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1.
Proc Natl Acad Sci U S A ; 121(7): e2315069121, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38315851

RESUMO

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.


Assuntos
Doenças Transmissíveis , Descoberta de Drogas , Camundongos , Animais , Interações Medicamentosas , Modelos Animais de Doenças , Sistema Enzimático do Citocromo P-450/metabolismo , Aceleração
2.
Toxicology ; 443: 152563, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32805335

RESUMO

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake.


Assuntos
Inseticidas/farmacocinética , Linfa/metabolismo , Nitrilas/farmacocinética , Permetrina/farmacocinética , Veia Porta/metabolismo , Piretrinas/farmacocinética , Administração Oral , Animais , Transporte Biológico , Inseticidas/sangue , Masculino , Nitrilas/sangue , Permetrina/sangue , Piretrinas/sangue , Ratos Sprague-Dawley
3.
Xenobiotica ; 50(12): 1434-1442, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32672501

RESUMO

The metabolism of bifenthrin (BIF), ß-cyfluthrin (CYFL), λ-cyhalothrin (CYHA), cyphenothrin (CYPH) and esfenvalerate (ESF) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 90, 21 and 15 days and from adult humans. Pyrethroid metabolism was also studied with some human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. All five pyrethroids were metabolised by adult (90 day old) rat hepatic microsomal CYP and CES enzymes and by cytosolic CES enzymes. The pyrethroids were also metabolised by human liver microsomes and cytosol. Some species differences were observed. Pyrethroid metabolism by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. CYFL, CYHA, CYPH and ESF were metabolised by rat plasma CES enzymes, whereas none of the pyrethroids were metabolised by human plasma. This study demonstrates that the ability of male rats to metabolise these pyrethroids by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. All pyrethroids were metabolised by some of the human expressed CYP enzymes studied and apart from BIF were also metabolised by CES enzymes.


Assuntos
Carboxilesterase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Piretrinas/metabolismo , Animais , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Nitrilas/metabolismo , Ratos
4.
Xenobiotica ; 49(5): 521-527, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-29779438

RESUMO

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.


Assuntos
Carboxilesterase/química , Sistema Enzimático do Citocromo P-450/química , Nitrilas/química , Permetrina/química , Piretrinas/química , Carboxilesterase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Nitrilas/farmacocinética , Permetrina/farmacocinética , Piretrinas/farmacocinética , Estereoisomerismo
5.
Clin Cancer Res ; 24(9): 2138-2147, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29437786

RESUMO

Purpose: Osimertinib is a third-generation inhibitor of the epidermal growth factor receptor used in treatment of non-small cell lung cancer. A full understanding of its disposition and capacity for interaction with other medications will facilitate its effective use as a single agent and in combination therapy.Experimental Design: Recombinant cytochrome P450s and liver microsomal preparations were used to identify novel pathways of osimertinib metabolism in vitro A panel of knockout and mouse lines humanized for pathways of drug metabolism were used to establish the relevance of these pathways in vivoResults: Although some osimertinib metabolites were similar in mouse and human liver samples there were several significant differences, in particular a marked species difference in the P450s involved. The murine Cyp2d gene cluster played a predominant role in mouse, whereas CYP3A4 was the major human enzyme responsible for osimertinib metabolism. Induction of this enzyme in CYP3A4 humanized mice substantially decreased circulating osimertinib exposure. Importantly, we discovered a further novel pathway of osimertinib disposition involving CPY1A1. Modulation of CYP1A1/CYP1A2 levels markedly reduced parent drug concentrations, significantly altering metabolite pharmacokinetics (PK) in humanized mice in vivoConclusions: We demonstrate that a P450 enzyme expressed in smokers' lungs and lung tumors has the capacity to metabolise osimertinib. This could be a significant factor in defining the outcome of osimertinib treatment. This work also illustrates how P450-humanized mice can be used to identify and mitigate species differences in drug metabolism and thereby model the in vivo effect of critical metabolic pathways on anti-tumor response. Clin Cancer Res; 24(9); 2138-47. ©2018 AACR.


Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cromatografia Líquida , Família 2 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Metaboloma , Metabolômica/métodos , Camundongos , Camundongos Transgênicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Toxicol Sci ; 158(2): 367-378, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28541575

RESUMO

Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics.


Assuntos
Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Fígado/efeitos dos fármacos , RNA Longo não Codificante/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Xenobióticos/toxicidade , Animais , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio , Clordano/toxicidade , Receptor Constitutivo de Androstano , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Fenobarbital/toxicidade , Regulação para Cima/efeitos dos fármacos
8.
Drug Metab Dispos ; 45(1): 17-22, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27756789

RESUMO

Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. It is a prodrug that is converted by several cytochrome P450 enzymes to a primary metabolite, N-desmethyltamoxifen (NDT), which is then further modified by CYP2D6 to a pharmacologically potent secondary metabolite, 4-hydroxy-N-desmethyltamoxifen (endoxifen). Antidepressants (ADs), which are often coprescribed to patients receiving tamoxifen, are also metabolized by CYP2D6 and evidence suggests that a drug-drug interaction between these agents adversely affects the outcome of tamoxifen therapy by inhibiting endoxifen formation. We evaluated this potentially important drug-drug interaction in vivo in mice humanized for CYP2D6 (hCYP2D6). The rate of conversion of NDT to endoxifen by hCYP2D6 mouse liver microsomes (MLMs) in vitro was similar to that of the most active members of a panel of 13 individual human liver microsomes. Coincubation with quinidine, a CYP2D6 inhibitor, ablated endoxifen generation by hCYP2D6 MLMs. The NDT-hydroxylation activity of wild-type MLMs was 7.4 times higher than that of hCYP2D6, whereas MLMs from Cyp2d knockout animals were inactive. Hydroxylation of NDT correlated with that of bufuralol, a CYP2D6 probe substrate, in the human liver microsome panel. In vitro, ADs of the selective serotonin reuptake inhibitor class were, by an order of magnitude, more potent inhibitors of NDT hydroxylation by hCYP2D6 MLMs than were compounds of the tricyclic class. At a clinically relevant dose, paroxetine pretreatment inhibited the generation of endoxifen from NDT in hCYP2D6 mice in vivo. These data demonstrate the potential of ADs to affect endoxifen generation and, thereby, the outcome of tamoxifen therapy.


Assuntos
Antidepressivos/farmacologia , Antineoplásicos Hormonais/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Microssomos Hepáticos/metabolismo , Tamoxifeno/análogos & derivados , Animais , Antineoplásicos Hormonais/sangue , Biotransformação , Cromatografia Líquida , Citocromo P-450 CYP2D6/genética , Interações Medicamentosas , Feminino , Técnicas de Introdução de Genes , Humanos , Técnicas In Vitro , Desentoxicação Metabólica Fase I , Camundongos , Microssomos Hepáticos/enzimologia , Tamoxifeno/sangue , Tamoxifeno/metabolismo , Espectrometria de Massas em Tandem
9.
Br J Cancer ; 115(12): 1530-1539, 2016 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-27824809

RESUMO

BACKGROUND: Although the nuclear factor-erythroid 2-related factor 2 (NRF2) pathway is one of the most frequently dysregulated in cancer, it is not clear whether mutational status is a good predictor of NRF2 activity. Here we utilise four members of the aldo-keto reductase (AKR) superfamily as biomarkers to address this question. METHODS: Twenty-three cell lines of diverse origin and NRF2-pathway mutational status were used to determine the relationship between AKR expression and NRF2 activity. AKR expression was evaluated in lung cancer biopsies and Cancer Genome Atlas (TCGA) and Oncomine data sets. RESULTS: AKRs were expressed at a high basal level in cell lines carrying mutations in the NRF2 pathway. In non-mutant cell lines, co-ordinate induction of AKRs was consistently observed following activation of NRF2. Immunohistochemical analysis of lung tumour biopsies and interrogation of TCGA data revealed that AKRs are enriched in both squamous cell carcinomas (SCCs) and adenocarcinomas that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKRs were also enriched in most other tumours. CONCLUSIONS: An AKR biomarker panel can be used to determine NRF2 status in tumours. Hyperactivation of the NRF2 pathway is far more prevalent in lung SCC than previously predicted by genomic analyses.


Assuntos
Aldeído Redutase/metabolismo , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Aldo-Ceto Redutases , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia
10.
Cancer Res ; 75(21): 4573-81, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26363009

RESUMO

Vemurafenib is a revolutionary treatment for melanoma, but the magnitude of therapeutic response is highly variable, and the rapid acquisition of resistance is frequent. Here, we examine how vemurafenib disposition, particularly through cytochrome P450-mediated oxidation pathways, could potentially influence these outcomes using a panel of knockout and transgenic humanized mouse models. We identified CYP3A4 as the major enzyme involved in the metabolism of vemurafenib in in vitro assays with human liver microsomes. However, mice expressing human CYP3A4 did not process vemurafenib to a greater extent than CYP3A4-null animals, suggesting that other pregnane X receptor (PXR)-regulated pathways may contribute more significantly to vemurafenib metabolism in vivo. Activation of PXR, but not of the closely related constitutive androstane receptor, profoundly reduced circulating levels of vemurafenib in humanized mice. This effect was independent of CYP3A4 and was negated by cotreatment with the drug efflux transporter inhibitor elacridar. Finally, vemurafenib strongly induced PXR activity in vitro, but only weakly induced PXR in vivo. Taken together, our findings demonstrate that vemurafenib is unlikely to exhibit a clinically significant interaction with CYP3A4, but that modulation of bioavailability through PXR-mediated regulation of drug transporters (e.g., by other drugs) has the potential to markedly influence systemic exposure and thereby therapeutic outcomes.


Assuntos
Transporte Biológico/fisiologia , Citocromo P-450 CYP3A/metabolismo , Indóis/farmacocinética , Receptores de Esteroides/agonistas , Sulfonamidas/farmacocinética , Acridinas/farmacologia , Animais , Disponibilidade Biológica , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Citocromo P-450 CYP3A/genética , Feminino , Masculino , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Knockout , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Vemurafenib
11.
Mol Cell Proteomics ; 14(3): 750-60, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25561501

RESUMO

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.


Assuntos
Aminoácidos/metabolismo , Enzimas/isolamento & purificação , Marcação por Isótopo/métodos , Fígado/enzimologia , Modelos Biológicos , Animais , Técnicas de Cultura de Células , Cromatografia Líquida , Grupo dos Citocromos a/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH-Ferri-Hemoproteína Redutase/genética , Espectrometria de Massas em Tandem
12.
PLoS One ; 9(11): e114055, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25427220

RESUMO

The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Acroleína/administração & dosagem , Acroleína/farmacologia , Antineoplásicos/administração & dosagem , Elementos de Resposta Antioxidante/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxinas/administração & dosagem , Citotoxinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo
13.
Drug Metab Dispos ; 42(6): 1022-30, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24671958

RESUMO

In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Deleção de Genes , Intestino Delgado/enzimologia , Microssomos Hepáticos/enzimologia , Preparações Farmacêuticas/metabolismo , Animais , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem
14.
J Proteome Res ; 13(2): 866-74, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24303842

RESUMO

The modulation of drug metabolism enzyme (DME) expression by therapeutic agents is a central mechanism of drug-drug interaction and should be assessed as early as possible in preclinical drug development. Direct measurement of DME levels is typically achieved by Western blotting, qPCR, or microarray, but these techniques have their limitations; antibody cross-reactivity among highly homologous subfamilies creates ambiguity, while discordance between mRNA and protein expression undermines observations. The aim of this study was to design a simple targeted workflow by combining in vivo SILAC and label-free proteomics approaches for quantification of DMEs in mouse liver, facilitating a rapid and comprehensive evaluation of metabolic potential at the protein level. A total of 197 peptides, representing 51 Phase I and Phase II DMEs, were quantified by LC-MS/MS using targeted high resolution single ion monitoring (tHR/SIM) with a defined mass-to-charge and retention time window for each peptide. In a constitutive androstane receptor (Car) activated mouse model, comparison of tHR/SIM-in vivo SILAC with Western blotting for analysis of the expression of cytochromes P450 was favorable, with agreement in fold-change values between methods. The tHR/SIM-in vivo SILAC approach therefore permits the robust analysis of multiple DME in a single protein sample, with clear utility for the assessment of the drug-drug interaction potential of candidate therapeutic compounds.


Assuntos
Enzimas/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Western Blotting , Cromatografia Líquida , Receptor Constitutivo de Androstano , Camundongos , Espectrometria de Massas em Tandem
15.
Drug Metab Dispos ; 38(2): 341-6, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19920056

RESUMO

The aldo-keto reductase (AKR) phase I drug metabolism enzyme superfamily is implicated in detoxification or bioactivation of a wide variety of carbonyl-bearing compounds. In this study, we have used antibodies raised against purified recombinant rat AKR isoforms 1A3, 1B4, 1C9, 1D2, and 7A1 to characterize the expression profile of these superfamily members in the rat and define their localization by immunohistochemistry. Western blotting showed that AKR1A3, AKR1B4, and AKR1C9 are ubiquitously expressed, whereas AKR1D2 and AKR7A1 are present in liver, adrenal gland, and kidney, with the latter also present in testis, spleen, and stomach. Immunohistochemical analysis of the kidney demonstrated the localization of AKR1A3 in proximal convoluted tubules, AKR1B4 in the loop of Henle, and AKR1C9 in the pars recta S3 segment of proximal tubules. We also report localization of AKR1B4 in the adrenal gland (parenchymal cells of the zona reticularis) and testis (Sertoli cells and late spermatids), of AKR1D2 in the liver (hepatocyte nuclei), and of AKR7A1 in the pancreatic duct and bronchiolar epithelium. Previous studies have shown that expression of AKR7A1 is induced in response to dietary administration of the phenolic antioxidants butylated hydroxyanisole and ethoxyquin. Here we identify AKR1B13 and AKR1D2 as further inducible members of the rat AKR superfamily.


Assuntos
Antioxidantes/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oxirredutases/genética , Oxirredutases/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Animais , Hidroxianisol Butilado/farmacologia , Etoxiquina/farmacologia , Feminino , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Carcinogenesis ; 30(9): 1571-80, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19608619

RESUMO

To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated > or = 2.0-fold. mRNA for aldo-keto reductase (AKR) 1B10, AKR1C1, AKR1C2 and AKR1C3 were induced to the greatest extent, showing increases of between 12- and 16-fold, whereas mRNA for glutamate-cysteine ligase catalytic and modifier subunits, NAD(P)H:quinone oxidoreductase-1 and haem oxygenase-1 (HMOX1) were induced between 2.0- and 4.8-fold. Knockdown of BACH1 increased HMOX1 135-fold but induced the other genes examined to a maximum of only 2.7-fold. Activation of NRF2, by KEAP1 knockdown, caused a 75% increase in the amount of glutathione in HaCaT cells and a 1.4- to 1.6-fold increase in their resistance to the electrophiles acrolein, chlorambucil and cumene hydroperoxide (CuOOH), as well as the redox-cycling agent menadione. Inhibition of glutathione synthesis during KEAP1 knockdown, by treatment with buthionine sulfoximine, abrogated resistance to acrolein, chlorambucil and CuOOH, but not to menadione. In contrast, knockdown of BACH1 did not increase glutathione levels or resistance to xenobiotics. Knockdown of NRF2 in HaCaT cells decreased glutathione to approximately 80% of normal homeostatic levels and similarly reduced their tolerance of electrophiles. Thus, the KEAP1-NRF2 pathway determines resistance to electrophiles and redox-cycling compounds in human keratinocytes through glutathione-dependent and glutathione-independent mechanisms. This study also shows that AKR1B10, AKR1C1 and AKR1C2 proteins have potential utility as biomarkers for NRF2 activation in the human.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Citoproteção , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/fisiologia , Neoplasias/prevenção & controle , 20-Hidroxiesteroide Desidrogenases/genética , Células Cultivadas , Perfilação da Expressão Gênica , Glutationa/metabolismo , Heme Oxigenase-1/genética , Humanos , Hidroxiesteroide Desidrogenases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Queratinas/genética , Elementos de Resposta , Transdução de Sinais
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