RESUMEN
Genetic variation in the gene encoding CYP2D6 is used to guide drug prescribing in clinical practice. However, genetic variants in CYP2D6 show substrate-specific effects that are currently not accounted for. With a systematic literature, we retrieved 22 original studies describing in vitro experiments focusing on CYP2D6 alleles (CYP2D6*1, *2, *10 and *17) and substrates. Allele activity (clearance of the allele of interest divided by the clearance of the wildtype) was extracted. The results support the hypothesis of the existence of substrate specificity of the CYP2D6*17-allele (higher debrisoquine clearance), a subtle effect of the CYP2D6*10-allele (lower dextromethorphan clearance) but no substrate-specific effect of the CYP2D6*2-allele. Although our results support substrate specificity, for most substrates data are too sparse and require further studies.
Asunto(s)
Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Alelos , Debrisoquina/farmacocinética , Variación Genética , Humanos , Isoenzimas/genética , Especificidad por SustratoRESUMEN
BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine. METHODS: We have expressed CYP2U1 in E. coli and performed UV-vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS. RESULTS: The UV-vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate FeIII state. Imidazole (Im) derivatives, such as miconazole, acted as FeIII ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to "reverse type I" difference UV-vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model. MAJOR CONCLUSION AND GENERAL SIGNIFICANCE: Our study describes for the first time the mode of interaction of several FeIII-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP.
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Familia 2 del Citocromo P450/química , Familia 2 del Citocromo P450/metabolismo , Modelos Moleculares , Ácido Araquidónico/química , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Biocatálisis , Debrisoquina/química , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Humanos , Imidazoles/química , Ácidos Láuricos/química , Ligandos , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Unión Proteica , Piridinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
OBJECTIVE To characterize polymorphisms of the gene for cytochrome P450 isozyme 2D50 (CYP2D50) and the disposition of 2 CYP2D50 probe drugs, dextromethorphan and debrisoquine, in horses. ANIMALS 23 healthy horses (22 Thoroughbreds and 1 Standardbred). PROCEDURES Single-nucleotide polymorphisms (SNPs) in CYP2D50 were identified. Disposition of dextromethorphan (2 mg/kg) and debrisoquine (0.2 mg/kg) were determined after oral (dextromethorphan) or nasogastric (debrisoquine) administration to the horses. Metabolic ratios of plasma dextromethorphan and total dextrorphan (dextrorphan plus dextrorphan-O-ß-glucuronide) and 4-hydroxydebrisoquine concentrations were calculated on the basis of the area under the plasma concentration-versus-time curve extrapolated to infinity for the parent drug divided by that for the corresponding metabolite. Pharmacokinetic data were used to categorize horses into the phenotypic drug-metabolism categories poor, extensive, and ultrarapid. Disposition patterns were compared among categories, and relationships between SNPs and metabolism categories were explored. RESULTS Gene sequencing identified 51 SNPs, including 27 nonsynonymous SNPs. Debrisoquine was minimally detected after oral administration. Disposition of dextromethorphan varied markedly among horses. Metabolic ratios for dextromethorphan ranged from 0.03 to 0.46 (mean, 0.12). On the basis of these data, 1 horse was characterized as a poor metabolizer, 18 were characterized as extensive metabolizers, and 3 were characterized as ultrarapid metabolizers. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that CYP2D50 is polymorphic and that the disposition of the probe drug varies markedly in horses. The polymorphisms may be related to rates of drug metabolism. Additional research involving more horses of various breeds is needed to fully explore the functional implication of polymorphisms in CYP2D50.
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Sistema Enzimático del Citocromo P-450/genética , Debrisoquina/metabolismo , Dextrometorfano/metabolismo , Caballos/genética , Polimorfismo de Nucleótido Simple , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Debrisoquina/análogos & derivados , Femenino , Caballos/metabolismo , Isoenzimas/genética , MasculinoRESUMEN
AIM: This study was aimed to functionally characterize four novel CYP2D6 alleles identified in Chinese Han population. MATERIALS & METHODS: CYP2D6 proteins of wild-type and the four novel variants along with CYP2D6.2 and CYP2D6.10 were heterologously expressed in yeast cells and the kinetic parameters were determined. RESULTS: Compared with CYP2D6.1 (frequency in Chinese 24.65%), CYP2D6.X (1.63%), CYP2D6.Y (1.50%), CYP2D6.Z (0.81%), CYP2D6.10 (52.53%) and CYP2D6.75 (0.13%) exhibited low activity at different degrees, whereas the kinetic parameters of CYP2D6.2 (11.06%) were much the same with CYP2D6.1. The novel allele CYP2D6.75 showed decreased enzyme activity. CONCLUSION: This is the first study to conduct functional analysis of CYP2D6 four novel alleles in Chinese Han population, which might be helpful for optimizing pharmacotherapy and the design of personalized medicine.
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Pueblo Asiatico , Citocromo P-450 CYP2D6/genética , Alelos , Citocromo P-450 CYP2D6/química , Debrisoquina/análogos & derivados , Debrisoquina/química , Variación Genética , Humanos , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genéticaRESUMEN
We tested the influence of four polymorphisms and gene duplication in CYP2D6 on in vivo enzyme activity in a Chilean mestizo population in order to identify the most relevant genetic profiles that account for observed phenotypes in this ethnic group. CYP2D6*2 (2850C>T), *3 (2549A>del), *4 (1846G>A), *17 (1023C>T) and gene duplication were determined by PCR-RFLP or PCRL in a group of 321 healthy volunteers. Individuals with different variant alleles were phenotyped by determining debrisoquine 4-hydroxylase activity as a metabolic ratio (MR) using a validated HPLC assay. Minor allele frequencies were 0.41, 0.01, 0.12 and 0.00 for CYP2D6*2, *3, *4 and *17 variants, respectively, and the duplication frequency was 0.003. Genotype analysis correlated with phenotypes in 18 of 23 subjects (78.3%). 11 subjects were extensive metabolizers (EM), 8 were intermediate metabolizers (IM), 2 were poor metabolizers (PM) and 2 were ultra-rapid metabolizers (UM) which is fairly coincident with expected phenotypes metabolic ratios ranged from 0.11 to 126.41. The influence of CYP2D6*3 was particularly notable, although only heterozygote carriers were present in our population. Individuals homozygous for *4 were always PM. As expected, the only subject with gene duplication was UM. In conclusion, there was a clear effect of genotype on observed CYP2D6 activity. Classification of EM, PM and UM through genotyping was useful to characterize CYP2D6 phenotype in the Chilean mestizo population.
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Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Adolescente , Adulto , Chile , Debrisoquina/metabolismo , Femenino , Duplicación de Gen , Frecuencia de los Genes , Estudios de Asociación Genética , Humanos , Indígenas Sudamericanos/genética , Cinética , Masculino , Polimorfismo Genético , Población Blanca/genética , Adulto JovenRESUMEN
BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin. METHODS: We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions. RESULTS: We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site. MAJOR CONCLUSION: Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model. GENERAL SIGNIFICANCE: These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.
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Sistema Enzimático del Citocromo P-450/química , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Western Blotting , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Simulación por Computador , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , Debrisoquina/química , Debrisoquina/metabolismo , Cinética , Espectrometría de Masas , Estructura Molecular , Oxidación-Reducción , Unión Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
INTRODUCTION: Chimeric mice with humanized livers were recently established by transplanting human hepatocytes. This mouse model that is repopulated with functional human hepatocytes could be a useful tool for investigating human hepatic cell biology and drug metabolism and for other preclinical applications. Successfully transplanting human hepatocytes into mice requires that recipient mice with liver failure do not reject these human cells and provide a suitable microenvironment (supportive niche) to promote human donor cell expansion and differentiation. To overcome the limitations of current mouse models, we used Alb-TRECK/SCID mice for in vivo human immature hepatocyte differentiation and humanized liver generation. METHODS: 1.5 µg/kg diphtheria toxin was administrated into 8-week-old Alb-TRECK/SCID mice, and the degree of liver damage was assessed by serum aspartate aminotransferase activity levels. Forty-eight hours later, mice livers were sampled for histological analyses, and the human donor cells were then transplanted into mice livers on the same day. Chimeric rate and survival rate after cell transplantation was evaluated. Expressions of human hepatic-related genes were detected. A human albumin enzyme-linked immunosorbent assay was performed after 50 days of transplantation. On day 60 after transplantation, drug metabolism was examined in mice. RESULTS: Both human primary fetal liver cells and hepatic stem cells were successfully repopulated in the livers of Alb-TRECK/SCID mice that developed lethal fulminant hepatic failure after administering diphtheria toxin; the repopulation rate in some mice was nearly 100%. Compared with human primary fetal liver cells, human hepatic stem cell transplantation rescued Alb-TRECK/SCID mice with lethal fulminant hepatic failure, and human hepatic stem cell-derived humanized livers secreted more human albumin into mouse sera and also functioned as a "human liver" that could metabolize the drugs ketoprofen and debrisoquine. CONCLUSION: Our model of a humanized liver in Alb-TRECK/SCID mice may provide for functional applications such as drug metabolism, drug to drug interactions, and promote other in vivo and in vitro studies.
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Hepatocitos/trasplante , Inactivación Metabólica/fisiología , Fallo Hepático Agudo/terapia , Regeneración Hepática/fisiología , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Quimera , Debrisoquina/metabolismo , Toxina Diftérica/administración & dosificación , Modelos Animales de Enfermedad , Hepatocitos/citología , Humanos , Cetoprofeno/metabolismo , Hígado/citología , Fallo Hepático Agudo/inducido químicamente , Ratones , Ratones Noqueados , Ratones SCID , Células Madre/metabolismo , Trasplante HeterólogoRESUMEN
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.
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Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromos b5/metabolismo , Animales , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Citocromos b5/genética , Debrisoquina/farmacocinética , Etanolaminas/farmacocinética , Femenino , Humanos , Masculino , Ratones Noqueados , Microsomas Hepáticos/metabolismo , Nifedipino/farmacocinética , Factores Sexuales , Triazolam/farmacocinéticaRESUMEN
Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous DA is unsettled. This study seeks to determine the ventilatory effects of increased availability of endogenous DA caused by inhibition of DA enzymatic breakdown. The peripheral inhibitor of MAO - debrisoquine, or COMT - entacapone, or both combined were injected to conscious rats. Ventilation and its responses to acute 8 % O(2) in N(2) were investigated in a whole body plethysmograph. We found that inhibition of MAO augmented the hyperventilatory response to hypoxia. Inhibition of COMT failed to influence the hypoxic response. However, simultaneous inhibition of both enzymes, the case in which endogenous availability of DA should increase the most, reversed the hypoxic augmentation of ventilation induced by MAO-inhibition. The inference is that when MAO alone is blocked, COMT takes over DA degradation in a compensatory way, which lowers the availability of DA, resulting in a higher intensity of the HVR. We conclude that MAO is the enzyme predominantly engaged in the chemoventilatory effects of DA. Furthermore, the findings imply that endogenous DA is inhibitory, rather than stimulatory, for hypoxic ventilation.
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Cuerpo Carotídeo/fisiopatología , Inhibidores de Catecol O-Metiltransferasa/farmacología , Dopamina/metabolismo , Hiperventilación/etiología , Hipoxia/fisiopatología , Inhibidores de la Monoaminooxidasa/farmacología , Respiración/efectos de los fármacos , Adaptación Fisiológica/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Catecol O-Metiltransferasa/fisiología , Catecoles/farmacología , Debrisoquina/farmacología , Dopamina/fisiología , Sinergismo Farmacológico , Hiperventilación/fisiopatología , Hiperventilación/prevención & control , Masculino , Monoaminooxidasa/fisiología , Nitrilos/farmacología , Pletismografía Total , Ratas , Ratas WistarRESUMEN
BACKGROUND & AIM: The CYP2D6 -1584C>G polymorphism (rs1080985) has been identified as a major factor for CYP2D6 expression and function, with the mutant -1584G promoter type being consistently associated with significantly greater expression than -1584C. It may therefore be associated with ultrarapid metabolism. The objective of the present study was to explore the relationship between the CYP2D6 -1584C>G polymorphism and the debrisoquine metabolic ratio in healthy volunteers in order to evaluate its potential impact on the ultrarapid CYP2D6 hydroxylation capacity. MATERIALS & METHODS: The CYP2D6 -1584C>G polymorphism was analyzed in 320 unrelated healthy individuals who were previously phenotyped for debrisoquine hydroxylation. RESULTS: The metabolic ratio (log10 mean ± standard deviation) of individuals with the -1584G allele was lower than that of individuals with the -1584C allele for carriers of one active CYP2D6 gene (-0.13 ± 0.33 and 0.17 ± 0.52, respectively; p < 0.05) or two active CYP2D6 genes (-0.32 ± 0.39 and -0.20 ± 0.44, respectively; p < 0.05). CONCLUSION: The presence of the -1584G allele in the promoter region of the CYP2D6 gene was related to a high CYP2D6 hydroxylation capacity.
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Citocromo P-450 CYP2D6/genética , Debrisoquina/uso terapéutico , Hidroxilación/genética , Alelos , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Voluntarios Sanos , Heterocigoto , Humanos , Hidroxilación/efectos de los fármacos , Polimorfismo Genético , Regiones Promotoras GenéticasRESUMEN
The penetration properties of drug-like molecules on human cell membranes are crucial for understanding the metabolism of xenobiotics and overall drug distribution in the human body. Here, we analyze partitioning of substrates of cytochrome P450s (caffeine, chlorzoxazone, coumarin, ibuprofen, and debrisoquine) and their metabolites (paraxanthine, 6-hydroxychlorzoxazone, 7-hydroxycoumarin, 3-hydroxyibuprofen, and 4-hydroxydebrisoquine) on two model membranes: dioleoylphosphatidylcholine (DOPC) and palmitoyloleoylphophatidylglycerol (POPG). We calculated the free energy profiles of these molecules and the distribution coefficients on the model membranes. The drugs were usually located deeper in the membrane than the corresponding metabolites and also had a higher affinity to the membranes. Moreover, the behavior of the molecules on the membranes differed, as they seemed to have a higher affinity to the DOPC membrane than to POPG, implying they have different modes of action in human (mostly PC) and bacterial (mostly PG) cells. As the xenobiotics need to pass through lipid membranes on their way through the body and the effect of some drugs might depend on their accumulation on membranes, we believe that detailed information of penetration phenomenon is important for understanding the overall metabolism of xenobiotics.
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Membrana Celular/química , Membrana Dobles de Lípidos/química , Preparaciones Farmacéuticas/metabolismo , Cafeína/química , Cafeína/metabolismo , Membrana Celular/metabolismo , Clorzoxazona/química , Clorzoxazona/metabolismo , Cumarinas/química , Cumarinas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Debrisoquina/química , Debrisoquina/metabolismo , Humanos , Ibuprofeno/química , Ibuprofeno/metabolismo , Simulación de Dinámica Molecular , Preparaciones Farmacéuticas/química , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Termodinámica , Xenobióticos/química , Xenobióticos/metabolismoRESUMEN
Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug-drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake. Debrisoquine showed low carrier-independent membrane permeability (P(e) of 0.01×10â»6 cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP+ (IC50 of 6.2 ± 0.8 µM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis-Menten kinetics (K(M) of 5.9 ± 1.5 µM and V(max) of 41.9 ± 4.5pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V(max) by 48, 63 and 91%, respectively, but did not affect the K(M). The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake. In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.
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Antihipertensivos/metabolismo , Debrisoquina/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Farmacogenética , Secuencia de Bases , Cartilla de ADN , Reacción en Cadena de la PolimerasaRESUMEN
CYP2D6 genotype and debrisoquine metabolic ratio (MR) were analyzed in 133 Nicaraguan Mestizos (NMs) and 260 Cubans divided into Cuban Mestizos (CMs) and White Cubans (WCs). The frequencies of poor metabolizers (MRî¶12.6) were 6% in NMs, 3.9% in CMs and 5.3% in WCs. The frequencies of ultrarapid metabolizers (MRîº0.1) were 0% in NMs, 2.3% in CMs and 5.3% in WCs. Mean (±s.d.) MR among extensive metabolizers (MR<12.6) was higher in NMs (1.5±1.6; n=118) than in CMs (1.0±1.3; n=124; P<0.001) and WCs (0.7±1.0; n=124; P<0.001). MR correlated with the 'activity score' of CYP2D6 genotypes (P<0.05; r=-0.55). Mean MR was higher among NMs than WCs and CMs for groups classified as 1 (P<0.05) or 2 (P<0.01) 'activity score'. In addition, mean (±s.d.) MR was higher among subjects carrying CYP2D6*17 than in CYP2D6 wt/wt (P<0.001). The CYP2D6*10 allele was higher in NMs (3.1%) than in CMs (0.8%; P<0.05) and WCs (0.4%; P<0.05). CYP2D6*17 allele was higher in CMs (10.2%) than WC (2.7%; P<0.005) and NMs (0%). Thus, the variability in CYP2D6 phenotypes found may be related to differences in allele frequency among groups (that is, CYP2D6*10 and *17 highest in NMs and CMs, respectively). However, the influence of environmental factors or alleles different than those studied here cannot be ruled out.
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Citocromo P-450 CYP2D6/genética , Debrisoquina/metabolismo , Etnicidad/genética , Adolescente , Adulto , Cuba , Femenino , Genotipo , Humanos , Hidroxilación , Masculino , Persona de Mediana Edad , Nicaragua , Fenotipo , Polimorfismo Genético , Adulto JovenRESUMEN
The objective of this study was to define CYP2D enzymes in marmoset (Callithrix jacchus) liver microsomes, both at the activity level using debrisoquine as the model substrate and at the protein level using antibodies raised to human CYP2D6. Marmoset liver microsomes were incubated with [(14)C]debrisoquine, and the structure of the generated metabolites was determined using liquid chromatography-tandem mass spectrometry and NMR. Marmoset liver microsomes were very effective in hydroxylating debrisoquine at various positions. Although 4-hydroxydebrisoquine was formed, in contrast to rat and human it was only a minor metabolite. Debrisoquine was more extensively hydroxylated in the 7, 5, 6, and 8 positions. In addition to the monohydroxylated metabolites, a dihydroxy metabolite, namely 6,7-dihydroxydebrisoquine, was identified. Finally, metabolites that had undergone ring opening were also detected but were not investigated further. Antibodies to CYP2D6 immunoreacted with protein in marmoset and human but not rat hepatic microsomes. In conclusion, we demonstrate that marmoset liver microsomes are effective in hydroxylating debrisoquine at various positions and that they contain a protein that is immunorelated to human CYP2D6.
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Citocromo P-450 CYP2D6/biosíntesis , Debrisoquina/metabolismo , Regulación Enzimológica de la Expresión Génica , Microsomas Hepáticos/metabolismo , Animales , Callithrix , Humanos , Masculino , Microsomas Hepáticos/enzimología , RatasRESUMEN
The ability to predict circulating human metabolites of a candidate drug before first-in-man studies are carried out would provide a clear advantage in drug development. A recent report demonstrated that while in vitro studies using human liver preparations reliably predict primary human metabolites in plasma, the predictability of secondary metabolites, formed by multiple reactions, was low, with total success rates of < or =65%. Here, we assess the use of chimeric mice with humanized liver as an animal model for the prediction of human metabolism in vivo. Metabolism studies with debrisoquine and (S)-warfarin demonstrated significantly higher concentrations of their primary human abundant metabolites in serum or plasma in chimeric mice than in control mice. Humanized chimeric mice were also capable of producing human-specific metabolites of several in-house compounds which were generated through more than one metabolism reaction. This model is closer to in vivo human physiology and therefore appears to have an advantage over in vitro systems in predicting complex metabolites in human plasma. However, prediction of human metabolites failed for other compounds which were highly metabolized in mice. Although requiring careful consideration of compound suitability, this model represents a potential tool for predicting human metabolites in combination with conventional in vitro systems.
Asunto(s)
Hepatocitos/metabolismo , Hígado/metabolismo , Fase II de la Desintoxicación Metabólica , Preparaciones Farmacéuticas/metabolismo , Quimera por Trasplante/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Debrisoquina/metabolismo , Regulación Gubernamental , Hepatocitos/citología , Hepatocitos/trasplante , Humanos , Hígado/citología , Hígado/enzimología , Fase I de la Desintoxicación Metabólica , Ratones , Modelos Animales , Ratas , Warfarina/metabolismoRESUMEN
Drug-drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (K(i)) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R-bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF-BUN, BUF-DBQ, BUN-DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the K(i) values estimated were similar to their K(m) values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration-time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF-DBQ and BUN-DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF-BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information.
Asunto(s)
Antagonistas Adrenérgicos alfa/farmacología , Antagonistas Adrenérgicos beta/farmacología , Debrisoquina/farmacología , Etanolaminas/farmacología , Propanolaminas/farmacología , Antagonistas Adrenérgicos alfa/farmacocinética , Antagonistas Adrenérgicos beta/farmacocinética , Animales , Debrisoquina/farmacocinética , Interacciones Farmacológicas , Etanolaminas/farmacocinética , Técnicas In Vitro , Hígado/metabolismo , Propanolaminas/farmacocinética , RatasRESUMEN
The potential involvement of sympathetic overactivity has been neglected in this population despite accumulating experimental and clinical evidence suggesting a crucial role of sympathetic activation for both progression of renal failure and the high rate of cardiovascular events in patients with chronic kidney disease. The contribution of sympathetic neural mechanisms to the occurrence of cardiac arrhythmias, the development of hypertension, and the progression of heart failure are well established; however, the exact mechanisms contributing to heightened sympathetic tone in patients with chronic kidney disease are unclear. This review analyses potential mechanisms underlying sympathetic activation in chronic kidney disease, the range of adverse consequences associated with this activation, and potential therapeutic implications resulting from this relationship.
Asunto(s)
Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/fisiopatología , Sistema Nervioso Simpático/fisiopatología , Animales , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/mortalidad , Catecolaminas/sangre , Clonidina/uso terapéutico , Debrisoquina/uso terapéutico , Modelos Animales de Enfermedad , Vías Eferentes/fisiopatología , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/epidemiología , Hipertensión/mortalidad , Riñón/inervación , Fallo Renal Crónico/epidemiología , Monoaminooxidasa/deficiencia , Monoaminooxidasa/metabolismo , Fibras Nerviosas/fisiología , Óxido Nítrico/fisiología , Norepinefrina/uso terapéuticoRESUMEN
Debrisoquine hydroxylation polymorphism is by far the most thoroughly studied genetic polymorphism of the CYP2D6 drug-metabolizing enzyme. Debrisoquine hydroxylation phenotype has been the most used test in humans to evaluate CYP2D6 activity. Two debrisoquine hydroxylation phenotypes have been described: poor and extensive metabolizers. A group with a very low debrisoquine metabolic ratio within the extensive metabolizers, named ultrarapid metabolizers, has also been distinguished. This CYP2D6 variability can be for a large part alternatively determined by genotyping, which appears to be of clinical importance given CYP2D6 involvement in the metabolism of a large number of commonly prescribed drugs. CYP2D6 pharmacogenetics may then become a useful tool to predict drug-related side effects, interactions or therapeutic failures. However, a number of reasons appear to have made research into this field lag behind. The present review focuses on the relevance of genetics and environmental factors for determining debrisoquine hydroxylation phenotype, as well as the relevance of CYP2D6 genetic polymorphism in psychiatric patients treated with antipsychotic drugs.
Asunto(s)
Antipsicóticos/farmacocinética , Citocromo P-450 CYP2D6/genética , Debrisoquina/metabolismo , Trastornos Mentales/tratamiento farmacológico , Farmacogenética , Antipsicóticos/efectos adversos , Antipsicóticos/uso terapéutico , Biomarcadores/análisis , Inhibidores del Citocromo P-450 CYP2D6 , Monitoreo de Drogas , Humanos , Hidroxilación , Trastornos Mentales/enzimología , Trastornos Mentales/metabolismo , Polimorfismo GenéticoRESUMEN
Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned from equine liver and expressed in a baculovirus expression system. The nucleotide sequence of CYP2D50 was highly homologous to that of human CYP2D6 and therefore the activity of the enzyme was characterized using dextromethorphan and debrisoquine, two isoform selective substrates for the human orthologue. CYP2D50 displayed optimal catalytic activity with dextromethorphan using molar ratios of CYP2D50 to NADPH CYP450 reductase of 1:15. Although CYP2D50 and CYP2D6 shared significant sequence homology, there were striking differences in the catalytic activity between the two enzymes. CYP2D50 dextromethorphan-O-demethylase activity was nearly 180-fold slower than the human counterpart, CYP2D6. Similarly, rates of formation of 4-hydroxydebrisoquine activity were 50-fold slower for CYP2D50 compared to CYP2D6. The results of this study demonstrate substantial interspecies variability in metabolism of substrates by CYP2D orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Caballos/genética , Hígado/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/genética , Debrisoquina/metabolismo , Dextrometorfano/metabolismo , Femenino , Caballos/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
OBJECTIVES: Our group has previously show that interindividual variability in CYP2D6 hydroxylation capacity was related to personality differences in cognitive social anxiety. Thus, we aimed to analyze whether this relationship between personality and CYP2D6 phenotype and genotype was found in a similar population of healthy volunteers from a different latitude and culture by using the same methodology. METHODS: A total of 253 university students and staff from Havana Psychiatric Hospital and Calixto García Medical School in Cuba completed the Karolinska Scales of Personality (KSP), and were evaluated on debrisoquine hydroxylation capacity and CYP2D6 genotypes. KSP scores were compared between four groups, divided according to their CYP2D6 metabolic capacity: one of poor and three of extensive metabolizers. Furthermore, KSP scores were compared between another four different groups divided according to their number of CYP2D6 active genes: zero, one, two, and more than two. RESULTS: In Cubans, the differences in cognitive social anxiety-related personality traits across the four CYP2D6 hydroxylation capacity groups were strikingly similar to those found in Spaniards. These differences also came out to be significant for psychic anxiety (p = 0.02) and socialization (p = 0.02). The same pattern of results obtained for the subscales of psychic anxiety, socialization, psychasthenia and inhibition of aggression with regard to phenotype in both the Cuban and Spanish studies were seen with regard to CYP2D6 genotypes. CONCLUSIONS: Corroborating these results further strengthens evidence of the relationship between CYP2D6 metabolic capacity and personality. In this population of healthy Cuban volunteers, the CYP2D6 hydroxylation capacity was related to the degree of anxiety and socialization. These results support the postulated reduction of serotonin in CYP2D6 poor metabolizers, which may be associated with a cluster of behavioral traits (e.g., anxiety, impulsivity). Thus, research is warranted to determine CYP2D6 functional implications for interindividual differences in vulnerability to neuropsychiatric diseases and drug response.