RESUMEN
The goal of this work was to investigate the use of MDCK (Madin-Darby canine kidney) cells as a possible tool for assessing the membrane permeability properties of early drug discovery compounds. Apparent permeability (Papp) values of 55 compounds with known human absorption values were determined using MDCK cell monolayers. For comparison, Papp values of the same compounds were also determined using Caco-2 cells, a well-characterized in vitro model of intestinal drug absorption. Monolayers were grown on 0. 4-microm Transwell-COL membrane culture inserts. MDCK cells were seeded at high density and cultured for 3 days, and Caco-2 cells were cultured under standard conditions for 21 to 25 days. Compounds were tested using 100 microM donor solutions in transport medium (pH 7.4) containing 1% DMSO. The Papp values in MDCK cells correlated well with those in Caco-2 cells (r2 = 0.79). Spearman's rank correlation coefficient for MDCK Papp and human absorption was 0.58 compared with 0.54 for Caco-2 Papp and human absorption. These results indicate that MDCK cells may be a useful tool for rapid membrane permeability screening.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Riñón/metabolismo , Algoritmos , Animales , Transporte Biológico , Células CACO-2 , Línea Celular , Perros , Humanos , Absorción Intestinal , Riñón/citología , Riñón/ultraestructura , Control de CalidadRESUMEN
The absorption of a drug compound through the human intestinal cell lining is an important property for potential drug candidates. Measuring this property, however, can be costly and time-consuming. The use of quantitative structure-property relationships (QSPRs) to estimate percent human intestinal absorption (%HIA) is an attractive alternative to experimental measurements. A data set of 86 drug and drug-like compounds with measured values of %HIA taken from the literature was used to develop and test a QSPR mode. The compounds were encoded with calculated molecular structure descriptors. A nonlinear computational neural network model was developed by using the genetic algorithm with a neural network fitness evaluator. The calculated %HIA (cHIA) model performs wells, with root-mean-square (rms) errors of 9.4%HIA units for the training set, 19.7%HIA units for the cross-validation (CV) set, and 16.0%HIA units for the external prediction set.
Asunto(s)
Absorción Intestinal , Farmacocinética , Humanos , Modelos Lineales , Modelos Biológicos , Estructura Molecular , Redes Neurales de la Computación , Relación Estructura-ActividadRESUMEN
A high-performance liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method is presented that allows rapid and accurate determination of amino acid chiral purity in a peptide. Peptides are hydrolyzed in hydrochloric acid-d1/acetic acid-d4 and then converted to diastereomers by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey's reagent). Mixtures of D- and L-amino acid diastereomeric pairs are resolved in one chromatographic separation using conventional reversed-phase high-performance liquid chromatography. Hydrolysis in a deuterated solvent is necessary because the original ratio of D-/L-amino acids present in a peptide changes during acid hydrolysis due to racemization. Peptide hydrolysis in deuterated acids circumvents this problem by labeling each amino acid that racemizes with one deuterium at the alpha-carbon. An increase in molecular mass of one atomic mass unit allows racemized amino acids to be distinguished from non-racemized amino acids by mass spectrometry. This procedure was used to determine the chiral purity of each amino acid in a purified, hexapeptide by-product (Arg-Lys-Lys-Asp-Val-Tyr) present in a kilogram batch of the synthetic pentapeptide, thymopentin (Arg-Lys-Asp-Val-Tyr).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Péptidos/aislamiento & purificación , Alanina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Aminoácidos/química , Deuterio , Dinitrobencenos/química , Hidrólisis , Indicadores y Reactivos , Datos de Secuencia Molecular , Péptidos/química , Conformación ProteicaRESUMEN
Fluorescence spectroscopy was applied to the development of sensitive analytical methods for the determination of thiazole and several congeners that contain substituted thiazole rings. Treatment to yield thionine, previously used spectrophotometrically to measure thiazole and fluorometrically only for sulfur determinations in inorganic systems, is further characterized and illustrated with the determination of the antibiotic thiopeptin. This method is selective for submicrogram quantities of thiazole rings in the presence of fused-ring derivatives and reduced analogs. It has a precision of +/- 2% RSD (n = 11) at the 15-ng/ml thiazole concentration level with a signal-to-noise ratio of 3:1. For thiopeptin, this method has an accuracy of 5% mean relative error (n = 8) over the 5--20-ppm range in medicated feed.
Asunto(s)
Tiazoles/análisis , Alimentación Animal/análisis , Fenómenos Químicos , Química , Espectroscopía de Resonancia Magnética , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
An analytical method is described for the determination of the avermectins in plasma based upon high-performance liquid chromatography of fluorescent derivatives of these compounds. The analyte is isolated by adsorption chromatography on Florisil, dehydrated in an acetic anhydride-pyridine mixture, and the fluorophore is further separated by chromatography on silica gel in advance of introduction into a reversed-phase system. This method, which can be applied to samples containing as little as 0.2 ng drug per ml, has an accuracy of 5% mean relative error and a precision of 8% relative standard deviation. A study and discussion of several factors which affect the analytical reaction are included.
Asunto(s)
Antihelmínticos , Ivermectina/análogos & derivados , Lactonas/sangre , Animales , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Disacáridos/sangre , Espectrometría de FluorescenciaRESUMEN
A previously published colorimetric method for determining ronidazole, (1-methyl-5-nitro-imidazol-2-yl)methyl carbamate, can be used as an analytical technique for measuring the stability of this drug in medicated feeds. Although the color reaction per se is not selective, elution profiles of ronidazole and its demonstrated hydrolytic degradation product, 1-methyl-2-hydroxymethyl-5-nitroimidazole, show that the chromatographic separation used in the sample preparation efficiently isolates the drug from the degradation product. No interference was found in feeds containing 0.010% ronidazole and up to 0.010% degradation product.
Asunto(s)
Alimentación Animal/análisis , Nitroimidazoles/análisis , Ronidazol/análisis , Animales , Colorimetría/métodos , PorcinosRESUMEN
A difference spectrophotometric analytical method was developed for the selective determination of p-hydroxybenzoic acid in the presence of its alkyl esters without prior separation. Based on the spectral shift to a shorter wavelength accompanyint carboxyl dissociation, the procedure measures as little as 2% of this acid in mixtures with the alkyl ester preservatives and has an accuracy of 2% mean relative error over the 0.16-12.0 microgram of p-hydroxybenzoic acid/ml range.