RESUMO
BACKGROUND: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). METHODS: To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. RESULTS: The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. CONCLUSIONS: iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Citocromo P-450 CYP3A , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Puromicina/metabolismo , Puromicina/farmacologiaRESUMO
Preparations of plasma-derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4ß-hydroxycholesterol-to-cholesterol (4ßHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1-fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8-2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold-increase (90% CI)) of both liver (1.3 (1.1-1.5), P value = 0.014) and nonliver (1.9 (1.6-2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre-dose) 4ßHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil-induced changes in plasma 4ßHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α-ethinylestradiol, and abemaciclib) area under the plasma concentration-time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma-derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.
Assuntos
Indutores do Citocromo P-450 CYP3A/administração & dosagem , Citocromo P-450 CYP3A/biossíntese , Modafinila/administração & dosagem , Biomarcadores/sangue , Citocromo P-450 CYP3A/sangue , Citocromo P-450 CYP3A/genética , Indutores do Citocromo P-450 CYP3A/efeitos adversos , Esquema de Medicação , Interações Medicamentosas , Indução Enzimática , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/enzimologia , Voluntários Saudáveis , Humanos , Hidroxicolesteróis/sangue , Biópsia Líquida , Fígado/enzimologia , Modafinila/efeitos adversos , Modelos Biológicos , Plasma/enzimologia , Proteômica , Rifampina/administração & dosagem , Rifampina/efeitos adversos , Fatores de TempoRESUMO
Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Proteínas de Peixes/biossíntese , Fundulidae , Bifenilos Policlorados/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP3A/genética , Tolerância a Medicamentos , Indução Enzimática , Feminino , Proteínas de Peixes/genética , Fundulidae/genética , Fundulidae/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Receptor de Pregnano X/genética , RNA Mensageiro/metabolismoRESUMO
Variability in P-glycoprotein (P-gp) efflux transporting activity was supposed to be involved in altered intestinal absorption and bioavailability of clopidogrel in patients; however, reliable evidence is still lacking. In this study, we sought to determine whether P-gp could play an important role in the metabolic activation of and platelet response to clopidogrel in mice. Abcb1a/1b knock-out (KO) and wild-type (WT) mice were used to evaluate differences in the intracellular accumulation of clopidogrel in the intestine, liver, and brain tissues and in systemic exposure of clopidogrel and its main metabolites as well as the mechanisms involved. Results indicated that, compared with WT mice, KO mice exhibited an 84% increase in systemic exposure of clopidogrel active thiol metabolite H4 and a 14.5% rise of suppression of ADP-induced platelet integrin αIIbß3 activation, paralleled by a 41% decrease in systemic exposure of clopidogrel due to enhanced systemic clearance. Furthermore, KO mice displayed a 45% increase in Cyp3a11 but a 23% decrease in Ces1 at their protein levels compared with WT mice. Concurrently, intracellular clopidogrel concentrations in the tissues examined did not differ significantly between KO and WT mice. We conclude that although P-gp does not transport clopidogrel and its major metabolites in mice, P-gp-deficient mice exhibit elevated formation of the active metabolite H4 and enhanced antiplatelet effect of clopidogrel through up-regulation of Cyp3a11 and down-regulation of Ces1, suggesting that P-gp activity may correlate inversely with the formation of H4 and antiplatelet efficacy of clopidogrel in clinical settings due to P-gp and CYP3A4 interplay.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Plaquetas/metabolismo , Clopidogrel/farmacologia , Citocromo P-450 CYP3A/biossíntese , Proteínas de Membrana/biossíntese , Inibidores da Agregação Plaquetária/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Plaquetas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Cytochrome P450 (CYP) enzymes are involved in the metabolism of many important endogenous substrates (steroids, melatonin), drugs and toxic xenobiotics. Their induction accelerates drug metabolism and elimination. The present study aimed at examining the inducing abilities of two antipsychotic drugs levomepromazine and clozapine for the main CYPs. METHODS: The experiments were performed using cryopreserved human hepatocytes. The hepatotoxicity of levomepromazine and clozapine was assessed after exposure to the neuroleptics (LDH test). CYP activities were measured in the incubation medium using the CYP-specific reactions: caffeine 3-N-demethylation (CYP1A1/2), diclofenac 4'-hydroxylation (CYP2C9), perazine N-demethylation (CYP2C19) and testosterone 6ß-hydroxylation (CYP3A4). In parallel, CYP mRNA levels were measured in neuroleptic-treated hepatocytes. RESULTS: The results indicate that levomepromazine and clozapine induce the expression of main CYP enzyme CYP3A4 in human hepatocytes. Levomepromazine and clozapine at concentrations of 2.5 and 10 µM, respectively, caused a significant increase in the mRNA level and activity of CYP3A4. Both neuroleptics did not produce any changes in CYP1A1/2, CYP2C9 and CYP2C19. CONCLUSION: Levomepromazine and clozapine induce CYP3A4 in human hepatocytes in vitro. Further in vivo studies are advisable to confirm the CYP3A4 induction by levomepromazine and clozapine in the liver, and to assess the effect of these drugs on their own metabolism and on the biotransformation of other co-administered drugs which are the CYP3A4 substrates.
Assuntos
Antipsicóticos/farmacologia , Clozapina/farmacologia , Citocromo P-450 CYP3A/biossíntese , Indução Enzimática/efeitos dos fármacos , Metotrimeprazina/farmacologia , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado , RNA Mensageiro/biossínteseRESUMO
1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 µM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration-response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration-response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.
Assuntos
Hepatócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênese , Mutagênicos/toxicidade , Alcaloides de Pirrolizidina/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Relação Dose-Resposta a Droga , Indução Enzimática , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Masculino , Testes para Micronúcleos , Estrutura Molecular , Ratos Sprague-Dawley , Medição de Risco , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Relação Estrutura-AtividadeRESUMO
The human hepatic cytochrome P-450 3A4 (CYP3A4), recognized as a multifunctional enzyme, has a wide range of substrates including commonly used drugs. Previous investigations demonstrated that the expression of CYP3A4 in human hepatocytes could be regulated by some nuclear receptors (NRs) at transcriptional level under diverse situations. The significance of oxygen on CYP3A4-mediated metabolism seems notable while the regulatory mode of CYP3A4 in the particular case still remains elusive. Recently, striking evidence has emerged that both CYP3A4 and its regulator NR could be inhibited by exposure to hypoxia. Therefore, it is of great importance to elucidate whether and how these NRs act in the transcriptional regulation of CYP3A4 in human hepatocytes under hypoxic conditions. In this review, we mainly summarized transcriptional regulation of the pivotal enzyme CYP3A4 by NRs and explored the possible regulatory pathways of CYP3A4 via these major NRs under hypoxia, expecting to provide favorable evidence for further clinical guidance under such pathological situations.
Assuntos
Hipóxia Celular/genética , Citocromo P-450 CYP3A/genética , Hepatócitos/enzimologia , Receptores Citoplasmáticos e Nucleares/genética , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/metabolismo , Regulação Enzimológica da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Widely advocated for their health benefits worldwide, herbal medicines (HMs) have evolved into a billion dollar generating industry. Much is known regarding their wellness inducing properties, prophylactic and therapeutic benefits for the relief of both minor to chronic ailment conditions given their long-standing use among various cultures worldwide. On the other hand, their equally meaningful chemistry, pharmacokinetic profile in humans, interaction and toxicity profile have been poorly researched and documented. Consequently, this review is an attempt to highlight the health benefits, pharmacokinetics, interaction, and toxicity profile of five globally famous HMs. A systematic literature search was conducted by browsing major scientific databases such as Bentham Science, SciFinder, ScienceDirect, PubMed, Google Scholar and EBSCO to include 196 articles. In general, ginsenosides, glycyrrhizin and curcumin demonstrate low bioavailability when orally administered. Ginkgo biloba L. induces both CYP3A4 and CYP2C9 and alters the AUC and Cmax of conventional medications including midazolam, tolbutamide, lopinavir and nifedipine. Ginsenosides Re stimulates CYP2C9, decreasing the anticoagulant activity of warfarin. Camellia sinensis (L.) Kuntze increases the bioavailability of buspirone and is rich in vitamin K thereby inhibiting the activity of anticoagulant agents. Glycyrrhiza glabra L. displaces serum bound cardiovascular drugs such as diltiazem, nifedipine and verapamil. Herbal medicine can directly affect hepatocytes leading to hepatoxicity based on both intrinsic and extrinsic factors. The potentiation of the activity of concurrently administered conventional agents is potentially lethal especially if the drugs bear dangerous side effects and have a low therapeutic window.
Assuntos
Medicina Herbária , Compostos Fitoquímicos/farmacocinética , Compostos Fitoquímicos/toxicidade , Fitoterapia , Área Sob a Curva , Disponibilidade Biológica , Citocromo P-450 CYP2C9/biossíntese , Citocromo P-450 CYP3A/biossíntese , Indução Enzimática , História do Século XXI , HumanosRESUMO
Induction of cytochrome P450 enzyme 3A (CYP3A) in response to pregnane X receptor (PXR) activators shows species-specific differences. To study the induction of human CYP3A in response to human PXR activators, we generated a double-humanized mouse model of PXR and CYP3A. CYP3A-humanized mice generated by using a mouse artificial chromosome (MAC) vector containing the entire genomic human CYP3A locus (hCYP3A-MAC mouse line) were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR, resulting in double-humanized mice (hCYP3A-MAC/hPXR mouse line). Oral administration of the human PXR activator rifampicin increased hepatic expression of CYP3A4 mRNA and triazolam (TRZ) 1'- and 4-hydroxylation activities, CYP3A probe activities, in the liver and intestine microsomes of hCYP3A-MAC/hPXR mice. The plasma concentration of TRZ after oral dosing was significantly decreased by rifampicin treatment in hCYP3A-MAC/hPXR mice but not in hCYP3A-MAC mice. In addition, mass spectrometry imaging analysis showed that rifampicin treatment increased the formation of hydroxy TRZ in the intestine of hCYP3A-MAC/hPXR mice after oral dosing of TRZ. The plasma concentration of 1'- and 4-hydroxy TRZ in portal blood was also increased by rifampicin treatment in hCYP3A-MAC/hPXR mice. These results suggest that the hCYP3A-MAC/hPXR mouse line may be a useful model to predict human PXR-dependent induction of metabolism of CYP3A4 substrates in the liver and intestine. SIGNIFICANCE STATEMENT: We generated a double-humanized mouse line for CYP3A and PXR. Briefly, CYP3A-humanized mice generated by using a mouse artificial chromosome vector containing the entire genomic human CYP3A locus were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR. Expression of CYP3A4 and metabolism of triazolam, a typical CYP3A substrate, in the liver of CYP3A/PXR-humanized mice were enhanced in response to rifampicin, a typical human PXR activator. Enhancement of triazolam metabolism in the intestine of CYP3A/PXR-humanized mice was firstly shown by combination of mass spectrometry imaging of sliced intestine and liquid chromatography with tandem mass spectrometry analysis of metabolite concentration in portal blood after oral dosing of triazolam.
Assuntos
Indutores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/biossíntese , Intestino Delgado/metabolismo , Fígado/metabolismo , Veia Porta/metabolismo , Receptor de Pregnano X/biossíntese , Animais , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Humanos , Intestino Delgado/efeitos dos fármacos , Fígado/efeitos dos fármacos , Espectrometria de Massas/métodos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Veia Porta/efeitos dos fármacosRESUMO
XueShuanTong, a lyophilized extract of Panax notoginseng roots (Sanqi) for intravenous administration, is extensively used as add-on therapy in the treatment of ischemic heart and cerebrovascular diseases and comprises therapeutically active ginsenosides. Potential for XueShuanTong-drug interactions was determined; the investigation focused on cytochrome P450 (CYP)3A induction and organic anion-transporting polypeptide (OATP)1B inhibition. Ginsenosides considerably bioavailable for drug interactions were identified by dosing XueShuanTong in human subjects and their interaction-related pharmacokinetics were determined. The CYP3A induction potential was determined by repeatedly dosing XueShuanTong for 15 days in human subjects and by treating cryopreserved human hepatocytes with circulating ginsenosides; midazolam served as a probe substrate. Joint inhibition of OATP1B by XueShuanTong ginsenosides was assessed in vitro, and the data were processed using the Chou-Talalay method. Samples were analyzed by liquid chromatography/mass spectrometry. Ginsenosides Rb1, Rd, and Rg1 and notoginsenoside R1 were the major circulating XueShuanTong compounds; their interaction-related pharmacokinetics comprised compound dose-dependent levels of systemic exposure and, for ginsenosides Rb1 and Rd, long terminal half-lives (32â57 and 58â307 h, respectively) and low unbound fractions in plasma (0.8%â2.9% and 0.4%â3.0%, respectively). Dosing XueShuanTong did not induce CYP3A. Based on the pharmacokinetics and inhibitory potency of the ginsenosides, XueShuanTong was predicted to have high potential for OATP1B3-mediated drug interactions (attributed chiefly to ginsenoside Rb1) suggesting the need for further model-based determination of the interaction potential for XueShuanTong and, if necessary, a clinical drug interaction study. Increased awareness of ginsenosides' pharmacokinetics and XueShuanTong-drug interaction potential will help ensure the safe use of XueShuanTong and coadministered synthetic drugs.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Medicamentos de Ervas Chinesas/farmacocinética , Ginsenosídeos/farmacocinética , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Raízes de Plantas/química , Administração Intravenosa , Adulto , Cromatografia Líquida , Composição de Medicamentos , Interações Medicamentosas , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Feminino , Ginsenosídeos/administração & dosagem , Ginsenosídeos/química , Voluntários Saudáveis , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Masculino , Espectrometria de Massas , Conformação Molecular , Adulto JovemRESUMO
Adenosine deaminases acting on RNA (ADARs) enzymes-catalyzing adenosine-to-inosine RNA editing possibly modulates gene expression and function. In this study, we investigated whether ADARs regulate the expression of human constitutive androstane receptor (CAR), which controls the expression of various drug-metabolizing enzymes. CAR mRNA and protein levels in human hepatocellular carcinoma-derived HepG2 cells were increased by knockdown of ADAR1 and slightly increased by ADAR2, indicating that ADARs negatively regulate CAR expression. Increased luciferase activity of a reporter plasmid containing the CYP3A4 promoter region by phenobarbital was augmented by transfection of siRNA for ADAR1 (siADAR1) but not by siADAR2. In addition, the knockdown of ADAR1 resulted in the enhanced induction of CYP2B6 and CYP3A4 mRNA by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and phenobarbital, respectively. These results suggest that ADAR1-mediated downregulation of CAR affects its downstream cytochrome P450 expression. When the transcription was inhibited by α-amanitin, the degradation of CAR mRNA was attenuated by knockdown of ADAR1, suggesting that the increase in CAR mRNA level by ADAR1 knockdown is a post-transcriptional event. Finally, we found that ADAR1 knockdown promotes the splicing of CAR as a mechanism of the increased expression of CAR by ADAR1 knockdown. In conclusion, this study revealed that ADAR1 plays a role in modulating xenobiotic metabolism potency via regulation of CAR. SIGNIFICANCE STATEMENT: This study revealed that adenosine deaminase acting on RNA 1 (ADAR1) and ADAR2, which catalyze adenosine-to-inosine RNA editing, downregulate the expression of constitutive androstane receptor (CAR) in human liver-derived cells by attenuating splicing. The downregulation of CAR by ADARs affected its downstream cytochrome P450 expression. ADARs would play a role in modulating xenobiotic metabolism potency via regulation of CAR.
Assuntos
Adenosina Desaminase/metabolismo , Regulação para Baixo , Fígado/citologia , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Biocatálise , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6/biossíntese , Citocromo P-450 CYP3A/biossíntese , Indução Enzimática , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Estabilidade de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
Pyrrolizidine alkaloids (PAs) are a class of hepatic toxins widely existing in plants. Cytochromes P450 (CYP) mediates PA bioactivation and toxicities in mammals. It has been reported that PAs can induce developmental toxicity, but systematic research is lacking. In this study, we investigated developmental toxicity of monocrotaline (MCT) in rats. Pregnant rats were administered with MCT (20 mg/kg) intragastrically from gestation day 9 to 20, followed by determination of changes in fetal growth, hepatic morphology, serum biochemical indices, and indicators of hepatocytes apoptosis. MCT was found to induce developmental toxicity and fetal hepatotoxicity, particularly in female fetuses. Metabolic activation was also studied by examination of bioactivation efficiency of MCT in fetal liver microsomes, serum MCT, pyrrole-protein adduction derived from MCT, and hepatic CYP3 A expression of fetuses in vivo. Male fetuses showed greater basal MCT bioactivation than that of female fetuses, but continuous exposure to MCT caused a selective CYP3 A induction in female fetuses, which may contribute to the sex difference in MCT-induced developmental toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Monocrotalina/toxicidade , Ativação Metabólica , Animais , Doença Hepática Induzida por Substâncias e Drogas/embriologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocromo P-450 CYP3A/biossíntese , Indução Enzimática , Feminino , Idade Gestacional , Fígado/embriologia , Fígado/metabolismo , Masculino , Monocrotalina/metabolismo , Gravidez , Ratos Wistar , Medição de Risco , Fatores SexuaisRESUMO
CYP3A probe drugs such as midazolam and endogenous markers, and plasma 4ß-hydroxycholesterol (4ß-OHC) and urinary 6ß-hydroxycortisol-to-cortisol ratios (6ß-OHC/C) have been used as markers of CYP3A induction in cynomolgus monkeys, as with humans. However, there is limited information on their sensitivity and ability to detect CYP3A induction, as most studies were evaluated only at a high dose of the inducer, rifampicin (RIF; 20 mg/kg). In the present study, the CYP3A induction by RIF over a range doses of 0.2, 2 and 20 mg/kg (n = 4) was examined using CYP3A probe drugs (midazolam, triazolam and alprazolam) and the plasma and urinary endogenous CYP3A markers (4ß-OHC and 6ß-OHC/C). The sensitivity and relationship for detecting CYP3A induction was compared among the markers. Four days repeated oral administration of rifampicin to cynomolgus monkeys reduced the area under the plasma concentration-time curve of all CYP3A probe drugs in a rifampicin dose-dependent manner. Although the endogenous CYP3A markers (4ß-OHC and 6ß-OHC/C) were also changed for the middle (2 mg/kg) and high (20 mg/kg) doses of rifampicin, the fold-changes were relatively small, and CYP3A induction could not be detected at the lowest dose of rifampicin (0.2 mg/kg). In conclusion, CYP3A probe drugs are more sensitive for detecting CYP3A induction than endogenous CYP3A markers in cynomolgus monkeys, even for a short experimental period.
Assuntos
Alprazolam/farmacologia , Indutores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/biossíntese , Midazolam/farmacologia , Rifampina/farmacologia , Triazolam/farmacologia , Alprazolam/sangue , Animais , Área Sob a Curva , Biomarcadores/sangue , Biomarcadores/urina , Indutores do Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hidrocortisona/análogos & derivados , Hidrocortisona/urina , Hidroxicolesteróis/sangue , Macaca fascicularis , Masculino , Midazolam/sangue , Rifampina/sangue , Triazolam/sangueRESUMO
Alternative splicing modulates gene function by creating splice variants with alternate functions or non-coding RNA activity. Naturally occurring variants of nuclear receptor (NR) genes with dominant negative or gain-of-function phenotypes have been documented, but their cellular roles, regulation, and responsiveness to environmental stress or disease remain unevaluated. Informed by observations that class I androgen and estrogen receptor variants display ligand-independent signaling in human cancer tissues, we questioned whether the function of class II NRs, like the vitamin D receptor (VDR), would also respond to alternative splicing regulation. Artificial VDR constructs lacking exon 3 (Dex3-VDR), encoding part of the DNA binding domain (DBD), and exon 8 (Dex8-VDR), encoding part of the ligand binding domain (LBD), were transiently transfected into DU-145 cells and stably-integrated into Caco-2 cells to study their effect on gene expression and cell viability. Changes in VDR promoter signaling were monitored by the expression of target genes (e.g. CYP24A1, CYP3A4 and CYP3A5). Ligand-independent VDR signaling was observed in variants lacking exon 8, and a significant loss of gene suppressor function was documented for variants lacking exon 3. The gain-of-function behavior of the Dex8-VDR variant was recapitulated in vitro using antisense oligonucleotides (ASO) that induce the skipping of exon 8 in wild-type VDR. ASO targeting the splice acceptor site of exon 8 significantly stimulated ligand-independent VDR reporter activity and the induction of CYP24A1 above controls. These results demonstrate how alternative splicing can re-program NR gene function, highlighting novel mechanisms of toxicity and new opportunities for the use of splice-switching oligonucleotides (SSO) in precision medicine.
Assuntos
Processamento Alternativo , Neoplasias do Colo/genética , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Receptores de Calcitriol/genética , Células CACO-2 , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática , Éxons , Terapia Genética/métodos , Humanos , Ligantes , Masculino , Oligonucleotídeos Antissenso/farmacologia , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Receptores de Calcitriol/metabolismo , Vitamina D3 24-Hidroxilase/biossíntese , Vitamina D3 24-Hidroxilase/genéticaRESUMO
To date, many questions about the extent and cause of pharmacokinetic (PK) variability of even the most widely studied and prescribed ß1-adrenergic receptor blockers, such as metoprolol and bisoprolol, remain unanswered. Given that there are still no published population pharmacokinetic (PopPK) analyses of bisoprolol in routinely treated patients with acute coronary syndrome (ACS), the aim of this study was to determine its PK variability in 71 Serbian patients with ACS. PopPK analysis was conducted using a nonlinear mixed-effects model (NONMEM), version 7.3.0 (Icon Development Solutions). In each patient, the same formulation of bisoprolol was administered once or twice daily at a total daily dose of 0.625-7.5 mg. We separately assessed the effects of 31 covariates on the PKs of bisoprolol, and our results indicated that only 2 covariates could have possible influence on the variability of the clearance of bisoprolol: the mean daily dose of the drug and smoking habits of patients. These findings suggest that possible autoinduction of drug metabolism by higher total daily doses and induction of cytochrome P450 isoform 3A4 (CYP3A4) by cigarette smoke in liver could be the potential causes of increased total clearance of bisoprolol in patients with ACS.
Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Antagonistas de Receptores Adrenérgicos beta 1/farmacocinética , Bisoprolol/farmacocinética , Modelos Biológicos , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/diagnóstico , Antagonistas de Receptores Adrenérgicos beta 1/administração & dosagem , Antagonistas de Receptores Adrenérgicos beta 1/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Bisoprolol/administração & dosagem , Bisoprolol/sangue , Citocromo P-450 CYP3A/biossíntese , Indução Enzimática , Feminino , Humanos , Fígado/enzimologia , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Dinâmica não Linear , Sérvia , Fumantes , Fumar/efeitos adversos , Fumar/sangueRESUMO
Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We report here on a bacterial whole-cells high-throughput method for the activity evaluation of human CYP1A2, 2A6, and 3A4, when sustained by NADPH cytochrome P450 oxidoreductase (CPR), in the absence or presence of cytochrome b5 (CYB5). This new assay consists of a microplate real-time fluorometric method, with direct measurement of metabolite formation, in a suspension of Escherichia coli BTC-CYP bacteria, a human CYP competent tester strain when incubated with specific fluorogenic substrates. Overall, the maximum turnover (kcat) velocities of the three human CYPs resulting from the whole-BTC cells assays were similar to those obtained when applying the corresponding standard reference membrane fractions assays. CYP activity screening with co-expression of CYB5 suggests an enhancing effect of CYB5 on the kcat of specific isoforms, when using the whole-BTC cells assay. Our results demonstrate that this new approach can offer an efficient high-throughput method for screening of CYP1A2, 2A6 and 3A4 activity and can be potentially applicable for other human CYPs. This can be of particular use for timely and efficient screening of chemical libraries or mutant libraries of CYP enzyme complex proteins, without the necessity for labor intensive isolation of subcellular fractions.
Assuntos
Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP2A6/biossíntese , Citocromo P-450 CYP3A/biossíntese , Escherichia coli/enzimologia , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP3A/genética , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , HumanosRESUMO
Essential oils (EOs) are extensively used in food industry, gastronomy and alternative medicine. They are multicomponent mixtures of bioactive compounds; hence, their potential for food-drug interactions is substantial. In this study, we investigated the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of pregnane X receptor (PXR) and expression of cytochrome P450 3A4 (CYP3A4), using human intestinal and hepatic in vitro models. All tested EOs activated PXR in intestinal LS180 cells transiently transfected with PXR, as revealed by a reporter gene assay. Consistently, all EOs induced CYP3A4 mRNA expression in PXR-transfected LS180 cells, primary human hepatocytes and wild-type hepatic progenitor HepaRG cells. EO-mediated induction of CYP3A4 mRNA expression was nullified in PXR-knock out HepaRG cells, suggesting the involvement of PXR in these effects. Collectively, we showed that EOs of culinary herbs and spices might be common activators of PXR and inducers of CYP3A4 at doses present in foods, thereby, they might have a potential for food-drug interactions. Follow-up studies are warranted to identify the bioactive constituents in the tested EOs.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Óleos Voláteis/farmacologia , Receptores de Esteroides/metabolismo , Especiarias/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Linhagem Celular , Citocromo P-450 CYP2B6/biossíntese , Citocromo P-450 CYP2B6/genética , Indução Enzimática/efeitos dos fármacos , Genes Reporter , Hepatócitos/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Receptor de Pregnano X , Cultura Primária de Células , Receptores de Esteroides/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Receptor de Pregnano X/fisiologia , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Regulação Alostérica , Animais , Cristalografia por Raios X , Indução Enzimática/fisiologia , Humanos , Receptor de Pregnano X/química , RatosAssuntos
Doenças dos Gânglios da Base/etiologia , Carbamazepina/uso terapêutico , Citocromo P-450 CYP3A/biossíntese , Risperidona/uso terapêutico , Adulto , Doenças dos Gânglios da Base/prevenção & controle , Preparações de Ação Retardada , Indução Enzimática/efeitos dos fármacos , Humanos , Masculino , Transtornos Psicóticos/tratamento farmacológico , Risperidona/administração & dosagem , Risperidona/metabolismoRESUMO
Rifampin demonstrated dose-dependent relative induction between cytochrome P (CYP)3A and P-glycoprotein (P-gp), organic anion transporting polypeptides (OATPs), or CYP2C9; P-gp, OATP, and CYP2C9 induction was one drug-drug interaction (DDI) category lower than that observed for CYP3A across a wide range of pregnane X receptor (PXR) agonism. The objective of this study was to determine if these relationships could be utilized to predict transporter induction by other CYP3A inducers (rifabutin and carbamazepine) and of another P-gp substrate, sofosbuvir. Healthy subjects received sofosbuvir and a six-probe drug cassette before and after 300 mg q.d. rifabutin or 300 mg b.i.d. carbamazepine. Induction of P-gp, CYP2C9, and decreased sofosbuvir exposure were successfully predicted by observed CYP3A induction. Carbamazepine induction of OATP was underpredicted, likely due to reported additional non-PXR agonism. The results demonstrate that the effect of a PXR agonist on CYP3A can be leveraged to inform on induction liability for other primarily PXR-regulated P450s/transporters, allowing for prioritization of targeted DDI assessments during new drug development.