RESUMO
Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.
Assuntos
Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Células Th17/efeitos dos fármacos , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Inflamação/metabolismo , Interleucina-17/metabolismo , Camundongos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Th17/metabolismoRESUMO
The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical development for oncology bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (â¼40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical development compound 46 (ABBV-744).
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Descoberta de Drogas/métodos , Piridinas/química , Piridinas/metabolismo , Pirróis/química , Pirróis/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Animais , Feminino , Células HeLa , Humanos , Camundongos , Camundongos SCID , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Piridinas/farmacologia , Pirróis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Novel conformationally constrained BET bromodomain inhibitors have been developed. These inhibitors were optimized in two similar, yet distinct chemical series, the 6-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (A) and the 1-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (B). Each series demonstrated excellent activity in binding and cellular assays, and lead compounds from each series demonstrated significant efficacy in in vivo tumor xenograft models.
Assuntos
Proteínas Nucleares/antagonistas & inibidores , Piridonas/química , Fatores de Transcrição/antagonistas & inibidores , Animais , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Camundongos , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Piridonas/farmacocinética , Piridonas/farmacologia , Piridonas/uso terapêutico , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Transplante HeterólogoRESUMO
The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.
Assuntos
Descoberta de Drogas , Proteínas/antagonistas & inibidores , Piridonas/farmacologia , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Transferência Ressonante de Energia de Fluorescência , Meia-Vida , Humanos , Espectrometria de Massas , Camundongos , Espectroscopia de Prótons por Ressonância Magnética , Piridonas/química , Piridonas/farmacocinética , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMO
Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.
Assuntos
Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Piridonas/química , Piridonas/farmacologia , Animais , Cristalografia por Raios X , Descoberta de Drogas , Compostos Macrocíclicos/farmacocinética , Estrutura Molecular , Piridonas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each.
Assuntos
Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida/normas , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Espectrometria de Massas/normas , Fenótipo , Proteínas Recombinantes/metabolismoRESUMO
A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Digoxina/farmacocinética , Medição de Risco , Animais , Transporte Biológico , Células CACO-2 , Cães , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , Células LLC-PK1 , Análise de Componente Principal , SuínosRESUMO
In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.
Assuntos
Digoxina/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Árvores de Decisões , Interações Medicamentosas , Humanos , Curva ROC , Estados Unidos , United States Food and Drug AdministrationRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba, which is one of the most frequently used herbal medicines, is commonly used in the management of several conditions, including memory impairment. Previously, it was reported to decrease the expression of peripheral benzodiazepine receptor and the biosynthesis of glucocorticoids, thereby regulating glucocorticoid levels. However, it is not known whether Ginkgo biloba extract regulates the function of the glucocorticoid receptor. AIM OF THE STUDY: We determined whether Ginkgo biloba extract and several of its chemical constituents affect the activity of human glucocorticoid receptor (hGR). MATERIALS AND METHODS: A hGR-dependent reporter gene assay was conducted in HepG2 human hepatocellular carcinoma cells and hGR target gene expression assays were performed in primary cultures of human hepatocytes. RESULTS: Multiple lots and concentrations of the extract and several of its chemical constituents (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide) did not increase hGR activity, as assessed by a cell-based luciferase reporter gene assay. The extract did not influence the expression of hGR target genes, including tyrosine aminotransferase (hTAT), constitutive androstane receptor (hCAR), or pregnane X receptor (hPXR), in primary cultures of human hepatocytes. Moreover, hGR antagonism by mifepristone (also known as RU486) did not attenuate the extent of induction of hCAR- and hPXR-regulated target genes CYP2B6 and CYP3A4 by Ginkgo biloba extract. CONCLUSION: Ginkgo biloba extract, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide are not activators of hGR. Furthermore, the extract does not influence the hGR-hCAR or the hGR-hPXR signaling pathway in primary cultures of human hepatocytes.
Assuntos
Ginkgo biloba , Hepatócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptores de Glucocorticoides/genética , Hidrocarboneto de Aril Hidroxilases/genética , Células Cultivadas , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Oxirredutases N-Desmetilantes/genética , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genéticaRESUMO
P-glycoprotein (P-gp) is the most widely studied drug transporter due to its potential role in drug disposition and efficacy, and drug-drug interactions (DDI). It is abundantly expressed in both the intestinal wall and blood-brain barrier where it serves as a drug permeability barrier while simultaneously facilitating drug elimination in the liver and kidney. It is also abundantly expressed in tumors where it can facilitate the elimination of chemotherapeutics, a phenomenon known as multidrug resistance (MDR). Clinically relevant DDIs involving P-gp are well documented; for example, the P-gp substrate, digoxin, exhibits toxicity when co-administered with a Pgp-inhibitor. This makes it essential to screen new chemical entities early in development for their potential to be a substrate and/or inhibitor of P-gp. Detailed in this unit is an in vitro protocol for assessing the P-gp substrate and inhibition potential of test compounds using the MDCK MDR1 and MDCK WT cell lines.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Interações Medicamentosas/fisiologia , Preparações Farmacêuticas/metabolismo , Transfecção/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Bioensaio/métodos , Técnicas de Cultura de Células , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Resistência a Múltiplos Medicamentos/fisiologia , Impedância Elétrica , Genes MDR , Humanos , Indicadores e ReagentesRESUMO
Time-dependent inhibition (TDI) of hepatic cytochrome P450 (CYP) enzymes is increasingly implicated in the majority of clinically relevant drug-drug interactions (DDIs). A time-dependent inhibitor or its reactive metabolite irreversibly inactivates CYP enzymes, thereby inhibiting the metabolism of other drugs. As the majority of marketed drugs are metabolized by CYP enzymes, their inhibition has important clinical consequences, such as in decreasing the metabolic clearance of a co-administered drug (victim drug). This could be life threatening, as such an effect narrows the therapeutic index for drugs such as warfarin and other potentially toxic agents. Therefore, it is essential to examine new chemical entities for their potential to cause TDI to minimize adverse drug reactions during human studies and use. This unit presents an in vitro procedure utilizing a nondilution method in human liver microsomes for determining the TDI potential of test compounds.
Assuntos
Antagonismo de Drogas , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Interações Medicamentosas , Ensaios Enzimáticos/métodos , Humanos , Técnicas de Diluição do Indicador , Indicadores e Reagentes , Concentração Inibidora 50 , Fatores de TempoRESUMO
Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6ß-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16α-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.
Assuntos
Ciclopentanos/farmacologia , Furanos/farmacologia , Ginkgolídeos/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Receptor Constitutivo de Androstano , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Receptor de Pregnano X , Ratos , Receptores Citoplasmáticos e Nucleares/agonistas , Especificidade da EspécieRESUMO
Conflicting data exist as to whether meclizine is an activator of human pregnane X receptor (hPXR). Therefore, we conducted a detailed, systematic investigation to determine whether meclizine affects hPXR activity by performing a cell-based reporter gene assay, a time-resolved fluorescence resonance energy transfer competitive ligand-binding assay, a mammalian two-hybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. In pregnane X receptor (PXR)-transfected HepG2 cells, meclizine activated hPXR to a greater extent than rat PXR. It bound to hPXR ligand-binding domain and recruited steroid receptor coactivator-1 to the receptor. Consistent with its hPXR agonism, meclizine increased hPXR target gene expression (CYP3A4) in human hepatocytes. However, it did not increase but decreased testosterone 6ß-hydroxylation, suggesting inhibition of CYP3A catalytic activity. Meclizine has also been reported to be an inverse agonist and antagonist of human constitutive androstane receptor (hCAR). Therefore, given that certain tissues (e.g., liver) express both hPXR and hCAR and that various genes are cross-regulated by them, we quantified the expression of a hCAR- and hPXR-regulated gene (CYP2B6) in cultured human hepatocytes treated with meclizine. This drug did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist-mediated increase in CYP2B6 mRNA and CYP2B6-catalyzed bupropion hydroxylation levels. These observations reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a hCAR reporter gene assay and mammalian two-hybrid assay. In conclusion, meclizine is a hPXR agonist, and it does not act as a hCAR inverse agonist or antagonist in cultured human hepatocytes.
Assuntos
Meclizina/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/agonistas , Receptores de Esteroides/metabolismo , Células Cultivadas , Receptor Constitutivo de Androstano , Relação Dose-Resposta a Droga , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Meclizina/farmacologia , Receptor de Pregnano X , Ligação Proteica/fisiologiaRESUMO
Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6ß-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.
Assuntos
Ginkgo biloba/química , Ginkgolídeos/farmacologia , Extratos Vegetais/farmacologia , Receptores de Esteroides/agonistas , Idoso , Animais , Sítios de Ligação/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes Reporter/genética , Ginkgolídeos/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lactonas/metabolismo , Lactonas/farmacologia , Masculino , Pessoa de Meia-Idade , Coativador 1 de Receptor Nuclear/genética , Coativador 1 de Receptor Nuclear/metabolismo , Extratos Vegetais/metabolismo , Receptor de Pregnano X , Carbonitrila de Pregnenolona/farmacologia , Ratos , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Rifampina/farmacologia , Esteroide Hidroxilases/metabolismo , Testosterona/metabolismo , TransfecçãoRESUMO
The aryl hydrocarbon receptor (AhR) signaling pathway regulates the production of CYP1B1 and CYP1A1, which catalyze the bioactivation of various procarcinogens. In the present study, we investigated the effect of Ginkgo biloba extract and some of its chemical constituents on CYP1B1 and CYP1A1 gene expression and AhR activity in cultured MCF-10A human mammary epithelial cells. Treatment of MCF-10A cells with noncytotoxic concentrations of G. biloba extract (25-300 microg/mL for 24 or 48 h) increased CYP1B1 and CYP1A1 mRNA expression, which was accompanied by an increase in CYP1-mediated ethoxyresorufin O-dealkylation activity. The inductive effects of G. biloba extract were attenuated by an AhR antagonist (3',4'-dimethoxyflavone). G. biloba extract (25-300 microg/mL) increased AhR-dependent reporter activity, as determined in MCF-10A cells transfected with an AhR-regulated luciferase reporter plasmid (pGudluc6.1). Bilobalide and ginkgolides A, B, C, and J were not responsible for the modulation of CYP1B1 and CYP1A1 gene expression or AhR activation by G. biloba extract. In contrast, quercetin increased CYP1B1 and CYP1A1 gene expression and activated AhR, whereas kaempferol and isorhamnetin suppressed constitutive CYP1B1 expression and antagonized AhR activation by benzo[a]pyrene. Overall, our findings provide an impetus for future investigations on the effect of G. biloba extract in CYP1-mediated chemical carcinogenesis.
Assuntos
Citocromo P-450 CYP1A1/genética , Sistema Enzimático do Citocromo P-450/genética , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Ginkgo biloba/química , Extratos Vegetais/farmacologia , Hidrocarboneto de Aril Hidroxilases , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP1B1 , DNA Complementar/genética , Células Epiteliais/enzimologia , Feminino , Genes Reporter , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/genética , Extratos Vegetais/isolamento & purificação , Plasmídeos , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
PURPOSE: We investigated a potential hepatoprotective role of d-cis diltiazem, l-cis diltiazem, thiamine and the combination d-cis diltiazem and thiamine against lipid peroxidation in a piglet liver microsomal model. A modified in vitro dichlorofluorescein assay was developed to assess the extent of peroxidative damage induced by reactive oxygen species in the piglet liver microsomal fraction. METHODS: Microsomal membrane fraction, obtained from 3 week old female piglets, was treated with either the biologically vasoactive d-cis diltiazem or the non-vasoactive stereoisomer l-cis diltiazem (5-1000 microM) for 1 hour at 37 degrees C followed by one hour incubation with the free radical generator AAPH (2,2'-azobis-(2-amidinopropane) dihydrochloride; 1 mM) to initiate lipid peroxidation. In a separate study, piglet liver microsomes were pre-treated with d-cis diltiazem (50 or 500 microM) and thiamine (10-100 microM) to assess the antioxidant activity of the combination. RESULTS: A dose dependant inhibition of membrane lipid peroxidation was observed with d-cis diltiazem (p<0.05) but not with l-cis diltiazem, suggesting that diltiazem is stereospecific in protecting against microsomal lipid peroxidation. Combining diltiazem with thiamine further protected microsomes against lipid peroxidation compared to use of individual drugs. CONCLUSION: We conclude that diltiazem and the combination of diltiazem and thiamine offers a hepatoprotective effect against free radicals.
Assuntos
Antioxidantes/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Diltiazem/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Tiamina/farmacologia , Animais , Antioxidantes/administração & dosagem , Bloqueadores dos Canais de Cálcio/administração & dosagem , Diltiazem/administração & dosagem , Diltiazem/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Fluoresceínas , Microssomos Hepáticos , Substâncias Protetoras/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Estereoisomerismo , Suínos , Tiamina/administração & dosagem , Tiamina/químicaRESUMO
Liver fatty acid binding protein has recently been shown to possess antioxidant properties but its role in liver disease, such as cholestasis, is not known. Since oxidative stress has been recognized as an important contributing factor in liver disease, we investigated the expression and antioxidative function of this protein using the bile-duct ligated model of cholestasis. Rats were divided into 3 groups: sham, bile-duct ligated and bile-duct ligated plus clofibrate. Animals were sacrificed at various time points after bile-duct ligation. RT-PCR and Western blot were used to analyze liver fatty acid binding protein expression. Cellular lipid peroxidation products were assessed by measuring thiobarbituric acid-reactive substances. Liver function was evaluated by measuring serum total bilirubin, alanine aminotransferase and ammonia. Liver fatty acid binding protein mRNA and protein levels were reduced to 51% and 20% of sham, respectively at 2 weeks following bile-duct ligation (p<0.05). The decreased liver fatty acid binding protein was associated with a statistical increase in hepatic lipid peroxidation products (224%) and decrease in hepatic function. Clofibrate treatment restored protein level and improved hepatic function. Clofibrate treatment also reduced hepatic lipid peroxidation products by 68% as compared with the bile-duct ligated group (p<0.05). Liver fatty acid binding protein likely has important antioxidant function during hepatocellular oxidative stress.
Assuntos
Colestase/fisiopatologia , Clofibrato/farmacologia , Proteínas de Ligação a Ácido Graxo/fisiologia , Hipolipemiantes/farmacologia , Estresse Oxidativo/fisiologia , Animais , Ductos Biliares , Western Blotting , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/metabolismo , Radicais Livres , Ligadura , Peroxidação de Lipídeos , Fígado/patologia , Testes de Função Hepática , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations > or =75 mug/ml and > or =750 mug/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 mug/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [(14)C]-leucine incorporation. At the level present in a modulating concentration (50 mug/ml) of the extract, ginkgolide A (0.55 mug/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.