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1.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366067

RESUMO

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Desmetilação/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Biotransformação/efeitos dos fármacos , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães , Corantes Fluorescentes/química , Humanos , Isoquinolinas/química , Cinética , Macaca fascicularis , Camundongos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura , Teofilina/análogos & derivados , Teofilina/farmacologia
2.
Toxicol Lett ; 313: 196-204, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31278966

RESUMO

Fipronil is a chiral insecticide employed worldwide in crops, control of public hygiene and control of veterinary pests. Humans can be exposed to fipronil through occupational, food, and environmental contamination. Therefore, the risk assessment of fipronil in humans is important to protect human health. Fipronil sulfone is the major metabolite formed during fipronil metabolism by humans. Since the CYP450 enzymes are the main ones involved in drug metabolism, the evaluation of their inhibition by fipronil and its main metabolite is important to predict drug-pesticide interactions. The aim of this work was to investigate the inhibition effects of rac-fipronil, S-fipronil, R-fipronil and fipronil sulfone on the main human CYP450 isoforms. The results showed that CYP2D6 is the only CYP450 isoform inhibited by these xenobiotics. In addition, no enantioselective differences were observed in the inhibition of CYP450 isoforms by fipronil and its individuals' enantiomers. Rac-fipronil, S-fipronil and R-fipronil are moderate CYP2D6 inhibitors showing a competitive inhibition profile. On the other hand, the metabolite fipronil sulfone showed to be a strong inhibitor of CYP2D6 also by competitive inhibition. These results highlight the importance of metabolite evaluation on pesticide safety since the metabolism of fipronil into fipronil sulfone increases the risk of pesticide-drug interactions for drugs metabolized by CYP2D6.


Assuntos
Inibidores do Citocromo P-450 CYP2D6/toxicidade , Citocromo P-450 CYP2D6/metabolismo , Praguicidas/toxicidade , Pirazóis/toxicidade , Citocromo P-450 CYP2D6/química , Inibidores do Citocromo P-450 CYP2D6/química , Relação Dose-Resposta a Droga , Interações de Medicamentos , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Biológicos , Praguicidas/química , Conformação Proteica , Pirazóis/química , Medição de Risco , Relação Estrutura-Atividade
3.
Toxicol Lett ; 313: 188-195, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284022

RESUMO

Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2-/- (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2-/- mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Ritmo Circadiano , Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Fotoperíodo , Estricnina/análogos & derivados , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ritmo Circadiano/genética , Cronoterapia Farmacológica , Células HEK293 , Humanos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Estricnina/administração & dosagem , Estricnina/metabolismo , Estricnina/farmacocinética , Estricnina/toxicidade
4.
Toxicol Lett ; 313: 66-76, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31201936

RESUMO

Mono-methylindoles (MMI) were described as agonists and/or antagonists of the human aryl hydrocarbon receptor (AhR). Here, we investigated the effects of MMI on AhR-CYP1A pathway in human hepatocytes and HepaRG cells derived from human progenitor hepatic cells. All MMI, except of 2-methylindole, strongly induced CYP1A1 and CYP1A2 mRNAs in HepaRG cells. Induction of CYP1A genes was absent in AhR-knock-out HepaRG cells. Consistently, CYP1A1 and CYP1A2 mRNAs and proteins were induced by all MMIs (except 2-methylindole), in human hepatocytes. The enzyme activity of CYP1A1 was inhibited by MMIs in human hepatocytes and LS180 colon cancer cells in a concentration-dependent manner (IC50 values from 1.2 µM to 23.8 µM and from 3.4 µM to 11.4 µM, respectively). Inhibition of CYP1A1 activity by MMI in human liver microsomes was much weaker as compared to that in intact cells. Incubation of parental MMI with human hepatocytes either diminished (4-methylindole, 6-methylindole) or enhanced (7-methylindole) their agonist effects on AhR in AZ-AHR reporter cells. In conclusion, overall effects of MMI on AhR-CYP1A pathway in human cells comprise the induction of CYP1A genes through AhR, the inhibition of CYP1A catalytic activity and possibly the metabolic transformation causing loss or gain of AhR agonist activity of parental compounds.


Assuntos
Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/biossíntese , Indutores das Enzimas do Citocromo P-450/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Hepatócitos/efeitos dos fármacos , Indóis/farmacologia , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Citocromo P-450 CYP1A1/genética , Relação Dose-Resposta a Droga , Indução Enzimática , Feminino , Hepatócitos/enzimologia , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Adulto Jovem
5.
J Agric Food Chem ; 67(22): 6177-6189, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083903

RESUMO

The aim of this work was to obtain phase II metabolites of cyanidin-3- O-glucoside and its aglycone using porcine liver enzymes. For this purpose, anthocyanins extracted from blackberry concentrate and containing mostly cyanidin-3- O-glucoside were incubated with the S9, microsomal, and cytosolic fractions of porcine liver. The reactions were targeted to the direction of the respective phase II transformation by the addition of activated cofactors. LC-MS n and LC-IMS-QTOF-MS analyses showed that one methylated, three glucuronidated and three sulfated metabolites of cyanidin-3- O-glucoside were generated. The aglycone, cyanidin, was sulfated and glucuronidated by the liver enzymes. In addition, both were glucuronidated and methylated simultaneously. The detected compounds and the generated data like exact masses, mass spectra, and CCS values may serve as a basis in the search for metabolites formed in vivo. As their effects are largely unexplored, the described synthesis may contribute to a better understanding of the metabolism of anthocyanins.


Assuntos
Antocianinas/síntese química , Glucosídeos/química , Microssomos Hepáticos/enzimologia , Extratos Vegetais/química , Rubus/química , Animais , Antocianinas/química , Biocatálise , Cromatografia Líquida de Alta Pressão , Frutas/química , Metilação , Microssomos Hepáticos/química , Estrutura Molecular , Suínos , Espectrometria de Massas em Tandem
6.
Toxicol Lett ; 312: 214-221, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31128210

RESUMO

Shikonin, a natural red colorant, is widely used for food garnishment and cosmetic ingredient in the world. Shikonin also possesses a variety of pharmacological actions, including anti-inflammation and anti-cancer activities. However, little is known about its effects on the UDP-glucuronosyltransferases (UGT) activity. Therefore, the aim of this study was to evaluate the effect of shikonin on the UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 activities via the human and rat liver microsomal assay and cocktail approach. The results showed shikonin inhibited human and rat liver microsomal UGT activity only in a dose-dependent manner. The further enzyme kinetic studies demonstrated that shikonin was not only a competitive inhibitor of human UGT1A1, UGT1A9, and UGT2B7, but also presented competitive inhibition on rat UGT1A1 and AZTG reactions. In conclusion, shikonin as a reversible inhibitor of UGT enzyme has a high-risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Naftoquinonas/toxicidade , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Inibidores Enzimáticos/toxicidade , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
7.
Int Immunopharmacol ; 72: 31-39, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30959369

RESUMO

Chromones are a group of natural substances with a diversity of biological activities. Herein we assessed the pharmacological potential of three chromones (1, 2 and 3) isolated from Dictyoloma vandellianum as anti-inflammatory agents using in vitro and in vivo approaches. During in vitro screening, the production of NO and cytokines by macrophages stimulated with LPS and IFN-γ was inhibited by all chromones at concentrations (5-20 µM) that did not induce cytotoxicity. Analysis of pharmacokinetic parameters (in vitro half-life and intrinsic clearance) using human liver microsomes revealed that 3 has a superior pharmacokinetic profile, compared to 1 and 2. Treatment with 3 (100 mg/kg, ip) did not affect the mice motor performance, while 1 and 2 induced motor deficit. Taking into account the pharmacokinetic profile and absence of motor impairment, 3 was selected for further pharmacological characterization. Corroborating the data from in vitro screening, treatment of cell cultures with 3 (5-20 µM) reduced TNF-α, IL-6 and IL-1ß production by stimulated macrophages. In the complete Freund's adjuvant-induced paw inflammation model in mice, 3 (25 and 50 mg/kg, ip) inhibited mechanical hyperalgesia, edema and cytokine production/release (IL-1ß, IL-6 and TNF-α). 3 (5-20 µM) also reduced the transcriptional activity of NF-κB in stimulated macrophages. Furthermore, treatment with RU486, a glucocorticoid receptor (GR) antagonist, partially prevented the inhibitory effect of 3 on macrophages, indicating that this chromone exerts its anti-inflammatory effects in part through the activation of GR. The results presented herein demonstrate the pharmacological potential of natural chromones, highlighting 3 as a possible candidate for the drug discovery process targeting new anti-inflammatory drugs.


Assuntos
Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Cromonas/farmacologia , Cromonas/uso terapêutico , Edema/tratamento farmacológico , Rutaceae , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Citocinas/imunologia , Edema/imunologia , Humanos , Fígado/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Microssomos Hepáticos/enzimologia , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Raízes de Plantas
8.
Molecules ; 24(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013570

RESUMO

GL-V9 is a prominent derivative of wogonin with a wide therapeutic spectrum and potent anti-tumor activity. The metabolism characteristics of GL-V9 remain unclear. This study aimed to clarify the metabolic pathway of GL-V9 and investigate the generation of its glucuronidation metabolites in vitro and in vivo. HPLC-UV-TripleTOF was used to identify metabolites. The main metabolite that we found was chemically synthesized and the synthetic metabolite was utilized as standard substance for the subsequent metabolism studies of GL-V9, including enzyme kinetics in liver microsomes of five different species and reaction phenotyping metabolism using 12 recombinant human UDP-glucuronosyltransferase (UGT) isoforms. Results indicated that the glucuronidation reaction occurred at C5-OH group, and 5-O-glucuronide GL-V9 is the only glucuronide metabolite and major phase II metabolite of GL-V9. Among 12 recombinant human UGTs, rUGT1A9 showed the strongest catalytic capacity for the glucuronidation reaction of GL-V9. rUGT1A7 and rUGT1A8 were also involved in the glucuronidation metabolism. Km of rUGT1A7-1A9 was 3.25 ± 0.29, 13.92 ± 1.05, and 4.72 ± 0.28 µM, respectively. In conclusion, 5-O-glucuronide GL-V9 is the dominant phase II metabolite of GL-V9 in vivo and in vitro, whose formation rate and efficiency are closely related to isoform-specific metabolism profiles and the distribution of UGTs in different tissues of different species.


Assuntos
Flavanonas , Glucuronídeos/química , Glucuronosiltransferase/química , Microssomos Hepáticos/enzimologia , Animais , Flavanonas/química , Flavanonas/farmacocinética , Glucuronídeos/farmacocinética , Glucuronosiltransferase/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
9.
Eur J Pharm Sci ; 132: 125-131, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30878380

RESUMO

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


Assuntos
Carboxilesterase/metabolismo , Haloperidol/análogos & derivados , Haloperidol/síntese química , Microssomos/enzimologia , Pró-Fármacos/síntese química , Estabilidade de Medicamentos , Ésteres , Haloperidol/metabolismo , Humanos , Técnicas In Vitro , Inativação Metabólica , Intestino Delgado/enzimologia , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Pró-Fármacos/metabolismo
10.
Eur J Pharm Sci ; 131: 177-194, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776468

RESUMO

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in 'suspension' for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.


Assuntos
Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1B1 , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Quinazolinonas , Antineoplásicos/farmacologia , Benzo(a)pireno/toxicidade , Bioensaio , Linhagem Celular , Cisplatino/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inibidores , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Quinazolinonas/química , Quinazolinonas/farmacologia
12.
Xenobiotica ; 49(10): 1183-1191, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30654691

RESUMO

1. l-Menthol, as a kind of monocyclic terpene, is widely used in inhalation formulations, food and tobacco. The purpose of this study was to investigate the pharmacokinetic behavior of l-menthol as well as its influence on the activities of cytochrome P450 enzymes. 2. The pharmacokinetic behaviors of l-menthol after inhalation (50 mg/kg) and intravenous injection (10 mg/kg) were investigated. A rat liver microsomal model was adopted to elucidate the inhibitory effect of l-menthol on CYP1A2, CYP2C11, CYP2D1/2, CYP2D4, CYP2E1 and CYP3A1 using phenacetin, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and testosterone as probe drugs, respectively. 3. The plasma concentration reached the Cmax within 1.0 h (inhalation) and descended with the T1/2 of 8.53 and 6.69 h for inhalation and i.v. administration, respectively. IC50 for inhibition of l-menthol on CYP 450 enzymes were 4.35 µM for 2D4, 8.67 µM for 1A2, 13.02 µM for 3A1, 14.78 µM for 2D1/2, 234.9 µM for 2C11 and 525.4 µM for 2E1, respectively. 4. The results illustrate the pharmacokinetic process of l-menthol in rats and provide information for further rational applications. l-Menthol had moderate inhibitions on CYP2D4 and 1A2, which might affect the disposition of medicines primarily dependent on these pathways.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Mentol , Microssomos Hepáticos/enzimologia , Administração por Inalação , Administração Intravenosa , Animais , Masculino , Mentol/farmacocinética , Mentol/farmacologia , Ratos , Ratos Sprague-Dawley
13.
Toxicol Appl Pharmacol ; 366: 64-74, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30685480

RESUMO

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.


Assuntos
Antineoplásicos/metabolismo , Citocromo-B(5) Redutase/deficiência , Citocromos b5/deficiência , Elipticinas/metabolismo , Hepatócitos/enzimologia , Fígado/enzimologia , Ativação Metabólica , Animais , Antineoplásicos/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo-B(5) Redutase/genética , Citocromos b5/genética , Adutos de DNA/metabolismo , Elipticinas/farmacologia , Genótipo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Fenótipo
14.
Xenobiotica ; 49(10): 1127-1132, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29676195

RESUMO

1. Sophocarpine is a biologically active component isolated from the foxtail-like sophora herb and seed that is often orally administered for the treatment of cancer and chronic bronchial asthma. However, whether sophocarpine affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. 2. In this study, the inhibitory effects of sophocarpine on the eight human liver CYP isoforms (CYP1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs). 3. The results indicate that sophocarpine could inhibit the activity of CYP3A4 and 2C9, with the IC50 values of 12.22 and 15.96 µM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that sophocarpine is not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2C9, with Ki values of 6.74 and 9.19 µM, respectively. Also, sophocarpine is a time-dependent inhibitor of CYP3A4 with Kinact/KI value of 0.082/21.54 µM-1 min-1. 4. The in vitro studies of sophocarpine with CYP isoforms suggested that sophocarpine has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.


Assuntos
Alcaloides , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/química , Microssomos Hepáticos/enzimologia , Alcaloides/química , Alcaloides/farmacologia , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Cinética
15.
Clin Pharmacol Ther ; 105(1): 131-141, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29737521

RESUMO

The ontogeny of hepatic uridine diphosphate-glucuronosyltransferases (UGTs) was investigated by determining their protein abundance in human liver microsomes isolated from 136 pediatric (0-18 years) and 35 adult (age >18 years) donors using liquid chromatography / tandem mass spectrometry (LC-MS/MS) proteomics. Microsomal protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by ∼8, 55, 35, 33, 8, and 3-fold from neonates to adults, respectively. The estimated age at which 50% of the adult protein abundance is observed for these UGT isoforms was between 2.6-10.3 years. Measured in vitro activity was generally consistent with the protein data. UGT1A1 protein abundance was associated with multiple single nucleotide polymorphisms exhibiting noticeable ontogeny-genotype interplay. UGT2B15 rs1902023 (*2) was associated with decreased protein activity without any change in protein abundance. Taken together, these data are invaluable to facilitate the prediction of drug disposition in children using physiologically based pharmacokinetic modeling as demonstrated here for zidovudine and morphine.


Assuntos
Genótipo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/enzimologia , Adolescente , Fatores Etários , Analgésicos Opioides/farmacologia , Antimetabólitos/farmacologia , Criança , Pré-Escolar , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Lactente , Recém-Nascido , Microssomos Hepáticos/efeitos dos fármacos , Morfina/farmacologia , Adulto Jovem , Zidovudina/farmacologia
16.
Mol Pharm ; 16(1): 382-392, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30517006

RESUMO

Testosterone (TST) and midazolam (MDZ) are widely used as probes to detect CYP3A4/5 activity, but the data acquired with these two substrates do not correlate well at the microsomal level (per milligram of microsomal protein), and the reason is unclear. In this study, CYP3A4/5 activity was probed with TST and MDZ at the microsomal and enzyme levels (per picomole of CYP3A4/5) in 72 human liver samples. Correlation coefficients were lower in Vmax and CLint at the microsomal level, as compared with those at the enzyme level ( Vmax 0.658 vs 0.883; CLint no correlation vs 0.796). Compared with TST, MDZ was found to correlate better with the content of CYP3A4/5 (no correlation vs 0.431) and CYP3A5 (no correlation vs 0.447), and huge variations in enzyme content existed among different genotypes, which explained the lower degree of correlation at the microsomal level. In addition, different genotypes had varying effects on activity at the enzyme level, whereas the difference between activity at the enzyme level probed with TST and that probed with MDZ was not obvious ( P > 0.05), indicating that the effect of gene polymorphisms on correlation between activity probed with these two substrates was limited at the enzyme level. In conclusion, our study demonstrates a high degree of correlation between CYP3A4/5 activity probed with TST and MDZ at the enzyme level but not at the microsomal level and allows us to correctly understand the influence of gene polymorphisms on the correlations.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Midazolam/farmacologia , Testosterona/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Genótipo , Humanos , Técnicas In Vitro , Cinética
17.
Xenobiotica ; 49(10): 1164-1172, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30488748

RESUMO

ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications. The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1. ZYTP1 IC50 for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC50 of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam. The mean IC50 values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3-58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC50 of depropylated metabolite was 11.95-24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC50 shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075 min-1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively. Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.


Assuntos
Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450 , Microssomos Hepáticos/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-30476594

RESUMO

Birds are exposed to many xenobiotics during their lifetime. For accurate prediction of xenobiotic-induced toxic effects on avian species, it is necessary to understand metabolic capacities in a comprehensive range of bird species. However, there is a lack of information about avian xenobiotic metabolizing enzymes (XMEs), particularly in wild birds. Uridine diphosphate glucuronosyltransferase (UGT) is an XME that plays an important role in phase II metabolism in the livers of mammals and birds. This study was performed to determine the characteristics of UGT1E isoform in avian species, those are related to mammals UGT 1A. To understand the characteristics of avian UGT1E isoforms, in vitro metabolic activity and genetic characteristics were investigated. Furthermore, mRNA expression levels of all chicken UGT1E isoforms were measured. On in vitro enzymatic analysis, the white-tailed eagle, great horned owl, and Humboldt penguin showed lower UGT-dependent activity than domestic birds. In synteny analysis, carnivorous birds were shown to have fewer UGT1E isoforms than herbivorous and omnivorous birds, which may explain why they have lower in vitro UGT activity. These observations suggested that raptors and seabirds, in which UGT activity is low, may be at high risk if exposed to elevated levels of xenobiotics in the environment. Phylogenetic analysis suggested that avian UGT1Es have evolved independently from mammalian UGT1As. We identified the important UGT isoforms, such as UGT1E13, and suspected their substrate specificities in avian xenobiotic metabolism by phylogenetic and quantitative real-time PCR analysis. This is the first report regarding the genetic characteristics and interspecies differences of UGT1Es in avian species.


Assuntos
Aves/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucuronosiltransferase/metabolismo , Acetaminofen/metabolismo , Analgésicos não Entorpecentes/metabolismo , Animais , Aves/metabolismo , Glucuronosiltransferase/genética , Microssomos Hepáticos/enzimologia , Filogenia , Especificidade da Espécie , Sintenia
19.
Artigo em Inglês | MEDLINE | ID: mdl-30368017

RESUMO

Many reef fishes are capable of feeding on chemically-defended benthic prey, such as soft (alcyonarian) corals; however, little is known about the molecular mechanisms that underpin allelochemical biotransformation and detoxification. Butterflyfishes (Chaetodon: Chaetdontidae) are a useful group for comparatively exploring links between biotransformation enzymes and diet, because they commonly feed on chemically defended prey. Moreover, diets of some species vary among geographic locations. This study compares gene expression, protein and enzymatic activity of key detoxification enzymes (cytochrome P450 (CYP) 2, 3, epoxide hydrolase, glutathione transferase and UDP-glucuronosyltransferase) in livers of four coral-feeding butterflyfish species between Australia and Hawaii, where these fishes differ in diet composition. For C. kleinii, C. auriga, and C. unimaculatus, we found higher CYP2 and CYP3 levels were linked to more allelochemically rich diets in Australia relative to Hawaii. For C. lunulatus from Hawaii CYP2 and CYP3 levels were 1 to 20-fold higher than C. lunulatus from Australia, possibly due to their predominant prey in Hawaii (Porities spp.) being richer in allelochemicals. UGT, GST and epoxide hydrolase varied between species and location and did not correspond to any specific dietary preference or location. Higher levels of CYP2 and CYP3A isozymes in species that feed on allelochemically-rich prey suggest that these biotransformation enzymes may be involved in detoxification of coral dietary allelochemicals in butterflyfishes.


Assuntos
Biotransformação , Recifes de Corais , Família 2 do Citocromo P450/metabolismo , Família 3 do Citocromo P450/metabolismo , Dieta/veterinária , Perciformes/metabolismo , Animais , Antozoários , Austrália , Expressão Gênica , Hawaii , Inativação Metabólica , Microssomos Hepáticos/enzimologia , Perciformes/fisiologia , Feromônios/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
20.
Pharmacology ; 103(3-4): 120-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30544127

RESUMO

Isofraxidin is a Coumarin compound widely distributed in plants, such as the Umbelliferae or Chloranthaceae, and it possesses numerous pharmacological activities. However, whether isofraxidin affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. In this study, the inhibitory effects of isofraxidin on the 8 human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes. The results showed that isofraxidin inhibited the activity of CYP1A2, 3A4, and 2E1, with IC50 values of 23.01, 15.49, and 15.98 µmol/L, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that isofraxidin was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP1A2 and 2E1, with Ki values of 7.91, 10.14, and 9.30 µmol/L, respectively. In addition, isofraxidin is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.047/12.33 µmol/L-1min-1. The in vitro studies of isofraxidin with CYP isoforms indicate that isofraxidin has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by -CYP1A2, 3A4, and 2E1. Further clinical studies are needed to evaluate the significance of this interaction.


Assuntos
Cumarínicos/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP2E1 , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450/farmacologia , Fígado/efeitos dos fármacos , Cumarínicos/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Inibidores do Citocromo P-450 CYP2E1/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Inibidores das Enzimas do Citocromo P-450/toxicidade , Interações de Medicamentos , Humanos , Cinética , Fígado/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia
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