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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638547

RESUMO

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.


Assuntos
Domínio Catalítico/genética , Citocromo P-450 CYP2C9/genética , Inativação Metabólica/genética , Losartan/metabolismo , Alelos , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Citocromo P-450 CYP2C9/metabolismo , Variação Genética/genética , Humanos , Inativação Metabólica/fisiologia , Conformação Proteica
2.
Sci Rep ; 11(1): 17081, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429480

RESUMO

CYP2C9, one of the most abundant hepatic cytochrome P450 enzymes, is involved in metabolism of 15-20% of clinically important drugs (warfarin, sulfonylureas, phenytoin, non-steroid anti-inflammatory drugs). To avoid adverse events and/or impaired drug-response, CYP2C9 pharmacogenetic testing is recommended. The impact of CYP2C9 polymorphic alleles (CYP2C9*2, CYP2C9*3) and phenoconverting non-genetic factors on CYP2C9 function and expression was investigated in liver tissues from Caucasian subjects (N = 164). The presence of CYP2C9*3 allele was associated with CYP2C9 functional impairment, and CYP2C9*2 influenced tolbutamide 4'-hydroxylase activity only in subjects with two polymorphic alleles, whereas the contribution of CYP2C8*3 was not confirmed. In addition to CYP2C9 genetic polymorphisms, non-genetic factors (co-medication with CYP2C9-specific inhibitors/inducers and non-specific factors including amoxicillin + clavulanic acid therapy or chronic alcohol consumption) contributed to the prediction of hepatic CYP2C9 activity; however, a CYP2C9 genotype-phenotype mismatch still existed in 32.6% of the subjects. Substantial variability in CYP2C9 mRNA levels, irrespective of CYP2C9 genotype, was demonstrated; however, CYP2C9 induction and non-specific non-genetic factors potentially resulting in liver injury appeared to modify CYP2C9 expression. In conclusion, complex implementation of CYP2C9 genotype and non-genetic factors for the most accurate estimation of hepatic CYP2C9 activity may improve efficiency and safety of medication with CYP2C9 substrate drugs in clinical practice.


Assuntos
Variação Biológica da População , Citocromo P-450 CYP2C9/genética , Fígado/metabolismo , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Consumo de Bebidas Alcoólicas/epidemiologia , Amoxicilina/farmacologia , Ácido Clavulânico/farmacologia , Citocromo P-450 CYP2C9/metabolismo , Etanol/farmacologia , Feminino , Humanos , Fígado/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Inibidores de beta-Lactamases/farmacologia
3.
Am J Hum Genet ; 108(9): 1735-1751, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314704

RESUMO

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Mutação de Sentido Incorreto , Medicamentos sob Prescrição/metabolismo , Saccharomyces cerevisiae/enzimologia , Xenobióticos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/genética , Ensaios Enzimáticos , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenitoína/química , Polimorfismo Genético , Medicamentos sob Prescrição/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Transgenes , Varfarina/química , Varfarina/metabolismo , Xenobióticos/química
4.
Methods Mol Biol ; 2342: 765-779, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272716

RESUMO

The potential for new chemical entities to inhibit the major cytochrome P450 (CYP) isoforms is routinely evaluated to minimize the risk of developing drugs with drug-drug interaction liabilities. CYP inhibition assays are routinely performed in a high-throughput format to efficiently screen large numbers of compounds. In evaluating a time-saving assay using diclofenac as the CYP2C9 probe substrate, a discrepancy was observed in which minimal inhibition was detected using diclofenac whereas using (S)-warfarin resulted in potent inhibition, supporting the presence of dual-binding sites in the relatively large CYP2C9 active site cavity.These observations provided further insights into explaining the reported ineffective inactivation of CYP2C9 for the pan-CYP inactivator 1-aminobenzotriazole (ABT). Mechanistic reversible and time-dependent inhibition experiments revealed that the ineffective CYP2C9 inactivation by ABT was also probe-dependent, with utilization of (S)-warfarin as the probe substrate resulting in more potent CYP2C9 inhibition by ABT compared to diclofenac. Addition of (S)-warfarin to the reversible and time-dependent inhibition experiments between ABT and diclofenac resulted in an attenuation of the inhibitory effects of ABT on CYP2C9-mediated diclofenac metabolism. Molecular docking studies further confirmed that (S)-warfarin and diclofenac preferentially bind in different regions of the CYP2C9 active site, with (S)-warfarin occupying a distal "warfarin-binding pocket" and diclofenac occupying a binding site close to the active heme moiety. ABT preferentially binds in the distal warfarin-binding pocket, supporting that diclofenac is minimally deterred from access to the CYP2C9 active site in the presence of ABT, thus resulting in minimal inactivation. Simultaneously docking of (S)-warfarin and ABT revealed that (S)-warfarin outcompetes ABT for the distal binding site and results in the binding of ABT to the CYP2C9 active site, supporting the observations of potent inactivation of CYP2C9 when (S)-warfarin is the probe substrate.These results highlight that probe selection is crucial when evaluating CYP inhibition potential, and it is recommended that multiple probes be utilized for CYP2C9, similar to the approach routinely employed for CYP3A4. Further, utilization of ABT as a pan-inhibitor of CYP activity for investigational compounds, both in vitro and in vivo, should be accompanied with the understanding that residual CYP-mediated oxidative metabolism could potentially be observed for CYP2C9 substrates and should not necessarily be attributed to non-P450-mediated metabolism.


Assuntos
Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Diclofenaco/farmacologia , Triazóis/farmacologia , Varfarina/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Diclofenaco/química , Interações Medicamentosas , Inativação Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Fatores de Tempo , Triazóis/química , Varfarina/química
5.
Food Chem Toxicol ; 153: 112278, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34019943

RESUMO

Bergamottin (BGM) is a major furanocoumarin constituent of grapefruit and is reported to have inhibitory effects on cytochrome P450 enzymes. This study investigated the chemical interactions between BGM and the enzyme CYP2C9. BGM exhibited time-, concentration-, and NADPH-dependent inhibition of CYP2C9. Co-incubation with diclofenac, a reversible inhibitor of CYP2C9, attenuated the time-dependent enzyme inhibition. Exhaustive dialysis did not restore enzyme activity post-inhibition. Glutathione (GSH) and catalase/superoxide dismutase failed to reverse BGM-induced CYP2C9 inactivation. A GSH trapping study suggested that BGM was metabolized to an epoxide and/or γ-ketoenal that may have been responsible for the enzyme inactivation. In conclusion, BGM can be characterized as a mechanism-based inactivator of CYP2C9 acting via the formation of an epoxide and/or γ-ketoenal.


Assuntos
Inibidores do Citocromo P-450 CYP2C9/farmacologia , Citocromo P-450 CYP2C9/metabolismo , Furocumarinas/farmacologia , Inibidores do Citocromo P-450 CYP2C9/metabolismo , Diclofenaco/farmacologia , Furocumarinas/metabolismo , Humanos , Microssomos Hepáticos/metabolismo
6.
Chem Biol Interact ; 343: 109498, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961833

RESUMO

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.


Assuntos
Inibidores do Citocromo P-450 CYP2C9/farmacologia , Miconazol/farmacologia , Fenitoína/metabolismo , Anticonvulsivantes/metabolismo , Antifúngicos/farmacologia , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Interações Medicamentosas , Humanos , Hidroxilação/efeitos dos fármacos , Cinética , Microssomos Hepáticos/metabolismo , Medição de Risco
7.
Pharm Biol ; 59(1): 532-536, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33915070

RESUMO

CONTEXT: Pogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination. OBJECTIVE: To investigate the effect of pogostone on the activity of human CYP450s. MATERIALS AND METHODS: The effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0-100 µM pogostone and various concentrations of substrates. RESULTS: Pogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC50 values of 11.41, 12.11, and 14.90 µM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the Ki value of 5.69 µM and the KI/Kinact value of 5.86/0.056/(µM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the Ki value of 6.46 and 7.67 µM and was not affected by the incubation time. DISCUSSION AND CONCLUSIONS: The inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.


Assuntos
Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP2E1/farmacologia , Inibidores do Citocromo P-450 CYP3A/farmacologia , Óleos Voláteis/farmacologia , Citocromo P-450 CYP2C9/efeitos dos fármacos , Citocromo P-450 CYP2C9/metabolismo , Inibidores do Citocromo P-450 CYP2C9/administração & dosagem , Citocromo P-450 CYP2E1/efeitos dos fármacos , Citocromo P-450 CYP2E1/metabolismo , Inibidores do Citocromo P-450 CYP2E1/administração & dosagem , Citocromo P-450 CYP3A/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Óleos Voláteis/administração & dosagem , Fatores de Tempo
8.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799598

RESUMO

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.


Assuntos
Aflatoxina B1/toxicidade , Efeito Fundador , Medições Luminescentes/métodos , Primaquina/toxicidade , Imagem com Lapso de Tempo/métodos , Xenobióticos/toxicidade , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células Hep G2 , Humanos , Concentração Inibidora 50 , Luciferases/genética , Luciferases/metabolismo , Luminescência , Camundongos
9.
Sci Rep ; 11(1): 6854, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767225

RESUMO

Colorectal cancer (CRC) represents the third leading cause of death among cancer patients below the age of 50, necessitating improved treatment and prevention initiatives. A crude methanol extract from the wood pulp of Artocarpus heterophyllus was found to be the most bioactive among multiple others, and an enriched extract containing 84% (w/v) artocarpin (determined by HPLC-MS-DAD) was prepared. The enriched extract irreversibly inhibited the activity of human cytochrome P450 CYP2C9, an enzyme previously shown to be overexpressed in CRC models. In vitro evaluations on heterologously expressed microsomes, revealed irreversible inhibitory kinetics with an IC50 value of 0.46 µg/mL. Time- and concentration-dependent cytotoxicity was observed on human cancerous HCT116 cells with an IC50 value of 4.23 mg/L in 72 h. We then employed the azoxymethane (AOM)/dextran sodium sulfate (DSS) colitis-induced model in C57BL/6 mice, which revealed that the enriched extract suppressed tumor multiplicity, reduced the protein expression of proliferating cell nuclear antigen, and attenuated the gene expression of proinflammatory cytokines (Il-6 and Ifn-γ) and protumorigenic markers (Pcna, Axin2, Vegf, and Myc). The extract significantly (p = 0.03) attenuated (threefold) the gene expression of murine Cyp2c37, an enzyme homologous to the human CYP2C9 enzyme. These promising chemopreventive, cytotoxic, anticancer and anti-inflammatory responses, combined with an absence of toxicity, validate further evaluation of A. heterophyllus extract as a therapeutic agent.


Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Artocarpus/química , Colite/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Extratos Vegetais/farmacologia , Madeira/química , Animais , Azoximetano/toxicidade , Colite/induzido quimicamente , Colite/patologia , Neoplasias Colorretais/patologia , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Células HCT116 , Humanos , Masculino , Lectinas de Ligação a Manose/química , Camundongos , Camundongos Endogâmicos C57BL , Lectinas de Plantas/química
10.
Biomed Pharmacother ; 138: 111459, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33706132

RESUMO

Silymarin is a mixture of flavonolignans isolated from the fruit of milk thistle (Silybum marianum (L.) Gaertner). Milk thistle extract is the active ingredient of several medications and dietary supplements to treat liver injury/diseases. After the oral administration, flavonolignans are extensively biotransformed, resulting in the formation of sulfate and/or glucuronide metabolites. Previous studies demonstrated that silymarin components form stable complexes with serum albumin and can inhibit certain cytochrome P450 (CYP) enzymes. Nevertheless, in most of these investigations, silybin was tested; while no or only limited information is available regarding other silymarin components and metabolites. In this study, the interactions of five silymarin components (silybin A, silybin B, isosilybin A, silychristin, and 2,3-dehydrosilychristin) and their sulfate metabolites were examined with human serum albumin and CYP (2C9, 2C19, 2D6, and 3A4) enzymes. Our results demonstrate that each compound tested forms stable complexes with albumin, and certain silymarin components/metabolites can inhibit CYP enzymes. Most of the sulfate conjugates were less potent inhibitors of CYP enzymes, but 2,3-dehydrosilychristin-19-O-sulfate showed the strongest inhibitory effect on CYP3A4. Based on these observations, the simultaneous administration of high dose silymarin with medications should be carefully considered, because milk thistle flavonolignans and/or their sulfate metabolites may interfere with drug therapy.


Assuntos
Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Albumina Sérica Humana/metabolismo , Silimarina/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligação Proteica/fisiologia , Silimarina/química , Silimarina/farmacologia , Sulfatos/química , Sulfatos/metabolismo , Sulfatos/farmacologia
11.
Toxicol Lett ; 343: 28-33, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33652071

RESUMO

Carfentanil is an ultra-potent opioid with an analgesic potency 10,000 times that of morphine but has received little scientific investigation. In the present study, the human cytochrome P450 (CYP) isozymes catalyzing the oxidative metabolism of carfentanil were investigated. Using UHPLC-HRMS, Michaelis-Menten kinetics of formation for three major metabolites norcarfentanil (M1), pharmaceutical active metabolite 4-[(1-oxopropyl)phenylamino]-1-(2-hydroxyl-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M11), and 4-[(1-oxopropyl)phenylamino]-1-(2-oxo-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M15) were determined. Isozymes catalyzing the formation of the low abundant, highly active metabolite 1-[2-(2-hydroxylphenyl)ethyl]-4-[(1-oxopropyl)phenylamino]-4-piperidinecarboxylic acid methyl ester (M13) were also identified. Selective P450 inhibition studies with pooled human liver microsomes (HLMs) and recombinant CYP isozymes suggested that metabolites M1, M11, and M15 were predominantly formed by isozyme CYP3A5, followed by CYP3A4. Isozymes CYP2C8 and CYP2C9 also made contributions but to a much lesser extent. Highly potent metabolite M13 was predominantly formed by isozyme CYP2C9, followed by CYP2C8. These findings indicate that CYP3A5, CYP3A4, CYP2C8 and CYP2C9 play a major role in the transformation of carfentanil to M1 (norcarfentanil), M11, M13 and M15 through N-dealkylation of piperidine ring, hydroxylation of phenethyl group and ketone formation on phenethyl linker by human liver micrsomes.


Assuntos
Analgésicos Opioides/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Fentanila/análogos & derivados , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP3A/genética , Fentanila/química , Fentanila/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Oxirredução
12.
Bioelectrochemistry ; 138: 107729, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33421896

RESUMO

Polymorphism is an important aspect in drug metabolism responsible for different individual response to drug dosage, often leading to adverse drug reactions. Here human CYP2C9 as well as its polymorphic variants CYP2C9*2 and CYP2C9*3 present in approximately 35% of the Caucasian population have been engineered by linking their gene to the one of D. vulgaris flavodoxin (FLD) that acts as regulator of the electron flow from the electrode surface to the haem. The redox properties of the immobilised proteins were investigated by cyclic voltammetry and electrocatalysis was measured in presence of the largely used anticoagulant drug S-warfarin, marker substrate for CYP2C9. Immobilisation of the CYP2C9-FLD, CYP2C9*2-FLD and CYP2C9*3-FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms and mid-point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3-FLD (2.73 ± 1.07 min-1) and CYP2C9*2-FLD (12.42 ± 2.17 min-1) compared to the wild type CYP2C9-FLD (18.23 ± 1.29 min-1). These differences in activity among the CYP2C9 variants are in line with the reported literature data, and this set the basis for the use of the bio-electrode for the measurement of the different catalytic responses towards drugs very relevant in therapy.


Assuntos
Biocatálise , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Engenharia de Proteínas , Citocromo P-450 CYP2C9/química , Eletroquímica , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Humanos
13.
Mol Cell Biochem ; 476(5): 2011-2020, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33515198

RESUMO

Cytochrome P450 2C9 (CYP2C9) is involved in the metabolism of cancer drugs and exogenous carcinogens. In our study, CYP2C9 was downregulated in multiple cohorts of human esophageal squamous cell carcinoma (ESCC). Until now, its role and epigenetic regulation of CYP2C9 repression in ESCC remain poorly understood. CYP2C9 repression in collected ESCC patient tumor tissues was demonstrated by RT-qPCR and Western blot. The histone acetylation level was carried out by the treatment of histone deacetylase inhibitor TSA and RNA interference. Epigenetic analysis revealed that the increased expression of CYP2C9 in KYSE-150 and TE1 cells was characterized by inhibition of HDAC8 and HDAC1, respectively. TSA decreased the levels of HDAC occupancy around CYP2C9 promoter region greatly. Overexpression of CYP2C9 reduced the invasion and migration of ESCC cells.


Assuntos
Movimento Celular , Citocromo P-450 CYP2C9/metabolismo , Regulação para Baixo , Neoplasias Esofágicas/enzimologia , Carcinoma de Células Escamosas do Esôfago/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/biossíntese , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP2C9/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Histona Desacetilases/genética , Humanos , Invasividade Neoplásica , Proteínas de Neoplasias/genética
14.
Pharmacol Res Perspect ; 9(1): e00718, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33508175

RESUMO

Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%-17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS-7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus-based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild-type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.


Assuntos
Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Substituição de Aminoácidos , Animais , Grupo com Ancestrais do Continente Asiático/genética , Baculoviridae/genética , Catálise , Linhagem Celular , Citocromo P-450 CYP2C9/química , Diclofenaco/metabolismo , Humanos , Insetos , Losartan/metabolismo , Modelos Moleculares , Polimorfismo Genético , Conformação Proteica , Proteínas Recombinantes/metabolismo , Tolbutamida/metabolismo
15.
Toxicol Lett ; 337: 111-120, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33232775

RESUMO

Quantifying variability in pharmacokinetics (PK) and toxicokinetics (TK) provides a science-based approach to refine uncertainty factors (UFs) for chemical risk assessment. In this context, genetic polymorphisms in cytochromes P450 (CYPs) drive inter-phenotypic differences and may result in reduction or increase in metabolism of drugs or other xenobiotics. Here, an extensive literature search was performed to identify PK data for probe substrates of the human polymorphic isoforms CYP2C9 and CYP2C19. Relevant data from 158 publications were extracted for markers of chronic exposure (clearance and area under the plasma concentration-time curve) and analysed using a Bayesian meta-regression model. Enzyme function (EF), driven by inter-phenotypic differences across a range of allozymes present in extensive and poor metabolisers (EMs and PMs), and fraction metabolised (Fm), were identified as exhibiting the highest impact on the metabolism. The Bayesian meta-regression model provided good predictions for such inter-phenotypic differences. Integration of population distributions for inter-phenotypic differences and estimates for EF and Fm allowed the derivation of CYP2C9- and CYP2C19-related UFs which ranged from 2.7 to 12.7, and were above the default factor for human variability in TK (3.16) for PMs and major substrates (Fm >60%). These results provide population distributions and pathway-related UFs as conservative in silico options to integrate variability in CYP2C9 and CYP2C19 metabolism using in vitro kinetic evidence and in the absence of human data. The future development of quantitative extrapolation models is discussed with particular attention to integrating human in vitro and in vivo PK or TK data with pathway-related variability for chemical risk assessment.


Assuntos
Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Toxicocinética , Algoritmos , Área Sob a Curva , Teorema de Bayes , Simulação por Computador , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C9/genética , Variação Genética , Humanos , Cinética , Fenótipo , Polimorfismo Genético , Medição de Risco , Xenobióticos/metabolismo
16.
Bioorg Chem ; 106: 104466, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33246603

RESUMO

This study concerns synthesis and evaluation of pharmacodynamic and pharmacokinetic profile for all four stereoisomers of MF-8 (5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione), the previously described, highly potent 5-HT7R ligand with antidepressant activity on mice. The combination of DFT calculations of 1H NMR chemical shifts with docking and dynamic simulations, in comparison to experimental screening results, provided prediction of the configuration for one of two present stereogenic centers. The experimental data for stereoisomers (MF-8A-MF-8D) confirmed the significant impact of stereochemistry on both, 5-HT7R affinity and antagonistic action, with Ki and Kb values in the range of 3-366 nM and 0.024-99 µM, respectively. We also indicated the stereochemistry-dependent influence of the tested compounds on P-glycoprotein efflux, absorption in Caco-2 model, metabolic pathway as well as CYP3A4 and CYP2C9 activities.


Assuntos
Hidantoínas/farmacocinética , Piperazinas/farmacocinética , Antagonistas da Serotonina/farmacocinética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Inibidores do Citocromo P-450 CYP3A/síntese química , Inibidores do Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Inibidores do Citocromo P-450 CYP3A/toxicidade , Teoria da Densidade Funcional , Estabilidade de Medicamentos , Humanos , Hidantoínas/síntese química , Hidantoínas/metabolismo , Hidantoínas/toxicidade , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperazinas/síntese química , Piperazinas/metabolismo , Piperazinas/toxicidade , Ligação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/síntese química , Antagonistas da Serotonina/metabolismo , Antagonistas da Serotonina/toxicidade , Estereoisomerismo
17.
Toxicol Lett ; 338: 10-20, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33253783

RESUMO

Meloxicam is a thiazole-containing NSAID that was approved for marketing with favorable clinical outcomes despite being structurally similar to the hepatotoxic sudoxicam. Introduction of a single methyl group on the thiazole results in an overall lower toxic risk, yet the group's impact on P450 isozyme bioactivation is unclear. Through analytical methods, we used inhibitor phenotyping and recombinant P450s to identify contributing P450s, and then measured steady-state kinetics for bioactivation of sudoxicam and meloxicam by the recombinant P450s to determine relative efficiencies. Experiments showed that CYP2C8, 2C19, and 3A4 catalyze sudoxicam bioactivation, and CYP1A2 catalyzes meloxicam bioactivation, indicating that the methyl group not only impacts enzyme affinity for the drugs, but also alters which isozymes catalyze the metabolic pathways. Scaling of relative P450 efficiencies based on average liver concentration revealed that CYP2C8 dominates the sudoxicam bioactivation pathway and CYP2C9 dominates meloxicam detoxification. Dominant P450s were applied for an informatics assessment of electronic health records to identify potential correlations between meloxicam drug-drug interactions and drug-induced liver injury. Overall, our findings provide a cautionary tale on assumed impacts of even simple structural modifications on drug bioactivation while also revealing specific targets for clinical investigations of predictive factors that determine meloxicam-induced idiosyncratic liver injury.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Meloxicam/metabolismo , Microssomos Hepáticos/enzimologia , Tiazinas/metabolismo , Ativação Metabólica , Anti-Inflamatórios não Esteroides/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Mineração de Dados , Aprendizado Profundo , Interações Medicamentosas , Registros Eletrônicos de Saúde , Feminino , Humanos , Inativação Metabólica , Cinética , Masculino , Meloxicam/toxicidade , Pessoa de Meia-Idade , Especificidade por Substrato , Tiazinas/toxicidade
18.
Drug Metab Pharmacokinet ; 36: 100364, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33341662

RESUMO

The inhibition of CYP2C9-mediated warfarin metabolism by acid or lactone forms of statin converted in the body and effects of CYP2C9 genetic variants on their inhibition are not fully understood. Here, the effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation were investigated in vitro. S-Warfarin 7-hydroxylase activities of human liver microsomes (HLMs), recombinant CYP2C9.1 (rCYP2C9.1), and rCYP2C9.3 (Ile359Leu variant) in the presence of statins were determined by high-performance liquid chromatography. Lactone forms of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin inhibited the activity of HLMs more potently than the corresponding acid forms, whereas fluvastatin acid showed stronger inhibition than fluvastatin lactone. When the effects of statins on rCYP2C9 variants were examined, inhibition profiles of acid versus lactone forms of statins except for fluvastatin were similar between rCYP2C9.1 and rCYP2C9.3. However, the degrees of inhibition by atorvastatin lactone, fluvastatin acid, fluvastatin lactone, lovastatin lactone, and pitavastatin lactone (Ki values) were significantly different between these variants. These results indicated that lactone forms of statins other than fluvastatin showed more potent inhibition of CYP2C9-catalyzed S-warfarin 7-hydroxylation than the corresponding acid forms. Furthermore, our results indicated that Ile359Leu substitution in CYP2C9 affected the inhibitory potencies of statins.


Assuntos
Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Microssomos Hepáticos/metabolismo , Variantes Farmacogenômicos/fisiologia , Varfarina/metabolismo , Ácidos/metabolismo , Catálise , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Feminino , Humanos , Hidroxilação/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Lactonas/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Varfarina/farmacologia
19.
Phytomedicine ; 81: 153416, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33321412

RESUMO

BACKGROUND: Bulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PURPOSE: This study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. RESULTS: We found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6'-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. CONCLUSIONS: These results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Suplementos Nutricionais , Interações Ervas-Drogas , Xanthorrhoeaceae/química , Inibidores do Citocromo P-450 CYP2C9/química , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Suplementos Nutricionais/efeitos adversos , Células Hep G2 , Humanos , Masculino , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Receptor de Pregnano X/química , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo
20.
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
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