RESUMEN
Glucuronidation is an important pathway for human drug metabolism. Four cloned and expressed human UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A9, and UGT2B15) were used to screen a series of three potential drug substrates differing only in position of the phenol moiety. The meta and para phenols, UK-156,037 and UK-157,147, were found to be substrates for UGT1A1 with K(m) values of 256 and 105 microM, respectively. The ortho phenol UK-157,261 was glucuronidated predominantly by UGT1A9 with a K(m) of 45 microM. The latter K(m) compares favorably with the known UGT1A9 substrate propofol (K(m) = 200 microM). In a series of competition experiments, UK-157,261 was shown to inhibit the glucuronidation of propofol by UGT1A9 with a K(i) value of 65 microM. This result indicates that even the most potent of these compounds is extremely unlikely to interact in the clinic with the glucuronidation of propofol. This study shows the utility of the expressed human UDP-glucuronosyltransferases in determining substrate structure-activity relationships and potential drug-drug interactions.
Asunto(s)
Benzopiranos/farmacología , Glucuronosiltransferasa/metabolismo , Sulfonas/farmacología , Animales , Benzopiranos/farmacocinética , Línea Celular , Clonación Molecular , Cricetinae , Interacciones Farmacológicas , Glucuronosiltransferasa/genética , Humanos , Sulfonas/farmacocinéticaRESUMEN
1. Pharmacokinetics were studied in mouse, rat, rabbit, dog and man after single intravenous and/or oral doses of sildenafil or [14C]-sildenafil (Viagra). 2. In man, absorption from the gastrointestinal tract was essentially complete. With the exception of male rat, Tmax occurred at approximately 1 h or less. Bioavailability was attenuated by pre-systemic hepatic metabolism in all species. 3. The volume of distribution was similar in rodents and humans (1-2 l/kg) but was greater in dog (5.2 l/kg), due to lower plasma protein binding (84 versus 94-96% respectively). 4. High clearance was the principal determinant of short elimination half-lives in rodents (0.4-1.3 h), whereas moderate clearance in dog and man resulted in longer half-lives (6.1 and 3.7 h respectively). Clearances were in agreement with in vitro metabolism rates by liver microsomes from the various species. 5. After single oral or intravenous doses of [14C]-sildenafil, the majority of radioactivity was excreted in the faeces of all species. No unchanged drug was detected in the excreta of man. 6. Five principal pathways of metabolism in all species were piperazine N-demethylation, pyrazole N-demethylation, loss of a two-carbon fragment from the piperazine ring (N,N'-deethylation), oxidation of the piperazine ring and aliphatic hydroxylation. Additional metabolites arose through combinations of these pathways. 7. Sildenafil was the major component detected in human plasma. Following oral doses, AUC(infinity) for the piperazine N-desmethyl and piperazine N,N'-desethyl metabolites were 55 and 27% that of parent compound respectively.
Asunto(s)
Inhibidores de Fosfodiesterasa/metabolismo , Inhibidores de Fosfodiesterasa/farmacocinética , Piperazinas/metabolismo , Piperazinas/farmacocinética , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Proteínas Sanguíneas/metabolismo , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión/métodos , Perros , Heces/química , Femenino , Semivida , Humanos , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Piperazinas/administración & dosificación , Piperazinas/análisis , Purinas , Pirimidinonas/análisis , Pirimidinonas/farmacocinética , Conejos , Ratas , Ratas Sprague-Dawley , Citrato de Sildenafil , Especificidad de la Especie , Sulfonas , Orina/químicaRESUMEN
A new unified assay for the determination of UDP-glucuronosyltransferase (UGT) activities has been developed. The resolution of [14C]uridine diphosphate glucuronic acid from radiolabeled glucuronides formed by incorporation of this radiolabel can now be achieved by a sensitive and rapid-gradient HPLC method which utilizes a radioactivity endpoint as a universal detection method. One important application of this method is the determination of kinetic parameters for cloned and expressed UGT isoforms with greater speed and precision than can be afforded by TLC methodology. Moreover, assays with 14C-labeled substrates indicate that gradient HPLC can easily resolve the substrate from the glucuronide products and present an alternative to the time-consuming optimization of conditions for organic phase extraction assays.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glucuronosiltransferasa/análisis , Animales , Radioisótopos de Carbono , Línea Celular , Glucuronosiltransferasa/metabolismo , Cinética , Radiofármacos , Especificidad por Sustrato , Uridina Difosfato Ácido Glucurónico/análisis , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
We describe a comprehensive retrospective analysis in which the abilities of several methods by which human pharmacokinetic parameters are predicted from preclinical pharmacokinetic data and/or in vitro metabolism data were assessed. The prediction methods examined included both methods from the scientific literature as well as some described in this report for the first time. Four methods were examined for their ability to predict human volume of distribution. Three were highly predictive, yielding, on average, predictions that were within 60% to 90% of actual values. Twelve methods were assessed for their utility in predicting clearance. The most successful allometric scaling method yielded clearance predictions that were, on average, within 80% of actual values. The best methods in which in vitro metabolism data from human liver microsomes were scaled to in vivo clearance values yielded predicted clearance values that were, on average, within 70% to 80% of actual values. Human t1/2 was predicted by combining predictions of human volume of distribution and clearance. The best t1/2 prediction methods successfully assigned compounds to appropriate dosing regimen categories (e.g., once daily, twice daily and so forth) 70% to 80% of the time. In addition, correlations between human t1/2 and t1/2 values from preclinical species were also generally successful (72-87%) when used to predict human dosing regimens. In summary, this retrospective analysis has identified several approaches by which human pharmacokinetic data can be predicted from preclinical data. Such approaches should find utility in the drug discovery and development processes in the identification and selection of compounds that will possess appropriate pharmacokinetic characteristics in humans for progression to clinical trials.
Asunto(s)
Farmacocinética , Animales , Disponibilidad Biológica , Semivida , Humanos , Tasa de Depuración Metabólica , Estudios RetrospectivosRESUMEN
Partially purified aldehyde oxidase (EC 1.2.3.1) has been prepared from human, rabbit, guinea pig, and baboon liver by heat treatment and precipitation with ammonium sulfate. The interaction of 35 substituted quinazolines and phthalazines with human liver enzyme has been studied using a spectrophotometric assay. Fifteen quinazoline and 14 phthalazine derivatives were found to be substrates for human liver aldehyde oxidase with Km values ranging from 5 to 500 microM. The substrate specificity of the quinazolines toward rabbit, guinea pig, and baboon liver aldehyde oxidase has also been investigated; the reaction of substituted phthalazines with mammalian liver enzyme has been reported previously (Beedham et al., 1990, Biochem. Pharmacol. 39, 1213-1221). Oxidation products of 2-substituted (4-substituted) quinazolines with rabbit liver aldehyde oxidase were identified by MS as 4-oxo (2-oxo)-quinazolines, respectively. In all cases, unsubstituted compounds gave the highest oxidation rates and the presence of lipophilic substituents presumably facilitated hydrophobic binding to the enzymes. However, there were marked differences in substrate specificity between human liver aldehyde oxidase and hepatic enzyme from rabbit, guinea pig, and baboon with the size of substrate being the differentiating factor. The molecular sizes of the substrates, estimated using calculated molar refractivities, ranked the size of the binding site of aldehyde oxidase in the order rabbit < guinea pig < baboon < man. Isoelectric points of the different aldehyde oxidase isozymes ranged from pH 5.10 for rabbit to 6.40 for the human liver isozyme. These results indicate that rabbit liver aldehyde oxidase shows marked differences from the human liver enzyme in its handling of quinazoline and phthalazine substrates.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Hígado/enzimología , Ftalazinas/metabolismo , Quinazolinas/metabolismo , Aldehído Oxidasa , Animales , Cobayas , Humanos , Cinética , Oxidación-Reducción , Papio , Conejos , Especificidad por SustratoRESUMEN
1. After administration of [14C]-carbazeran by oral gavage to guinea pigs, 48% of the dosed radioactivity was recovered in urine and 46% in faeces after 144 h. 2. The major urinary metabolite was identified by infrared spectroscopy and mass spectrometry as the O-glucuronide of O-desmethyl-4-oxocarbazeran, O-demethylation having taken place at the 6- or 7-position. The corresponding aglycone was identified as the major faecal metabolite. 3. A minor metabolite in urine was identified as the 4-oxo-4'-hydroxy derivative of carbazeran. 4. In animals pretreated with hydralazine, an aldehyde oxidase inhibitor, less radioactivity was extracted in the urine and a significant decrease was observed in the levels of the major urinary metabolite. 5. These results show that aldehyde oxidase plays a key role in the metabolism of carbazeran in guinea pig as it does in man. The similarity of man and guinea pig liver aldehyde oxidase has been observed previously in vitro.
Asunto(s)
Carbamatos/farmacocinética , Hidralazina/farmacología , Aldehído Oxidasa , Aldehído Oxidorreductasas/metabolismo , Animales , Biotransformación , Carbamatos/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Heces/química , Cobayas , Hidrólisis , Hígado/efectos de los fármacos , Hígado/enzimología , MasculinoAsunto(s)
Cardiotónicos/análisis , Piridinas/análisis , Quinolonas/análisis , Animales , Cardiotónicos/sangre , Cardiotónicos/orina , Cromatografía Líquida de Alta Presión , Óxidos N-Cíclicos/análisis , Óxidos N-Cíclicos/sangre , Perros , Indicadores y Reactivos , Piridinas/sangre , Piridinas/orina , Quinolonas/sangre , Quinolonas/orina , Ratas , Estándares de Referencia , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
1. Bioanalysis is traditionally associated with the development phase of drugs; its use in discovery programmes is often ignored but can have a major impact. 2. Pharmacokinetic studies conducted in conjunction with pharmacology screening can provide additional information to that considered in conventional structure activity relationships. Such factors as half-life and bioavailability can be critical in designing improved drugs. 3. Analytical methods in discovery programmes may differ from those used in later development work: for instance bioassay allows a common assay system for a large number of project compounds. Moreover its use, when combined with conventional methods, such as h.p.l.c., allows active metabolites to be readily detected. 4. Bioanalytical data generated in discovery and pre-clinical programmes are a valuable guide to early clinical programmes. Plasma concentration-response data from these programmes can be compared with those obtained in man. Such comparisons are particularly valuable during the phase one-initial dose escalation study. To maximize this it is our practice to generate pharmacokinetic data between each dose increase.
Asunto(s)
Interpretación Estadística de Datos , Toma de Decisiones , Evaluación Preclínica de Medicamentos/métodos , Evaluación de Medicamentos/métodos , Animales , Ensayos Clínicos Fase II como Asunto/métodos , Diseño de Fármacos , Humanos , FarmacocinéticaRESUMEN
Molybdenum hydroxylase activity in guinea pig liver has been compared with that of marker enzymes in mitochondria (succinate dehydrogenase), microsomes (glucose-6-phosphatase) and cytosol (lactate dehydrogenase). Aldehyde oxidase activity was highest in the cytosol, with about 10-fold activity of xanthine oxidase. Significant molybdenum hydroxylase activity was found in mitochondria with minimal levels in microsomes. Mitochondrial and cytosolic aldehyde oxidase varied in substrate specificity and electrophoretic mobility with two major bands in each fraction, one of which was common to cytosol and mitochondria.
Asunto(s)
Aldehído Oxidorreductasas/análisis , Isoenzimas , Mitocondrias/enzimología , Oxigenasas de Función Mixta/análisis , Molibdeno/metabolismo , Aldehído Oxidasa , Animales , Biomarcadores , Cobayas , Masculino , Microsomas/enzimología , Conejos , Ratas , Fracciones Subcelulares/enzimología , Xantina OxidasaRESUMEN
A method is described for the determination of UK-57,400 (I), a 6-substituted quinoline cardiotonic, and its pyridyl-N-oxide in dog plasma. The analytes are selectively retained from plasma (1 ml) on a solid-phase extraction column and eluted with 1 ml of methanol. After evaporation to dryness, the residue is reconstituted in 100 microliters of the mobile phase. Chromatography is carried out on a Spherisorb 5 microns phenyl high-performance liquid chromatography column, with ultraviolet detection. Calibration curves are linear for concentrations from 10 to 100 ng ml-1. For I, the coefficients of variation at highest and lowest concentrations are 1 and 14%, respectively, while the corresponding figures for the pyridyl-N-oxide metabolite are 4 and 10%, respectively. Sample recovery from extraction is greater than 90%. The limit of detection is 4 ng ml-1 for both analytes.
Asunto(s)
Cardiotónicos/sangre , Piridinas/sangre , Quinolonas/sangre , Animales , Cromatografía Líquida de Alta Presión , Perros , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrofotometría UltravioletaRESUMEN
The nonapeptide Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln has been reported as a model substrate for an aspartyl protease produced by the human immunodeficiency virus (HIV-1). Cleavage of this peptide at the Tyr-Pro linkage to produce tetra- and pentapeptide fragments is the basis of high-performance liquid chromatographic assays to detect HIV-1 protease activity. Confirmation of the cleavage site has been proved by using microbore liquid chromatography coupled to a dynamic fast atom bombardment interface. Comparison with fortified control incubates indicates that an approximate stoichiometric amount of the tetrapeptide was formed from the nonapeptide, confirming that the cleavage of the substrate by HIV-1 protease is both specific and quantitative.
Asunto(s)
Proteasa del VIH , Péptidos/análisis , Prolina/análisis , Tirosina/análisis , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Proteasa del VIH/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría UltravioletaRESUMEN
The interaction of a series of 1-substituted phthalazine derivatives with partially purified aldehyde oxidase from rabbit, guinea-pig and baboon liver, and with bovine milk xanthine oxidase, has been investigated. Of the 18 compounds examined, rabbit liver aldehyde oxidase metabolized 10, whereas guinea-pig and baboon liver enzyme oxidized 13 and 14, respectively. Where metabolites were characterized, oxidation was shown to occur at position four of the phthalazine ring. Km values ranged from 0.003 to 1.8 mM. In contrast, most compounds were competitive inhibitors of bovine milk xanthine oxidase with Ki values ranging from 0.015 to 1.3 mM; the cationic derivative 2-methylphthalazinium iodide was oxidized to 2-methyl-1-phthalazinone by both aldehyde oxidase and, with a much reduced affinity, by xanthine oxidase. In terms of structure-metabolism relationships, Vmax values were relatively insensitive to the electronic effects of substituents, but a trend for the more lipophilic derivatives to show increased affinities (Km and Vmax/Km) towards aldehyde oxidase could be seen. However, calculations of molecular size revealed a species-dependent cut-off threshold above which compounds were not metabolized. Results suggest that the relative size of the active site for hepatic aldehyde oxidase is in the order baboon greater than guinea-pig greater than rabbit, and that in spatial terms the active site of bovine milk xanthine oxidase is similar to that of baboon liver aldehyde oxidase. Thus, the binding site of rabbit liver aldehyde oxidase, a widely used source of the oxidase, is apparently more restricted than in some other species.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Molibdeno , Ftalazinas/metabolismo , Piridazinas/metabolismo , Aldehído Oxidasa , Animales , Sitios de Unión/efectos de los fármacos , Cobayas , Cinética , Hígado/enzimología , Espectrometría de Masas , Sondas Moleculares , Oxidación-Reducción , Papio , Ftalazinas/farmacología , Conejos , Espectrofotometría Infrarroja , Especificidad por Sustrato , Xantina Oxidasa/metabolismoRESUMEN
The activity of the molybdenum hydroxylase, aldehyde oxidase, was determined in crude homogenates and (NH4)2SO4 fractions prepared from guinea pig liver, lung, kidney, intestine, spleen and heart. Xanthine oxidase was also measured in (NH4)2SO4 fractions. In each case, xanthine oxidase levels were lower than those of aldehyde oxidase; activity of the latter enzyme was highest in the liver, whereas xanthine oxidase was predominant in the small intestine. There was no significant difference in the activity of either molybdenum hydroxylase between tissues taken from male and female guinea pigs.
Asunto(s)
Aldehído Oxidorreductasas/farmacocinética , Xantina Oxidasa/farmacocinética , Aldehído Oxidasa , Aldehído Oxidorreductasas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cobayas , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Masculino , Miocardio/enzimología , Bazo/enzimología , Distribución Tisular , Xantina Oxidasa/metabolismoRESUMEN
The activity of hepatic aldehyde oxidase from rabbit, guinea pig, rat, marmoset, dog, baboon and man was investigated in vitro with charged and uncharged N-heterocyclic substrates: Km and Vmax values were determined for phthalazine, 6,7-dimethoxy-1-[-4-(ethylcarbamoyloxy)piperidino]phthalazine (carbazeran), quinine and quinidine. The oxidation of N-phenylquinolinium chloride to N-phenyl-2-quinolone and N-phenyl-4-quinolone was followed spectrophotometrically. Rat or dog liver showed low and negligible enzyme activity respectively, whereas baboon liver contained a highly active aldehyde oxidase. Enzyme from marmoset and guinea pig liver had the closest spectrum of activity to human liver aldehyde oxidase. Unlike that from man, rabbit hepatic aldehyde oxidase was refractory towards carbazeran and converted N-phenylquinolinium chloride predominantly to the 2-quinolone. N-Phenyl-4-quinolone was the major oxidation product with enzyme from guinea pig, marmoset, baboon and man.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Hígado/enzimología , Especificidad de la Especie , Aldehído Oxidasa , Aldehído Oxidorreductasas/análisis , Animales , Callitrichinae , Perros , Cobayas , Humanos , Papio , Ftalazinas/análisis , Ftalazinas/metabolismo , Quinidina/análisis , Quinidina/metabolismo , Compuestos de Quinolinio/análisis , Compuestos de Quinolinio/metabolismo , Conejos , Ratas , Ratas Endogámicas , Espectrofotometría UltravioletaRESUMEN
The metabolism of carbazeran has been investigated in vitro using liver cytosol from dog, baboon and man. Carbazeran was not metabolized in cytosol prepared from dog liver but was rapidly metabolized to a single product in baboon- and human-liver cytosol. The product was identified as 4-hydroxy carbazeran. The enzyme responsible for the 4-hydroxylation of carbazeran in vitro was shown by the use of inhibitors to be liver aldehyde oxidase. Species differences in the metabolism of carbazeran in vitro correlate well with studies in vivo; these showed that following an oral dose to man and baboon, the compound was almost completely cleared via pre-systemic 4-hydroxylation, whereas in the dog, this metabolic route appeared unimportant.