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 XenoenxertoRESUMO
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 , Desintoxicação Metabólica Fase I , Camundongos , Microssomos Hepáticos/enzimologia , Tamoxifeno/sangue , Tamoxifeno/metabolismo , Espectrometria de Massas em TandemRESUMO
This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.
Assuntos
Membrana Celular/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Microssomos Hepáticos/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Relatório de Pesquisa , Animais , Sistema Enzimático do Citocromo P-450/química , Retículo Endoplasmático/metabolismo , Humanos , Estrutura Secundária de ProteínaRESUMO
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 , VemurafenibRESUMO
The acetylenic tricyclic bis(cyanoenone) TBE-31 is a highly potent cysteine targeting compound with a reversible covalent mode of action; its best-characterized target being Kelch-like ECH-associated protein-1 (Keap1), the cellular sensor for oxidants and electrophiles. TBE-31 reacts with cysteines of Keap1, impairing its ability to target nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) for degradation. Consequently, Nrf2 accumulates and orchestrates cytoprotective gene expression. In this study we investigated the pharmacokinetic and pharmacodynamic properties of TBE-31 in C57BL/6 mice. After a single oral dose of 10 µmol/kg (â¼200 nmol/animal), the concentration of TBE-31 in blood exhibited two peaks, at 22.3 nM and at 15.5 nM, 40 min and 4 h after dosing, respectively, as determined by a quantitative stable isotope dilution LC-MS/MS method. The AUC0-24h was 195.5 h/nmol/l, the terminal elimination half-life was 10.2 h, and the kel was 0.068 h(-1). To assess the pharmacodynamics of Nrf2 activation by TBE-31, we determined the enzyme activity of its prototypic target, NAD(P)H: quinone oxidoreductase 1 (NQO1) and found it elevated by 2.4- and 1.5-fold in liver and heart, respectively. Continuous feeding for 18 days with diet delivering the same daily doses of TBE-31 under conditions of concurrent treatment with the immunosuppressive agent azathioprine had a similar effect on Nrf2 activation without any indications of toxicity. Together with previous reports showing the cytoprotective effects of TBE-31 in animal models of carcinogenesis, our results demonstrate the high potency, efficacy and suitability for chronic administration of cysteine targeting reversible covalent drugs.
Assuntos
Fator 2 Relacionado a NF-E2/agonistas , Fenantrenos/farmacocinética , Fenantrenos/toxicidade , Administração Oral , Animais , Relação Dose-Resposta a Droga , Feminino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fenantrenos/administração & dosagem , Taxa de Sobrevida , Distribuição TecidualRESUMO
Variability in drug pharmacokinetics is a major factor in defining drug efficacy and side effects. There remains an urgent need, particularly with the growing use of polypharmacy, to obtain more informative experimental data predicting clinical outcomes. Major species differences in multiplicity, substrate specificity, and regulation of enzymes from the cytochrome P450-dependent mono-oxygenase system play a critical role in drug metabolism. To develop an in vivo model for predicting human responses to drugs, we generated a mouse, where 31 P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene families were exchanged for their relevant human counterparts. The model has been improved through additional humanization for the nuclear receptors constitutive androgen receptor and pregnane X receptor that control the expression of key drug metabolizing enzymes and transporters. In this most complex humanized mouse model reported to date, the cytochromes P450 function as predicted and we illustrate how these mice can be applied to predict drug-drug interactions in humans.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Modelos Animais , Preparações Farmacêuticas/metabolismo , Transdução de Sinais/fisiologia , Animais , Citocromo P-450 CYP3A/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/administração & dosagem , Transdução de Sinais/efeitos dos fármacosRESUMO
The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased â¼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.
Assuntos
Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromos b5/metabolismo , Animais , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Citocromos b5/genética , Debrisoquina/farmacocinética , Etanolaminas/farmacocinética , Feminino , Humanos , Masculino , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Nifedipino/farmacocinética , Fatores Sexuais , Triazolam/farmacocinéticaRESUMO
The relative contribution of hepatic compared with intestinal oxidative metabolism is a crucial factor in drug oral bioavailability and therapeutic efficacy. Oxidative metabolism is mediated by the cytochrome P450 mono-oxygenase system to which cytochrome P450 reductase (POR) is the essential electron donor. In order to study the relative importance of these pathways in drug disposition, we have generated a novel mouse line where Cre recombinase is driven off the endogenous Cyp1a1 gene promoter; this line was then crossed on to a floxed POR mouse. A 40 mg/kg dose of the Cyp1a1 inducer 3-methylcholanthrene (3MC) eliminated POR expression in both liver and small intestine, whereas treatment at 4 mg/kg led to a more targeted deletion in the liver. Using this approach, we have studied the pharmacokinetics of three probe drugs--paroxetine, midazolam, nelfinavir--and show that intestinal metabolism is a determinant of oral bioavailability for the two latter compounds. The Endogenous Reductase Locus (ERL) mouse represents a significant advance on previous POR deletion models as it allows direct comparison of hepatic and intestinal effects on drug and xenobiotic clearance using lower doses of a single Cre inducing agent, and in addition minimizes any cytotoxic effects, which may compromise interpretation of the experimental data.
Assuntos
Integrases/fisiologia , Mucosa Intestinal/metabolismo , Microssomos Hepáticos/metabolismo , Midazolam/metabolismo , Nelfinavir/metabolismo , Paroxetina/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Feminino , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Midazolam/farmacocinética , Nelfinavir/farmacocinética , Paroxetina/farmacocinéticaRESUMO
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/metabolismoRESUMO
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 â¼80% 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 & dosagemRESUMO
The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolized by a range of cytochrome P450s, including five anticancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent; AUC increased (75-245%) and clearance decreased (35-72%) for phenacetin, metoprolol, and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics were measured for: cyclophosphamide [maximum plasma concentration (Cmax) and terminal half-life increased 55% and 40%, respectively], tamoxifen (AUClast and Cmax increased 370% and 233%, respectively), and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data provide strong evidence that both hepatic and extrahepatic Cyb5 levels are an important determinant of in vivo drug disposition catalyzed by a range of cytochrome P450s, including currently prescribed anticancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man.
Assuntos
Antineoplásicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Animais , Meia-Vida , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismoRESUMO
We previously described the development of genetic models to study the in vivo functions of the hepatic cytochrome P450 (P450) system, through the hepatic deletion of either cytochrome P450 oxidoreductase [POR; HRN (hepatic reductase null) line] or cytochrome b(5) [HBN (hepatic cytochrome b(5) null) line]. However, HRN mice still exhibit low levels of mono-oxygenase activity in spite of the absence of detectable reductase protein. To investigate whether this is because cytochrome b(5) and cytochrome b(5) reductase can act as the sole electron donor to the P450 system, we crossed HRN with HBN mice to generate a line lacking hepatic expression of both electron donors (HBRN). HBRN mice exhibited exacerbation of the phenotypic characteristics of the HRN line: liver enlargement, hepatosteatosis, and increased expression of certain P450s. Also, drug metabolizing activities in vitro were further reduced relative to the HRN model, in some cases to undetectable levels. Pharmacokinetic studies in vivo demonstrated that midazolam half-life, C(max), and area under the concentration-time curve were increased, and clearance was decreased, to a greater extent in the HBRN line than in either the HBN or HRN model. Microsomal incubations using NADPH concentrations below the apparent K(m) of cytochrome b(5) reductase, but well above that for POR, led to the virtual elimination of 7-benzyloxyquinoline turnover in HRN samples. These data provide strong evidence that cytochrome b(5)/cytochrome b(5) reductase can act as a sole electron donor to the P450 system in vitro and in vivo.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Fígado/enzimologia , Animais , Citocromo-B(5) Redutase/genética , Citocromos b5/genética , Elétrons , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/enzimologia , Midazolam/farmacocinéticaRESUMO
This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.
Assuntos
Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Preparações Farmacêuticas/metabolismo , Alelos , Animais , Citocromos b5/metabolismo , Camundongos , Camundongos Knockout , NADPH-Ferri-Hemoproteína Redutase/genética , Células Tumorais CultivadasRESUMO
NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2',5'-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k(cat)) of 105 s(-1) and 88 s(-1), respectively, for mosquito and human CPR. However, the inhibitory effects of 2',5'-ADP on activity were different; the IC(50) value of AgCPR for 2',5'-ADP was significantly higher (6-10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2',5'-ADP. This was confirmed by isothermal titration calorimetry where binding of 2',5'-ADP to AgCPR (K(d)â=â410±18 nM) was â¼10 fold weaker than human CPR (K(d)â=â38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K(d)â=â83±2.0 nM) than the equivalent human domain (K(d)â=â23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC(50)â=â28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of 'smart' insecticides and synergists that selectively target mosquito CPR.
Assuntos
Difosfato de Adenosina/metabolismo , Anopheles/enzimologia , Mononucleotídeo de Flavina/metabolismo , Proteínas de Insetos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calorimetria , Citocromos c/metabolismo , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Dados de Sequência Molecular , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Microsomal cytochrome b (5) is a ubiquitous, 15.2 kDa haemoprotein implicated in a number of cellular processes such as fatty acid desaturation, drug metabolism, steroid hormone biosynthesis and methaemoglobin reduction. As a consequence of these functions this protein has been considered essential for life. Most of the ascribed functions of cytochrome b (5), however, stem from in vitro studies and for this reason we have carried out a germline deletion of this enzyme. We have unexpectedly found that cytochrome b (5) null mice were viable and fertile, with pups being born at expected Mendelian ratios. However, a number of intriguing phenotypes were identified, including altered drug metabolism, methaemoglobinemia and disrupted steroid hormone homeostasis. In addition to these previously identified roles for this protein, cytochrome b (5) null mice displayed skin defects closely resembling those observed in autosomal recessive congenital ichthyosis and retardation of neonatal development, indicating that this protein, possibly as a consequence of its role in the de novo biosynthesis of unsaturated fatty acids, plays a central role in skin development and neonatal nutrition. Results from fatty acid profile analysis of several tissues suggest that cytochrome b (5) plays a role controlling saturated/unsaturated homeostasis. These data demonstrate that regional concentrations of unsaturated fatty acids are controlled by endogenous metabolic pathways and not by diet alone.
Assuntos
Citocromos b5/genética , Modelos Animais de Doenças , Ácidos Graxos Insaturados/metabolismo , Doenças Genéticas Inatas/genética , Ictiose/genética , Camundongos Knockout , Animais , Feminino , Doenças Genéticas Inatas/patologia , Homeostase , Humanos , Ictiose/patologia , Masculino , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Pele/patologiaRESUMO
We demonstrated recently that cytochrome b(5) plays an important in vivo role in hepatic cytochrome P450 (P450) function [J Biol Chem 283:31385-31393, 2008]. We have now generated a model in which cytochrome b(5) has been deleted in all tissues [cytochrome b(5) complete null (BCN)], which surprisingly results in a viable mouse despite the putative in vivo roles of this protein in lipid and steroid hormone metabolism and the reduction of methemoglobin. In contrast to the liver-specific deletion, complete deletion of cytochrome b(5) leads to a neonatal increase in the expression of many hepatic P450s at both the protein and mRNA level. In extrahepatic tissues, some changes in P450 expression were also observed that were isoform-dependent. In vitro cytochrome P450 activities in liver, kidney, lung, and small intestine of BCN mice were determined for a range of model substrates and probe drugs; a profound reduction in the metabolism of some substrates, particularly in lung, kidney, and small intestine, was observed. In vivo, the metabolism of metoprolol was significantly altered in BCN mice, in contrast to the previous finding in the liver-specific cytochrome b(5) deletion, suggesting that extrahepatic cytochrome b(5) plays a significant role in its disposition. Testicular Cyp17 hydroxylase and lyase activities were also significantly reduced by cytochrome b(5) deletion, leading to significantly lower levels of testicular testosterone. The BCN mouse provides an additional model system with which to further investigate the functions of cytochrome b(5), particularly in extrahepatic tissues.
Assuntos
Citocromos b5/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Sequência de Bases , Cromatografia Líquida , Primers do DNA , Masculino , Camundongos , Camundongos Knockout , Espectrometria de Massas em TandemRESUMO
In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/deficiência , Citocromos b5/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Citocromos b5/genética , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , NADP/metabolismo , Preparações Farmacêuticas/metabolismoRESUMO
The increasing number of transgenic or gene knockout mouse models generated for use in drug metabolism studies has meant that a greater understanding of the function and substrate specificities of murine cytochromes P450 (P450s) has become essential, particularly with the recent advances in "humanized" mouse models. In this study, we have heterologously expressed nine murine P450s--Cyp1a1, Cyp1a2, Cyp1b1, Cyp2a4, Cyp2b20, Cyp2c29, Cyp2d22, Cyp2e1, and Cyp3a11--individually with human P450 oxidoreductase to generate functional monooxygenase systems in Escherichia coli. We have identified a suitable fluorogenic probe for each P450 and determined the apparent kinetic parameters. These probes have enabled the screening of a panel of 31 test compounds classified as "drugs," "natural compounds," "endogenous compounds," and "pesticides" by measurement of IC(50), thus allowing the comparison of binding affinities. Human P450s CYP2C9, CYP2D6, and CYP3A4 were also included in the study to enable direct comparisons to be made with the mouse enzymes. Although there were general similarities between human and mouse P450s, perhaps the most significant finding in this study was the observation that, despite 77% amino acid identity, Cyp2d22 and CYP2D6 were remarkably dissimilar in a range of enzymatic properties, with potentially serious implications for pharmacokinetic studies using CYP2D substrates. The data presented in this study provide a solid foundation with which to assess the degree of similarity (or difference) between mouse and human P450s involved in xenobiotic metabolism and can be used as a basis for further studies.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar , Humanos , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.
Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP2D6/química , Quinidina/farmacologia , Alanina/química , Ácido Aspártico/química , Sítios de Ligação , Ligação Competitiva , Citocromo P-450 CYP2D6/genética , Sistema Enzimático do Citocromo P-450/química , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Ácido Glutâmico/química , Humanos , Cinética , Espectrometria de Massas , Oxigenases de Função Mista/química , Modelos Moleculares , Mutação , Oxirredutases O-Desmetilantes/química , Ligação Proteica , Estrutura Terciária de Proteína , Quinidina/química , Espectrofotometria , Especificidade por SubstratoRESUMO
Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1' hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on K(m) values are observed for dextromethorphan O-demethylation than for bufuralol 1' hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120-->Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.