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1.
Chem Biol Interact ; 330: 109247, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866466

RESUMO

This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.


Assuntos
Fígado/metabolismo , Oxazinas/metabolismo , Frações Subcelulares/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Feminino , Herbicidas/química , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Ratos , Fatores Sexuais , Estereoisomerismo
2.
Chem Biol Interact ; 330: 109228, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827518

RESUMO

This study aimed at exploring the potential mechanism of decreased in vivo exposure of the antiplatelet agent, ticagrelor and its active metabolite, AR-C124910XX, mediated by tea polyphenols, which was first revealed by our previous study, as well as predicting the in vivo drug-drug interaction (DDI) potential utilizing an in vitro to in vivo extrapolation (IVIVE) approach. The bidirectional transport and uptake kinetics of ticagrelor were determined using Caco-2 cells. Inhibition potency of major components of tea polyphenols, epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were obtained from Caco-2 cells, human intestinal and hepatic microsomes (HIMs and HLMs) in vitro. A mean efflux ratio of 2.28 ± 0.38 and active uptake behavior of ticagrelor were observed in Caco-2 cell studies. Further investigation showed that the IC50 values of EGCG and EGC on the uptake of ticagrelor were 42.0 ± 5.1 µM (95% CI 31.9-54.8 µM) and 161 ± 13 µM (95% CI 136-191 µM), respectively. EGCG and EGC also displayed moderate to weak reversible inhibition on the formation of AR-C124910XX and the inactive metabolite, AR-C133913XX in HIMs and HLMs, while no clinically significant time-dependent inhibition was observed for either compound. IVIVE indicated a significant inhibition effect of EGCG on the uptake process of ticagrelor, while no potential DDI risk was found based on microsomal data. A 45% decrease in ticagrelor in vivo exposure was mechanistically predicted by incorporating intestinal and hepatic metabolism as well as intestinal absorption. This dual inhibition of tea polyphenols on ticagrelor revealed the underlying potential of transporter-enzyme interplay, in which the altered uptake process was more critical.


Assuntos
Modelos Teóricos , Polifenóis/farmacologia , Chá/química , Ticagrelor/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Absorção Intestinal/efeitos dos fármacos , Cinética , Microssomos Hepáticos/metabolismo , Inibidores da Agregação de Plaquetas/farmacocinética , Antagonistas do Receptor Purinérgico P2Y/farmacocinética , Ticagrelor/metabolismo , Ticagrelor/farmacocinética
3.
Chem Biol Interact ; 329: 109147, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32738202

RESUMO

Acacetin is a natural flavonoid that is widely distributed in plants and possesses numerous pharmacological activities. The aim of the present study was to investigate the effects of acacetin on the activities of the cytochrome P450 family members CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1, and CYP3A2 in rat liver microsomes in vitro and rats in vivo to evaluate potential herb-drug interactions by using a cocktail approach. Phenacetin, bupropion, tolbutamide, dextromethorphan, chlorzoxazone, and midazolam were chosen as the probe substrates. An ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the probe substrates and their metabolites. In vitro, the mode of acacetin inhibition of CYP2B1, CYP2C11, and CYP2E1 was competitive, while mixed inhibition was observed for CYP1A2 and CYP3A2. The Ki values in this study were less than 8.32 µM. In vivo, the mixed probe substrates were administered by gavage after daily intraperitoneal injection with 50 mg/kg acacetin or saline for 2 weeks. The main pharmacokinetic parameters, area under the plasma concentration-time curve (AUC), plasma clearance (CL), and maximum plasma concentration (Cmax) of the probe substrates were significantly different in the experimental group than in the control group. Overall, the in vitro and in vivo results indicated that acacetin would be at high risk to cause toxicity and drug interactions via cytochrome P450 inhibition.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/metabolismo , Animais , Área Sob a Curva , Sistema Enzimático do Citocromo P-450/química , Flavonas/química , Flavonas/farmacocinética , Meia-Vida , Concentração Inibidora 50 , Cinética , Masculino , Microssomos Hepáticos/metabolismo , Curva ROC , Ratos , Ratos Sprague-Dawley
4.
Chem Biol Interact ; 328: 109192, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32712081

RESUMO

Many natural products are prodrugs which are biotransformed and activated after oral administration. The investigation of gastrointestinal and hepatic biotransformation can be facilitated by in vitro screening methods. This study compares two widely used in vitro models for hepatic biotransformation: 1) human S9 fractions and 2) human liver microsomes and cytosolic fractions in a two-step sequence, with the purpose of identifying differences in the biotransformation of medicagenic acid, the putative precursor of active metabolites, responsible for the medicinal effects of the herb Herniaria hirsuta. The combination of liquid chromatography coupled to high-resolution mass spectrometry with subsequent suspect and non-target data analysis allowed the identification of thirteen biotransformation products, four of which are reported here for the first time. Eight biotransformation products resulting from oxidative Phase I reactions were identified. Phase II conjugation reactions resulted in the formation of three glucuronidated and two sulfated biotransformation products. No major differences could be observed between incubations with human liver S9 or when utilizing human microsomal and cytosolic fractions. Apart from two metabolites, both methods rendered the same qualitative metabolic profile, with minor quantitative differences. As a result, both protocols applied in this study can be used to study in vitro human liver biotransformation reactions.


Assuntos
Microssomos Hepáticos/metabolismo , Triterpenos/metabolismo , Biotransformação , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Frações Subcelulares/metabolismo , Fatores de Tempo , Triterpenos/química
5.
Toxicol Lett ; 332: 7-13, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32615244

RESUMO

Root canal sealers are commonly used to endodontically treat teeth with periapical infections. Some root canal sealers based on epoxy resin contain bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE). The presence of these chemicals is of concern due to the close contact to the blood stream at the apex and the long setting times of up to 24 h. These chemicals, or any of their degradation products or metabolites, can then exert their toxic effects before being excreted. This study aimed to identify the phase I in vitro biotransformation products of BADGE and BFDGE using human liver microsomes. During incubation with microsomal fractions, the epoxides were rapidly hydrolysed in a NADPH independent manner resulting in the formation of BADGE.2H2O and BFDGE.2H2O. Further, oxidative reactions, such as hydroxylation and carboxylation, generated other BADGE metabolites, such as BADGE.2H2O-OH and BADGE.H2O.COOH, respectively. For BFDGE, further oxidation of BFDGE.2H2O led to the newly reported carboxylic acid, BFDGE.H2O.COOH. In total, three specific metabolites have been identified which can serve in future human biomonitoring studies of BADGE and BFDGE.


Assuntos
Compostos Benzidrílicos/farmacocinética , Compostos de Epóxi/farmacocinética , Fígado/metabolismo , Materiais Restauradores do Canal Radicular/farmacocinética , Compostos Benzidrílicos/toxicidade , Biotransformação , Ácidos Carboxílicos/metabolismo , Compostos de Epóxi/toxicidade , Feminino , Humanos , Hidroxilação , Masculino , Microssomos Hepáticos/metabolismo , NADP/metabolismo , Oxirredução , Materiais Restauradores do Canal Radicular/toxicidade
6.
Toxicol Appl Pharmacol ; 401: 115104, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32531296

RESUMO

Nitrofurans (5-nitro-2-hydrazonylfuran as pharmacophore) are a group of widely used antimicrobial drugs but also associated to a variety of side effects. The molecular mechanisms that underlie the cytotoxic effects of nitrofuran drugs are not yet clearly understood. One-electron reduction of 5-nitro group by host enzymes and ROS production via redox cycling have been attributed as mechanisms of cell toxicity. However, the current evidence suggests that nitrofuran ROS generation by itself is uncapable to explain the whole toxic effects associated to nitrofuran consumption, proposing a nitro-reduction independent mechanism of toxicity. In the present work, a series of nitrated and non-nitrated derivatives of nitrofuran drugs were synthesized and evaluated in vitro for their cytotoxicity, ROS-producing capacity, effect on GSH-S-transferase and antibacterial activity. Our studies showed that in human cells non-nitrated derivatives were less toxic than parental drugs but, unexpectedly preserved the ability to generate intracellular ROS in similar amounts to nitrofurans despite not entering into a redox cycle mechanism. In addition, some non-nitrated derivatives although being uncapable to generate ROS exhibited the highest cell toxicity among all derivatives. Inhibition of cytosolic glutathione-S-transferase activity by some derivatives was also observed. Finally, only nitrofuran derivatives displayed antibacterial effect. Results suggest that the combined 2-hydrazonylfuran moiety, redox cycling of 5-nitrofuran, and inhibitory effects on antioxidant enzymes, would be finally responsible for the toxic effects of the studied nitrofurans on mammalian cells.


Assuntos
Antibacterianos/toxicidade , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Nitrofuranos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Células A549 , Animais , Antibacterianos/química , Células HCT116 , Células HEK293 , Células HL-60 , Células Hep G2 , Humanos , Masculino , Nitrofuranos/química , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
7.
Toxicology ; 440: 152478, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32437779

RESUMO

Thiazoles are biologically active aromatic heterocyclic rings occurring frequently in natural products and drugs. These molecules undergo typically harmless elimination; however, a hepatotoxic response can occur due to multistep bioactivation of the thiazole to generate a reactive thioamide. A basis for those differences in outcomes remains unknown. A textbook example is the high hepatotoxicity observed for sudoxicam in contrast to the relative safe use and marketability of meloxicam, which differs in structure from sudoxicam by the addition of a single methyl group. Both drugs undergo bioactivation, but meloxicam exhibits an additional detoxification pathway due to hydroxylation of the methyl group. We hypothesized that thiazole bioactivation efficiency is similar between sudoxicam and meloxicam due to the methyl group being a weak electron donator, and thus, the relevance of bioactivation depends on the competing detoxification pathway. For a rapid analysis, we modeled epoxidation of sudoxicam derivatives to investigate the impact of substituents on thiazole bioactivation. As expected, electron donating groups increased the likelihood for epoxidation with a minimal effect for the methyl group, but model predictions did not extrapolate well among all types of substituents. Through analytical methods, we measured steady-state kinetics for metabolic bioactivation of sudoxicam and meloxicam by human liver microsomes. Sudoxicam bioactivation was 6-fold more efficient than that for meloxicam, yet meloxicam showed a 6-fold higher efficiency of detoxification than bioactivation. Overall, sudoxicam bioactivation was 15-fold more likely than meloxicam considering all metabolic clearance pathways. Kinetic differences likely arise from different enzymes catalyzing respective metabolic pathways based on phenotyping studies. Rather than simply providing an alternative detoxification pathway, the meloxicam methyl group suppressed the bioactivation reaction. These findings indicate the impact of thiazole substituents on bioactivation is more complex than previously thought and likely contributes to the unpredictability of their toxic potential.


Assuntos
Meloxicam/metabolismo , Tiazinas/metabolismo , Ativação Metabólica , Biotransformação , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Elétrons , Compostos de Epóxi/metabolismo , Humanos , Hidroxilação , Técnicas In Vitro , Cinética , Redes e Vias Metabólicas/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Tiazóis/metabolismo
8.
J Chromatogr A ; 1622: 461160, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32450990

RESUMO

The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the α-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation.


Assuntos
Cromatografia Líquida/métodos , Flúor/química , Glutationa/análise , Espectrometria de Massas em Tandem/métodos , Glutationa/química , Humanos , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo
9.
Xenobiotica ; 50(10): 1220-1227, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32369392

RESUMO

Horses are exposed to various kinds of medication, however, there are limited determinations of plasma clearance (CLp) for the drugs used due to the high cost of equine in vivo studies.Many of the CLp values generated come from the equine sports industry for determining drug plasma screening limits in the control of medications at the time of competition.The kinetics of omeprazole metabolism were investigated in freshly isolated and cryopreserved equine hepatocytes and hepatic microsomes (n = 3 horses).The Vmax, Km and intrinsic clearance (CLint) of omeprazole were determined via the substrate depletion method as well as Km values for the formation of three metabolites.The CLint values were extrapolated to in vivo hepatic plasma clearance (CLH) using the well stirred and parallel tube models.Clp for omeprazole was successfully predicted using freshly isolated or cryopreserved equine hepatocytes, while microsomes under-predicted.Equine microsomes were used to perform a drug-drug interaction (DDI) study between omeprazole and chloramphenicol. The average inhibitor constant Ki, assuming competitive inhibition, was 15.4 ± 5 µM.To the authors' knowledge, this is the first report showing the successful extrapolation of drug CLp in the horse using equine hepatocytes and the prediction of a DDI using microsomes.


Assuntos
Cavalos/metabolismo , Omeprazol/metabolismo , Animais , Criopreservação , Interações Medicamentosas , Hepatócitos , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Omeprazol/farmacologia
10.
Mol Pharmacol ; 97(6): 392-401, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234810

RESUMO

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the µ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the µ-opioid receptor most efficaciously because it has the ability to cross cell membranes.


Assuntos
Indazóis/química , Paroxetina/química , Pirimidinas/química , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Animais , Western Blotting , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Feminino , Células HEK293 , Humanos , Indazóis/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Pirimidinas/farmacologia
11.
Xenobiotica ; 50(8): 939-946, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32238050

RESUMO

Osthol, a pharmacologically active ingredient in various traditional Chinese medicines, is predominantly metabolized by CYP2C9. It may be co-administered with other drugs which are metabolized by CYP2C9 in clinical medicine. However, CYP2C9*1/*2/*3 genotype on the pharmacokinetics of osthole and its metabolic diversity between rat and human are unclear.In this study, we investigated the effects of osthole on enzyme activity of CYP2C11/CYP2C9 in rat liver microsomes (RLMs) and human liver microsomes (HLMs), to distinguish metabolic manner of osthole in different species. Interestingly, we found that osthole inhibits the activity of CYP2C11 in a non-competitive manner in RLMs, while inhibits CYP2C9 activity in a competitive manner in pooled HLMs. Then, the effects of CYP2C9*1/*2/*3 allele on the pharmacokinetics of osthole were identified. In human CYP2C9 isoform, the Ki value of 21.93 µM (CYP2C9*1), 18.10 µM (CYP2C9*2), 13.12 µM (CYP2C9*3) indicate that there are individual differences in the inhibition of osthole on CYP2C9 activity.We investigated how the indomethacin pharmacokinetics was affected by osthole in SD rat. To estimate the area under the curve (AUC), maximum plasma concentration (Cmax) and apparent clearance (CL/F), indomethacin (10 mg/kg) was given orally combined with osthole (20 mg/kg) in adult SD rat. We found the value of PK on indomethacin, such as the AUC0-∞, was from 176.40 ± 17.29 to 173.74 ± 27.69 µg/ml h-1, Cmax from 9.02 ± 1.24 to 9.89 ± 0.82 µg/ml and CL/F from 0.11 ± 0.01 to 0.12 ± 0.04 mg/kg/h which were unsignificantly changed compared with the control groups. However, the Tmax was prolonged from 2.00 ± 0.00 h to 7.33 ± 1.15 h, and T1/2 increased from 8.38 ± 2.30 h to 11.37 ± 2.11 h. These results indicate that osthole could potentially affect the metabolism of indomethacin in vivo.


Assuntos
Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indometacina/farmacocinética , Animais , Citocromo P-450 CYP2C9/metabolismo , Humanos , Indometacina/metabolismo , Medicina Tradicional Chinesa , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley
12.
PLoS One ; 15(4): e0230975, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287278

RESUMO

Feline infectious peritonitis (FIP) is a systemic, fatal, viral-induced, immune-mediated disease of cats caused by feline infectious peritonitis virus (FIPV). Mefloquine, a human anti-malarial agent, has been shown to inhibit FIPV in vitro. As a first step to evaluate its efficacy and safety profile as a potential FIP treatment for cats, mefloquine underwent incubation in feline, canine and common brush-tailed possum microsomes and phase I metabolism cofactors to determine its rate of phase I depletion. Tramadol was used as a phase I positive control as it undergoes this reaction in both dogs and cats. Using the substrate depletion method, the in vitro intrinsic clearance (mean ± S.D.) of mefloquine by pooled feline and common brush-tailed possum microsomes was 4.5 ± 0.35 and 18.25 ± 3.18 µL/min/mg protein, respectively. However, phase I intrinsic clearance was too slow to determine with canine microsomes. Liquid chromatography-mass spectrometry (LC-MS) identified carboxymefloquine in samples generated by feline microsomes as well as negative controls, suggesting some mefloquine instability. Mefloquine also underwent incubation with feline, canine and common brush-tailed possum microsomes and phase II glucuronidative metabolism cofactors. O-desmethyltramadol (ODMT or M1) was used as a positive control as it undergoes a phase II glucuronidation reaction in these species. The rates of phase II mefloquine depletion by microsomes by all three species were too slow to estimate. Therefore mefloquine likely undergoes phase I hepatic metabolism catalysed by feline and common brush-tailed possum microsomes but not phase II glucuronidative metabolism in all three species and mefloquine is not likely to have delayed elimination in cats with clinically normal, hepatic function.


Assuntos
Antimaláricos/metabolismo , Mefloquina/metabolismo , Microssomos Hepáticos/metabolismo , Trichosurus/metabolismo , Animais , Antimaláricos/farmacocinética , Antivirais/metabolismo , Antivirais/farmacocinética , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/veterinária , Calicivirus Felino , Gatos , Coronavirus Felino , Cães , Reposicionamento de Medicamentos/veterinária , Peritonite Infecciosa Felina/tratamento farmacológico , Peritonite Infecciosa Felina/metabolismo , Peritonite Infecciosa Felina/virologia , Técnicas In Vitro , Mefloquina/farmacocinética , Taxa de Depuração Metabólica , Especificidade da Espécie
13.
J Med Chem ; 63(8): 4047-4068, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32275432

RESUMO

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.


Assuntos
Benzimidazóis/administração & dosagem , Benzimidazóis/química , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
14.
J Med Chem ; 63(8): 4171-4182, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32285676

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent worldwide, causing serious liver complications, including nonalcoholic steatohepatitis. Recent findings suggest that peripheral serotonin (5-hydroxytryptamine, 5HT) regulates energy homeostasis, including hepatic lipid metabolism. More specifically, liver-specific 5HT2A knockout mice exhibit alleviated hepatic lipid accumulation and hepatic steatosis. Here, structural modifications of pimavanserin (CNS drug), a 5HT2A antagonist approved for Parkinson's disease, led us to synthesize new peripherally acting 5HT2A antagonists. Among the synthesized compounds, compound 14a showed good in vitro activity, good liver microsomal stability, 5HT subtype selectivity, and no significant inhibition of CYP and hERG. The in vitro and in vivo blood-brain barrier permeability study proved that 14a acts peripherally. Compound 14a decreased the liver weight and hepatic lipid accumulation in high-fat-diet-induced obesity mice. Our study suggests new therapeutic possibilities for peripheral 5HT2A antagonists in NAFLD.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Desenho de Fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Antagonistas do Receptor 5-HT2 de Serotonina/síntese química , Antagonistas do Receptor 5-HT2 de Serotonina/uso terapêutico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ratos Sprague-Dawley , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia
15.
Xenobiotica ; 50(10): 1149-1157, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32283993

RESUMO

WCK 771 (INN: levonadifloxacin) is a novel antibacterial agent belonging to benzoquinolizine subclass of fluoroquinolones which is under clinical development as a parenteral formulation and its prodrug WCK 2349 (INN: alalevonadifloxacin) as an oral option. Both the drugs have been approved recently in India based on phase III trial completed for ABSSSI.In vitro CYP inhibition potential of levonadifloxacin and its sulfate metabolite (WCK 2146) were assessed in this study. The inhibitory effects of levonadifloxacin and its sulfate metabolite were assessed for seven key human liver CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 using human liver microsome (HLM) employing validated LC-MS/MS method.The results showed that levonadifloxacin and its metabolite did not inhibit enzyme activity of any of the key CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) even at supra therapeutic concentrations (12-24X, Clinical Cmax: 25-35µg/mL).These in vitro CYP inhibition studies of levonadifloxacin and its sulfate metabolite indicate lack of potential for pharmacokinetic drug interactions of levonadifloxacin when co-administered with drugs which are substrate of these isoforms. Therefore, further clinical studies evaluating CYP mediated drug-drug interactions are not warranted for levonadifloxacin and alalevonadifloxacin.


Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Alanina , Antibacterianos/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fluoroquinolonas/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Microssomos Hepáticos/metabolismo
16.
Xenobiotica ; 50(10): 1180-1201, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32338108

RESUMO

Ethanol, as a small-molecule organic compound exhibiting both hydrophilic and lipophilic properties, quickly pass through the biological barriers. Over 95% of absorbed ethanol undergoes biotransformation, the remaining amount is excreted unchanged, mainly with urine and exhaled air.The main route of ethyl alcohol metabolism is its oxidation to acetaldehyde, which is converted into acetic acid with the participation of cytosolic NAD+ - dependent alcohol (ADH) and aldehyde (ALDH) dehydrogenases. Oxidative biotransformation pathways of ethanol also include reactions catalyzed by the microsomal ethanol oxidizing system (MEOS), peroxisomal catalase and aldehyde (AOX) and xanthine (XOR) oxidases. The resulting acetic acid can be activated to acetyl-CoA by the acetyl-CoA synthetase (ACS).It is also possible, to a much smaller extent, non-oxidative routes of ethanol biotransformation including its esterification with fatty acids by ethyl fatty acid synthase (FAEES), re-esterification of phospholipids, especially phosphatidylcholines, with phospholipase D (PLD), coupling with sulfuric acid by alcohol sulfotransferase (SULT) and with glucuronic acid using UDP-glucuronyl transferase (UGT, syn. UDPGT).The intestinal microbiome plays a significant role in the ethanol biotransformation and in the initiation and progression of liver diseases stimulated by ethanol and its metabolite - acetaldehyde, or by lipopolysaccharide and ROS.


Assuntos
Biotransformação/fisiologia , Etanol/metabolismo , Acetaldeído , Catalase/metabolismo , Humanos , Taxa de Depuração Metabólica , Redes e Vias Metabólicas , Microssomos Hepáticos/metabolismo , Oxirredução
17.
Xenobiotica ; 50(10): 1202-1207, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32338127

RESUMO

Peimine is a major component of Fritillaria ussuriensis, which is a widely used herb in pediatric. It is very common in Chinese traditional medicine to combine with two or more herbs in the clinic. To investigate the effect of peimine on the activity of cytochrome P450 enzymes (CYP450) is necessary for the clinical application of peimine.The effects of peimine on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro in human liver microsomes (HLMs) with the specific inhibitors as positive control and without peimine or inhibitors as negative control. The enzyme kinetic parameters were calculated.It was found that peimine inhibited the activity of CYP3A4, 2E1, and 2D6 in a concentration-dependent manner with the IC50 values of 13.43, 21.93, and 22.46 µM, respectively. The inhibition of CYP3A4 was performed in a non-competitive manner with the Ki value of 6.49 µM, and the inhibition of CYP2E1 and 2D6 was performed in a competitive manner with Ki values of 10.76 and 11.95 µM. Additionally, peimine inhibited the activity of CYP3A4 in a time-dependent manner with the KI/Kinact value of 6.17/0.049 min-1 µM-1.Peimine inhibited the activity of CYP3A4, 2E1, and 2D6, which indicated the potential interaction between peimine and drugs metabolized by CYP3A4, 2E1, and 2D6. Further studies are needed to verify the drug-drug interaction and the in vivo effects.


Assuntos
Cevanas/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Fígado/metabolismo , Microssomos Hepáticos/metabolismo
18.
Ecotoxicol Environ Saf ; 194: 110338, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32135376

RESUMO

2,2',3,5',6-Pentachlorobiphenyl (PCB95) is known as a persistent pollutant that was found in eggs in China. PCB 95 can be metabolized into OH-PCB95 and MeO-PCB95 in liver microsomes. However, the toxicity and its mechanism of PCB95 or its metabolites have been little studied on laying hens. Herein, chicken embryo liver cells of laying hens were selected and treated with different levels of PCB95 and its two metabolites, and the EC50 of PCB95, OH-PCB95, MeO-PCB95 was 80.85, 4.81 and 107.04 µg/mL respectively, indicating that OH-PCB95 is much more cytotoxic than PCB95 or MeO-PCB95. Targeted metabolomics was further used to study the effects of the parent compound and its metabolites on cell metabolism. The results showed that four primary types of glycerophospholipids were down-regulated after exposure to PCB95 and its metabolites, especially PE and PS (60% more than the control for PCB95, 40% for OH-PCB95, and less than 40% for MeO-PCB95). KEGG pathway analysis based on amino acid metabolism showed that PCB95 may mainly interfere with the amino acids involved in immune regulation (phenylalanine and tyrosine), and OH-PCB95 may be associated with genetic disoders (cysteine, methionine and purine metabolism). However, the metabolic pathways induced by MeO-PCB95 are quite different from those induced by PCB95 and OH-PCB95, affecting mainly D-glutamine and D-glutamate metabolism, alanine and glutamate metabolism, and arginine and proline metabolism; these pathways mainly regulate the elimination of excess purines and are involved in the synthesis of the amino acids required by cells. These results showed that OH-PCB95 has the highest toxicity on chicken embryo liver cells and MeO-PCB95 could be a detoxification product of PCB95 and OH-PCB95. This study contributes to the understanding of the different effects of PCB95 and its metabolites on cellular metabolism, and the data are helpful in evaluating the hepatotoxic effects of these compounds.


Assuntos
Poluentes Ambientais/toxicidade , Bifenilos Policlorados/toxicidade , Aminoácidos/metabolismo , Animais , Embrião de Galinha , Galinhas/metabolismo , China , Ovos , Poluentes Ambientais/metabolismo , Feminino , Hepatócitos/metabolismo , Fígado/metabolismo , Redes e Vias Metabólicas , Metabolômica , Metionina/metabolismo , Microssomos Hepáticos/metabolismo , Testes de Toxicidade
19.
Xenobiotica ; 50(9): 1052-1063, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32118505

RESUMO

Cytochromes P450 (CYPs) catalyze a great number of metabolic reactions that have profound effects on the biological activities of xenobiotics and endobiotics. In this study, we aimed to characterize rhythmic expressions of drug-metabolizing CYPs using synchronized hepatoma cells, and to investigate the potential roles of cis-elements of circadian clock system (E-box, D-box and RevRE or RORE) in generating the rhythms.Serum was used to synchronize circadian cycles and to induce circadian gene expression in cultured hepatoma cells (HepRG and HepG2 cells). Regulation of CYP genes by circadian clock components was investigated by performing luciferase reporter, overexpression and knockdown experiments. mRNA and protein expression were determined by qPCR and Western blotting assays, respectively.Of ten major drug-metabolizing CYP genes, six are rhythmically expressed (CYP1A2, 2B6, 2C8, 2D6, 2E1 and 3A4), whereas other four are non-rhythmic (CYP1B1, 2A6, 2C9 and 2C19).The E-box binding protein BMAL1 directly controls the rhythmic expression of CYP1A2. Rhythmic expressions of CYP2E1 and CYP3A4 are generated via both E-box and D-box elements. The RevRE binding protein REV-ERBα contributes to rhythmic oscillations in CYP2B6 and CYP2C8.In conclusion, rhythmic expressions of five human CYPs (CYP1A2, 2B6, 2C8, 2E1 and 3A4) are generated and regulated by E-box-, D-box-, and/or RevRE-acting clock components. Our findings may have implications for understanding chronopharmacokinetic events in humans.


Assuntos
Relógios Circadianos/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Humanos , Microssomos Hepáticos/metabolismo
20.
J Food Sci ; 85(4): 1285-1291, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32157685

RESUMO

Green tea is widely consumed as a beverage and/or dietary supplement worldwide, resulting in the difficulty to avoid the comedication with ticagrelor for acute coronary syndrome (ACS) patients receiving antiplatelet therapy. This study was designed to investigate the effect of the most abundant content in green tea, tea polyphenols on the oral and intravenous pharmacokinetics of ticagrelor in rats and its in vitro metabolism. Rats were orally treated with either saline or tea polyphenol extracts (TPEs) dissolved in saline once daily for 6 consecutive days. On day 6, after the last dose of saline or TPE, ticagrelor was given to the rats orally or intravenously. Plasma samples were collected for pharmacokinetic analysis. Human liver and intestinal microsomes were then used to investigate the inhibition by TPE, as well as its major constituents on the metabolism of ticagrelor to its two metabolites, AR-C124910XX and AR-C133913XX. Apparent kinetic constants and inhibition potency (IC50 ) for each metabolic pathway of each compound were estimated. Oral study indicated that exposure of ticagrelor and AR-C124910XX was significantly decreased after TPE administration, while no significant differences were observed in pharmacokinetic parameters after intravenous administration of ticagrelor. TPE effectively inhibited the metabolism of ticagrelor in vitro, with epigallocatechin-3-gallate as the major constituent responsible for the observed inhibitory effects in human liver microsomes and intestinal microsomes (IC50 = 4.23 ± 0.18 µM). Caution should be taken for ACS patients receiving ticagrelor therapy with daily drinking of green tea. PRACTICAL APPLICATION: Potential interactions between tea polyphenols and ticagrelor were revealed for the first time. Results can provide suggestions for clinicians to optimize the dosing of ticagrelor while they are in the face of ACS patients receiving ticagrelor therapy, who also take green tea or its related products in their daily life.


Assuntos
Microssomos Hepáticos/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacocinética , Polifenóis/farmacocinética , Chá/química , Ticagrelor/farmacocinética , Adenosina/análogos & derivados , Administração Intravenosa , Administração Oral , Animais , Catequina/análogos & derivados , Masculino , Microssomos Hepáticos/metabolismo , Inibidores da Agregação de Plaquetas/administração & dosagem , Polifenóis/administração & dosagem , Polifenóis/química , Ratos , Ticagrelor/administração & dosagem , Ticagrelor/metabolismo
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