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1.
PLoS One ; 15(9): e0237809, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915792

RESUMO

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.


Assuntos
Canalículos Biliares/efeitos dos fármacos , Desenvolvimento de Medicamentos/métodos , Hepatócitos/efeitos dos fármacos , Cultura Primária de Células/métodos , Quimeras de Transplante , Animais , Canalículos Biliares/citologia , Canalículos Biliares/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Ciclosporina/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Xenobiotica ; 50(11): 1275-1284, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32400275

RESUMO

First dose prediction is challenging in neonates. Our objective in this proof-of-concept study was to perform a pharmacokinetic (PK) bridging study from juvenile mice to neonates for drugs metabolized by CYP3A. We selected midazolam and clindamycin as model drugs. We developed juvenile mice population PK models using NONMEM. The PK parameters of these two drugs in juvenile mice were used to bridge PK parameters in neonates using different correction methods. The bridging results were evaluated by the fold-error of 0.5- to 1.5-fold. Simple allometry with and without a correction factor for maximum lifespan potential could be used for a bridging of clearance (CL) and volume of distribution (Vd), respectively, from juvenile mice to neonates. Simulation results demonstrated that for midazolam, 100% of clinical studies for which both the predictive CL and Vd were within 0.5- to 1.5-fold of the observed. For clindamycin, 75% and 100% of clinical studies for which the predictive CL and Vd were within 0.5- to 1.5-fold of the observed. A PK bridging of drugs metabolized by CYP3A is feasible from juvenile mice to neonates. It could be a complement to the ADE and PBPK models to support the first dose in neonates.


Assuntos
Simulação por Computador , Citocromo P-450 CYP3A/metabolismo , Animais , Clindamicina/farmacocinética , Camundongos , Midazolam/farmacocinética , Modelos Biológicos
3.
PLoS One ; 15(5): e0233010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396581

RESUMO

Methamphetamine use has increased over the past decade and the first use of methamphetamine is most often when women are of reproductive age. Methamphetamine accumulates in the liver; however, little is known about the effect of methamphetamine use on hepatic drug metabolism. Methamphetamine was administered on 3 occassions to female Dunkin Hartley guinea pigs of reproductive age, mimicking recreational drug use. Low doses of test drugs caffeine and midazolam were administered after the third dose of methamphetamine to assess the functional activity of cytochrome P450 1A2 and 3A, respectively. Real-time quantitative polymerase chain reaction was used to quantify the mRNA expression of factors involved in glucocorticoid signalling, inflammation, oxidative stress and drug transporters. This study showed that methamphetamine administration decreased hepatic CYP1A2 mRNA expression, but increased CYP1A2 enzyme activity. Methamphetamine had no effect on CYP3A enzyme activity. In addition, we found that methamphetamine may also result in changes in glucocorticoid bioavailability, as we found a decrease in 11ß-hydroxysteroid dehydrogenase 1 mRNA expression, which converts inactive cortisone into active cortisol. This study has shown that methamphetamine administration has the potential to alter drug metabolism via the CYP1A2 metabolic pathway in female guinea pigs. This may have clinical implications for drug dosing in female methamphetamine users of reproductive age.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metanfetamina/administração & dosagem , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/sangue , Estimulantes do Sistema Nervoso Central/toxicidade , Citocromo P-450 CYP1A2/genética , Feminino , Cobaias , Humanos , Taxa de Depuração Metabólica , Redes e Vias Metabólicas/efeitos dos fármacos , Metanfetamina/sangue , Metanfetamina/toxicidade , Modelos Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Toxicol Appl Pharmacol ; 396: 115000, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275916

RESUMO

The pharmacokinetics of Tacrolimus is characterized by a high interindividual variability that is mainly explained by pharmacogenetics biomarkers. The aims were to develop a population pharmacokinetic model (Pk pop) taking into account post-transplant phases (PTP), CYP3A4*1B, CYP3A4*22 and CYP3A5*3 polymorphisms on Tac pharmacokinetics in adult kidney transplant patients. The Pk pop study was performed using a nonparametric approach (Pmetrics*). The influence of covariates (age, weight, sex, hematocrit and CYP3A4*1B, CYP3A4*22 and CYP3A5*3 polymorphisms) was tested on the model's Pk parameters. The performance of the final model was assessed using an external dataset. A one-compartment model (Vd: volume of distribution, CL: Tac Clearance) was found to correctly describe the evolution of the C0/D regardless of the PTP. The influence of the covariates has shown that only the CYP3A4*1B and CYP3A4*22 polymorphisms were significantly associated only with CL, regardless of PTP (p = .04 and 0.02, respectively). Only the CYP3A4*22 polymorphism influenced CL during early PTP (P1: the first three months, p = .02). During the late PTP (P2: >3 months), only CYP3A4 polymorphisms were found to affect CL (p = .03 for both). The external validation of the final model, including both CYP3A4 polymorphisms, showed an acceptable predictive performance during P1 and P2. We developed and validated a tac Pk pop model including both CYP3A4*22 and CYP3A4*1B polymorphisms, taking into account PTP. This model was very useful in the Tac dose proposal in this population on any PT day but could not be used in other organ transplants due to pharmacokinetic differences.


Assuntos
Citocromo P-450 CYP3A/genética , Imunossupressores/farmacocinética , Transplante de Rim , Tacrolimo/farmacocinética , Adulto , Estudos Transversais , Citocromo P-450 CYP3A/metabolismo , Feminino , Técnicas de Genotipagem , Humanos , Masculino , Taxa de Depuração Metabólica/genética , Pessoa de Meia-Idade , Testes Farmacogenômicos , Polimorfismo Genético/genética , Tunísia , Adulto Jovem
6.
Chem Biol Interact ; 324: 109062, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198087

RESUMO

Ginsenoside Rg1 is an active ingredient extracted from the roots of ginsenoside, and an α-naphthylisothiocyanate (ANIT)-induced rat model of intrahepatic cholestasis was used to investigate the protective effect of Rg1 on cholestasis. 48 SD male rats were randomly divided into 6 groups: control group, model group, UDCA group (ursodeoxycholic acid), low-dose Rg1 group (10 mg/kg), medium-dose Rg1 group (20 mg/kg) and high-dose Rg1 group (40 mg/kg). The model group, the UDCA group and all the Rg1 group were then intragastrically administered with 80 mg/kg ANIT, and the control group were given equal volume of olive oil. Then the pathological changes in liver tissue were observed, the secretion of bile in the bile duct was measured, and the biochemical markers in serum were quantified, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transfer peptidase (GTP) and the content of total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA). The contents of inflammatory mediators in serum were quantified, including tumor necrosis factor (TNF-α), γ-interferon (IFN-γ) and interleukin-1ß (IL-1ß). The contents of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in liver homogenate were quantified. Expression of farnesoid X receptor (FXR), transporters and metabolic enzymes in liver tissue was monitored. Rg1 treatment improved liver tissue pathological damage, promoted bile secretion and significantly reduced serum levels of the intrahepatic cholestasis markers ALT, AST, ALP, GTP, TBIL, DBIL and TBA. Rg1 increased the activity of SOD and GSH-Px in liver homogenate, while, reducing the serum levels of MDA and inflammatory mediators. Rg1 also regulated the expression of FXR, bile acid transporters and metabolic enzymes. Overall, Rg1 alleviated liver injury by improving secretion of bile and normalizing the activity of enzymes in the serum. The protective mechanism appeared to be related to the activation of FXR and regulation of liver transporters and metabolic enzymes.


Assuntos
Colestase Intra-Hepática/tratamento farmacológico , Ginsenosídeos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Substâncias Protetoras/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , 1-Naftilisotiocianato , Animais , Bile/metabolismo , Biomarcadores/metabolismo , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/patologia , Citocromo P-450 CYP3A/metabolismo , Citocinas/metabolismo , Glucuronosiltransferase/metabolismo , Glutationa Peroxidase/metabolismo , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Ratos Sprague-Dawley , Sulfotransferases/metabolismo , Superóxido Dismutase/metabolismo
7.
Xenobiotica ; 50(9): 1032-1042, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32129697

RESUMO

1. The absorption, distribution, metabolism, elimination, and drug-drug interaction (DDI) potential of the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib was characterised in vitro.2. Rucaparib showed moderate cellular permeability, moderate human plasma protein binding (70.2%), and slow metabolism in human liver microsomes (HLMs). In HLMs, cytochrome P450 (CYP) 1A2 and CYP3A contributed to the metabolism of rucaparib to its major metabolite M324 with estimated fractions of metabolism catalysed by CYP (fm,CYP) of 0.27 and 0.64, respectively. Rucaparib reversibly inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3As (IC50, 3.55, 12.9, 5.42, 41.6, and 17.2-22.9 µM [2 substrates], respectively), but not CYP2B6 or CYP2C8 (>190 µM). No time-dependent inhibition of any CYP was observed. In cultured human hepatocytes, rucaparib showed concentration-dependent induction of CYP1A2 mRNA and downregulation of CYP3A4 and CYP2B6 mRNA. In transfected cells expressing drug transporters, rucaparib was a substrate for P-gp and BCRP, but not for OATP1B1, OATP1B3, OAT1, OAT3, or OCT2. Rucaparib inhibited P-gp and BCRP (IC50, 169 and 55 µM, respectively) and slightly inhibited OATP1B1, OATP1B3, OAT1, and OAT3 (66%, 58%, 58%, and 42% inhibition, respectively) at 300 µM. Rucaparib inhibited OCT1, OCT2, MATE1, and MATE2-K (IC50, 4.3, 31, 0.63, and 0.19 µM, respectively).3. DDI risk assessment using static models suggested potential CYP-related DDIs, with rucaparib as a perpetrator. Caution is advised when co-administering rucaparib with sensitive substrates of MATEs, OCT1, and OCT2.


Assuntos
Indóis/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/metabolismo , Transporte Biológico , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Humanos , Indóis/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos , Proteínas de Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo
8.
Food Chem ; 319: 126578, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32187570

RESUMO

For clementine juice, previous data indicate a possible food-drug interaction with substrates of key enzymes responsible for drug metabolism (i.e. cytochrome P450 [CYP] 3A4, CYP1A2). However, which compounds in clementine juice are responsible for these effects are unknown. Therefore, we aimed to identify the compounds in clementine juice provoking metabolic enzyme inhibition or induction. The results demonstrated that the flavonoid fraction of clementine juice provoked induction of several genes and inhibition of both CYP3A4 and CYP1A2, matching effects observed with whole clementine juice. CYP1A2 inhibition and induction can most likely be attributed to nobiletin, sinensetin, and tangeretin. Tangeretin was the only compound causing CYP3A4 induction while CYP3A4 inhibition was most likely the result of additive or synergistic effects caused by several compounds. Thus, whenever evaluating the clinical relevance of clementine interactions, flavonoid contents should be reported because these might explain differences between cultivars and harvests.


Assuntos
Citrus/química , Flavonas/farmacocinética , Flavonoides/farmacocinética , Interações Alimento-Droga , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Inibidores do Citocromo P-450 CYP1A2/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Flavonoides/análise , Frutas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo
9.
J Med Chem ; 63(7): 3701-3712, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32160459

RESUMO

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.


Assuntos
Benzopiranos/farmacologia , Receptor de Pregnano X/agonistas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Benzopiranos/metabolismo , Benzopiranos/toxicidade , Linhagem Celular Tumoral , Cristalografia por Raios X , Citocromo P-450 CYP3A/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X/metabolismo
10.
PLoS One ; 15(2): e0229654, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106262

RESUMO

Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William's E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14-25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura , Hepatócitos/citologia , Hepatócitos/metabolismo , Albuminas/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Ureia/metabolismo
11.
Arch Biochem Biophys ; 682: 108283, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001245

RESUMO

Hydroxylation activity at the 6ß-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6ß-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6ß-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6ß-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6ß-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6ß-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Hormônios/metabolismo , Esteroides/metabolismo , Catálise , Humanos , Hidrocortisona/metabolismo , Hidroxilação , Cinética , Microssomos Hepáticos/metabolismo , Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , Testosterona/metabolismo
12.
Chemistry ; 26(28): 6214-6223, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32049373

RESUMO

The hydroxylation of nonreactive C-H bonds can be easily catalyzed by a variety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically. However, understanding the regio- and stereoselectivities of substrates hydroxylated by P450s remains a great challenge. Herein, we use a multi-scale modeling approach to investigate the selectivity of testosterone (TES) and dihydrotestosterone (DHT) hydroxylation catalyzed by two important P450s, CYP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHT were predicted by dockings and molecular dynamics simulations, in which the experimentally identified sites of metabolism of TES/DHT can access to the catalytic center. The regio- and stereoselectivities of TES/DHT hydroxylation were further evaluated by quantum mechanical and ONIOM calculations. For CYP19A1, we found that sites 1ß, 2ß and 19 can access the catalytic center, with the intrinsic reactivity 2ß>1ß>19. However, our ONIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT, which is consistent with the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlying the selectivity of TES/DHT hydroxylation mediated by CYP3A4 and CYP19A1 and is helpful for understanding the selectivity of other substrates that are hydroxylated by P450s.


Assuntos
Aromatase/metabolismo , Citocromo P-450 CYP3A/metabolismo , Di-Hidrotestosterona/química , Testosterona/metabolismo , Aromatase/química , Catálise , Citocromo P-450 CYP3A/química , Humanos , Hidroxilação , Cinética , Oxirredução , Testosterona/química
13.
Xenobiotica ; 50(8): 913-918, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32026737

RESUMO

Pachymic acid is a wildly used traditional Chinese medicine with various pharmacological features. It also exists in many drugs which are wildly used in pediatric.The effect of pachymic acid on the activity of eight major CYP isoforms was investigated in human liver microsomes.The effects of pachymic acid on eight human liver CYP isoforms (i.e. 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs), and the enzyme kinetic parameters were calculated.The activity of CP3A4, 2E1, and 2C9 was inhibited by pachymic acid in a concentration-dependent manner with IC50 values of 15.04, 27.95, and 24.22 µM, respectively. Pachymic acid is a non-competitive inhibitor of CYP3A4, with the Ki value of 6.47 µM. While the inhibition of CYP2E1 and 2C9 was performed in a competitive manner, with the Ki value of 11.96 and 10.94 µM, respectively. Moreover, the inhibition of CYP3A4 was in a time-dependent manner with the KI/Kinact value of 7.77/0.048 min-1 µM-1.The in vitro inhibitory effect of pachymic acid on the activity of CYP3A4, 2E1, and 2C9 indicated the potential drug-drug interaction with the drugs that metabolized by CYP3A4, 2E1, and 2C9. Further clinical and in vivo studies are needed to evaluate the significance of this interaction.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Triterpenos/farmacologia , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Humanos , Microssomos Hepáticos/metabolismo
14.
Xenobiotica ; 50(8): 929-938, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32065000

RESUMO

We assessed the contribution of CYP2C19 and CYP3A4 metabolic activity to the ADP-induced platelet aggregation 1h and 24h after a loading dose of 60 mg prasugrel or 180 mg ticagrelor in patients with ST-elevation myocardial infarction (STEMI). Further, we assessed the contribution of CYP2C19 polymorphisms and medication to the CYP enzymatic activity.Patients with STEMI were randomly assigned to the treatment with prasugrel (n = 51) or ticagrelor (n = 46). Metabolic activity of CYP2C19 and CYP3A4 was assessed by the rate of 5-hydroxylation and sulfoxidation of lansoprazole. Further, patients were genotyped for CYP2C19 *2 and *17 alleles.In prasugrel-treated patients, high ADP-induced platelet reactivity 1h after the loading dose positively correlated with 5OH-lansoprazole/lansoprazole ratio (r = 0.44, p = 0.002), a marker of CYP2C19 metabolic activity, and negatively with lansoprazole-sulfone/lansoprazole ratio, which reflects CYP3A4 metabolic activity (r = -0.35, p = 0.018).CYP2C19 poor metabolizers had lower 5OH-lansoprazole/lansoprazole ratio and higher lansoprazole-sulfone/lansoprazole ratio, but without any effect on the ADP-induced platelet reactivity. The treatment with amiodarone, a CYP3A4 inhibitor, influenced neither the metabolic ratios nor the ADP-induced platelet reactivity.The CYP3A4 and CYP2C19 metabolic activity is associated with ADP-induced platelet reactivity in prasugrel-treated, but not ticagrelor-treated patients with STEMI.


Assuntos
Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Cloridrato de Prasugrel/farmacologia , Ticagrelor/farmacologia , Difosfato de Adenosina/metabolismo , Feminino , Humanos , Masculino , Cloridrato de Prasugrel/uso terapêutico , Infarto do Miocárdio com Supradesnível do Segmento ST/tratamento farmacológico , Ticagrelor/uso terapêutico
15.
PLoS One ; 15(2): e0229106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106230

RESUMO

In vitro studies of drug toxicity and drug-drug interactions are crucial for drug development efforts. Currently, the utilization of primary human hepatocytes (PHHs) is the de facto standard for this purpose, due to their functional xenobiotic response and drug metabolizing CYP450 enzyme metabolism. However, PHHs are scarce, expensive, require laborious maintenance, and exhibit lot-to-lot heterogeneity. Alternative human in vitro platforms include hepatic cell lines, which are easy to access and maintain, and induced pluripotent stem cell (iPSC) derived hepatocytes. In this study, we provide a direct comparison of drug induced CYP3A4 and PXR expression levels of PHHs, hepatic cell lines Huh7 and HepG2, and iPSC derived hepatocyte like cells. Confluently cultured Huh7s exhibited an improved CYP3A4 expression and were inducible by up to 4.9-fold, and hepatocytes differentiated from human iPSCs displayed a 3.3-fold CYP3A4 induction. In addition, an increase in PXR expression levels was observed in both hepatic cell lines and iPSC derived hepatocytes upon rifampicin treatment, whereas a reproducible increase in PXR expression was not achieved in PHHs. Our results indicate that both hepatoma originated cell lines and iPSCs may provide alternative sources to primary hepatocytes, providing reliable and reproducible results for CYP3A4/PXR metabolism, upon in vitro maturation. This study may serve as a guide for the selection of suitable and feasible in vitro platforms for drug-drug interaction and toxicology studies.


Assuntos
Indutores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Hepatócitos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Receptor de Pregnano X/metabolismo , Reprodutibilidade dos Testes , Testes de Toxicidade/métodos
16.
Pharmacol Res Perspect ; 8(1): e00561, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32003945

RESUMO

Synthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS-135. In this study, STS-135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra-performance liquid chromatography. STS-135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS-135's oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a Km value of 11.4 µmol/L. Thus, it was concluded that STS-135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS-135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS-135-induced cardiovascular toxicity.


Assuntos
Adamantano/análogos & derivados , Citocromo P-450 CYP3A/metabolismo , Indóis/farmacocinética , Microssomos/metabolismo , Adamantano/química , Adamantano/farmacocinética , Encéfalo/citologia , Cromatografia Líquida de Alta Pressão , Humanos , Indóis/química , Intestinos/citologia , Fígado/citologia , Oxirredução
17.
J Agric Food Chem ; 68(5): 1257-1265, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31927919

RESUMO

Bedaquiline (TMC-207) is a recently approved drug for the treatment of multidrug-resistant tuberculosis (MDR-TB). Moreover, there is a present and growing concern for natural-product-mediated drug interaction, as these are inadvertently taken by patients as a dietary supplement, food additive, and medicine. In the present study, we investigated the impact of 20 plant-based natural products, typically phenolics, on in vivo oral bedaquiline pharmacokinetics, as previous studies are lacking. Three natural phenolics were identified that can significantly enhance the oral exposure of bedaquiline upon coadministration. We further investigated the possible role of all of the phytochemicals on in vitro P-glycoprotein (P-gp) induction and inhibition and CYP3A4 inhibition in a single platform as bedaquiline is the substrate for both P-gp and CYP3A4. In conclusion, curcumin, CC-I (3',5-dihydroxyflavone-7-O-ß-d-galacturonide-4'-O-ß-d-glucopyranoside), and 6-gingerol should not be coadministered with bedaquiline to avoid untoward drug interactions and, subsequently, its dose-dependent adverse effects.


Assuntos
Antituberculosos/farmacocinética , Diarilquinolinas/farmacocinética , Suplementos Nutricionais/efeitos adversos , Interações Alimento-Droga , Fenóis/efeitos adversos , Extratos Vegetais/efeitos adversos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antituberculosos/administração & dosagem , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Diarilquinolinas/administração & dosagem , Suplementos Nutricionais/análise , Feminino , Humanos , Fenóis/administração & dosagem , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Resistente a Múltiplos Medicamentos/metabolismo
18.
Chin J Nat Med ; 18(1): 57-69, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31955824

RESUMO

Diterpenoid lactones (DLs), a group of furan-containing compounds found in Dioscorea bulbifera L. (DB), have been reported to be associated with hepatotoxicity. Different hepatotoxicities of these DLs have been observed in vitro, but reasonable explanations for the differential hepatotoxicity have not been provided. Herein, the present study aimed to confirm the potential factors that contribute to varied hepatotoxicity of four representative DLs (diosbulbins A, B, C, F). In vitro toxic effects were evaluated in various cell models and the interactions between DLs and CYP3A4 at the atomic level were simulated by molecular docking. Results showed that DLs exhibited varied cytotoxicities, and that CYP3A4 played a modulatory role in this process. Moreover, structural variation may cause different affinities between DLs and CYP3A4, which was positively correlated with the observation of cytotoxicity. In addition, analysis of the glutathione (GSH) conjugates indicated that reactive intermediates were formed by metabolic oxidation that occurred on the furan moiety of DLs, whereas, GSH consumption analysis reflected the consistency between the reactive metabolites and the hepatotoxicity. Collectively, our findings illustrated that the metabolic regulation played a crucial role in generating the varied hepatotoxicity of DLs.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocromo P-450 CYP3A/metabolismo , Dioscorea/toxicidade , Medicamentos de Ervas Chinesas/toxicidade , Furanos/toxicidade , Cromatografia Líquida , Dioscorea/química , Medicamentos de Ervas Chinesas/química , Furanos/química , Células Hep G2 , Humanos , Espectrometria de Massas , Simulação de Acoplamento Molecular , Estrutura Molecular
19.
Biochemistry ; 59(6): 766-779, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31961139

RESUMO

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the ß1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.


Assuntos
Sítio Alostérico/fisiologia , Citocromo P-450 CYP3A/metabolismo , Bicamadas Lipídicas/metabolismo , Midazolam/metabolismo , Simulação de Dinâmica Molecular , Nanopartículas/metabolismo , Ansiolíticos/química , Ansiolíticos/metabolismo , Citocromo P-450 CYP3A/química , Humanos , Bicamadas Lipídicas/química , Lipídeos/química , Midazolam/química , Nanopartículas/química , Estrutura Secundária de Proteína
20.
Biopharm Drug Dispos ; 41(1-2): 64-71, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31925794

RESUMO

Osthol (OST) has a wide range of pharmacological effects and has long been used in clinical medicine in China. Previous studies have indicated that osthol has weak inhibitory effects on CYP3A4 in human liver microsomes. The aim of the present study was to investigate the inhibition of Cyp3a by osthol in rats in vivo. A substrate assay was used to corroborate the inhibitory effect on Cyp3a by osthol in rats, and the substrate probe (midazolam) was detected by high-performance liquid chromatography (HPLC). Semi-quantitative RT-PCR (SqRT-PCR) analysis was used to study the effect of osthol on Cyp3a1 and Cyp3a2 mRNA expression and Western blot analysis was used to investigate the effect of OST on Cyp3a1 and Cyp3a2 protein expression. Our study confirmed the inhibitory effect of osthol on Cyp3a and indicated that the inhibitory effect on Cyp3a was stronger in the group receiving multiple doses compared with the single dose group. The SqRT-PCR analysis results showed that medium and high doses of osthol (20 and 40 mg/kg, respectively) had an inhibitory effect on Cyp3a1 mRNA expression but not on Cyp3a2 mRNA expression. Western blot analysis results indicated that the inhibitory effect of the medium and high osthol doses on Cyp3a1 and Cyp3a2 protein expression was significantly different. It was also demonstrated that the inhibitory effect of osthol on Cyp3a in rats resulted from the comprehensive effect of the direct inhibition of the Cyp3a enzyme, as well as the down-regulation of its mRNA and protein expression level.


Assuntos
Cumarínicos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Animais , Área Sob a Curva , Cumarínicos/química , Citocromo P-450 CYP3A/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Estrutura Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley
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