RESUMEN
An automated Cobas Fara method was developed determining the activity of recombinant M. thermophila laccase (rMtL). The chromogenic substrate used was syringaldazine. Under aerobic conditions, rMtL catalyses the oxidation of syringaldazine forming tetrametoxy-azo bis methylene quinone. The developed violet colour was measured kinetically at 530 nm as an expression of the enzyme activity, rMtL is a very sensitive oxidoreductase, therefore many factors had to be carefully controlled in order to get a robust analytical assay. In order to stabilize rMtL, PEG 6000 was added to the enzyme dilution medium. Furthermore, Triton X-I00 was included in the enzyme incubation solution.The analytical as well as technical conditions have been optimized, resulting in a method with good precision, sensitivity and speed of analysis. The Michaelis-Menten constant, K(m), was determined to be 22muM syringaldazine. LOQ was determined to be 0.010 Uml(-1), LOD to be 0.0002 Uml(-1) The analytical range of the enzyme dilution curve was from 0.01 to 0.044 Uml(-1) The repeatability was 1.9%, the reproducibility 3.1%. Testing the robustness of the method showed that the most sensible factors in the rMtL analysis in decreasing range were: incubation temperature, concentration of Triton X-I00, molarity and pH of the incubation buffer, and finally the concentration of syringaldazine.
RESUMEN
EXP921, 5,5-bis(4-pyridinylmethyl)-5H-cyclopenta[2,1-b:3,4-b']-dipyridine, was a potential drug candidate for the improvement of cognitive performance in patients with Alzheimer's-type dementia. It has been shown to improve cognitive performance in rodent and primate models of learning and memory. To characterize the disposition of EXP921, the pharmacokinetics and metabolism of this compound were studied in rats after oral and intravenous administrations. EXP921 exhibited good bioavailability, 43% at 3 mg/kg and 61% at 10 mg/kg and was rapidly eliminated with a terminal half-life ranging from 1.28 to 2.29 hr after oral doses. Absorption from oral doses was rapid, as peak plasma levels were reached within 1 hr. A major metabolite was identified in plasma as the pyridinyl mono-N-oxide of EXP921. This metabolite (EXP696) was rapidly formed, and significant levels were detected in rat plasma after oral or intravenous administration. Its terminal half-life was slightly longer than that of EXP921. EXP696 was found to be reduced back to EXP921, demonstrating that the N-oxidation at the pyridyl ring is reversible. The interconversion favored the oxidation of EXP921 to EXP696. Two additional metabolites were identified in rat plasma at doses higher than or equal to 30 mg/kg. They result from despicolylation, followed by hydroxylation in the cyclopentane ring.
Asunto(s)
Nootrópicos/farmacocinética , Piridinas/farmacocinética , Administración Oral , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/psicología , Animales , Esquema de Medicación , Semivida , Inyecciones Intravenosas , Masculino , Tasa de Depuración Metabólica , Estructura Molecular , Nootrópicos/metabolismo , Piridinas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
HPCE was evaluated for the identification of different serine protease analogues. This work represents a separation of two serine proteases, Savinase (SAV) (Novo Nordisk A/S, Bagsvaerd, Denmark) and a Savinase analogue, both consisting of 269 amino acids and having identical isoelectric points. The only difference between the two protease variants is that a methionine is substituted with a serine, M222S. The separation was only made feasible by the HPCE/micellar electrokinetic capillary chromatography (MECC) technique using sodium dodecyl sulfate (SDS) dynamically as the surfactant. This paper shows that it is possible to employ HPCE/MECC to separate Savinase and a Savinase variant with no alteration in net charge.
Asunto(s)
Serina Endopeptidasas/aislamiento & purificación , Bacillus/enzimología , Tampones (Química) , Acción Capilar , Cromatografía/instrumentación , Cromatografía/métodos , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Fermentación , Indicadores y Reactivos , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Serina Endopeptidasas/química , Dodecil Sulfato de SodioRESUMEN
An automated flow injection method has been developed for the determination of microbial peroxidase activity. The substrate used was hydrogen peroxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) was used as the electron donor. In the presence of hydrogen peroxide, peroxidase catalyses the dehydrogenation of ABTS, resulting in the formation of a resonance-stabilized radical cation of ABTS. The green-blue colour formed, recorded at 418 nm, is taken as a measure of the peroxidase activity. The general technical conditions and the general enzymic kinetics have been optimized. Conditions for activation and stabilization of the enzyme were found, e.g., ammonium sulfate acts as a peroxidase activator. The resulting method has a good precision, sensitivity and speed.
Asunto(s)
Autoanálisis/métodos , Bacterias/enzimología , Fermentación , Análisis de Inyección de Flujo/métodos , Peroxidasa de Rábano Silvestre/metabolismo , Peróxido de Hidrógeno/metabolismo , Ácidos Sulfónicos/metabolismo , Autoanálisis/estadística & datos numéricos , Benzotiazoles , Tampones (Química) , Calcio/farmacología , Electroquímica , Estabilidad de Enzimas , Análisis de Inyección de Flujo/estadística & datos numéricos , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Cinética , Fosfatos , Sensibilidad y EspecificidadRESUMEN
A selective and sensitive high-performance liquid chromatographic assay for a novel cognitive enhancer, X9121 (I), and its mono N-oxide metabolite, XG696 (II), in dog plasma has been developed. Compounds I, II and internal standard (I.S.) were first extracted from dog plasma using a solid-phase Bond Elut Certify I 10-ml LRC reservoir extraction cartridge. Chromatographic separation of I, II and I.S. was conducted on a reversed-phase Zorbax Stable Bond cyano column. Ammonium acetate buffer (0.05 M, pH 6)-acetonitrile-triethylamine (75:25:0.1, v/v) was used as the mobile phase. Detection of all three compounds was by UV light absorbance at 313 nm. Using 0.5 ml of dog plasma for extraction, the minimum quantifiable limit was 10 ng/ml and the assay was linear from 10 to 5400 ng/ml. The coefficients of variation for intra-day precision ranged from 2.2 to 8.5% for I and from 2.5 to 9.8% for II. The coefficients of variation for the inter-day precision for these two compounds ranged from 2.6 to 9.0% and from 3.6 to 16.2%, respectively. The absolute percent differences for the accuracy results were within 11.0% of the spiked concentrations. Compounds I and II were stable in frozen plasma at -20 degrees C for at least 67 days.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Óxidos N-Cíclicos/sangre , Piridinas/sangre , Animales , Cognición/efectos de los fármacos , Perros , Femenino , Piridinas/metabolismo , Piridinas/farmacología , Reproducibilidad de los Resultados , Espectrofotometría UltravioletaRESUMEN
DuP 532, 2-propyl-4-pentafluoroethyl-1-([2'-(1H-tetrazol-5-yl)biph eny l-4-]methyl) imidazole-5-carboxylic acid, is an orally active, non-peptide angiotensin II (AII) receptor antagonist. DuP 532 is more potent and longer acting than losartan, another AII receptor antagonist currently undergoing phase III clinical trials. The pharmacokinetics and the effect of the salt form on the bioavailability of DuP 532 were determined in rats and dogs. In rats, the absolute oral bioavailability and half-life averaged 8.0% and 3.5 h, respectively, after the sodium bicarbonate solution and 7.6% and 3.6 h, respectively, after the methyl cellulose suspension. In dogs, the absolute oral bioavailability averaged 13.4% after the sodium bicarbonate solution and 11.9% after hard gelatin capsules containing the neat powder. The data demonstrated that there were no differences in bioavailability between the free acid and the sodium salt of DuP 532 after oral administration to rats and dogs. The in vitro metabolism of 14C-DuP 532 was evaluated with rat, dog, and human liver microsomes. HPLC analyses with UV and radiochemical flow detection showed that recovery of DuP 532 was greater than 99%, suggesting that there was little if any metabolism by liver microsomal enzymes. Therefore, the low oral bioavailability in rats was probably due to poor absorption of DuP 532 from the GI tract rather than extensive metabolism.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Imidazoles/farmacocinética , Microsomas Hepáticos/metabolismo , Tetrazoles/farmacocinética , Absorción , Animales , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada , Perros , Formas de Dosificación , Semivida , Humanos , Imidazoles/administración & dosificación , Imidazoles/metabolismo , Técnicas In Vitro , Inyecciones Intravenosas , Masculino , Ratas , Ratas Sprague-Dawley , Análisis de Regresión , Tetrazoles/administración & dosificación , Tetrazoles/metabolismoRESUMEN
[R(+),S(-)]-Cyclophosphamide [(R,S)-CP] is an anticancer drug, containing a chiral phosphorous atom, which is prepared and used clinically as the racemic mixture. A new high-performance liquid chromatographic assay suitable for pharmacokinetic studies of CP enantiomers in plasma has been reported recently by this laboratory (Reid et al., Anal Chem 61: 441-446, 1989). Briefly, the assay involves ethyl acetate extraction of CP enantiomers from plasma followed by derivatization to diastereomers in a two-step process utilizing chloral and (+)-naproxen acid chloride. Chromatographic analysis was performed on a reversed phase (ODS) column with detection at 232 nm. In the present study, preliminary results on the applicability of this assay to pharmacokinetic studies are presented. Several rabbits were used to compare the influence of i.p., i.v., and oral routes of administration on the stereoselective disposition of (R,S)-CP. Following i.p. administration, S-CP was cleared faster than R-CP. Following oral administration, only R-CP was detectable in plasma, while i.v. administration resulted in minor or no stereoselective disposition. These results indicated that there was a marked stereoselective metabolism of the S-CP enantiomer, with the i.p. and oral routes producing the greatest differences due to first-pass metabolism. Incubation of rabbit-liver microsomes with (R,S)-CP demonstrated that the monooxygenase system can exhibit marked stereoselectivity in its metabolism of CP. The ratio of R-CP to S-CP in the incubation medium increased during the incubation period from 1:1 initially to 4.5:1 after 60 min. The results from the experiments with rabbits indicate that the first-pass metabolism of this drug is highly stereoselective; in contrast, cancer patients who had received (R,S)-CP as an i.v. infusion showed no stereoselectivity in the elimination of the enantiomers. Pharmacokinetic studies with cancer patients, receiving (R,S)-CP as an oral dose, are in progress in order to determine if stereoselective first-pass metabolism of this drug also occurs in humans.
Asunto(s)
Ciclofosfamida/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Ciclofosfamida/administración & dosificación , Ciclofosfamida/metabolismo , Femenino , Humanos , Técnicas In Vitro , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Masculino , Microsomas Hepáticos/metabolismo , Conejos , EstereoisomerismoRESUMEN
Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Anticuerpos Monoclonales , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/metabolismo , Animales , Complejo Antígeno-Anticuerpo , Cromatografía de Afinidad , Sistema Enzimático del Citocromo P-450/inmunología , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Hidroxilación , Cinética , Masculino , Metilcolantreno/farmacología , Microsomas Hepáticos/efectos de los fármacos , Oxigenasas de Función Mixta/inmunología , Oxigenasas de Función Mixta/aislamiento & purificación , Fenobarbital/farmacología , Ratas , Ratas EndogámicasRESUMEN
We have cloned and sequenced one of the two genes encoding a 255 nucleotide small nuclear RNA from the fission yeast Schizosaccharomyces pombe. Based on the presence of four regions of primary sequence conservation and a predicted secondary structure similar to that previously proposed for human U3, we conclude that this molecule is the fission yeast homologue of this mammalian snRNA. The 5' one-third of fission yeast U3 is, however, unable to form a single stable hairpin as proposed for this region of the human RNA, but rather folds into two stem-loop structures. By analogy to fission yeast U3, we propose revised secondary structures containing two hairpins for this portion of the U3-like snRNAs from Saccharomyces cerevisiae and Dictyostelium discoideum. Thus, our data suggest that the structure of U3 snRNA has diverged in lower and higher eukaryotes.
Asunto(s)
Evolución Biológica , Genes Fúngicos , ARN Nuclear Pequeño/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
Incubation of prostaglandin E1 (PGE1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at omega-2, omega-1, and omega carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the omega-1 and omega-2 hydroxylation of PGE1 and PGE2. Additionally, a small amount of product with chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome b5 into the reconstituted system did not enhance the rate of PGE1 hydroxylation and had no effect on the ratio of products formed. The Km value for the omega-1 and omega-2 hydroxylation of PGE1 with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 microM; the Vmax's (nmol product min-1 nmol P-450-1) were 2.1 and 1.1 for the omega-1 and omega-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the omega-2 hydroxylation of PGEs observed in rabbit liver microsomes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Microsomas Hepáticos/enzimología , Prostaglandinas/metabolismo , Alprostadil/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Dinoprostona , Etanol/farmacología , Hidroxilación , Imidazoles/farmacología , Cinética , Miconazol/farmacología , NADP/metabolismo , Proadifeno/farmacología , Prostaglandinas E/metabolismo , Conejos , Safrol/farmacologíaRESUMEN
The effects of methylcholanthrene (MC) treatment of male rats on the regioselectivity of hydroxylation of prostaglandins E1 and E2 (PGE1 and PGE2) by liver microsomes, supplemented with NADPH or H2O2, was examined. In the presence of NADPH, control microsomes catalyzed the hydroxylation at omega-1 (C19) and at omega-(C20) sites with minimal formation of novel monohydroxy metabolites of PGE1 and PGE2, referred to as compounds X1 and X2, respectively. Similarly, H2O2 supported the 19-hydroxylation and the formation of compounds X1 and X2, but yielded only minimal amounts of 20-hydroxy products. With NADPH, MC-treated microsomal incubations demonstrated only minor quantitative change in the 19- and 20-hydroxylation as compared with controls, but showed a 7- to 11-fold increase in formation of compound X1 and a 10-fold increase in formation of X2. By contrast with H2O2, MC-treatment increased by about 3-fold the 19- and 20-hydroxylation of PGE1 and by 35- to 46-fold the formation of X1; similarly, there was an approximate 2-fold increase in 19- and 20-hydroxylation of PGE2 and a 10-fold increase in formation of X2. These findings suggest that several monooxygenases are involved in catalyzing the hydroxylation at the various sites of the PGE molecule. Inhibitors of monooxygenases (SKF 525A, alpha-naphthoflavone, and imidazole derivatives) provided further evidence that the hydroxylation at the three sites of PGEs is catalyzed by different P-450 monooxygenases. It is striking that the inhibitors had a much lesser effect on the 20-hydroxylation of PGE1 as compared with other sites of hydroxylation. Structural identification of compounds X1 and X2 was elucidated as follows. Resistance of the PGB derivative of X1 to periodate oxidation and mass fragmentation analysis of the t-butyldimethylsilyl ether methyl ester, placed the hydroxylation at C17 or C18. Finally, mass fragmentation of trimethylsilyl ether methyl ester PGB derivatives of X1 and X2 provided conclusive evidence that X1 and X2 are 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively. The above findings indicate that the high regioselectivity of hydroxylation of PGE1 and PGE2, resulting in the formation of 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively, is catalyzed by P-450 isozyme(s) which are induced by MC, possibly by P-450c.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Metilcolantreno/farmacología , Microsomas Hepáticos/metabolismo , NADP/metabolismo , Prostaglandinas E/metabolismo , Alprostadil , Animales , Cromatografía Líquida de Alta Presión , Dinoprostona , Cromatografía de Gases y Espectrometría de Masas , Hidroxilación , Técnicas In Vitro , Masculino , Ratas , Ratas EndogámicasRESUMEN
Previous studies suggested that rabbit liver microsomes contain cytochrome P-450 monooxygenase(s) with low affinity for (omega-1)-hydroxylation and high affinity for omega-hydroxylation of prostaglandins (Theoharides, A. D., and Kupfer, D. (1981) J. Biol. Chem. 256, 2168-2175). The current investigation describes the isolation from livers of untreated rabbits of a cytochrome P-450 catalyzing, with regioselectivity, the omega-hydroxylation of prostaglandins E1 and E2. The isolation of the enzyme involved enrichment of the omega-hydroxylase activity by polyethylene glycol 8000 fractionation, followed by ion-exchange high performance liquid chromatography. Based on Mr of 59,000-60,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated enzyme is referred to as P-450 form 7. This P-450 exhibits a low spin spectrum (lambda max = 417 nm) and a difference spectrum of the CO-reduced complex versus reduced (lambda max = 451 nm). For catalytic activity, the P-450 form 7 was reconstituted with NADPH-P-450 reductase, cytochrome b5, and lipid. There was no activity in the absence of the reductase, and deletion of cytochrome b5 yielded a minimal amount of product (heme could not substitute for cytochrome b5), demonstrating an absolute requirement for these components.