RESUMEN
Oligonucleotide microarrays were used to study the variability of pharmacokinetics and drug metabolism (PKDM)-related gene expression in 75 normal human livers. The objective was to define and use absorption, distribution, metabolism and excretion (ADME) gene expression variability to discern co-regulated genes and potential surrogate biomarkers of inducible gene expression. RNA was prepared from donor tissue and hybridized on Agilent microarrays against an RNA mass balanced pool from all donors. Clustering of PKDM gene sets revealed donors with distinct patterns of gene expression that grouped genes known to be regulated by the nuclear receptor, pregnane X-receptor (PXR). Fold range metrics and frequency distributions from the heterogeneous human population were used to define the variability of individual PKDM genes in the 75 human livers and were placed in context by comparing expression data with basal ADME gene expression variability in an inbred and diet/environment controlled population of 27 Rhesus livers. The most variable genes in the hepatic transcriptome were mainly related to drug metabolism, intermediary metabolism, inflammation and cell cycle control. Unique patterns of expression across 75 individuals of inducible ADME gene expression allowed their expression to be correlated with the expression of many other genes. Correlated genes for AhR, CAR and PXR responsive genes (CYP1A2, CYP2B6 and CYP3A4) were identified that may be co-regulated and, therefore, provide clues to the identity of surrogate gene or protein markers for CYP induction. In conclusion, microarrays were used to define the variable expression of hepatic ADME genes in a diverse human population, the expression variability of ADME genes was compared with the expression variability in an inbred population of Rhesus monkeys, and genes were defined that may be co-regulated with important inducible CYP genes.
Asunto(s)
Perfilación de la Expresión Génica , Hígado/metabolismo , Modelos Biológicos , Transcripción Genética , Xenobióticos/metabolismo , Animales , Análisis por Conglomerados , Humanos , Inactivación Metabólica , Macaca mulatta , Biología de Sistemas , Xenobióticos/farmacocinéticaRESUMEN
Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin, marker substrates for cytochrome P450 3A4 are commonly used within the pharmaceutical industry to screen new chemical entities as inhibitors of CYP3A4 in a high-throughput manner to predict the potential for drug-drug interactions. However, it has been observed that inhibition data obtained with a given CYP3A4 probe substrate may not correlate well with results from a different probe. As a consequence, the choice of the probe compound becomes an important consideration in such screens. In the present study, kinetic interactions between either two of the above three substrates were evaluated, and three-dimensional nonlinear regression analysis was performed to understand the kinetic mechanisms of drug interaction. Our results demonstrate that the kinetic interaction between each pair of substrates does not appear to be competitive and that the interactions are characterized by an unchanged or a decrease in both apparent K(m) (a = 0.21-0.72, a change of K(m) in the absence of the effector) and V(max) (alpha and beta = 0.09-0.75, changes of V(max) in the absence of the effector). These data suggest that 1) the three substrates bind to different domains; 2) at least two substrates can coexist in the active site of CYP3A4; and 3) the two bound substrates interact kinetically with each other (e.g., through steric hindrance), thereby leading to a change in both apparent kinetic parameters and partial inhibition. Selection of multiple substrates, which are shown not to be competitive, is necessary to accurately predict CYP3A4 inhibition and the potential for drug-drug interaction.
Asunto(s)
Cumarinas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Colorantes Fluorescentes/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Oxigenasas de Función Mixta/metabolismo , Quinolinas/metabolismo , Testosterona/metabolismo , Baculoviridae/genética , Sitios de Unión , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Interacciones Farmacológicas , Cinética , Oxigenasas de Función Mixta/antagonistas & inhibidores , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Algoritmos , Animales , Inhibidores Enzimáticos del Citocromo P-450 , Activación Enzimática/fisiología , Humanos , CinéticaRESUMEN
RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.
Asunto(s)
ARN/análisis , Espectrometría de Fluorescencia/métodos , Secuencia de Bases , Biotecnología/métodos , VIH/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
In-vitro studies were conducted to assess the impact of CYP2C9 genotype on the metabolism (methyl hydroxylation) and pharmacokinetics of celecoxib, a novel cyclooxygenase-2 inhibitor and CYP2C9 substrate. When compared to cDNA-expressed wild-type CYP2C9 (CYP2C9*1), the Vmax/Km ratio for celecoxib methyl hydroxylation was reduced by 34% and 90% in the presence of recombinant CYP2C9*2 and CYP2C9*3, respectively. These data indicated that the amino acid substitution at position 359 (Ile to Leu) elicited a more pronounced effect on the metabolism of celecoxib than did a substitution at position 144 (Arg to Cys). The Vmax/Km ratio was also decreased in microsomes of livers genotyped CYP2C9*1/*2 (47% decrease, mean of two livers), or CYP2C9*1/*3 (59% decrease, one liver). In all cases, these changes were largely reflective of a decrease in Vmax, with a minimal change in Km. Based on simulations of the in-vitro data obtained with the recombinant CYP2C9 proteins, it was anticipated that the pharmacokinetics of celecoxib (as a much as a five-fold increase in plasma AUC) would be altered (versus CYP2C9*1/*1 subjects) in subjects genotyped heterozygous or homozygous for the CYP2C9*2 (Cys144) or CYP2C9*3 (Leu359) allele. In a subsequent clinical study, the AUC of celecoxib was increased (versus CYP2C9*1/*1 subjects) approximately 2.2-fold (range, 1.6-3-fold) in two CYP2C9*1/*3 subjects and one CYP2C9*3/*3 subject receiving a single oral dose (200 mg) of the drug. In contrast, there was no significant change in celecoxib AUC in two subjects genotyped CYP2C9*1/*2.
Asunto(s)
Alelos , Hidrocarburo de Aril Hidroxilasas , Inhibidores de la Ciclooxigenasa/farmacocinética , Sistema Enzimático del Citocromo P-450/genética , Isoenzimas/antagonistas & inhibidores , Hígado/metabolismo , Microsomas Hepáticos/enzimología , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Sulfonamidas/farmacocinética , Administración Oral , Adulto , Celecoxib , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Citocromo P-450 CYP2C9 , Cartilla de ADN/química , Genotipo , Humanos , Hidroxilación , Proteínas de la Membrana , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Prostaglandina-Endoperóxido Sintasas , PirazolesRESUMEN
Halothane causes a mild form of liver injury in guinea pigs that appears to model the hepatotoxicity seen in approximately 20% of patients treated with this drug. In previous studies, it was concluded that the increased susceptibility of some outbred guinea pigs to halothane-induced liver injury is not caused by their inherent ability to metabolize halothane to form toxic levels of trifluoroacetylated protein adducts in the liver. In this study, we reevaluated the role of trifluoroacetylated protein adducts in halothane-induced liver injury in guinea pigs. Male outbred Hartley guinea pigs were treated with halothane intraperitoneally. On the basis of serum alanine aminotransferase levels and liver histology, treated animals were designated as being susceptible, mildly susceptible, or resistant to halothane. Immunoblot studies with the use of anti-trifluoroacetylated antibodies showed that susceptible guinea pigs for the most part had higher levels of trifluoroacetylated protein adducts in the liver 48 h after treatment with halothane than did less susceptible animals. In support of this finding, the level of trifluoroacetylated protein adducts detected immunochemically in the sera of treated guinea pigs correlated with sera levels of alanine aminotransferase activity. In addition, the levels of cytochrome P450 2A-related protein but not those of other cytochrome P450 isoforms, measured by immunoblot analysis with isoform-specific antibodies, correlated with the amount of trifluoroacetylated protein adducts detected in the livers of guinea pigs 8 h after halothane administration. The results of this study indicate that the susceptibility of outbred guinea pigs to halothane-induced liver injury is related to an enhanced ability to metabolize halothane in the liver to form relatively high levels of trifluoroacetylated protein adducts. They also suggest that cytochrome P450 2A-related protein might have a major role in catalyzing the formation of trifluoroacetylated protein adducts in the liver of susceptible guinea pigs. Similar mechanisms may be important in humans.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Halotano/toxicidad , Proteínas/metabolismo , Ácido Trifluoroacético/química , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Cobayas , Humanos , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Most cytochrome P450 (P450 or CYP)-catalyzed reactions are adequately described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), which are usually determined by a saturation profile of velocity of product formation versus substrate concentration. In turn, these parameters may be used to predict pharmacokinetics. However, some P450 enzymes exhibit atypical or non-Michaelis-Menten kinetics, due largely to substrate inhibition at higher concentrations of substrate. Although the mechanism of substrate inhibition is unknown, ignoring it and truncating the data can lead to erroneous estimates of kinetic parameters. In the present study, 13 P450 marker substrates were examined with 10 recombinant P450 proteins, and 6 were found, to varying degrees, to exhibit substrate inhibition. To understand the nature of the inhibition, a kinetic model was proposed (assuming that two binding sites exist on the enzyme) and used to fit the experimental data. The derived data indicated that 1) the K(I) values (substrate inhibition) were approximately 1.2- to 10-fold greater than the respective K(S) values; 2) both K(S) and K(I) values may be affected by the interaction of the two bound substrates within the enzyme, exhibited by a factor alpha (alpha = 5.1-23.3); and 3) enzyme activity was inhibited markedly (39-97%) at excess concentrations of the substrates (beta = 0.03-0.61). These findings suggest that substrates have access to both the inhibitory site and catalytic site simultaneously (K(I) > K(S)). Furthermore, the two sites, in the presence of substrate, can interact with each other. Therefore, the degree of inhibition of the enzyme is dependent on the concentration of the substrate (usually >K(I)) that sufficiently occupies the inhibitory site.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Antineoplásicos/metabolismo , Unión Competitiva , Catálisis , Celecoxib , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/química , Dextrometorfano/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Humanos , Cinética , Oxazinas/metabolismo , Progesterona/metabolismo , Pirazoles , Especificidad por Sustrato , Sulfonamidas/metabolismoRESUMEN
In many cases, CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrates that coexist at the active site. In the present study, we observed that alpha-naphthoflavone (alpha-NF) exhibited a differential effect on CYP3A4-mediated product formation as shown by an increase and decrease, respectively, of the carboxylic acid (P(2)) and omega-3-hydroxylated (P(1)) metabolites of losartan, while losartan was found to be an inhibitor of the formation of the 5,6-epoxide of alpha-NF. Thus, to address this problem, a kinetic model was developed on the assumption that CYP3A4 can accommodate two distinct and independent binding domains for the substrates within the active site, and the resulting velocity equations were employed to predict the kinetic parameters for all possible enzyme-substrate species. Our results indicate that the predicted values had a good fit with the experimental observations. Therefore, the kinetic constants can be used to adequately describe the nature of the metabolic interaction between the two substrates. Applications of the model provide some new insights into the mechanism of drug-drug interactions at the level of CYP3A4.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Modelos Biológicos , Benzoflavonas/metabolismo , Sitios de Unión , Citocromo P-450 CYP3A , Cinética , Losartán/metabolismo , Especificidad por SustratoRESUMEN
In this communication, we report that suspension cultures of Sf21 insect cells, co-infected with baculovirus containing the cDNA for a single cytochrome P450 and NADPH-cytochrome P450 oxidoreductase, can be employed successfully as "bioreactors" for the synthesis of milligram quantities of cytochrome P450-generated metabolite(s). Three standard or probe substrates for the human P450s were chosen for the initial biosynthetic experiments: testosterone, diazepam, and diclofenac. Testosterone (100 microM, 2.88 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to 6beta-hydroxytestosterone (2.3 mg) and 15beta-hydroxytestosterone (0.18 mg). Diazepam (100 microM, 2.9 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to temazepam (1.1 mg), N-demethyldiazepam (0.35 mg), and oxazepam (0.15 mg). Diclofenac (100 microM, 3.18 mg/100 ml), added to a 100-ml CYP2C9 bioreactor, was converted to 4'-hydroxydiclofenac (2.6 mg). Since the goal for the development of the bioreactors was to provide a platform for both the production and subsequent purification of milligram quantities of P450-generated metabolite(s), a second 100-ml CYP2C9 bioreactor was used for the large-scale production and subsequent purification of 4'-hydroxydiclofenac. After 55 h of incubation, 7.95 mg of diclofenac was converted to 4.35 mg of 4'-hydroxydiclofenac, while 3.55 mg of unchanged diclofenac remained in the bioreactor. Using a simple preparative HPLC method, approximately 2.2 mg of 4'-hydroxydiclofenac and 1.9 mg of diclofenac were recovered from this experiment (28% yield). These results indicate clearly that suspension cultures of Sf21 insect cells coexpressing a cytochrome P450 and NADPH-cytochrome P450 oxidoreductase can be used effectively as bioreactors for the production and subsequent purification of milligram quantities of P450-derived metabolite(s).
Asunto(s)
Reactores Biológicos , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Baculoviridae , Técnicas de Cultivo de Célula/métodos , Sistema Enzimático del Citocromo P-450/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismoRESUMEN
In vitro studies were conducted to identify the cytochromes P450 (CYP) involved in the oxidative metabolism of celecoxib. The hydroxylation of celecoxib conformed to monophasic Michaelis-Menten kinetics (mean +/- S.D., n = 4 livers, K(m) = 3.8 +/- 0.95 microM, V(max) = 0.70 +/- 0.45 nmol/min/mg protein) in the presence of human liver microsomes, although substrate inhibition was significant at higher celecoxib concentrations. The treatment of a panel of human liver microsomal samples (n = 16 subjects) with antibodies against CYP2C9 and CYP3A4 inhibited the formation of hydroxy celecoxib by 72 to 92% and 0 to 27%, respectively. The presence of both antibodies in the incubation suppressed the activity by 90 to 94%. In addition, the formation of hydroxy celecoxib significantly correlated with CYP2C9-selective tolbutamide methyl hydroxylation (r = 0.92, P <. 001) and CYP3A-selective testosterone 6beta-hydroxylation (r = 0.55, P <.02). In contrast, correlation with activities selective for other forms of CYP was weak (r = 0.46). Chemical inhibition studies showed that ketoconazole (selective for CYP3A4) and sulfaphenazole (selective for CYP2C9) inhibited the formation of hydroxy celecoxib in a concentration-dependent manner, whereas potent inhibitors selective for other forms of CYP did not show any significant effect over a range of 1 to 10 microM. In agreement, cDNA-expressed CYP2C9 catalyzed the formation of hydroxy celecoxib with an apparent K(m) value (microM) and a V(max) value (pmol/min/pmol recombinant CYP) of 5.9 and 21.7, whereas a higher K(m) value (18.2) and a lower V(max) value (1.42) were obtained with rCYP3A4. It is concluded that methyl hydroxylation of celecoxib is primarily catalyzed by human liver microsomal CYP2C9, although CYP3A4 also plays a role.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Inhibidores de la Ciclooxigenasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Microsomas Hepáticos/enzimología , Proteínas de Plantas/metabolismo , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/metabolismo , Sulfonamidas/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Biotransformación , Celecoxib , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Humanos , Hidroxilación , Técnicas In Vitro , Cinética , Espectrometría de Masas , Ratones , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , NADP/metabolismo , Oxidación-Reducción , Pirazoles , Espectrofotometría Ultravioleta , Esteroide Hidroxilasas/biosíntesis , Esteroide Hidroxilasas/genética , Sulfonamidas/antagonistas & inhibidoresRESUMEN
Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Indanos/farmacología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Piperazinas/farmacología , Sitios de Unión , Catálisis , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Simulación por Computador , Bromuro de Cianógeno/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Diálisis , Escherichia coli/citología , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Hidroxilación , Indanos/química , Indanos/metabolismo , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismo , Modelos Químicos , Modelos Moleculares , NADP/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
Cytochrome P-450 (CYP) 3A4 is an inordinately important CYP enzyme that catalyzes the metabolism of a vast array of clinically used drugs. Microsomal proteins of Spodoptera frugiperda (Sf21) insect cells infected with recombinant baculoviruses encoding CYP3A4 cDNA were used to immunize mice and to develop a monoclonal antibody (mAb(3A4a)) specific to CYP3A4 through the use of hybridoma technology. The mAb is both a potent inhibitor and a strong binder of CYP3A4. One and 5 microl (0.5 and 2.5 microM IgG(2a)) of the mAb mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6beta-hydroxylation by 95 and 99%, respectively, and, to a lesser extent, cross-inhibited CYP3A5 and CYP3A7 activity. mAb(3A4a) exhibited no cross-reactivity with any of the other recombinant human CYP isoforms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) in the course of CYP reaction phenotyping and Western immunoblot analyses. The potency of mAb-induced inhibition is insensitive to substrate concentration in human liver microsomes. Therefore, mAb(3A4a) was used to assess the quantitative role of CYP3A4/5 to the metabolism of testosterone and diazepam in five human liver microsomes. The results showed that CYP3A4 and CYP3A5 contribute >95% to both testosterone 6beta-hydroxylation and diazepam 3-hydroxylation and 52 to 73% to diazepam N-demethylation, respectively. In addition, mAb(3A4a) significantly inhibited testosterone 6beta-hydroxylase activity in rhesus monkey liver microsomes to a degree equal to that observed with CYP3A4 in human liver microsomes. By comparison, no inhibition of testosterone 6beta-hydroxylase activity was observed in the presence of dog, rat, and mouse liver microsomes. The selectivity of ketoconazole, a chemical inhibitor of CYP3A4, was probed with mAb(3A4a) and was shown to be highly concentration dependent in the diazepam N-demethylation by human liver microsomes. The results demonstrate that inhibitory and immunoblotting mAb(3A4a) can offer a precise and useful tool for quantitative identification of CYP3A4/5 in the metabolism of drugs in clinical use and drugs in development.
Asunto(s)
Anticuerpos Monoclonales , Sistema Enzimático del Citocromo P-450/inmunología , Oxigenasas de Función Mixta/inmunología , Preparaciones Farmacéuticas/metabolismo , Adulto , Animales , Western Blotting , Reacciones Cruzadas , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , ADN Complementario/fisiología , Diazepam/metabolismo , Perros , Femenino , Haplorrinos , Humanos , Cetoconazol/farmacología , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Fenotipo , Unión Proteica , Ratas , Especificidad de la Especie , Spodoptera/virología , Testosterona/metabolismoRESUMEN
Cytochrome P450 3A4 (CYP3A4) plays a prominent role in the metabolism of a vast array of drugs and xenobiotics and exhibits broad substrate specificities. Most cytochrome P450-mediated reactions follow simple Michaelis-Menten kinetics. These parameters are widely accepted to predict pharmacokinetic and pharmacodynamic consequences in vivo caused by exposure to one or multiple drugs. However, CYP3A4 in many cases exhibits allosteric (sigmoidal) characteristics that make the Michaelis constants difficult to estimate. In the present study, diazepam, temazepam and nordiazepam were employed as substrates of CYP3A4 to propose a kinetic model. The model hypothesized that CYP3A4 contains two substrate-binding sites in a single active site that are both distinct and co-operative, and the resulting velocity equation had a good fit with the sigmoidal kinetic observations. Therefore, four pairs of the kinetic estimates (KS1, kalpha, KS2, kbeta, KS3, kdelta, KS4 and kgamma) were resolved to interpret the features of binding affinity and catalytic ability of CYP3A4. Dissociation constants KS1 and KS2 for two single-substrate-bound enzyme molecules (SE and ES) were 3-50-fold greater than KS3 and KS4 for a two-substrate-bound enzyme (SES), while respective rate constants kdelta and kgamma were 3-218-fold greater than kalpha and kbeta, implying that access and binding of the first molecule to either site in an active pocket of CYP3A4 can enhance the binding affinity and reaction rate of the vacant site for the second substrate. Thus our results provide some new insights into the co-operative binding of two substrates in the inner portions of an allosteric CYP3A4 active site.
Asunto(s)
Sitio Alostérico , Sistema Enzimático del Citocromo P-450/metabolismo , Diazepam/metabolismo , Oxigenasas de Función Mixta/metabolismo , Modelos Químicos , Adulto , Regulación Alostérica , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Catálisis , Línea Celular , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/inmunología , Diazepam/análogos & derivados , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Cinética , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/inmunología , Nordazepam/metabolismo , Oxidación-Reducción , Temazepam/metabolismoRESUMEN
The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Sistema Enzimático del Citocromo P-450/metabolismo , Diclofenaco/farmacocinética , Glutatión/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Bilis/metabolismo , Biotransformación , Células Cultivadas , Cromatografía Liquida , Femenino , Humanos , Masculino , Espectrometría de Masas , Microsomas Hepáticos/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/química , Ticrinafeno/química , Animales , Baculoviridae/genética , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Activación Enzimática , Vectores Genéticos , Humanos , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Esteroide Hidroxilasas/antagonistas & inhibidores , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/aislamiento & purificaciónRESUMEN
Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya1 gene (gstA2) in isolated rat hepatocytes and rat liver in vivo. Transient transfection experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acetyltransferase (CAT) constructs containing a palindromic GRE (pGRE) and three GRE hexanucleotide half-sites between -1.6 and -1.1 kb of the 5'-flanking region of gstA2 were repressed >50% by GC when induced with polyaromatic hydrocarbon. This pGRE, if either mutated or deleted, significantly reduces GC responsiveness of the gene to 20-30%; no effect of GC was observed with CAT constructs containing -1.15 kb of the 5'-flanking region. The dexamethasone concentration dependence of the repression was consistent with involvement of the GC receptor and was antagonized by RU38486. Electrophoretic mobility shift assays demonstrated that pGRE formed a specific DNA/protein complex, which was prevented by the addition of excess unlabeled or mouse mammary tumor virus GRE but not by unrelated or mutated gstA2 GRE double-stranded oligonucleotides. This complex was supershifted by incubation of nuclear extracts containing GC receptor with anti-GC receptor globulins. Constructs containing multiple copies of pGRE sequence were either nonresponsive or positively responsive (three copies) to GC. Luciferase constructs containing -1.62 to -1.03 kb of the 5'-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated receptor activated the constructs that were positively responsive to GC but did not mediate the negative effect in constructs containing 1.6 kb of 5'-flanking region. We conclude that pGRE and half-site GREs of gstA2 participate in regulation of this gene; however, a second unidentified responsive element must exist between -1.03 and -0.164 kb, resulting in repression of gstA2 expression.
Asunto(s)
Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Transferasa/genética , Receptores de Glucocorticoides/fisiología , Animales , Benzo(a)Antracenos/farmacología , Relación Dosis-Respuesta a Droga , Luciferasas/genética , Ratones , Mifepristona/farmacología , Regiones Promotoras Genéticas , RatasRESUMEN
Cytochrome P4502C9 (CYP2C9) is largely responsible for terminating the anticoagulant effect of racemic warfarin via hydroxylation of the pharmacologically more potent S-enantiomer to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of three allelic variants, CYP2C9*1, CYP2C9*2 and CYP2C9*3. Both CYP2C9*2 and CYP2C9*3 exhibit altered catalytic properties in vitro relative to the wild-type enzyme. In the present study, a patient was genotyped who had proven unusually sensitive to warfarin therapy and could tolerate no more than 0.5 mg of the racemic drug/day. PCR-amplification of exons 3 and 7 of the CYP2C9 gene, followed by restriction digest or sequence analysis, showed that this individual was homozygous for CYP2C9*3. In addition, patient plasma warfarin enantiomer ratios and urinary 7-hydroxywarfarin enantiomer ratios were determined by chiral-phase high performance liquid chromotography in order to investigate whether either parameter might be of diagnostic value in place of a genotypic test. Control patients receiving 4-8 mg warfarin/day exhibited plasma S:R ratios of 0.50 +/- 0.25:1, whereas the patient on very low-dose warfarin exhibited an S:R ratio of 3.9:1. In contrast, the urinary 7-hydroxywarfarin S:R ratio of 4:1 showed the same stereoselectivity as that reported for control patients. Therefore, expression of CYP2C9*3 is associated with diminished clearance of S-warfarin and a dangerously exacerbated therapeutic response to normal doses of the racemic drug. Analysis of the plasma S:R warfarin ratio may serve as a useful alternative test to genotyping for this genetic defect.
Asunto(s)
Anticoagulantes/farmacología , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Warfarina/farmacología , Anticoagulantes/farmacocinética , Citocromo P-450 CYP2C9 , Sistema Enzimático del Citocromo P-450/metabolismo , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Estereoisomerismo , Esteroide Hidroxilasas/metabolismo , Warfarina/farmacocinéticaRESUMEN
PURPOSE: To study the transcription regulation of rat glutathione S-transferase Ya (rGSTya) subunit gene expression by chemopreventive agents. METHODS: The effects of chemopreventive agents; tamoxifen, genistein, oltipraz, indole-3-carbinol, and various isothiocyanates-sulforaphane, PMITC, PEITC, PBITC, and PPITC, on the transcriptional activation of rGSTya were investigated in cell culture. These were accomplished with a stable human hepatoma Hep G2 cell line transfected with a 1.6 kilobase (kb) 5'-flanking region of the rGSTya fused with the chloramphenicol acetyltransferase (CAT) reporter gene. Concentration-effect relationship and the kinetics of gene activation following treatments of the cells with different chemopreventive agents were carried out by quantitating CAT reporter protein using ELISA. Northern blot analysis of total RNA on the expression of CAT mRNA as well as potential transcription factors such as c-Jun, c-Fos, and LFR-1 were performed. RESULTS: Treatment of the cells with increasing concentrations of different chemopreventive agents resulted in corresponding increases in the gene expression of CAT reporter protein. Kinetically, induction of CAT protein was seen as early as 3 hr and peaked at about 20 hr. Northern blot analysis revealed an increase in CAT mRNA transcripts and these mRNA inductions in general were in agreement with those quantitated by the production of CAT reporter protein. Induction of the transcription factor, c-Jun mRNA was observed with sulforaphane. CONCLUSIONS: These results show that different chemopreventive agents transcriptionally activate rGSTya CAT in a time and dose-dependent fashion.