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1.
Toxicol Lett ; 315: 9-13, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31408697

RESUMO

Cytochrome P450 mediated metabolism is the rate-limiting step of elimination for many drugs. CYP3A4 is the most abundant hepatic isoform and CYP3A4/5 metabolize the largest fraction of drugs. Pharmacogenetic studies have not been able to characterize population variability in CYP3A4 activity because few variant alleles associated with aberrant enzyme activity have been found. Substrate probes such as midazolam and testosterone have been utilized in-vivo and in-vitro to determine catalytic activity of these enzymes, but they suffer from several limitations. Eplerenone, an aldosterone antagonist, is also metabolized by CYP3A enzymes, and it has the potential to be an excellent substrate probe for CYP3A4/5. Eplerenone's primary metabolite, 6 beta-hydroxyeplerenone is formed preferentially via CYP3A4, however, the relative contribution of CYP3A5 to the 21-hydroxyeplerenone metabolite formation is unknown. Through in-vitro microsomal incubations with recombinant CYP3A4 and CYP3A5 enzymes, we identified their relative contributions to 21-hydroxyeplerenone metabolism. The 21-hydroxy metabolite is formed preferentially via CYP3A5 Vmax/KM (3.3) versus CYP3A4 Vmax/KM (1.9). Based on these findings, eplerenone has the potential to serve as an in-vivo substrate probe for CYP3A4 by monitoring 6-beta-hydroxy metabolite formation as well as CYP3A4/5 by monitoring 21-hydroxy metabolite formation.


Assuntos
Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Eplerenona/metabolismo , Eplerenona/farmacologia , Hipertensão/tratamento farmacológico , Anti-Hipertensivos/uso terapêutico , Humanos , Microssomos/metabolismo
2.
Exp Parasitol ; 200: 61-66, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30946841

RESUMO

Anthelmintic and in particular macrocyclic lactone (ML) resistance is a widespread problem in trichostrongyloid parasitic nematodes, yet mechanisms of ML resistance are still poorly understood. In the absence of target-site changes in resistant parasite field populations, increased drug extrusion and xenobiotic metabolism have been implicated in modification of susceptibility to MLs. In addition to P-glycoproteins, cytochrome P450 monooxygenases (CYPs) were considered to be involved in ML resistance. CYPs are highly divergent in nematodes with about 80 genes in the model organism Caenorhabditis elegans. Using larval development assays in the C. elegans model, piperonyl butoxide (PBO) and a temperature-sensitive variant of the emb-8 cytochrome reductase were used for chemical and genetic ablation of CYP activity. Additionally, a loss-of-function variant of cyp-14A5 was characterized to determine whether increased expression of this CYP in an ivermectin (IVM)-tolerant C. elegans line might be related to the phenotype. In a preliminary experiment with PBO, susceptibility to 5 nM IVM was synergistically increased by PBO. However, effects of genetic ablation of CYP activity on the EC50 values were small (1.5-fold decrease) for IVM and not significant for moxidectin (MOX). However, due to the steep concentration-response-curves, there were again strong differences between the wild-type and the CYP deficient genotype at individual IVM but not MOX concentrations. Although these results suggest small but significant effects on the susceptibility level of C. elegans to IVM, the cyp14A5 gene proposed by a previous study as candidate was ruled out since it was neither IVM/MOX inducible nor did a strain with a loss-of-function allele show increased susceptibility to either drug. In conclusion, the effect of the CYP system on IVM susceptibility in C. elegans is at best low while effects on MOX susceptibility were not detected. The previously suggested candidate cyp14A5 could be excluded to be involved in ML metabolism.


Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Lactonas/farmacologia , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/farmacologia , Relação Dose-Resposta a Droga , Ivermectina/farmacologia , Modelos Logísticos , Macrolídeos/farmacologia , Butóxido de Piperonila/farmacologia , Reação em Cadeia da Polimerase em Tempo Real
3.
Drug Metab Dispos ; 47(3): 257-268, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30567881

RESUMO

Cyclophosphamide (CP), a prodrug that is enzymatically converted to the cytotoxic 4-hydroxycyclophosphamide (4OHCP) by hepatic enzymes, is commonly used in both human and veterinary medicine to treat cancers and modulate the immune system. We investigated the metabolism of CP in humans, dogs, cats, and mice using liver microsomes; apparent K M, V max, and intrinsic clearance (V max/K M) parameters were estimated. The interspecies and intraspecies variations in kinetics were vast. Dog microsomes were, on average, 55-fold more efficient than human microsomes, 2.8-fold more efficient than cat microsomes, and 1.2-fold more efficient than mouse microsomes at catalyzing CP bioactivation. These differences translated to cell-based systems. Breast cancer cells exposed to 4OHCP via CP bioactivation by microsomes resulted in a stratification of cytotoxicity that was dependent on the species of microsomes measured by IC50: dog (31.65 µM), mouse (44.95 µM), cat (272.6 µM), and human (1857 µM). The contributions of cytochrome P450s, specifically, CYP2B, CYP2C, and CYP3A, to CP bioactivation were examined: CYP3A inhibition resulted in no change in 4OHCP formation; CYP2B inhibition slightly reduced 4OHCP in humans, cats, and mice; and CYP2C inhibition drastically reduced 4OHCP formation in each species. Semiphysiologic modeling of CP metabolism using scaled metabolic parameters resulted in simulated data that closely matched published pharmacokinetic profiles, determined by noncompartmental analysis. The results highlight differential CP metabolism delineated by species and demonstrate the importance of metabolism on CP clearance.


Assuntos
Antineoplásicos Alquilantes/farmacocinética , Ciclofosfamida/farmacocinética , Imunossupressores/farmacocinética , Modelos Biológicos , Pró-Fármacos/farmacocinética , Animais , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/uso terapêutico , Doenças do Gato/tratamento farmacológico , Doenças do Gato/imunologia , Gatos , Linhagem Celular Tumoral , Ciclofosfamida/metabolismo , Ciclofosfamida/uso terapêutico , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Cães , Feminino , Humanos , Imunossupressores/metabolismo , Imunossupressores/uso terapêutico , Masculino , Camundongos , Microssomos Hepáticos , Neoplasias/tratamento farmacológico , Oxirredução/efeitos dos fármacos , Pró-Fármacos/metabolismo , Pró-Fármacos/uso terapêutico
4.
Int J Nanomedicine ; 13: 8561-8575, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30587978

RESUMO

Introduction and objective: Currently, carbon nanostructures are vastly explored materials with potential for future employment in biomedicine. The possibility of employment of diamond nanoparticles (DN), graphene oxide (GO) or graphite nanoparticles (GN) for in vivo applications raises a question of their safety. Even though they do not induce a direct toxic effect, due to their unique properties, they can still interact with molecular pathways. The objective of this study was to assess if DN, GO and GN affect three isoforms of cytochrome P450 (CYP) enzymes, namely, CYP1A2, CYP2D6 and CYP3A4, expressed in the liver. Methods: Dose-dependent effect of the DN, GO and GN nanostructures on the catalytic activity of CYPs was examined using microsome-based model. Cytotoxicity of DN, GO and GN, as well as the influence of the nanostructures on mRNA expression of CYP genes and CYP-associated receptor genes were studied in vitro using HepG2 and HepaRG cell lines. Results: All three nanostructures interacted with the CYP enzymes and inhibited their catalytic activity in microsomal-based models. CYP gene expression at the mRNA level was also downregulated in HepG2 and HepaRG cell lines. Among the three nanostructures, GO showed the most significant influence on the enzymes, while DN was the most inert. Conclusion: Our findings revealed that DN, GO and GN might interfere with xenobiotic and drug metabolism in the liver by interactions with CYP isoenzymes responsible for the process. Such results should be considered if DN, GO and GN are used in medical applications.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/farmacologia , Diamante/química , Regulação para Baixo/genética , Grafite/química , Fígado/metabolismo , Nanoestruturas/química , Sobrevivência Celular , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Fluorescência , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrodinâmica , Isoenzimas/metabolismo , Nanoestruturas/ultraestrutura
5.
J Nanobiotechnology ; 16(1): 17, 2018 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-29463260

RESUMO

BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.


Assuntos
Antineoplásicos Hormonais/administração & dosagem , Bacteriófago P22/enzimologia , Neoplasias da Mama/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Tamoxifeno/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Bacteriófago P22/química , Biocatálise , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Terapia Enzimática , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Estrogênicos/metabolismo , Tamoxifeno/farmacologia
6.
Xenobiotica ; 48(11): 1164-1172, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29082813

RESUMO

1. Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects. 2. Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC50 data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole. 3. Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1'-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2. 4. The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Safrol/farmacocinética , Safrol/toxicidade , Cromatografia Líquida de Alta Pressão/métodos , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Glutationa/metabolismo , Humanos , Inativação Metabólica , Concentração Inibidora 50 , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Safrol/metabolismo , Espectrometria de Massas em Tandem
7.
Drug Metab Dispos ; 45(6): 676-685, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28283499

RESUMO

Ortho Tri-Cyclen, a two-drug cocktail comprised of ethinylestradiol and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450 (P450), recombinant uridine 5'-diphospho-glucuronosyltransferases, and human liver microsomes in the presence and absence of selective P450 inhibitors were conducted. It was found that CYP3A4 plays a key role in NGMN metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm value, the predicted plasma concentration versus time area under the curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of the reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm value of 1. The data presented in this paper will lead to better understanding and management of NGMN-based drug-drug interactions when norgestimate is coadministered with CYP3A4 modulators.


Assuntos
Anticoncepcionais Orais Sintéticos/farmacologia , Anticoncepcionais Orais Sintéticos/farmacocinética , Norgestrel/análogos & derivados , Acetilação , Cromatografia Líquida , Anticoncepcionais Orais Sintéticos/química , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Interações de Medicamentos , Humanos , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Norgestrel/química , Norgestrel/farmacocinética , Norgestrel/farmacologia , Oximas/química , Oximas/farmacocinética , Oximas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Espectrometria de Massas em Tandem
8.
Seishin Shinkeigaku Zasshi ; 117(1): 49-55, 2015.
Artigo em Japonês | MEDLINE | ID: mdl-26514046

RESUMO

Pharmacokinetics is the field dedicated to investigating the absorption, distribution, metabolism and excretion of drugs. Absorption of drugs is affected when they are taken together with a meal. Depending on the drug, the area under the concentration curve is affected by whether a medication is taken before or after a meal. Combined use of drugs with a high plasma protein binding fraction may be dangerous, since drug efficacy is impacted by efficiency, which in turn is affected by the degree to which it binds to proteins. Even more significant is the issue of "drug/drug" interactions that arise due to inhibition of the cytochrome P450 (CYP) hepatic microsomal enzyme system. Some antidepressants, such as paroxetine and fluvoxamine, are strong inhibitors of the CYP system. In the case of a medication that depends on renal clearance for elimination, caution is required when taking such a drug if it influences renal function. When a medicinal effect changes, pharmacodynamic changes must also be considered.


Assuntos
Antidepressivos/farmacologia , Sistema Enzimático do Citocromo P-450/farmacologia , Interações de Medicamentos , Farmacocinética , Psicotrópicos/farmacologia , Animais , Humanos , Inibidores de Captação de Serotonina/farmacologia
9.
Environ Sci Pollut Res Int ; 21(2): 862-71, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23828728

RESUMO

The human lipid regulator gemfibrozil (GEM) has been shown to induce peroxisome proliferation in rodents leading to hepatocarcinogenesis. Since GEM is found at biological active concentrations in the aquatic environment, the present study investigates the effects of this drug on the yellow European eel (Anguilla anguilla). Eels were injected with different concentrations of GEM (0.1 to 200 µg/g) and sampled 24- and 96-h post-injection. GEM was shown to inhibit CYP1A, CYP3A and CYP2K-like catalytic activities 24-h post-injection, but at 96-h post-injection, only CYP1A was significantly altered in fish injected with the highest GEM dose. On the contrary, GEM had little effect on the phase II enzymes examined (UDP-glucuronyltransferase and glutathione-S-transferase). Peroxisome proliferation inducible enzymes (liver peroxisomal acyl-CoA oxidase and catalase) were very weakly induced. No evidence of a significant effect on the endocrine system of eels was observed in terms of plasmatic steroid levels or testosterone esterification in the liver.


Assuntos
Anguilla/fisiologia , Enguias/fisiologia , Genfibrozila/toxicidade , Peroxissomos/metabolismo , Poluentes Químicos da Água/toxicidade , Anguilla/metabolismo , Animais , Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Glutationa Transferase/metabolismo , Humanos , Hipolipemiantes/toxicidade , Fígado/efeitos dos fármacos , Fígado/enzimologia , Oxirredução , Oxirredutases/metabolismo
10.
Proc Natl Acad Sci U S A ; 109(38): 15206-11, 2012 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-22949643

RESUMO

Worldwide, increasing numbers of insects have evolved resistance to a wide range of pesticides, which hampers their control in the field and, therefore, threatens agriculture. Members of the carboxylesterase and cytochrome P450 monooxygenase superfamilies are prominent candidates to confer metabolic resistance to pyrethroid insecticides. Both carboxylesterases and P450 enzymes have been shown to be involved in pyrethroid resistance in Australian Helicoverpa armigera, the noctuid species possessing by far the most reported resistance cases worldwide. However, specific enzymes responsible for pyrethroid resistance in field populations of this species have not yet been identified. Here, we show that the resistance toward fenvalerate in an Australian strain of H. armigera is due to a unique P450 enzyme, CYP337B3, which arose from unequal crossing-over between two parental P450 genes, resulting in a chimeric enzyme. CYP337B3 is capable of metabolizing fenvalerate into 4'-hydroxyfenvalerate, which exhibits no toxic effect on susceptible larvae; enzymes from the parental P450 genes showed no detectable fenvalerate metabolism. Furthermore, a polymorphic H. armigera strain could be bred into a susceptible line possessing the parental genes CYP337B1 and CYP337B2 and a resistant line possessing only CYP337B3. The exclusive presence of CYP337B3 in resistant insects of this strain confers a 42-fold resistance to fenvalerate. Thus, in addition to previously documented genetic mechanisms of resistance, recombination can also generate selectively advantageous variants, such as this chimeric P450 enzyme with an altered substrate specificity leading to a potent resistance mechanism.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/farmacologia , Resistência a Medicamentos , Lepidópteros/efeitos dos fármacos , Nitrilos/farmacologia , Piretrinas/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Inibidores Enzimáticos/farmacologia , Epitopos/química , Heme/química , Inseticidas/farmacologia , Lepidópteros/metabolismo , Conformação Molecular , Dados de Sequência Molecular , Controle de Pragas , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Temperatura Ambiente
11.
Langenbecks Arch Surg ; 396(8): 1245-53, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21887579

RESUMO

PURPOSE: Epoxyeicosatrienoic acids (EETs) are known to modulate proliferation and angiogenesis in vitro. Tissue levels of EETs are regulated by the cytochrome P450 (CYP) epoxygenases that generate them as well as by the soluble epoxide hydrolase metabolizes them to their less active diols. The aim of this study was to determine the effect of locally administered EETs (11,12- and 14,15-EETs) and the selective sEH inhibitor (sEHI) trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on wound healing in vivo. METHODS: Standardized full thickness dermal wounds were created on the dorsum of hairless mouse ears. Wound epithelialization was directly viewed and measured using intravitalmicroscopy and computerized planimetry every second day until healing was complete. Wound sections were analyzed by immunostaining for endothelial lineage marker CD31, vascular endothelial growth factor (VEGF), and angiogenic cytokine stromal cell-derived factor (SDF) 1α on days 2, 4, and 13. RESULTS: Treatment with EETs and t-AUCB, respectively, significantly accelerated wound epithelialization and neovascularization by synergistic upregulation of SDF1α and VEGF in vivo. CONCLUSIONS: These findings demonstrated that exogenous CYP-derived EETs and globally decreased EET hydrolysis by sEH inhibition significantly accelerated wound epithelialization and neovascularization in unimpaired healing wounds. Given that hypoxia induces CYP expression and subsequently EET-dependent angiogenesis, EETs and sEHIs provide a promising new class of therapeutics for ischemic non-healing wounds.


Assuntos
Sistema Enzimático do Citocromo P-450/farmacologia , Orelha Externa/lesões , Neovascularização Fisiológica/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Análise de Variância , Animais , Modelos Animais de Doenças , Orelha Externa/patologia , Epitélio/efeitos dos fármacos , Epitélio/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Pelados , Distribuição Aleatória , Valores de Referência , Sensibilidade e Especificidade , Cicatrização/fisiologia , Ferimentos e Lesões/patologia
12.
Rev. Rol enferm ; 34(7/8): 532-540, jul.-ago. 2011. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-89796

RESUMO

En general, el metabolismo de fármacos engloba reacciones de fase 1 o funcionalización (principalmente oxidativas) y reacciones de conjugación (biosintésis) de fase 2. Las reacciones de fase 1 inactivan la sustancia (fármaco) original mediante la introducción de un grupo funcional en la molécula. En las reacciones de fase 2 se crea un enlace covalente entre el grupo funcional con alguna de las siguientes moléculas: ácido glucurónico, sulfato, glutatión, aminoácidos o ácido acético. El proceso de conjugación genera compuestos con una polaridad incrementada y, además, los inactiva y pueden ser excretados más rápidamente a través de la orina o las heces. La transformación metabólica de un fármaco es de naturaleza enzimática y aunque cualquier tejido puede – inicialmente– poseer actividad metabólica, los sistemas participantes en este proceso se localizan principalmente en el hígado. Las enzimas de fase 1 se ubican en el retículo endoplásmico y las de fase 2 tienen localización principalmente citosólica en los hepatocitos. El sistema enzimático de monooxigenasas del citocromo P450 es la piedra angular y el más importante en las reacciones metabólicas. Este sistema se divide en familias y subfamilias de enzimas que se clasifican en relación con la similitud en las secuencias de los aminoácidos de las diferentes proteínas. En la actualidad, no hay duda de que las reacciones metabólicas catalizadas por las isoenzimas CYPs presentan importantes variaciones, sometidas a estudio dentro de la disciplina de la farmacogenética. Además, la respuesta farmacológica puede verse afectada por multitud de factores tales como las interacciones farmacológicas, dieta del paciente o consumo de tabaco entre otros. Dichos factores modulan la actividad de los distintos citocromos mediante procesos de inducción o inhibición metabólica, que pueden desencadenar reacciones adversas, graves en algunos casos(AU)


In general, drug biotransformation reactions are classified as either phase 1 functionalization (mainly oxidative) reactions or phase 2 conjugation (biosynthetic) reactions. Phase 1 reactions introduce or expose a functional group on the parent compound and generally result in the loss of pharmacological activity. Phase 2 reactions lead to the formation of a covalent linkage between a functional group on the parent compound with glucuronic acid, sultate, glutathione, amino acids, or acetate. These highly polar conjugates are generally inactive and are excreted rapidly in the urine and feces. The metabolic conversion of drugs is enzymatic in nature. The enzyme systems involved in the biotransformation of drugs are localized in the liver, although every tissue examined has some metabolic activity. The enzymes involved in phase 1 reactions are located primarily in the endoplasmic reticulum while the phase 2 conjugation enzymes are mainly cytosolic. The cytochrome P450 monooxigenase system is the cornerstone and the major catalyst of drug biotransformation reactions. The CYP superfamily is divided into families and subfamilies of enzymes that are defined on the basis of similarities in the amino acid sequence. Currently, no doubt exists that the CYP-mediated reactions typically show pronounced interindividual variability that is evaluated under the scope of the pharmacogenetic discipline. In addition, therapeutic outcome may be influenced by a number of factors such as concomitant drug administration, diet or smoking, among others. It may regulate drug metabolism through modulation (induction or inhibition) of the activities of CYPs and hence, it may provoke the occurrence of severe adverse drug reactions(AU)


Assuntos
Humanos , Masculino , Feminino , Preparações Farmacêuticas/síntese química , Preparações Farmacêuticas/metabolismo , Tratamento Farmacológico/enfermagem , Tratamento Farmacológico , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/farmacologia , Farmacogenética/métodos , Farmacogenética/educação , Farmacogenética/organização & administração
14.
J Cardiovasc Pharmacol ; 57(3): 294-301, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21266918

RESUMO

OBJECTIVE: 20-Hydroxyeicosatetraenoic acid (20-HETE), a [omega]-hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A, may play a role in the cardiovascular system. It is well known that cytochrome P450 [omega]-hydroxylase inhibitors markedly reduced the cardiac ischemia reperfusion injury. However, the direct effect of 20-HETE on cardiomyocytes is still poorly investigated. Here, we studied the effect of 20-HETE on cardiomyocyte apoptosis and the apoptosis-associated signaling pathways. METHODS AND RESULTS: The cardiomyocyte apoptosis was measured by fluorescein isothiocyanate conjugated annexin V/propidium iodide double staining cytometry, indicating that the percentage of early apoptotic cells increased from 15.6% +/- 2.6% to 25.5% +/- 2.5% in control and 20-HETE-treated cells, respectively. The mitochondrial membrane potential ([DELTA][PSI]m) was measured by detecting the ratio of JC-1 green/red emission intensity. A significant decrease in the ratio was observed after treatment with 20-HETE for 24 hours in comparison with control group, suggesting the disruptive effect of 20-HETE on mitochondrial [DELTA][PSI]m. In addition, 20-HETE stimulated caspase-3 activity and Bax mRNA expression in cardiomyocytes. In contrast, the Bcl-2 mRNA levels were significantly decreased by 20-HETE treatment. CONCLUSION: These results demonstrate that 20-HETE induces cardiomyocyte apoptosis by activation of several intrinsic apoptotic pathways. The 20-HETE-induced apoptosis could contribute to the cytochrome P450 [omega]-hydroxylase-dependent cardiac injure during cardiac ischemia-reperfusion.


Assuntos
Amidinas/farmacologia , Apoptose/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Ácidos Hidroxieicosatetraenoicos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Ácido Araquidônico/metabolismo , Benzimidazóis , Carbocianinas , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP4A/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Corantes Fluorescentes , Genes bcl-2/efeitos dos fármacos , Hidroxilação/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais , Coloração e Rotulagem , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/efeitos dos fármacos
15.
Med Hypotheses ; 76(3): 328-31, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21106301

RESUMO

Results of epidemiological studies suggest that, after one controls for the number of cigarettes smoked, women have a three times higher risk of getting lung cancer than men. Although the mechanism(s) explaining this gender-dependent difference in lung cancer risk is not known, it is thought that endocrine factors may play an important role. Normal human bronchial epithelial cells contain estrogen receptors and synthesize 17ß-estradiol (E(2)) and estrone (E(1)), which can undergo further metabolism into the catechol estrogens, 4-hydroxyestradiol (4-OHE(2)) and 4-hydroxyestrone (4-OHE(1)), respectively. Catechol estrogens are formed from E(2) by the actions of cytochrome p450 1B1 (CYP1B1). CYP1B1 is present in normal human bronchial epithelial) cells, and its activity is increased by cigarette smoking. Both 4-OHE(1) and 4-OHE(2) are mutagenic and carcinogenic and may exert their biological effects by inducing DNA adducts in cancer-related genes, including the tumor suppressor gene p53 and the proto-oncogene K-ras. Women with lung cancer have a different p53 mutational spectrum and a higher frequency of K-ras mutations than do men with lung cancer. Both clinical and basic research studies support the hypothesis that E(2) and cigarette smoking are cofactors in lung carcinogenesis in women. More specifically, cigarette smoke stimulates metabolism of E(2) into the genotoxic metabolites, 4-OHE(1) and 4-OHE(2,) which interact with DNA in cancer-related genes, including the tumor suppressor gene, p53, and the proto-oncogene K-ras, two genes frequently mutated in patients with lung cancer. E(2) may stimulate cellular proliferation and enhance tumor growth.


Assuntos
Estradiol/metabolismo , Estrogênios/metabolismo , Neoplasias Pulmonares/epidemiologia , Fumar/efeitos adversos , Adenocarcinoma/epidemiologia , Adenocarcinoma de Pulmão , Animais , Carcinógenos/farmacologia , Sistema Enzimático do Citocromo P-450/farmacologia , Adutos de DNA/farmacologia , Estradiol/farmacologia , Feminino , Terapia de Reposição Hormonal/efeitos adversos , Humanos , Masculino , Camundongos , Proto-Oncogenes/genética , Fatores de Risco , Fatores Sexuais
16.
Cancer Metastasis Rev ; 29(4): 723-35, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20941528

RESUMO

Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Neoplasias/metabolismo , Animais , Ácido Araquidônico/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Redes e Vias Metabólicas , Neoplasias/enzimologia
17.
J Clin Invest ; 120(9): 3127-36, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20739751

RESUMO

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.


Assuntos
Células-Tronco Pluripotentes Induzidas , Hepatopatias , Fígado/metabolismo , Adulto , Idoso , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Glicogênio/metabolismo , Glicogênio/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Biológicos
18.
Lab Invest ; 90(9): 1325-38, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20458282

RESUMO

Cholangiocarcinomas are devastating cancers that are resistant to chemotherapies. Resveratrol, a food-derived polyphenol with antitumorigenic properties, can regulate the expression of cytochrome p450 1b1 (Cyp1b1), which may confer chemoresistance in various cancers. Our aims were to assess the effects of resveratrol on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents and show an association between Cyp1b1 expression and chemosensitivity. Cholangiocarcinoma cell lines were treated with resveratrol before the addition of 5-fluorouracil (5-FU), gemcitabine, or mitomycin C. Cell proliferation and apoptosis were assessed by MTS assays and Annexin staining. Resveratrol effects on cholangiocarcinoma tumor sensitivity to 5-FU was assessed in an in vivo xenograft model using Mz-ChA-1 cells. After resveratrol treatment, Cyp1b1 expression was assessed by real-time PCR and immunoblotting. Stable-transfected cell lines with Cyp1b1 expression knocked down (Mz-Cyp1b1) were used to assess sensitivity to chemotherapeutic agents by MTS assays and Annexin staining and in a xenograft model using Mz-ChA-1 and Mz-Cyp1b1 cells, respectively. For each chemotherapeutic agent, co-treatment with resveratrol in vitro decreased cell proliferation and increased apoptosis to a greater extent than with the chemotherapeutic agent alone. In vivo, 5-FU+resveratrol decreased tumor size and increased TUNEL staining to a greater extent than 5-FU alone. In parallel, resveratrol decreased Cyp1b1 expression in Mz-ChA-1 cells and in cholangiocarcinoma tumors. Mz-Cyp1b1 cells were more sensitive to chemotherapeutic agents in vitro than mock-transfected cells, and Mz-Cyp1b1-induced tumors were more susceptible to 5-FU treatment. We suggest that resveratrol treatment may be a useful adjunct therapy to improve chemosensitivity in cholangiocarcinoma.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/patologia , Sistema Enzimático do Citocromo P-450/farmacologia , Desoxicitidina/análogos & derivados , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Camundongos , Camundongos Nus , Mitomicina/farmacologia , Mitomicina/uso terapêutico , Resveratrol , Estilbenos
19.
Am J Physiol Regul Integr Comp Physiol ; 299(1): R325-33, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20427718

RESUMO

High-salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% NaCl (HS) or 0.45% NaCl (NS) diet for 4-5 wks, concentration-response curves for ACh, 5'-N-ethylcarboxamidoadenosine (NECA; adenosine analog) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate [CGS-21680; A(2A) adenosine receptor (A(2A) AR) agonist] were obtained with N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide inhibitor, 10(-4) M), methylsulfonyl-propargyloxyphenylhexanamide [MS-PPOH; a CYP (cytochrome P-450) epoxygenase blocker, 10(-5) M including CYP2J2], 12-(3-adamantan-1-yl-ureido)dodecanoic acid [AUDA; soluble epoxide hydrolase (sEH) blocker, 10(-5) M], dibromo-dodecenyl-methylsulfimide [DDMS; CYP omega-hydroxylase (CYP4A blocker), 10(-5) M], glibenclamide (K(ATP) channel blocker; 10(-5) M) and 5-hydroxydecanoate (5-HD; mitochondrial-K(ATP) channel blocker, 10(-4) M). HS dose response to ACh (10(-7) - 10(-5) M) was not different from NS (P > 0.05). Relaxation to 10(-6) M NECA was greater in the HS group (28.4 +/- 3.9%) than in the NS group (4.1 +/- 2.3%). Relaxation to 10(-6) M CGS-21680 was also greater in HS (27.9 +/- 4.5%) than in NS (4.9 +/- 2.2%). L-NAME was able to block the dose response of ACh (10(-7) - 10(-5) M) equally in both HS and NS (P > 0.05), whereas L-NAME did not block CGS-21680-induced response in HS. In HS the CGS-21680 response was greatly reduced by MS-PPOH (to 4.7 +/- 2.0%) and 5-HD (to 8.9 +/- 2.2%), and also abolished by glibenclamide (-1.0 +/- 5.9%). In NS, the CGS-21680 response was increased by AUDA (to 26.3 +/- 3.4%) and DDMS (to 27.2 +/- 3.0%). Compared with NS, HS vessels showed increased CYP2J2 and A(2A) AR expression (46 and 74% higher, respectively) but decreased sEH, CYP4A, and A(1) AR expression (75, 30, and 55% lower, respectively). These data suggest that in mice fed NS-containing diet, upregulation of arterial A(1) receptor causes vasoconstriction via increased sEH and CYP4A proteins. However, in mice fed HS-containing diet, upregulation of A(2A) receptor protein triggers vascular relaxation through ATP-sensitive (K(+)) channels via upregulation of CYP2J2 enzyme.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Acetilcolina/farmacologia , Adamantano/análogos & derivados , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Amidas/farmacologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Citocromo P-450 CYP4A/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Ácidos Decanoicos , Epóxido Hidrolases/farmacologia , Hidroxiácidos , Ácidos Láuricos , Camundongos , Camundongos Endogâmicos C57BL , NG-Nitroarginina Metil Éster/farmacologia , Fenetilaminas , Cloreto de Sódio na Dieta/farmacologia , Vasoconstrição/efeitos dos fármacos
20.
Biochem Pharmacol ; 80(1): 122-8, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20206139

RESUMO

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency (V(max)/K(m)), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1'-hydroxylase activities (R(2)=0.98; P<0.0001) and CYP2D6 contents (R(2)=0.77; P=0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg-CYP2D6) mouse models showed that Tg-CYP2D6 mice receiving the same dose of 5-MeO-DMT (20mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg-CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.


Assuntos
Citocromo P-450 CYP2D6/metabolismo , Metoxidimetiltriptaminas/metabolismo , Metoxidimetiltriptaminas/farmacocinética , Inibidores da Monoaminoxidase/farmacologia , Psicotrópicos/metabolismo , Animais , Área Sob a Curva , Bufotenina/metabolismo , Citocromo P-450 CYP2D6/genética , Sistema Enzimático do Citocromo P-450/farmacologia , Relação Dose-Resposta a Droga , Genótipo , Meia-Vida , Harmalina/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Metoxidimetiltriptaminas/farmacologia , Metilação/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Monoaminoxidase/metabolismo , Pargilina/metabolismo , Fenótipo , Polimorfismo Genético/efeitos dos fármacos , Psicotrópicos/farmacologia
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