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1.
Xenobiotica ; 51(1): 61-71, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32813611

RESUMO

UR-1102, a novel uricosuric agent for treating gout, has been confirmed to exhibit a pharmacological effect in patients. We clarified its metabolic pathway, estimated the contribution of each metabolic enzyme, and assessed the impact of genetic polymorphisms using human in vitro materials. Glucuronide, sulfate and oxidative metabolites of UR-1102 were detected in human hepatocytes. The intrinsic clearance by glucuronidation or oxidation in human liver microsomes was comparable, but sulfation in the cytosol was much lower, indicating that the rank order of contribution was glucuronidation ≥ oxidation > sulfation. Recombinant UGT1A1 and UGT1A3 showed high glucuronidation of UR-1102. We took advantage of a difference in the inhibitory sensitivity of atazanavir to the UGT isoforms and estimated the fraction metabolised (fm) with UGT1A1 to be 70%. Studies using recombinant CYPs and CYP isoform-specific inhibitors showed that oxidation was mediated exclusively by CYP2C9. The effect of UGT1A1 and CYP2C9 inhibitors on UR-1102 metabolism in hepatocytes did not differ markedly between the wild type and variants.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Glucuronosiltransferase/metabolismo , Gota/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Glucuronídeos/metabolismo , Gota/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxazinas/metabolismo , Piridinas/metabolismo
2.
Xenobiotica ; 51(1): 105-114, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32820679

RESUMO

Mycophenolic acid (MPA) has become a cornerstone of immunosuppressive therapy, in particular for transplant patients. In the gastrointestinal tract, the liver and the kidney, MPA is mainly metabolized into phenyl-ß-d glucuronide (MPAG). Knowledge about the interactions between MPA/MPAG and membrane transporters is still fragmented. The aim of the present study was to explore these interactions with the basolateral hepatic MRP4 transporter. The inhibition of the MRP4-driven transport by various drugs which can be concomitantly prescribed was also evaluated. In vitro experiments using vesicles overexpressing MRP4 showed an ATP-dependent transport of MPAG driven by MRP4 (Michaelis-Menten constant of 233.9 ± 32.8 µM). MPA was not effluxed by MRP4. MRP4-mediated transport of MPAG was inhibited (from -43% to -84%) by ibuprofen, cefazolin, cefotaxime and micafungin. An in silico approach based on molecular docking and molecular dynamics simulations rationalized the mode of binding of MPAG to MRP4. The presence of the glucuronide moiety in MPAG was highlighted as key, being prone to make electrostatic and H-bond interactions with specific residues of the MRP4 protein chamber. This explains why MPAG is a substrate of MRP4 whereas MPA is not.


Assuntos
Glucuronídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ácido Micofenólico/análogos & derivados , Transporte Biológico , Hepatócitos/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Acoplamento Molecular , Ácido Micofenólico/metabolismo
3.
Xenobiotica ; 50(11): 1285-1300, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32394778

RESUMO

Viloxazine is currently being developed as a treatment for attention deficit/hyperactivity disorder (ADHD). The aim of these studies is to update the understanding of the rat and human metabolism and the in vitro drug-drug interaction profile of viloxazine to a degree where it meets current regulatory standards for such investigations. In vivo absorption-distribution-metabolism-excretion (ADME) studies demonstrated that in humans 5-hydroxylation followed by glucuronidation is the major metabolic route. This route was also seen as a minor route in rats where the major route is O-deethylation with subsequent sulfation. In humans, the 5-hydoxylation pathway is mediated by CYP2D6. An estimate for the fraction of the metabolism via this pathway suggests a PM/EM difference of <2-fold, making it highly unlikely that this will be an issue of clinical significance. Viloxazine forms a unique N-carbamoyl glucuronide in humans. The chemical reactivity characteristics of this metabolite are similar to stable glucuronide conjugates and dissimilar from acyl glucuronides; therefore, it is regarded as a stable Phase II conjugate. In vitro drug-drug interaction (DDI) testing indicates that viloxazine is not a significant inhibitor or inducer of CYPs and transporters with the exception of CYP1A2.


Assuntos
Antidepressivos de Segunda Geração/farmacologia , Interações Medicamentosas , Viloxazina/farmacologia , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronídeos/metabolismo , Humanos , Microssomos Hepáticos/metabolismo
4.
Ecotoxicol Environ Saf ; 197: 110611, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32294595

RESUMO

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.


Assuntos
Hepatócitos/enzimologia , Fitoestrógenos/metabolismo , Salmo salar/metabolismo , Animais , Aquicultura , Biotransformação , Cromatografia Líquida , Genisteína/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Isoflavonas/metabolismo , Microssomos Hepáticos/enzimologia , Soja/química
5.
Biochem Pharmacol ; 175: 113916, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179043

RESUMO

The transport of UDP-glucuronic acid (UDPGA), a co-substrate of UDP-glucuronosyltransferase (UGT), to the intraluminal side of the endoplasmic reticulum (ER) is an essential step in the glucuronidation of exogenous and endogenous compounds. According to a previous study, the expression of recombinant SLC35B1, SLC35B4, or SLC35D1, nucleotide sugar transporters, in V79 cells has the potential to transport UDPGA into the lumen of microsomes. The purpose of this study is to examine whether the transport of UDPGA by these transporters substantially affects UGT activity. Since the knockdown of UDP-glucose 6-dehydrogenase, a synthetase of UDPGA, in HEK293 cells stably expressing UGT1A1 (HEK/UGT1A1 cells) resulted in a significant decrease in 4-methylumbelliferone (4-MU) glucuronosyltransferase activity, supplementation of a sufficient amount of UDPGA is required for UGT activity. By performing qRT-PCR using cDNA samples from 21 human liver samples, we observed levels of the SLC35B1 and SLC35D1 mRNAs that were 15- and 14-fold higher, respectively, than the levels of the SLC35B4 mRNA, and SLC35B1 showed the largest (37-fold) interindividual variability. Interestingly, 4-MU glucuronosyltransferase activity was significantly decreased upon the knockdown of SLC35B1 in HEK/UGT1A1 cells, and this phenomenon was also observed in HepaRG cells. Using siRNAs targeting 23 different SLC35 subfamilies, the knockdown of SLC35B1 and SLC35E3 decreased 4-MU glucuronosyltransferase activity in HEK/UGT1A1 cells. However, the 4-MU glucuronosyltransferase activity was not altered by SLC35E3 knockdown in HepaRG cells, suggesting that SLC35B1 was the main transporter of UDPGA into the ER in the human liver. In conclusion, SLC35B1 is a key modulator of UGT activity by transporting UDPGA to the intraluminal side of the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Proteínas de Transporte de Monossacarídeos/deficiência , Uridina Difosfato Ácido Glucurônico/metabolismo , Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes/métodos , Glucuronídeos/genética , Glucuronosiltransferase/genética , Células HEK293 , Hepatócitos/metabolismo , Humanos , Proteínas de Transporte de Monossacarídeos/genética , Uridina Difosfato Ácido Glucurônico/genética
6.
Xenobiotica ; 50(8): 906-912, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32005083

RESUMO

Wogonin, one of the flavonoids isolated from Scutellaria baicalensis, exhibits some beneficial bioactivities, including anti-inflammatory and anticancer effects, and is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in humans. In the present study, wogonin glucuronidation was examined in the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice using a kinetic analysis.The kinetics of wogonin glucuronidation by liver microsomes followed the biphasic model in all species examined. CLint values (x-intercept) based on v versus V/[S] plots were rats > humans ≈ monkeys > mice > dogs. The kinetics of intestinal microsomes fit the Michaelis-Menten model for humans, monkeys, rats, and mice and the substrate inhibition model for dogs. CLint values were rats > monkeys > mice > dogs > humans. The tissue dependence of CLint values was liver microsomes > intestinal microsomes for humans, dogs, and rats, and liver microsomes ≈ intestinal microsomes for monkeys and mice.These results demonstrated that the metabolic abilities of UGT enzymes toward wogonin in the liver and intestines markedly differ among humans, monkeys, dogs, rats, and mice, and suggest that species differences are closely associated with the biological effects of wogonin.


Assuntos
Flavanonas/metabolismo , Extratos Vegetais/metabolismo , Animais , Cães , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Intestinos , Cinética , Fígado/metabolismo , Macaca fascicularis/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Ratos
7.
Artigo em Inglês | MEDLINE | ID: mdl-32058315

RESUMO

Mitragyna speciosa (kratom) is a drug that is increasingly used recreationally and "therapeutically", in the absence of medical supervision. The drug has been associated with a growing number of fatalities, and although its medicinal properties as an atypical opioid require further study, there are legitimate concerns regarding its unregulated use. Mitragynine is the most widely reported alkaloid within the plant, although more than forty other alkaloids have been identified. 7-Hydroxymitragynine is reported to have greater abuse liability due to its increased potency relative to mitragynine. In this report, biomarkers for mitragynine were investigated using liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS). Speciociliatine and speciogynine were identified as alternative biomarkers, often exceeding the concentration of mitragynine in unhydrolyzed urine. 9-O-Demethylmitragynine and 7-hydroxymitragynine were identified in unhydrolyzed urine in 75% and 63% of the cases. Deconjugation of phase II metabolites using chemical hydrolysis was not suitable due to degradation of the Mitragyna alkaloids. Enzymatic hydrolysis was evaluated using three traditional glucuronidases, four sulfatases and four recombinant enzymes. Although enzymatic hydrolysis increased the concentration of 16-carboxymitragynine, it had nominal benefit for other metabolites. Deconjugation of urine was not necessary due to the abundance of parent drug (mitragynine), its diastereoisomers (speciociliatine and speciogynine) or metabolites (9-O-demethylmitragynine and 7-hydroxymitragynine).


Assuntos
Biomarcadores/urina , Mitragyna/metabolismo , Oxindois/urina , Extratos Vegetais/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Cromatografia Líquida de Alta Pressão , Glucuronídeos/análise , Glucuronídeos/metabolismo , Hidrólise , Metaboloma , Mitragyna/química , Extratos Vegetais/análise , Alcaloides de Triptamina e Secologanina/análise , Sulfatases/análise , Sulfatases/metabolismo , Espectrometria de Massas em Tandem
8.
Artigo em Inglês | MEDLINE | ID: mdl-31999978

RESUMO

20-hydroxyeicosatetraenoic acid (20-HETE) is an arachidonic acid metabolite which is known to increase platelet aggregation and cardiovascular risk. In this study, nine non-steroidal anti-inflammatory drugs (NSAIDs) selected by chemical structures were screened to determine their effects on the glucuronidation of 20-HETE using human liver microsomes (HLMs). Then, the combined effects of the selected NSAID and genetic polymorphisms in UDP-glucuronosyltransferase (UGT) were investigated. Among the tested NSAIDs, diclofenac was the strongest inhibitor of 20-HETE glucuronidation with an IC50 value of 3.5 µM. Celecoxib, naproxen, mefenamic acid, ibuprofen, and indomethacin showed modest inhibition with IC50 values of 77, 91, 190, 208, and 220 µM, respectively, while acetylsalicylic acid, rofecoxib, and meloxicam did not inhibit 20-HETE glucuronidation. Glucuronidation of 20-HETE by UGT2B7 and UGT1A9 recombinant enzymes was significantly inhibited by indomethacin, mefanemic acid, diclofenac, ibuprofen, naproxen, and celecoxib (P < 0.001). In addition, diclofenac exhibited a competitive inhibition mechanism with the Km value of 20-HETE glucuronidation increasing from 23.5 µM to 62 µM in the presence of 3.5 µM diclofenac. Diclofenac further decreased 20-HETE glucuronidation in HLMs carrying UGT2B7*2 alleles compared with the wild-type HLMs. The results from this study would be useful in understanding the alteration of 20-HETE levels in relation to NSAID and UGT genetic polymorphisms.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Glucuronídeos/metabolismo , Ácidos Hidroxieicosatetraenoicos/química , Microssomos Hepáticos/química , Adulto , Anti-Inflamatórios não Esteroides/química , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Pessoa de Meia-Idade , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , República da Coreia , Relação Estrutura-Atividade
10.
Eur J Pharm Sci ; 143: 105195, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31852629

RESUMO

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5'-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Piperazinas/farmacocinética , para-Aminobenzoatos/farmacocinética , Ativação Metabólica , Animais , Cães , Feminino , Glucuronídeos/metabolismo , Glutationa/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Piperazinas/sangue , Piperazinas/urina , Ratos Sprague-Dawley , para-Aminobenzoatos/sangue , para-Aminobenzoatos/urina
11.
J Vet Med Sci ; 82(2): 153-161, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31839622

RESUMO

Zearalenone (ZON), produced by Fusarium fungi, exhibits estrogenic activity. Livestock can be exposed to ZON orally through contaminating feeds such as cereals, leading to reproductive disorders such as infertility and miscarriage via endocrine system disruption. However, the details of ZON metabolism remain unclear, and the mechanism of its toxicity has not been fully elucidated. In this study, we investigated the kinetics of ZON absorption and metabolism in rat segmented everted intestines. ZON absorption was confirmed in each intestine segment 60 min after application to the mucosal buffer at 10 µM. Approximately half of the absorbed ZON was metabolized to α-zearalenol, which tended to be mainly glucuronidated in intestinal cells. In the proximal intestine, most of the glucuronide metabolized by intestinal cells was excreted to the mucosal side, suggesting that the intestine plays an important role as a first drug metabolism barrier for ZON. However, in the distal intestine, ZON metabolites tended to be transported to the serosal side. Glucuronide transported to the serosal side could be carried via the systemic circulation to the local tissues, where it could be reactivated by deconjugation. These results are important with regard to the mechanism of endocrine disruption caused by ZON.


Assuntos
Glucuronídeos/metabolismo , Absorção Intestinal/fisiologia , Zearalenona/metabolismo , Animais , Feminino , Mucosa Intestinal/metabolismo , Masculino , Gravidez , Ratos Sprague-Dawley , Zearalenona/farmacocinética , Zeranol/análogos & derivados , Zeranol/metabolismo , Zeranol/farmacocinética
12.
Anal Bioanal Chem ; 412(2): 335-342, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31788715

RESUMO

Triclosan (TCS) is an antibacterial and antifungal compound found in many hygiene products, including toothpaste, soap, and detergents. However, this molecule can act as an endocrine disruptor and can induce harmful effects on human health and the environment. In this study, triclosan was biotransformed in vitro using human and rat liver fractions, to evaluate oxidative metabolism, the formation of reactive metabolites via the detection of GSH adducts, as well as glucuronide and sulfate conjugates using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). A deuterated analog of triclosan was also employed for better structural elucidation of specific metabolic sites. Several GSH adducts were found, either via oxidative metabolism of triclosan or its cleavage product, 2,4-dichlorophenol. We also detected glucuronide and sulfated conjugates of triclosan and its cleaved product. This study was aimed at understanding the routes of detoxification of this xenobiotic, as well as investigating any potential pathways related to additional toxicity via reactive metabolite formation. Graphical abstract.


Assuntos
Anti-Infecciosos Locais/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Triclosan/metabolismo , Animais , Glucuronídeos/metabolismo , Glutationa/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Sulfatos/metabolismo
13.
J Steroid Biochem Mol Biol ; 197: 105518, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31704245

RESUMO

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 µM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 µM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 µM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.


Assuntos
Epitestosterona/metabolismo , Glucuronídeos/metabolismo , Fígado/metabolismo , Testosterona/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Humanos , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo
14.
Toxicol Lett ; 320: 46-51, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31812603

RESUMO

Pterostilbene (PT) is a natural stilbene common in small berries and food supplements, possessing numerous pharmacological activities. However, whether PT can affect the activities of UDP-glucuronosyltransferases (UGT) enzymes remains unclear. The aim of the present study was to investigate the effect of PT on UGT activities and to quantitatively evaluate the food-drug interaction potential due to UGT inhibition. Our data indicated that PT exhibited potent inhibition against HLM, UGT1A6, UGT1A9, UGT2B7, and UGT2B15, moderate inhibition against UGT1A1, UGT1A3, UGT1A8, and UGT2B4, negligible inhibition against UGT1A4, UGT1A7, UGT1A10, and UGT2B17. Further kinetic investigation demonstrated that PT exerted potent noncompetitive inhibition 4-MU glucuronidation by UGT1A9, with IC50 and Ki values of 0.92 µM and 0.52 ± 0.04 µM, respectively. Quantitative prediction study suggested that coadministration of PT supplements at 100 mg/day or higher doses may result in at least a 50% increase in the AUC of drugs predominantly cleared by UGT1A9. Thus, the coadministration of PT supplements and drugs primarily cleared by UGT1A9 may result in potential drug interaction, and precautions should be taken when coadministration of PT supplements and drugs metabolized by UGT1A9.


Assuntos
Suplementos Nutricionais/efeitos adversos , Inibidores Enzimáticos/toxicidade , Interações Alimento-Droga , Glucuronosiltransferase/antagonistas & inibidores , Estilbenos/toxicidade , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Cinética , Taxa de Depuração Metabólica , Desintoxicação Metabólica Fase II , Modelos Biológicos , Medição de Risco , Estilbenos/farmacocinética
15.
Xenobiotica ; 50(7): 776-782, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31755346

RESUMO

Quantitative aspects of in vitro phase II glucuronidative metabolism of O-desmethyltramadol (O-DSMT or M1), the active metabolite of the analgesic drug tramadol, by feline, canine and common brush-tailed possum hepatic microsomes are described.Whilst previous studies have focused on the phase I conversion of tramadol to M1, this is the first report in which the phase II glucuronidative metabolic pathway of M1 has been isolated by an in vitro comparative species study.Using the substrate depletion method, microsomal phase II glucuronidative in vitro intrinsic clearance (Clint) of M1 was determined.The in vitro Clint (mean ± SD) by pooled common brush-tailed possum microsomes was 9.9 ± 1.7 µL/min/mg microsomal protein whereas the in vitro Clint by pooled canine microsomes was 1.9 ± 0.07 µL/min/mg microsomal protein. The rate of M1 depletion by feline microsomes, as measured solely by high pressure liquid chromatography, was too slow to determine. Liquid chromatography-mass spectrometry identified O-DSMT glucuronide in samples generated from all three species' microsomes, although the amount detected under the feline condition was minimal.This study indicates that M1 likely undergoes in vitro phase II glucuronidation by canine and common brush-tailed possum microsomes and, to a minor extent, by feline microsomes. The rate of depletion of M1 by phase I metabolism was also undertaken.When incubated with phase I co-factors and common brush-tailed possum microsomes or canine microsomes, M1 had an in vitro Clint of 47.6 and 22.8 µL/min/mg microsomal protein, respectively. However, due to a lack of CYP2B-like activity in the feline liver, unsurprisingly, M1 did not deplete when incubated with feline microsomes. Consequently, major M1 elimination pathways, using feline microsomes, were not determined."


Assuntos
Tramadol/análogos & derivados , Animais , Gatos , Cães , Glucuronídeos/metabolismo , Humanos , Taxa de Depuração Metabólica , Microssomos/metabolismo , Tramadol/metabolismo , Trichosurus/metabolismo
16.
Eur J Pharm Sci ; 141: 105118, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669387

RESUMO

Beagle dog is a standard animal model for evaluating nonclinical pharmacokinetics of new drug candidates. Glucuronidation in intestine and liver is an important first-pass drug metabolic pathway, especially for phenolic compounds. This study evaluated the glucuronidation characteristics of several 7-hydroxycoumarin derivatives in beagle dog's intestine and liver in vitro. To this end, glucuronidation rates of 7-hydroxycoumarin (compound 1), 7-hydroxy-4-trifluoromethylcoumarin (2), 6-methoxy-7-hydroxycoumarin (3), 7-hydroxy-3-(4-tolyl)coumarin (4), 3-(4-fluorophenyl)coumarin (5), 7-hydroxy-3-(4-hydroxyphenyl)coumarin (6), 7-hydroxy-3-(4-methoxyphenyl)coumarin (7), and 7-hydroxy-3-(1H-1,2,4-tirazole)coumarin (8) were determined in dog's intestine and liver microsomes, as well as recombinant dog UGT1A enzymes. The glucuronidation rates of 1, 2 and 3 were 3-10 times higher in liver than in small intestine microsomes, whereas glucuronidation rates of 5, 6, 7 and 8 were similar in microsomes from both tissues. In the colon, glucuronidation of 1 and 2 was 3-5 times faster than in small intestine. dUGT1A11 glucuronidated efficiently all the substrates and was more efficient catalyst for 8 than any other dUGT1A. Other active enzymes were dUGT1A2 that glucuronidated efficiently 2, 3, 4, 5, 6 and 7, while dUGT1A10 glucuronidated efficiently 1, 2, 3, 4, 5 and 7. Kinetic analyses revealed that the compounds' Km values varied between 1.1 (dUGT1A10 and 2) and 250 µM (dUGT1A7 and 4). The results further strengthen the concept that dog intestine has high capacity for glucuronidation, and that different dUGT1As mediate glucuronidation with distinct substrates selectivity in dog and human.


Assuntos
Colo/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Intestino Delgado/metabolismo , Fígado/metabolismo , Umbeliferonas/metabolismo , Animais , Cães , Humanos , Microssomos/metabolismo
17.
J Hazard Mater ; 381: 120999, 2020 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-31430640

RESUMO

The fascinating collection and evaluation of natural products with enormous structural and chemical diversity can contribute to ensure human health and inspire potential drug discovery. We reported the identification of 14-(R)-hydroxy-gelsenicine (HGE), a new component from poisonous honey, which has recently caused multiple serious intoxications and deaths up on consumption. The prevalence, toxicity, toxicokinetics and metabolic profile of HGE were evaluated through in vitro and in vivo analyses. HGE is a very toxic substance and shows significant gender difference with LD50 of 0.125 mg kg-1 and 0.295 mg kg-1 for the female and male mice, respectively. Toxicokinetics test indicates that HGE has good bioavailability in rats, and is metabolized extensively, in which hydroxylation, reduction, N-demethyl ether and glucuronication are the major metabolic pathways. Additionally, HGE shows specific neurotoxicity by enhancing the binding of γ-aminobutyric acid (GABA) to its receptors. We found that flumazenil, a selective antagonist of GABA receptor, could effectively increase the survival of the tested animals, which provides a potential therapy for future clinical applications.


Assuntos
Mel/toxicidade , Alcaloides Indólicos/toxicidade , Neurotoxinas/toxicidade , Animais , Antídotos/farmacologia , Disponibilidade Biológica , Feminino , Flumazenil/farmacologia , Moduladores GABAérgicos/farmacologia , Glucuronídeos/metabolismo , Hidroxilação , Alcaloides Indólicos/farmacocinética , Dose Letal Mediana , Masculino , Camundongos Endogâmicos ICR , Neurotoxinas/farmacocinética , Picrotoxina/farmacologia , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismo
18.
Xenobiotica ; 50(4): 380-388, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31233374

RESUMO

1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects.2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer.3. NADPH-supplemented HLM generated six glycyrol metabolites (M1-M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7-M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7.4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.


Assuntos
Flavonoides/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem
19.
J Pharm Biomed Anal ; 178: 112972, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31727359

RESUMO

Silybum marianum (milk thistle) is a medicinal plant used for producing the hepatoprotective remedy silymarin. Its main bioactive constituents, including silybin and related flavonolignans, can be metabolized directly by phase II conjugation reactions. This study was designed to identify UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of six silymarin flavonolignans, namely silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. UHPLC-MS analyses showed that all of the tested compounds, both individually and in silymarin, were glucuronidated by human liver microsomes, and that glucuronidation was the main metabolic transformation in human hepatocytes. Further, each compound was glucuronidated by multiple recombinant human UGT enzymes. UGTs 1A1, 1A3, 1A8 and 1A9 were able to conjugate all of the tested flavonolignans, and some of them were also metabolized by UGTs 1A6, 1A7, 1A10, 2B7 and 2B15. In contrast, no glucuronides were produced by UGTs 1A4, 2B4, 2B10 and 2B17. With silymarin, we found that UGT1A1 and, to a lesser extent UGT1A9, were primarily responsible for the glucuronidation of the flavonolignan constituents. It is concluded that the metabolism of silymarin flavonolignans may involve multiple UGT enzymes, of which UGT1A1 appears to play the major role in the glucuronidation. These results may be relevant for future research on the metabolism of flavonolignans in humans.


Assuntos
Flavonolignanos/metabolismo , Glucuronosiltransferase/metabolismo , Silimarina/metabolismo , Adulto , Células Cultivadas , Glucuronídeos/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Cardo-Mariano/metabolismo , Silibina/metabolismo , Silimarina/análogos & derivados
20.
Drug Metab Dispos ; 47(12): 1372-1379, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31578207

RESUMO

Ginseng is known to have inhibitory effects on UGT1A9 activity. However, little is known about the inhibitory effects of ginsenosides, the major active compounds in ginseng, on UGT1A9 activity. In vitro investigation of UGT1A9 inhibition by ginsenosides was carried out using human liver microsomes (HLMs). Among 10 ginsenosides, ginsenoside Rc was the strongest inhibitor of UGT1A9-mediated mycophenolic acid glucuronidase activity. Further inhibition kinetic studies using HLMs suggested that ginsenoside Rc competitively and noncompetitively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities, with K i values of 2.83 and 3.31 µM, respectively. Next, to investigate whether the inhibitory effect of ginsenoside Rc is specific to the UGT1A9 isoform, we studied the inhibitory potency of ginsenoside Rc on nine human uridine diphospho-glucuronosyltransferase (UGT) activities using recombinant human UGT isoforms. Ginsenoside Rc exhibited a 12.9-fold selectivity (which was similar to niflumic acid at 12.5-fold) for UGT1A9 inhibition. Ginsenoside Rc at 50 µM also inhibited none of the other UGT isoform-specific activities above 12.0%, except for UGT1A9 (>91.5%) in HLMs, indicating that ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities. Considering lower plasma concentrations (0.01 µM) of ginsenoside Rc in healthy subjects and no induction potential on UGT isoforms, ginsenoside Rc does not cause pharmacokinetic drug interactions with other coadministered drugs metabolized by UGT1A9. SIGNIFICANCE STATEMENT: Ginsenoside Rc selectively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities in human liver microsomes and recombinant uridine diphospho-glucuronosyltransferase (UGT) isoforms. It exhibited a 12.9-fold selectivity for UGT1A9 inhibition. Therefore, ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities, such as niflumic acid.


Assuntos
Inibidores Enzimáticos/farmacologia , Ginsenosídeos/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Inibidores Enzimáticos/química , Ginsenosídeos/química , Glucuronídeos/metabolismo , Humanos , Técnicas In Vitro , Isoenzimas , Cinética , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Ácido Micofenólico/farmacologia , Propofol/farmacologia
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