP2B6 with maximal rate constant of inactivation (kinact) and half-maximal inactivator concentration (KI) values of 0.062 min-1 and 1.52 µM, respectively. No time-dependent inhibition by vicagrel was noted for CYP2C19. For UGT, negligible to moderate inhibition by vicagrel was observed with IC50 values of >50.0, >50.0, 28.2, 8.7, >50.0 and 28.2 µM for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of UGT1A6-catalyzed N-Acetylserotonin ß-D-glucuronidation with a Ki value of 5.6 µM. No time-dependent inhibition by vicagrel was noted for UGT1A6. PBPK simulation indicated that neither altered AUC nor Cmax of bupropion and S-mephenytoin was observed in the presence of vicagrel. Our study provides inhibitory constants for future DDI prediction between vicagrel and drug substrates of CYP2B6, CYP2C19 and UGT1A6. In addition, our simulation suggests the lack of clinically important DDI between vicagrel and bupropion or S-mephenytoin.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Fenilacetatos/farmacologia , Tiofenos/farmacologia , Bupropiona/administração & dosagem , Bupropiona/farmacocinética , Simulação por Computador , Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/administração & dosagem , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Citocromo P-450 CYP2C19/metabolismo , Inibidores do Citocromo P-450 CYP2C19/administração & dosagem , Inibidores do Citocromo P-450 CYP2C19/farmacologia , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Glucuronosiltransferase/metabolismo , Humanos , Técnicas In Vitro , Cinética , Mefenitoína/administração & dosagem , Mefenitoína/farmacocinética , Fenilacetatos/administração & dosagem , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacologia , Tiofenos/administração & dosagem
2.
Methods Mol Biol ; 2303: 63-70, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626370

RESUMO

Hyaluronan (HA) is a component of the extracellular matrix that is involved in many physiological and pathological processes. As HA modulates several functions (i.e., cell proliferation and migration, inflammation), its presence in the tissues can have positive or negative effects. HA synthases (HAS) are a family of three isoenzymes located on the plasma membrane that are responsible for the production of such polysaccharide and, therefore, their activity is critical to determine the accumulation of HA in tissues. Here, we describe a nonradioactive method to quantify the HAS enzymatic activity in crude cellular membrane preparation.


Assuntos
Glucuronosiltransferase/metabolismo , Membrana Celular , Matriz Extracelular , Receptores de Hialuronatos , Hialuronan Sintases , Ácido Hialurônico
3.
Methods Mol Biol ; 2303: 453-468, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626400

RESUMO

The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating ß-1,4 and ß-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Miofibroblastos , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico , Miofibroblastos/metabolismo
4.
Cells ; 10(12)2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34944101

RESUMO

Chondroitin sulfate (CS) is a major component of the extracellular matrix found to be abnormally accumulated in several types of cancer tissues. Previous studies have indicated that CS synthases and modification enzymes are frequently elevated in human gliomas and are associated with poor prognosis. However, the underlying mechanisms of CS in cancer progression and approaches for interrupting its functions in cancer cells remain largely unexplored. Here, we have found that CS was significantly enriched surrounding the vasculature in a subset of glioma tissues, which was akin to the perivascular niche for cancer-initiating cells. Silencing or overexpression of the major CS synthase, chondroitin sulfate synthase 1 (CHSY1), significantly regulated the glioma cell invasive phenotypes and modulated integrin expression. Furthermore, we identified CD44 as a crucial chondroitin sulfate proteoglycan (CSPG) that was modified by CHSY1 on glioma cells, and the suppression of CS formation on CD44 by silencing the CHSY1-inhibited interaction between CD44 and integrin ß1 on the adhesion complex. Moreover, we tested the CS-specific binding peptide, resulting in the suppression of glioma cell mobility in a fashion similar to that observed upon the silencing of CHSY1. In addition, the peptide demonstrated significant affinity to CD44, promoted CD44 degradation, and suppressed integrin ß1 expression in glioma cells. Overall, this study proposes a potential regulatory loop between CS, CD44, and integrin ß1 in glioma cells, and highlights the importance of CS in CD44 stability. Furthermore, the targeting of CS by specific binding peptides has potential as a novel therapeutic strategy for glioma.


Assuntos
Sulfatos de Condroitina/metabolismo , Glioma/metabolismo , Glioma/patologia , Receptores de Hialuronatos/metabolismo , Integrina beta1/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Glioma/irrigação sanguínea , Glioma/genética , Glucuronosiltransferase/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Enzimas Multifuncionais/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Peptídeos/metabolismo , Fenótipo , Proteólise
5.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639166

RESUMO

Coumarins, natural products abundant in Melilotus albus, confer features in response to abiotic stresses, and are mainly present as glycoconjugates. UGTs (UDP-glycosyltransferases) are responsible for glycosylation modification of coumarins. However, information regarding the relationship between coumarin biosynthesis and stress-responsive UGTs remains limited. Here, a total of 189 MaUGT genes were identified from the M. albus genome, which were distributed differentially among its eight chromosomes. According to the phylogenetic relationship, MaUGTs can be classified into 13 major groups. Sixteen MaUGT genes were differentially expressed between genotypes of Ma46 (low coumarin content) and Ma49 (high coumarin content), suggesting that these genes are likely involved in coumarin biosynthesis. About 73.55% and 66.67% of the MaUGT genes were differentially expressed under ABA or abiotic stress in the shoots and roots, respectively. Furthermore, the functions of MaUGT68 and MaUGT186, which were upregulated under stress and potentially involved in coumarin glycosylation, were characterized by heterologous expression in yeast and Escherichia coli. These results extend our knowledge of the UGT gene family along with MaUGT gene functions, and provide valuable findings for future studies on developmental regulation and comprehensive data on UGT genes in M. albus.


Assuntos
Cumarínicos/metabolismo , Genoma de Planta , Glucuronosiltransferase/metabolismo , Melilotus/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Difosfato de Uridina/metabolismo , Cromossomos de Plantas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Glucuronosiltransferase/genética , Glicosilação , Melilotus/genética , Melilotus/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/genética , Transcriptoma
6.
Molecules ; 26(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34500611

RESUMO

Human natural killer-1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.


Assuntos
Antígenos CD57/biossíntese , Glucuronosiltransferase/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Epitopos/metabolismo , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Polissacarídeos/metabolismo
7.
J Agric Food Chem ; 69(32): 9249-9258, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34357767

RESUMO

Resveratrol (RES) suffers from poor water solubility and extensive metabolism, which lead to low bioavailability. A phospholipid complex (PC) containing RES and a UDP-glucuronosyltransferase (UGT) inhibitor was prepared to address these two limiting factors, thereby improving RES bioavailability. First, 11 natural active ingredients metabolized by similar enzyme subtypes to RES were screened in a glucuronidation assay in liver microsomes. Then, glycyrrhetinic acid (GA), the strongest inhibitor, was prepared with RES in a PC. RES-PC was prepared as a control. As expected, the water solubility and the cumulative dissolution of RES were significantly enhanced by RES-PC and RES/GA-PC. Compared with the RES group, the AUC0-10 of RES and resveratrol-3-glucuronide (R-3-G) in the RES/GA-PC group showed increases of 2.49- and 1.70-fold, respectively, with the proportion of RES absorption to total absorption increasing 1.45 times. These results demonstrated that RES/GA-PC could improve the bioavailability of RES by increasing its water solubility and inhibiting its glucuronidation.


Assuntos
Glucuronosiltransferase , Microssomos Hepáticos , Disponibilidade Biológica , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/metabolismo , Resveratrol/metabolismo , Solubilidade , Água/metabolismo
8.
Molecules ; 26(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34361561

RESUMO

Glucuronidation is a Phase 2 metabolic pathway responsible for the metabolism and excretion of testosterone to a conjugate testosterone glucuronide. Bioavailability and the rate of anabolic steroid testosterone metabolism can be affected upon UGT glucuronidation enzyme alteration. However, there is a lack of information about the in vitro potential assessment of UGT2B17 inhibition by salicylic acid. The purpose of this study is to investigate if UGT2B17 enzyme activity is inhibited by salicylic acid. A UGT2B17 assay was developed and validated by HPLC using a C18 reversed phase column (SUPELCO 25 cm × 4.6 mm, 5 µm) at 246 nm using a gradient elution mobile phase system: (A) phosphate buffer (0.01 M) at pH = 3.8, (B) HPLC grade acetonitrile and (C) HPLC grade methanol. The UGT2B17 metabolite (testosterone glucuronide) was quantified using human UGT2B17 supersomes by a validated HPLC method. The type of inhibition was determined by Lineweaver-Burk plots. These were constructed from the in vitro inhibition of salicylic acid at different concentration levels. The UGT2B17 assay showed good linearity (R2 > 0.99), acceptable recovery and accuracy (80-120%), good reproducibility and acceptable inter and intra-assay precision (<15%), low detection (6.42 and 2.76 µM) and quantitation limit values (19.46 and 8.38 µM) for testosterone and testosterone glucuronide respectively, according to ICH guidelines. Testosterone and testosterone glucuronide were found to be stable up to 72 h in normal laboratory conditions. Our investigational study showed that salicylic acid uncompetitively inhibited UGT2B17 enzyme activity. Thus, drugs that are substrates for the UGT2B17 enzyme have negligible potential effect of causing interaction with salicylic acid in humans.


Assuntos
Glucuronosiltransferase , Antígenos de Histocompatibilidade Menor , Ácido Salicílico/farmacologia , Testosterona/análogos & derivados , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Testosterona/metabolismo
9.
Clin Pharmacol Ther ; 110(5): 1389-1400, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34390491

RESUMO

The applications of translational modeling of local drug concentrations in various organs had a sharp increase over the last decade. These are part of the model-informed drug development initiative, adopted by the pharmaceutical industry and promoted by drug regulatory agencies. With respect to the kidney, the models serve as a bridge for understanding animal vs. human observations related to renal drug disposition and any consequential adverse effects. However, quantitative data on key drug-metabolizing enzymes and transporters relevant for predicting renal drug disposition are limited. Using targeted and global quantitative proteomics, we determined the abundance of multiple enzymes and transporters in 20 human kidney cortex samples. Nine enzymes and 22 transporters were quantified (8 for the first time in the kidneys). In addition, > 4,000 proteins were identified and used to form an open database. CYP2B6, CYP3A5, and CYP4F2 showed comparable, but generally low expression, whereas UGT1A9 and UGT2B7 levels were the highest. Significant correlation between abundance and activity (measured by mycophenolic acid clearance) was observed for UGT1A9 (Rs = 0.65, P = 0.004) and UGT2B7 (Rs = 0.70, P = 0.023). Expression of P-gp ≈ MATE-1 and OATP4C1 transporters were high. Strong intercorrelations were observed between several transporters (P-gp/MRP4, MRP2/OAT3, and OAT3/OAT4); no correlation in expression was apparent for functionally related transporters (OCT2/MATEs). This study extends our knowledge of pharmacologically relevant proteins in the kidney cortex, with implications on more prudent use of mechanistic kidney models under the general framework of quantitative systems pharmacology and toxicology.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Córtex Renal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Proteômica/métodos , Sistema Enzimático do Citocromo P-450/genética , Bases de Dados Factuais , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Rim/metabolismo , Cinética , Proteínas de Membrana Transportadoras/genética
10.
Zhongguo Zhong Yao Za Zhi ; 46(13): 3410-3421, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34396762

RESUMO

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 µL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 µL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 µL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 µL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 µL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 µL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 µL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 µL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 µL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 µL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 µL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 µL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 µL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.


Assuntos
Glucuronosiltransferase , Microssomos Hepáticos , Animais , Benzofuranos , Cumarínicos , Cães , Glucuronídeos , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Cinética , Camundongos , Microssomos Hepáticos/metabolismo , Fenótipo , Ratos , Especificidade da Espécie , Suínos , Porco Miniatura/metabolismo
11.
Xenobiotica ; 51(10): 1146-1154, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34423713

RESUMO

Lamotrigine is a phenyltriazine anticonvulsant used to treat epilepsy and bipolar disorder, with species-dependent metabolic profiles. In this study, we investigated the metabolism of lamotrigine in chimeric NOG-TKm30 mice transplanted with human hepatocytes (humanised-liver mice).Substantial lamotrigine N2-glucuronidation activities were observed in the liver microsomes from humanised-liver mice, humans, marmosets, and rabbits, compared to those from monkeys, minipigs, guinea pigs, rats, and mice. Lamotrigine N2-glucuronidation activities in the liver microsomes from humanised-liver mice were dose-dependently inhibited by hecogenin, a specific inhibitor of the human UGT1A4.The major metabolite in the hepatocytes from humanised-liver mice and humans was lamotrigine N2-glucuronide, whereas that in mouse hepatocytes was lamotrigine N2-oxide. After a single oral dose of lamotrigine (10 mg/kg), the plasma levels of N2-glucuronide, N5-glucuronide, and N2-methyl were higher in humanised-liver mice compared to that in NOG-TKm30 mice. Lamotrigine N2-glucuronide was the most abundant metabolite in the urine in humanised-liver mice, similar to that reported in humans; whereas, lamotrigine N2-oxide was predominantly excreted in the urine in NOG-TKm30 mouse.These results suggest that humanised-liver mice may be a suitable animal model for studying the UGT1A4 mediated-lamotrigine metabolism.


Assuntos
Anticonvulsivantes , Glucuronosiltransferase , Animais , Anticonvulsivantes/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Cobaias , Lamotrigina/metabolismo , Fígado/metabolismo , Redes e Vias Metabólicas , Camundongos , Microssomos Hepáticos/metabolismo , Coelhos , Ratos , Suínos , Porco Miniatura/metabolismo
12.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443505

RESUMO

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18-11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.


Assuntos
Glutationa/metabolismo , Isotiocianatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Sulfóxidos/farmacologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Glucuronosiltransferase/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Humanos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Neoplasias da Bexiga Urinária/enzimologia
13.
Methods Mol Biol ; 2342: 301-338, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272700

RESUMO

Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation.


Assuntos
Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Vias de Eliminação de Fármacos , Interações Medicamentosas , Humanos , Cinética , Fenótipo , Especificidade por Substrato
14.
Methods Mol Biol ; 2342: 443-479, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272704

RESUMO

There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.


Assuntos
Enzimas/metabolismo , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Microssomos Hepáticos/enzimologia , Técnicas de Cultura de Células , Células Cultivadas , Enzimas/genética , Glucuronosiltransferase/genética , Hepatócitos/metabolismo , Humanos , Cinética , Variantes Farmacogenômicos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
15.
Methods Mol Biol ; 2342: 595-629, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272707

RESUMO

This chapter provides regulatory perspectives on how to translate in vitro drug metabolism findings into in vivo drug-drug interaction (DDI) predictions and how this affects the decision of conducting in vivo DDI evaluation. The chapter delineates rationale and analyses that have supported the recommendations in the U.S. Food and Drug Administration (FDA) DDI guidances in terms of in vitro-in vivo extrapolation of cytochrome P450 (CYP) inhibition-mediated DDI potential for investigational new drugs and their metabolites as substrates or inhibitors. The chapter also describes the framework and considerations to assess UDP-glucuronosyltransferase (UGT) inhibition-mediated DDI potential for drugs as substrates or inhibitors. The limitations of decision criteria and further improvements needed are also discussed. Case examples are provided throughout the chapter to illustrate how decision criteria have been utilized to evaluate in vivo DDI potential from in vitro data.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/metabolismo , Legislação de Medicamentos/organização & administração , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Interações Medicamentosas , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/química , Humanos , Cinética , Guias de Prática Clínica como Assunto , Estados Unidos , United States Food and Drug Administration/legislação & jurisprudência
16.
Methods Mol Biol ; 2342: 695-707, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272713

RESUMO

New molecular entities (NMEs) are evaluated using a rigorous set of in vitro and in vivo studies to assess their safety and suitability for testing in humans. Regulatory health authorities require that therapeutic and supratherapeutic doses be administered, by the intended route of administration, to two nonclinical species prior to human testing. The purpose of these studies is to identify potential target organ toxicity and to determine if the effects are reversible. Liver is a potential site for toxicity caused by orally administered NMEs due to high exposure during first pass after oral administration. A range of clinical chemistry analytes are routinely measured in both nonclinical and clinical studies to evaluate and monitor for hepatotoxicity. While bilirubin itself circulates within a wide range of concentrations in many animal species and humans, without causing adverse effects and possibly providing benefits, bilirubin is one of the few readily monitored circulating biomarkers that can provide insight into liver function. Therefore, any changes in plasma or urine bilirubin levels must be carefully evaluated. Changes in bilirubin may occur as a result of adaptive nontoxic changes or severe toxicity. Examples of adaptive nontoxic changes in liver function, which may elevate direct (conjugated) and/or indirect (unconjugated) bilirubin above baseline levels, include reversible inhibition of UGT1A1-mediated bilirubin metabolism and OATP1B1-, OATP1B3-, or MRP2-mediated transport. Alternatively, hepatocellular necrosis, hypoalbuminuria, or cholestasis may also lead to elevation of bilirubin; in some cases, these effects may be irreversible.This chapter aims to demonstrate application of enzyme kinetic principles in understanding the risk of bilirubin elevation through inhibition of multiple processes-involving both enzymes and transporters. In the sections that follow, we first provide a brief summary of bilirubin formation and disposition. Two case examples are then provided to illustrate the enzyme kinetic studies needed for risk assessment and for identifying the mechanisms of bilirubin elevation. Caveats of methods and data interpretation are discussed in these case studies. The data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in enzyme kinetics to the reader.


Assuntos
Bilirrubina/análise , Glucuronosiltransferase/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Bilirrubina/sangue , Bilirrubina/urina , Cães , Desenvolvimento de Medicamentos , Humanos , Concentração Inibidora 50 , Cinética , Ratos , Bibliotecas de Moléculas Pequenas/efeitos adversos
17.
Biomed Chromatogr ; 35(11): e5205, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34192355

RESUMO

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.


Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Imidazóis/farmacologia , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Cromatografia Líquida de Alta Pressão , Interações Medicamentosas , Glucuronosiltransferase/química , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Himecromona/análise , Himecromona/metabolismo , Cinética , Isoformas de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triflupromazina/análise , Triflupromazina/metabolismo
18.
BMC Plant Biol ; 21(1): 245, 2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34051740

RESUMO

BACKGROUND: Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of ß-glucuronosyltransferase genes/enzymes. RESULTS: Here, we have characterized two ß-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of ß-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size. CONCLUSIONS: These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.


Assuntos
Arabidopsis/enzimologia , Galactanos/metabolismo , Glucuronosiltransferase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Esterificação , Glucuronosiltransferase/genética , Monossacarídeos/metabolismo , Pectinas/metabolismo , Fenótipo , Polissacarídeos/metabolismo , Sementes/enzimologia , Sementes/genética
19.
J Pharm Pharmacol ; 73(3): 388-397, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33793880

RESUMO

OBJECTIVES: To determine the kinetics of the formation of 10,11-dihydro-10-hydroxy-carbazepine (MHD)-O-glucuronide in human liver microsomes (HLMs), human intestine microsomes (HIMs), human kidney microsomes (HKMs) and recombinant human UDP-glucuronosyltransferase (UGTs), and identify the primary UGT isoforms catalyzing the glucuronidation of MHD. METHODS: The kinetics of the glucuronidation of MHD was determined in HLMs, HIMs as well as HKMs. Screening assays with 13 recombinant human UGTs, inhibition studies and correlation analysis were performed to identify the main UGTs involved in the glucuronidation of MHD. KEY FINDINGS: MHD-O-glucuronide was formed in HLMs, HIMs as well as HKMs, HLMs showed the highest intrinsic clearance of MHD. Among 13 recombinant human UGTs, UGT2B7 and UGT1A9 were identified to be the principal UGT isoforms mediating the glucuronidation of MHD, while UGT1A4 played a partial role. In addition, inhibition studies and correlation analysis further confirmed that UGT2B7 and UGT1A9 participated in the formation of MHD-O-glucuronide. CONCLUSIONS: MHD could be metabolized by UGTs in the liver, intestine and kidney, and the hepatic glucuronidation was the critical metabolic pathway. UGT2B7 and UGT1A9 were the primary UGT isoforms mediating the formation of MHD-O-glucuronide in the liver.


Assuntos
Carbamazepina/análogos & derivados , Glucuronosiltransferase/metabolismo , Microssomos/metabolismo , Carbamazepina/metabolismo , Glucuronídeos , Humanos , Intestinos/citologia , Isoenzimas , Rim/metabolismo , Microssomos Hepáticos/metabolismo
20.
PLoS One ; 16(4): e0250605, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33891666

RESUMO

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.


Assuntos
Glucuronosiltransferase/genética , Hiperbilirrubinemia Hereditária/patologia , Vírus 40 dos Símios/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Linhagem Celular Tumoral , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucuronosiltransferase/deficiência , Glucuronosiltransferase/metabolismo , Humanos , Hiperbilirrubinemia Hereditária/genética , Fígado/metabolismo , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Distribuição Tecidual , Ativação Transcricional
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