RESUMO
Ciprofol is a novel short-acting intravenous anaesthetic developed in China that is mainly metabolized by cytochrome P450 2B6 (CYP2B6) and uridine diphosphate glucuronosyltransferase 1A9 (UGT1A9). Currently, insufficient evidence is available to support drugâdrug interactions between ciprofol and CYP2B6 inactivators. Here, we established a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to assess the concentration of ciprofol and investigated the effects of psoralen and clopidogrel on the metabolism of ciprofol in liver microsomes and rats. In rat and human liver microsomes, the median inhibitory concentration (IC50) values of psoralen were 63.31 µmol·L-1 and 34.05 µmol·L-1, respectively, showing mild inhibitory effects on ciprofol metabolism, whereas the IC50 values of clopidogrel were 6.380 µmol·L-1 and 2.565 µmol·L-1, respectively, with moderate inhibitory effects. SD rats were randomly divided into three groups: psoralen (27 mg·kg-1), clopidogrel (7.5 mg·kg-1), and the same volume of 0.5% carboxy methyl cellulose. After 7 days, all rats were injected with 2.4 mg·kg-1 ciprofol. Compared with the control group, the AUC and MRT values of ciprofol in the psoralen and clopidogrel groups were significantly greater, whereas the CL values were significantly lower. In addition, the durations of loss of righting reflex (LORR) in the psoralen and clopidogrel groups were 16.1% and 23.0% longer than that in the control group, respectively. In conclusion, psoralen and clopidogrel inhibit ciprofol metabolism to different degrees and prolong the duration of LORR in rats.
Assuntos
Clopidogrel , Citocromo P-450 CYP2B6 , Microssomos Hepáticos , Ratos Sprague-Dawley , Animais , Humanos , Clopidogrel/metabolismo , Clopidogrel/farmacologia , Ratos , Microssomos Hepáticos/metabolismo , Masculino , Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Ticlopidina/metabolismo , Ticlopidina/farmacologia , Ticlopidina/análogos & derivados , Ficusina/farmacologia , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Interações Medicamentosas , Fenilacetatos , TiofenosRESUMO
BACKGROUND AND OBJECTIVES: A genetic algorithm (GA) approach was developed to predict drug-drug interactions (DDIs) caused by cytochrome P450 2C8 (CYP2C8) inhibition or cytochrome P450 2B6 (CYP2B6) inhibition or induction. Nighty-eight DDIs, obtained from published in vivo studies in healthy volunteers, have been considered using the area under the plasma drug concentration-time curve (AUC) ratios (i.e., ratios of AUC of the drug substrate administered in combination with a DDI perpetrator to AUC of the drug substrate administered alone) to describe the extent of DDI. METHODS: The following parameters were estimated in this approach: the contribution ratios (CRCYP2B6 and CRCYP2C8, i.e., the fraction of the dose metabolized via CYP2B6 or CYP2C8, respectively) and the inhibitory or inducing potency of the perpetrator drug (IRCYP2B6, IRCYP2C8 and ICCYP2B6, for inhibition of CYP2B6 and CYP2C8, and induction of CYP2B6, respectively). The workflow consisted of three main phases. First, the initial estimates of the parameters were estimated through GA. Then, the model was validated using an external validation. Finally, the parameter values were refined via a Bayesian orthogonal regression using all data. RESULTS: The AUC ratios of 5 substrates, 11 inhibitors and 19 inducers of CYP2B6, and the AUC ratios of 19 substrates and 23 inhibitors of CYP2C8 were successfully predicted by the developed methodology within 50-200% of observed values. CONCLUSIONS: The approach proposed in this work may represent a useful tool for evaluating the suitable doses of a CYP2C8 or CYP2B6 substrates co-administered with perpetrators.
Assuntos
Algoritmos , Área Sob a Curva , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C8 , Interações Medicamentosas , Interações Medicamentosas/fisiologia , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2B6/genética , Humanos , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP2C8/genética , Inibidores do Citocromo P-450 CYP2C8/farmacologia , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Inibidores do Citocromo P-450 CYP2B6/farmacocinética , Teorema de BayesRESUMO
PURPOSE: Midostaurin, approved for treating FLT-3-mutated acute myeloid leukemia and advanced systemic mastocytosis, is metabolized by cytochrome P450 (CYP) 3A4 to two major metabolites, and may inhibit and/or induce CYP3A, CYP2B6, and CYP2C8. Two studies investigated the impact of midostaurin on CYP substrate drugs and oral contraceptives in healthy participants. METHODS: Using sentinel dosing for participants' safety, the effects of midostaurin at steady state following 25-day (Study 1) or 24-day (Study 2) dosing with 50 mg twice daily were evaluated on CYP substrates, midazolam (CYP3A4), bupropion (CYP2B6), and pioglitazone (CYP2C8) in Study 1; and monophasic oral contraceptives (containing ethinylestradiol [EES] and levonorgestrel [LVG]) in Study 2. RESULTS: In Study 1, midostaurin resulted in a 10% increase in midazolam peak plasma concentrations (Cmax), and 3-4% decrease in total exposures (AUC). Bupropion showed a 55% decrease in Cmax and 48-49% decrease in AUCs. Pioglitazone showed a 10% decrease in Cmax and 6% decrease in AUC. In Study 2, midostaurin resulted in a 26% increase in Cmax and 7-10% increase in AUC of EES; and a 19% increase in Cmax and 29-42% increase in AUC of LVG. Midostaurin 50 mg twice daily for 28 days ensured that steady-state concentrations of midostaurin and the active metabolites were achieved by the time of CYP substrate drugs or oral contraceptive dosing. No safety concerns were reported. CONCLUSION: Midostaurin neither inhibits nor induces CYP3A4 and CYP2C8, and weakly induces CYP2B6. Midostaurin at steady state has no clinically relevant PK interaction on hormonal contraceptives. All treatments were well tolerated.
Assuntos
Bupropiona , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP3A , Interações Medicamentosas , Midazolam , Estaurosporina , Humanos , Área Sob a Curva , Bupropiona/farmacocinética , Bupropiona/administração & dosagem , Anticoncepcionais Orais/administração & dosagem , Anticoncepcionais Orais/farmacologia , Anticoncepcionais Orais/farmacocinética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP3A/metabolismo , Combinação de Medicamentos , Etinilestradiol/farmacocinética , Etinilestradiol/administração & dosagem , Etinilestradiol/farmacologia , Voluntários Saudáveis , Levanogestrel/farmacocinética , Levanogestrel/administração & dosagem , Levanogestrel/farmacologia , Midazolam/farmacocinética , Midazolam/administração & dosagem , Pioglitazona/farmacologia , Pioglitazona/administração & dosagem , Pioglitazona/farmacocinética , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Estaurosporina/farmacocinética , Estaurosporina/administração & dosagem , Masculino , Feminino , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-IdadeRESUMO
Ciprofol is a novel intravenous anesthetic agent. Its major glucuronide metabolite, M4, is found in plasma and urine. However, the specific isoforms of UDP-glucuronosyltransferases (UGTs) that metabolize ciprofol to M4 remain unknown. This study systematically characterized UGTs that contribute to the formation of M4 using human liver microsomes (HLM), human intestinal microsomes (HIM), and human recombinant UGTs. The inhibitory potential of ciprofol and M4 against major human UGTs and cytochrome P450 enzymes (P450s) was also explored. In vitro-in vivo extrapolation (IVIVE) and physiologically-based pharmacokinetic (PBPK) simulations were performed to predict potential in vivo drug-drug interactions (DDIs) caused by ciprofol. Glucuronidation of ciprofol followed Michaelis-Menten kinetics in both HLM and HIM with apparent Km values of 345 and 412 µM, Vmax values of 2214 and 444 nmol min-1·mg protein-1, respectively. The in vitro intrinsic clearances (CLint = Vmax/Km) for ciprofol glucuronidation by HLM and HIM were 6.4 and 1.1 µL min-1·mg protein-1, respectively. Human recombinant UGT studies revealed that UGT1A9 is the predominant isoform mediating M4 formation, followed by UGT1A7, with UGT1A8 playing a minor role. Ciprofol competitively inhibited CYP1A2 (Ki = 12 µM) and CYP2B6 (Ki = 4.7 µM), and noncompetitively inhibited CYP2C19 (Ki = 29 µM). No time-dependent inhibition by ciprofol was noted for CYP1A2, CYP2B6, or CYP2C19. In contrast, M4 showed limited or no inhibitory effects against selected P450s. Neither ciprofol nor M4 inhibited UGTs significantly. Initial IVIVE suggested potential ciprofol-mediated inhibition of CYP1A2, CYP2B6, and CYP2C19 inhibition in vivo. However, PBPK simulations showed no significant effect on phenacetin, bupropion, and S-mephenytoin exposure or peak plasma concentration. Our findings are pertinent for future DDI studies of ciprofol as either a perpetrator or victim drug.
Assuntos
Citocromo P-450 CYP1A2 , Microssomos Hepáticos , Humanos , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Microssomos Hepáticos/metabolismo , Glucuronosiltransferase/metabolismo , Interações Medicamentosas , CinéticaRESUMO
PURPOSE: The inhibitory effect of Apatinib on cytochrome P450 (CYP450) enzymes has been studied. However, it is unknown whether the inhibition is related to the major metabolites, M1-1, M1-2 and M1-6. METHODS: A 5-in-1 cocktail system composed of CYP2B6/Cyp2b1, CYP2C9/Cyp2c11, CYP2E1/Cyp2e1, CYP2D6/Cyp2d1 and CYP3A/Cyp3a2 was used in this study. Firstly, the effects of APA and its main metabolites on the activities of HLMs, RLMs and recombinant isoforms were examined. The reaction mixture included HLMs, RLMs or recombinant isoforms (CYP3A4.1, CYP2D6.1, CYP2D6.10 or CYP2C9.1), analyte (APA, M1-1, M1-2 or M1-6), probe substrates. The reactions were pre-incubated for 5 min at 37 °C, followed by the addition of NAPDH to initiate the reactions, which continued for 40 min. Secondly, IC50 experiments were conducted to determine if the inhibitions were reversible. The reaction mixture of the "+ NADPH Group" included HLMs or RLMs, 0 to 100 of µM M1-1 or M1-2, probe substrates. The reactions were pre-incubated for 5 min at 37 °C, and then NAPDH was added to initiate reactions, which proceeded for 40 min. The reaction mixture of the "- NADPH Group" included HLMs or RLMs, probe substrates, NAPDH. The reactions were pre-incubated for 30 min at 37 °C, and then 0 to 100 µM of M1-1 or M1-2 was added to initiate the reactions, which proceeded for 40 min. Finally, the reversible inhibition of M1-1 and M1-2 on isozymes was determined. The reaction mixture included HLMs or RLMs, 0 to 10 µM of M1-1 or M1-2, probe substrates with concentrations ranging from 0.25Km to 2Km. RESULTS: Under the influence of M1-6, the activity of CYP2B6, 2C9, 2E1 and 3A4/5 was increased to 193.92%, 210.82%, 235.67% and 380.12% respectively; the activity of CYP2D6 was reduced to 92.61%. The inhibitory effects of M1-1 on CYP3A4/5 in HLMs and on Cyp2d1 in RLMs, as well as the effect of M1-2 on CYP3A in HLMs, were determined to be noncompetitive inhibition, with the Ki values equal to 1.340 µM, 1.151 µM and 1.829 µM, respectively. The inhibitory effect of M1-1 on CYP2B6 and CYP2D6 in HLMs, as well as the effect of M1-2 on CYP2C9 and CYP2D6 in HLMs, were determined to be competitive inhibition, with the Ki values equal to 12.280 µM, 2.046 µM, 0.560 µM and 4.377 µM, respectively. The inhibitory effects of M1-1 on CYP2C9 in HLMs and M1-2 on Cyp2d1 in RLMs were determined to be mixed-type, with the Ki values equal to 0.998 µM and 0.884 µM. The parameters could not be obtained due to the atypical kinetics of CYP2E1 in HLMs under the impact of M1-2. CONCLUSIONS: M1-1 and M1-2 exhibited inhibition for several CYP450 isozymes, especially CYP2B6, 2C9, 2D6 and 3A4/5. This observation may uncover potential drug-drug interactions and provide valuable insights for the clinical application of APA.
Assuntos
Citocromo P-450 CYP3A , Microssomos Hepáticos , Piridinas , Humanos , Ratos , Animais , Microssomos Hepáticos/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP2E1/metabolismo , Isoenzimas/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2B6/metabolismo , NADP/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
Ciprofol (HSK3486) is a novel intravenous agent for general anesthesia. In humans, HSK3486 mainly undergoes glucuronidation to form M4 [fraction of clearance (fCL): 62.6%], followed by the formation of monohydroxylated metabolites that further undergo glucuronidation and sulfation to produce M5-1, M5-2, M5-3, and M3 (summed fCL: 35.2%). However, the complete metabolic pathways of HSK3486 in humans remain unclear. In this study, by comparison with chemically synthesized reference standards, three monohydroxylated metabolites [M7-1, 4-hydroxylation with an unbound intrinsic clearance (CLint,u) of 2211 µl/min/mg; M7-2, ω-hydroxylation with a CLint,u of 600 µl/min/mg; and M7-3, (ω-1)-hydroxylation with a CLint,u of 78.4 µl/min/mg] were identified in human liver microsomes, and CYP2B6 primarily catalyzed their formation. In humans, M7-1 was shown to undergo glucuronidation at the 4-position and 1-position by multiple UDP-glucuronosyltransferases (UGTs) to produce M5-1 and M5-3, respectively, or was metabolized to M3 by cytosolic sulfotransferases. M7-2 was glucuronidated at the ω position by UGT1A9, 2B4, and 2B7 to form M5-2. UGT1A9 predominantly catalyzed the glucuronidation of HSK3486 (M4). The CLint,u values for M4 formation in human liver and kidney microsomes were 1028 and 3407 µl/min/mg, respectively. In vitro to in vivo extrapolation analysis suggested that renal glucuronidation contributed approximately 31.4% of the combined clearance. In addition to HSK3486 glucuronidation (M4), 4-hydroxylation (M7-1) was identified as another crucial oxidative metabolic pathway (fCL: 34.5%). Further attention should be paid to the impact of CYP2B6- and UGT1A9-mediated drug interactions and gene polymorphisms on the exposure and efficacy of HSK3486. SIGNIFICANCE STATEMENT: This research elucidates the major oxidative metabolic pathways of HSK3486 (the formation of three monohydroxylated metabolites: M7-1, M7-2, M7-3) as well as definitive structures and formation pathways of these monohydroxylated metabolites and their glucuronides or sulfate in humans. This research also identifies major metabolizing enzymes responsible for the glucuronidation (UGT1A9) and oxidation (CYP2B6) of HSK3486 and characterizes the mechanism of extrahepatic metabolism. The above information is helpful in guiding the safe use of HSK3486 in the clinic.
Assuntos
Glucuronosiltransferase , Microssomos Hepáticos , Humanos , Citocromo P-450 CYP2B6/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Difosfato de Uridina/metabolismoRESUMO
BACKGROUND: Maternal obesity (MO) increases fetal androgen concentrations, the prevalence of macrosomia, and predisposes offspring to metabolic dysfunction in later life, especially males. These risks may be, in part, the result of increased liver-specific androgen signalling pathway activity in utero. Androgen signalling activity can be suppressed by androgen metabolism via cytochrome P450 (CYP) isoenzymes (CYP2B6, CYP3A) or through inhibition of the full-length androgen receptor (AR-FL) via the antagonistic isoform, AR-45. We hypothesised MO impairs CYP enzyme activity and AR-45 expression in male fetal livers, thereby enhancing activity of androgen signalling pathways. METHODS: Nine months prior to pregnancy, nulliparous female baboons were assigned to either ad libitum control or high fat diet. At 165 day (d) gestation (term, 180 d) fetal liver was collected (n = 6/sex/group). CYP activity was quantified using functional assays; subcellular AR expression was measured using Western blot. RESULTS: CYP2B6 and CYP3A activity, and nuclear expression of AR-45, was reduced in MO males only. Nuclear AR-45 expression was inversely related with fetal body weight of MO males only. CONCLUSIONS: Reduced CYP2B6 and CYP3A activity in conjunction with decreased nuclear AR-45 expression may enhance liver androgen signalling in males from MO pregnancies, thereby increasing the risk of macrosomia, as well as metabolic dysfunction in later life.
Assuntos
Androgênios , Obesidade Materna , Humanos , Feminino , Gravidez , Masculino , Androgênios/metabolismo , Obesidade Materna/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Macrossomia Fetal/metabolismo , Receptores Androgênicos/metabolismo , Fígado/metabolismo , IsoenzimasRESUMO
Methadone is cleared predominately by hepatic cytochrome P450 (CYP) 2B6-catalyzed metabolism to inactive metabolites. CYP2B6 also catalyzes the metabolism of several other drugs. Methadone and CYP2B6 are susceptible to pharmacokinetic drug-drug interactions. Use of natural products such as herbals and other botanicals is substantial and growing, and concomitant use of prescription medicines and non-prescription herbals is common and may result in interactions, often precipitated by CYP inhibition. Little is known about herbal product effects on CYP2B6 activity, and CYP2B6-catalyzed methadone metabolism. We screened a family of natural product compounds used in traditional medicines, herbal teas, and synthetic analogs of compounds found in plants, including kavalactones, flavokavains, chalcones and gambogic acid, for inhibition of expressed CYP2B6 activity and specifically inhibition of CYP2B6-mediated methadone metabolism. An initial screen evaluated inhibition of CYP2B6-catalyzed 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation. Hits were further evaluated for inhibition of racemic methadone metabolism, including mechanism of inhibition and kinetic constants. In order of decreasing potency, the most effective inhibitors of methadone metabolism were dihydromethysticin (competitive, K i 0.074 µM), gambogic acid (noncompetitive, K i 6 µM), and 2,2'-dihydroxychalcone (noncompetitive, K i 16 µM). Molecular modeling of CYP2B6-methadone and inhibitor binding showed substrate and inhibitor binding position and orientation and their interactions with CYP2B6 residues. These results show that CYP2B6 and CYP2B6-catalyzed methadone metabolism are inhibited by certain natural products, at concentrations which may be clinically relevant. SIGNIFICANCE STATEMENT: This investigation identified several natural product constituents which inhibit in vitro human recombinant CYP2B6 and CYP2B6-catalyzed N-demethylation of the opioid methadone. The most potent inhibitors (K i) were dihydromethysticin (0.074 µM), gambogic acid (6 µM) and 2,2'-dihydroxychalcone (16 µM). Molecular modeling of ligand interactions with CYP2B6 found that dihydromethysticin and 2,2'-dihydroxychalcone bound at the active site, while gambogic acid interacted with an allosteric site on the CYP2B6 surface. Natural product constituents may inhibit CYP2B6 and methadone metabolism at clinically relevant concentrations.
Assuntos
Produtos Biológicos , Chalconas , Metadona , Humanos , Metadona/farmacocinética , Citocromo P-450 CYP2B6/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Microssomos Hepáticos/metabolismoRESUMO
Lekethromycin (LKMS) is a synthetic macrolide compound derivative intended for use as a veterinary medicine. Since there have been no in vitro studies evaluating its potential for drug-drug interactions related to cytochrome P450 (CYP450) enzymes, the effect of the inhibitory mechanisms of LKMS on CYP450 enzymes is still unclear. Thus, this study aimed to evaluate the inhibitory effects of LKMS on dog CYP450 enzymes. A cocktail approach using ultra-performance liquid chromatography-tandem mass spectrometry was conducted to investigate the inhibitory effect of LKMS on canine CYP450 enzymes. Typical probe substrates of phenacetin, coumarin, bupropion, tolbutamide, dextromethorphan, chlorzoxazone, and testosterone were used for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4, respectively. This study showed that LKMS might not be a time-dependent inhibitor. LKMS inhibited CYP2A6, CYP2B6, and CYP2D6 via mixed inhibition. LKMS exhibited mixed-type inhibition against the activity of CYP2A6 with an inhibition constant (Ki) value of 135.6 µΜ. LKMS inhibited CYP2B6 in a mixed way, with Ki values of 59.44 µM. A phenotyping study based on an inhibition assay indicated that CYP2D6 contributes to the biotransformation of LKMS. A mixed inhibition of CYP2D6 with Ki values of 64.87 µM was also observed. Given that this study was performed in vitro, further in vivo studies should be conducted to identify the interaction between LKMS and canine CYP450 enzymes to provide data support for the clinical application of LKMS and the avoidance of adverse interactions between other drugs.
Assuntos
Citocromo P-450 CYP2D6 , Espectrometria de Massas em Tandem , Cães , Animais , Cromatografia Líquida , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2D6/farmacologia , Microssomos Hepáticos/metabolismo , Inibidores das Enzimas do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismoRESUMO
Cyclophosphamide, an oxazaphosphorine prodrug is frequently used in treatment of neuroblastoma, which is one of the most prevalent solid organ malignancies in infants and young children. Cytochrome P450 2B6 (CYP2B6) is the major catalyst and CYP2C19 is the minor enzyme in bioactivation and inactivation pathways of cyclophosphamide. CYP-mediated metabolism may contribute to the variable pharmacokinetics of cyclophosphamide and its toxic byproducts leading to insufficient response to the therapy and development of clinically significant side effects. The aim of the study was to reveal the contribution of pharmacogenetic variability in CYP2B6 and CYP2C19 to the treatment efficacy and cyclophosphamide-induced side effects in pediatric neuroblastoma patients under cyclophosphamide therapy (N = 50). Cyclophosphamide-induced hematologic toxicities were pivotal in all patients, whereas only moderate hepatorenal toxicity was developed. The patients' CYP2B6 metabolizer phenotypes were associated with the occurrence of lymphopenia, thrombocytopenia, and monocytopenia as well as of liver injury, but not with kidney or urinary bladder (hemorrhagic cystitis) toxicities. Furthermore, the patients' age (< 1.5 years, P = 0.03) and female gender (P ≤ 0.02), but not CYP2B6 or CYP2C19 metabolizer phenotypes appeared as significant prognostic factors in treatment outcomes. Our results may contribute to a better understanding of the impact of CYP2B6 variability on cyclophosphamide-induced side effects.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neuroblastoma , Humanos , Criança , Feminino , Pré-Escolar , Lactente , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/genética , Ciclofosfamida/efeitos adversos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/induzido quimicamenteRESUMO
Background and Objectives: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections present significant public health challenges worldwide. The management of these infections is complicated by the need for antiviral and antiretroviral therapies, which are influenced by drug metabolism mediated by metabolic enzymes and transporters. This study focuses on the gene expression of CYP2B6, CYP3A4, and ABCB1 transporters in patients with HIV, HCV, and HIV/HCV co-infection, aiming to assess their potential association with the choice of therapy, patohistological and clinical parameters of liver damage such as the stage of liver fibrosis, serum levels of ALT and AST, as well as the grade of liver inflammation and other available biochemical parameters. Materials and Methods: The study included 54 patients who underwent liver biopsy, divided into HIV-infected, HCV-infected, and co-infected groups. The mRNA levels of CYP2B6, CYP3A4, and ABCB1 was quantified and compared between the groups, along with the analysis of liver fibrosis and inflammation levels. Results: The results indicated a significant increase in CYP2B6 mRNA levels in co-infected patients, a significant association with the presence of HIV infection with an increase in CYP3A4 mRNA levels. A trend towards downregulation of ABCB1 expression was observed in patients using lamivudine. Conclusions: This study provides insight into gene expression of CYP2B6 CYP3A4, and ABCB1 in HIV, HCV, and HIV/HCV co-infected patients. The absence of correlation with liver damage, inflammation, and specific treatment interventions emphasises the need for additional research to elucidate the complex interplay between gene expression, viral co-infection, liver pathology, and therapeutic responses in these particular patients population.
Assuntos
Coinfecção , Infecções por HIV , Hepatite C , Humanos , Hepacivirus/genética , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/uso terapêutico , Hepatite C/tratamento farmacológico , Cirrose Hepática/complicações , Inflamação/complicaçõesRESUMO
Cytochrome P450 2B6 (CYP2B6) is responsible for the metabolism of â¼7% of marketed drugs. The in vitro drug interaction studies guidance for industry issued by the FDA stipulates that drug sponsors need to evaluate whether the investigated drugs interact with the major drug-metabolizing P450s including CYP2B6. Therefore, there has been greater attention to the development of predictive models for CYP2B6 inhibitors and substrates. In this study, conventional machine learning and deep learning models were developed to predict CYP2B6 inhibitors and substrates. Our results showed that the best CYP2B6 inhibitor model yielded the AUC values of 0.95 and 0.75 with the 10-fold cross-validation and the test set, respectively, and the best CYP2B6 substrate model produced the AUC values of 0.93 and 0.90 with the 10-fold cross-validation and the test set, respectively. The generalization ability of the CYP2B6 inhibitor and substrate models was assessed by using the external validation sets. Several significant substructural fragments relevant to CYP2B6 inhibitors and substrates were detected via frequency substructure analysis and information gain. In addition, the applicability domain of the models was defined by employing a nonparametric method based on the probability density distribution. We anticipate that our results would be useful for the prediction of potential CYP2B6 inhibitors and substrates in the early stage of drug discovery.
Assuntos
Inibidores do Citocromo P-450 CYP2B6 , Sistema Enzimático do Citocromo P-450 , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or ß-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five- to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, whereas the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, whereas the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.
Assuntos
Citocromo P-450 CYP3A , Rifampina , Humanos , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Rifampina/farmacologia , Rifampina/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte/metabolismo , RNA Mensageiro/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismoRESUMO
Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine potential drug-drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory effects of CBD and 7-OH-CBD were tested in microsomes from HEK293 cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3'-hydroxycotinine (3HC), were 0.27 ± 0.060, 0.23 ± 0.14, and 0.21 ± 0.14 µM, respectively, for CYP2A6; and 0.26 ± 0.17 and 0.029 ± 0.0050 µM for cotinine and nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ± 0.18, 0.16 ± 0.08, and 0.78 ± 0.23 µM for cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ± 0.44 and 0.11 ± 0.030 µM for cotinine and nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ± 0.06 µM) of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco addiction and cessation.
Assuntos
Canabidiol , Canabinoides , Nicotina , Humanos , Canabidiol/farmacologia , Canabinoides/metabolismo , Canabinoides/farmacologia , Cotinina/metabolismo , Citocromo P-450 CYP2A6/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Microssomos Hepáticos/metabolismo , Nicotina/farmacologia , Nicotina/metabolismoRESUMO
INTRODUCTION: Drug metabolism during pregnancy is a complex process that involves maternal, placental and fetal sites of metabolism. Indeed, there is a lack of clarity provided from drug metabolism in human pregnancy due to ethical limitations. Large animal models of human pregnancy provide an opportunity to quantify activity of phase 1 drug metabolism mediated by cytochrome P450 (CYP) enzymes in the maternal, placental, and fetal compartments. Herein, we have validated a comprehensive assay to quantify maternal, placental, and fetal CYP activity. METHODS: Isolated microsomes from sheep maternal liver, placenta, and fetal liver (140d gestation, term = 150d) were incubated with CYP-specific probe drugs to quantify the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A. Inhibition studies were performed to validate specificity of probe drugs. The validated assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1 and CYP3A were active in maternal liver. In contrast, only CYP1A2, CYP2C8 and CYP2D6 were active in the placenta, whereas CYP2B6, CYP2C8 and CYP2D6 were active in the fetal liver. Of the placental-specific CYPs validated, CYP1A2 increased in type A compared with type D placentomes, whereas CYP2C8 activity increased in type B compared with type A and C. DISCUSSION: This study has established conditions for compartment-specific CYP activity in the sheep maternal-placental-fetal unit using a validated and standardised experimental workflow. Compartment- and placentome type-specific CYP activity are important considerations when examining drug metabolism in the maternal-placental-fetal unit and in determining the impact of pregnancy complications.
Assuntos
Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2E1 , Animais , Feminino , Gravidez , Cromatografia Líquida , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Fígado , Microssomos Hepáticos/metabolismo , Placenta/metabolismo , Ovinos , Espectrometria de Massas em TandemRESUMO
Cytochrome P450 2B6 (CYP2B6) is a highly polymorphic human enzyme involved in the metabolism of many clinically relevant drugs, environmental toxins, and endogenous molecules with disparate structures. Over the last 20-plus years, in silico and in vitro studies of CYP2B6 using various ligands have provided foundational information regarding the substrate specificity and structure-function relationship of this enzyme. Approaches such as homology modeling, X-ray crystallography, molecular docking, and kinetic activity assays coupled with CYP2B6 mutagenesis have done much to characterize this originally neglected monooxygenase. However, a complete understanding of the structural details that make new chemical entities substrates of CYP2B6 is still lacking. Surprisingly little in vitro data has been obtained about the structure-function relationship of amino acids identified to be in the CYP2B6 active site. Since much attention has already been devoted to elucidating the function of CYP2B6 allelic variants, here we review the salient findings of in silico and in vitro studies of the CYP2B6 structure-function relationship with a deliberate focus on the active site. In addition to summarizing these complementary approaches to studying structure-function relationships, we note gaps/challenges in existing data such as the need for more CYP2B6 crystal structures, molecular docking results with various ligands, and data coupling CYP2B6 active site mutagenesis with kinetic parameter measurement under standard expression conditions. Harnessing in silico and in vitro techniques in tandem to understand the CYP2B6 structure-function relationship will likely offer further insights into CYP2B6-mediated metabolism. SIGNIFICANCE STATEMENT: The apparent importance of cytochrome P450 2B6 (CYP2B6) in the metabolism of various xenobiotics and endogenous molecules has grown since its discovery with many in silico and in vitro studies offering a partial description of its structure-function relationship. Determining the structure-function relationship of CYP2B6 is difficult but may be aided by thorough biochemical investigations of the CYP2B6 active site that provide a more complete pharmacological understanding of this important enzyme.
Assuntos
Simulação de Dinâmica Molecular , Oxirredutases N-Desmetilantes , Humanos , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Domínio Catalítico , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/metabolismoRESUMO
The primary mode of metabolism of nicotine is via the formation of cotinine by the enzyme CYP2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine, but can also form cotinine-N-glucuronide and cotinine-N-oxide (COX). The goal of this study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (P450) enzymes were used in cotinine-N-oxidation reactions using pooled human liver microsomes (HLMs). COX formation was monitored by ultrahigh-pressure liquid chromatography-tandem mass spectrometry and enzyme kinetic analysis was performed using microsomes from P450-overexpressing human embryonic kidney 293 (HEK293) cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLMs when using inhibitors of CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A4. Microsomes from cells overexpressing CYP2A6 or CYP2C19 exhibited similar N-oxidation activity against cotinine, with maximum reaction rate over Michaelis constant values (intrinsic clearance) of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations (P < 0.05) were observed between COX formation and genetic variants in CYP2C19 (*2 and *17 alleles) in HLMs. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies. SIGNIFICANCE STATEMENT: This study is the first to elucidate the enzymes responsible for cotinine-N-oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify nicotine metabolic ratio in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.
Assuntos
Cotinina , Nicotina , Humanos , Cotinina/metabolismo , Nicotina/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Cinética , Citocromo P-450 CYP2B6/metabolismo , Células HEK293 , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Microssomos Hepáticos/metabolismoRESUMO
Chiral polychlorinated biphenyls (PCBs) have atropisomers that have different axial chiralities and exist as racemic mixtures. However, biochemical processes often result in the unequal accumulation of these atropisomers in organisms. This phenomenon leads to enantiospecific toxicity enhancement or reduction because either of the atropisomers mainly affects toxicity expression. Enantioselective accumulation is caused by cytochrome P450 (CYP, P450) monooxygenases, especially the CYP2B subfamilies. Therefore, this study investigates the metabolism of a chiral PCB in vitro. Both atropisomers isolated from racemic 2,2',3,4,4',5',6-heptachlorobiphenyl (CB183) were metabolized by human CYP2B6, but not rat CYP2B1. This may be due to the difference in the size of the substrate-binding cavities of CYP2B6 and CYP2B1. The stable accommodation of (-)-CB183 in the cavity without any steric hindrance explained the preferential metabolism of (-)-CB183 compared to (+)-CB183. Two hydroxylated metabolites, 3'-OH-CB183 and 5-OH-CB183, were identified. The docking study showed that the 3'-position of the trichlorophenyl ring closely approaches the heme of CYP2B6. To our knowledge, this is the first study to elucidate the structural basis of chiral PCB metabolism by P450 isozymes. These results will help promote the precise toxicity evaluation of chiral PCBs and provide an explanation of the structural basis of chiral PCB metabolism.
Assuntos
Bifenilos Policlorados , Animais , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme , Humanos , Hidroxilação , Isoenzimas/metabolismo , Bifenilos Policlorados/química , Ratos , EstereoisomerismoRESUMO
N6-Methyladenosine (m6A) modification is the most prevalent RNA modification in mammals. We have recently demonstrated that inhibition of m6A modification by 3-deazaadenosine results in an increase in the expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, and CYP2C8 in human liver-derived cells. In the present study, we aimed to clarify the mechanism of m6A-mediated regulation of CYP2B6 expression. RNA immunoprecipitation using an anti-m6A antibody revealed that CYP2B6 mRNA in human liver and hepatocarcinoma-derived HepaRG cells was m6A-modified around the stop codon. In contrast to the treatment with 3-deazaadenosine, double knockdown of methyltransferase like (METTL) 3 and METTL14 (METTL3/14) resulted in a decrease in the levels of CYP2B6 mRNA in Huh-7 and HepaRG cells and a decrease in bupropion hydroxylase activity, a marker activity of CYP2B6, in HepaRG cells. The stability of CYP2B6 mRNA was not influenced by siMETTL3/14. Reporter assays using the plasmids containing the last exon or 5'-flanking region of CYP2B6 indicated that reporter activities were not influenced by knockdown of METTL3/14. The expression levels of the constitutive androstane receptor, pregnane X receptor, and retinoid X receptor, which are the nuclear receptors regulating the transcription of CYP2B6, were not influenced by siMETTL3/14. The chromatin immunoprecipitation and formaldehyde-assisted enrichment of regulatory elements assays revealed that H3K9me2, a repressive histone marker, was enriched in the vicinity of the upstream region of CYP2B6, and knockdown of METTL3/14 induced the condensation of the chromatin structure in this region. In conclusion, we demonstrated that METTL3/14 upregulated CYP2B6 expression by altering the chromatin status.
Assuntos
Cromatina , Citocromo P-450 CYP2B6 , Humanos , Adenosina/farmacologia , Adenosina/metabolismo , Bupropiona , Cromatina/genética , Códon de Terminação , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C8/genética , Formaldeído , Histonas/metabolismo , Metilação , Metiltransferases/genética , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
20(S)-Protopanaxadiol [20(S)-PPD] is a fully deglycosylated ginsenoside metabolite produced by the gut microbiota in the gastrointestinal tract. Although diverse pharmacological effects have been reported, information on the pharmacokinetic interactions of 20(S)-PPD with cytochrome P450s (CYPs) remains limited. Therefore, the inhibitory potential of 20(S)-PPD on CYP enzymes, which mainly contribute to drug pharmacokinetics, was investigated in this study. The inhibitory effect of 20(S)-PPD was strong for CYP3A4 and moderate for CYP2B6 in human liver microsomes. 20(S)-PPD inhibited Cyp3a and Cyp2b in mouse liver microsomes with a potency similar to that in humans. The solubility of 20(S)-PPD in the artificial intestinal fluid was close to IC50 values of Cyp3a and Cyp2b in the mouse intestine. Systemic exposure to buspirone (Cyp3a specific substrate) and bupropion (Cyp2b specific substrate) increased significantly, whereas the area under the plasma concentration-time curve (AUC) ratio of metabolite to parent drug decreased significantly when co-administered with 20(S)-PPD in mice. The pharmacokinetics of felodipine, a widely used anti-hypertensive agent metabolized mainly by Cyp3a, was also altered following 20(S)-PPD treatment in mice. In conclusion, 20(S)-PPD likely affects the in vivo metabolism of CYP3A4 or CYP2B6 substrates, suggesting a need for careful attention when concomitantly administering 20(S)-PPD with other medications. This study will broaden our understanding of ginseng and products containing precursor ginsenosides of 20(S)-PPD for safer and more efficient use in humans.