RESUMEN
Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis-trans isomerization and the presence of four either intramolecular or intermolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophilin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, we documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 microM at 5 degrees C, but decreased by more than an order of magnitude at 37 degrees C. Moreover, CsA partitioned into non-hydrogen bond donating solvents linearly as a function of increasing temperature, suggestive of a significant conformational change. However, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the two major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane permeability of CsA do not originate from changes in the peptide backbone conformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret this behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a change would result in lower peptide desolvation energy, thereby promoting partitioning into cellular membranes. We contend that changes in membrane penetration result from alterations in the hydration state of CsA and are not related to the interconversion of the defined conformations.
Asunto(s)
Ciclosporina/metabolismo , Células CACO-2 , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Humanos , Espectroscopía de Resonancia Magnética , Solubilidad , Temperatura , Agua/químicaRESUMEN
The transepithelial transport of a synthetic peptide combinatorial library containing 375,000 individual peptides was assessed using Caco-2 cell monolayers in order to screen for permeability and deliverability. A series of 150 pools, each containing 2500 tripeptide sequences, were applied to the apical side of Caco-2 monolayers. Basolateral side samples were collected after 4 h and screened by capillary high-pressure liquid chromatography. The majority of pools showed no permeable species, due to low solubility, limited permeability and extensive metabolism. Several pools contained permeable structure, and transport proved reproducible with passage number and time. Permeable structures were identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). To discriminate between isobaric structures, several tripeptides were resynthesized and tested as discrete compounds. For example, 1-2% D-Phe-D-Ala-D-Ser-OH was transported across the Caco-2 cell monolayer with a Papp value of 0.35-0.69 x 10(-6) cm/s, which is comparable with the permeability of amino acids (Leu, Papp = 0.30 x 10(-6) cm/s) and dipeptides (L-Val-L-Val, Papp = 0.18 x 10(-6) cm/s) (Lennernas, H., Palm, K., Fagerholm, U., Artursson, P., 1996. Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo. Int. J. Pharm. 127, 103-107; Tamura, K., Bhatnagar, P.K., Takata, J.S., Lee, C.P., Smith, P.L., Borchardt, R.T., 1996. Metabolism, uptake, and transepithelial transport of the diastereomers of Val-Val in the human intestinal cell line Caco-2. Pharm. Res. 13, 1213-1218). These studies demonstrate the techniques used to screen combinatorial libraries for permeability across Caco-2 cells and structurally identify the resulting compounds. Such methodology can be of importance in the achievement of structure-permeability relationships, useful in the design of pharmaceutically bioavailable drugs.
Asunto(s)
Células CACO-2/metabolismo , Péptidos/farmacocinética , Administración Oral , Administración Tópica , Disponibilidad Biológica , Epitelio/metabolismo , Cromatografía de Gases y Espectrometría de Masas , HumanosRESUMEN
PURPOSE: The relative contribution of the intestinal mucosa, liver and lung to the in vivo disposition of propofol in the rat was investigated. METHODS: Propofol (4.9-5.1 mg.kg-1) was administered to groups of rats (n = 4) via the intra-arterial, intravenous, hepatic portal venous and oral routes. The AUC's of propofol were estimated and the fractions of the administered dose escaping first pass metabolism by the gut wall (fG), liver (fH) and lung (fL) were calculated. In addition, transport experiments were carried out using Caco-2 cell monolayers to rule out the possibility that intestinal permeability is limiting the oral absorption of propofol. RESULTS: Values for fG, fH and fL were the following: 0.21 +/- 0.07, 0.61 +/- 0.13, and 0.82 +/- 0.09, respectively. The apparent permeability coefficient of propofol across Caco-2 cell monolayers was 24.2 +/- 0.3 x 10(-6) cm.sec-1, which is similar to the apparent permeability coefficient obtained for propranolol (30.7 +/- 1.7 x 10(-6) cm.sec-1), a compound known to easily cross the intestinal epithelial membranes. The formation of propofol glucuronide, a major metabolite of propofol, could not be demonstrated during the flux experiments across the Caco-2 cell monolayers. CONCLUSIONS: The intestinal mucosa is the main site of first pass metabolism following oral administration of propofol in the rat. Intestinal metabolism could therefore also contribute to the systemic clearance of propofol.
Asunto(s)
Anestésicos Intravenosos/farmacocinética , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Propofol/farmacocinética , Administración Oral , Anestésicos Intravenosos/administración & dosificación , Anestésicos Intravenosos/sangre , Animales , Área Bajo la Curva , Células CACO-2 , Epitelio/metabolismo , Humanos , Inyecciones Intraarteriales , Inyecciones Intravenosas , Masculino , Vena Porta/fisiología , Propofol/administración & dosificación , Propofol/sangre , Ratas , Ratas WistarRESUMEN
Transport of cyclosporin A (CsA) across Caco-2 cells is modulated by its directional efflux, mediated by a p-glycoprotein-like pump (Augustijns et al., Biochem. Biophys. Res. Comm. 197:360-365, 1994). In addition to this unidirectional flux, oxidative metabolism of CsA by cytochrome P450 is likely to influence the absorption of this cyclic peptide across intestinal mucosa. Thus, metabolism of CsA in the in vitro Caco-2 cell culture system was investigated. Formation of several metabolites was observed during the course of CsA transport across Caco-2 cell monolayers. Results from LC/MS/MS experiments revealed that the major metabolite was 1eta-hydroxy CsA (M-17), one of the three major metabolites produced by CYP3A4 present in both the liver and small intestine in humans. Preincubation of Caco-2 cell monolayers with troleandomycin, a specific inhibitor for the microsomal CYP3A protein, reduced the formation of the metabolite M-17, suggesting that an enzyme that functionally resembles CYP3A is responsible for the formation of this metabolite. However, formation of only the M-17 metabolite suggests that the isozyme present in the Caco-2 cells is distinct from CYP3A4, which also catalyzes the formation of significant quantities of the metabolites 9gamma-hydroxy cyclosporin A (M-1) and 4N-desmethyl cyclosporin A (M-21) from CsA. Interestingly, the amount of M-17 accumulating on the apical (AP) side was much greater than that on the basolateral (BL) side during the AP --> BL transport of CsA across Caco-2 cell monolayers. This is consistent with p-glycoprotein pump-mediated efflux of the metabolite to the apical side. Furthermore, formation of the M-17 metabolite on the AP side of cell monolayers during the AP --> BL transport of CsA was much greater than that during the BL --> AP transport. This result suggests that the p-glycoprotein efflux pump causes an increase in the metabolism of CsA during the course of its AP --> BL transport by effectively slowing down the transport of CsA molecules across Caco-2 cells. Thus, Caco-2 cells serve as an excellent model to dissect the relative roles played by p-glycoprotein-mediated efflux and CYP3A-catalyzed oxidation in modulating the overall absorption of CsA and other such compounds.
Asunto(s)
Ciclosporina/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Inmunosupresores/metabolismo , Oxigenasas de Función Mixta/metabolismo , Antibacterianos/farmacología , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Ciclosporina/análisis , Ciclosporina/química , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/análisis , Humanos , Inmunosupresores/análisis , Inmunosupresores/química , Técnicas In Vitro , Oxigenasas de Función Mixta/análisis , Oxidación-Reducción , Troleandomicina/farmacologíaRESUMEN
The transepithelial transport and uptake of chloroquine were studied in cultured human intestinal Caco-2 cell layers, to investigate whether a specific mechanism facilitates the flux of chloroquine. Due to ionization of chloroquine at the pH of the intestinal lumen, the fraction of the neutral form, which is required for partitioning into biological membranes, is very low, while oral bioavailability has been reported to be nearly complete. Several observations, such as concentration-dependent uptake and temperature-dependent transepithelial flux, suggest the presence of carrier mediated transport. However, alternative mechanisms may be invoked to explain these observations. It is suggested that concentration dependence can originate from ion-trapping in acidic compartments of the cell or non-specific binding to cell components, while temperature-dependent transport can, at least partly, be explained by the temperature dependence of the acid dissociation constants of chloroquine. No differences were observed in the transepithelial flux of the enantiomers of chloroquine. pH-dependent uptake as well as pH-dependent transepithelial transport suggest that the translocation of chloroquine occurs according to the fraction of neutral molecules. From the data obtained in this study, it is concluded that chloroquine crosses the gastrointestinal barrier by passive diffusion. The extensive area of the gastrointestinal tract probably compensates for the low fraction of the neutral molecule. An interesting finding of this study was the concentration-dependent increase in transepithelial electrical resistance across monolayers incubated with chloroquine at the apical side.
Asunto(s)
Antimaláricos/metabolismo , Cloroquina/metabolismo , Mucosa Intestinal/metabolismo , Cloruro de Amonio/farmacología , Transporte Biológico , Células CACO-2/metabolismo , Humanos , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
A cultured human intestinal epithelial (Caco-2) cell monolayer was used to study the transport and metabolism of delta sleep-inducing peptide [DSIP (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu)]. DSIP is of interest because it has been reported to be capable of permeating biological barriers (e.g. blood-brain barrier), and this property has been related to its solution conformation. When applied to the apical (AP) side of Caco-2 cell monolayers, DSIP was rapidly metabolized (8.2 +/- 1.1% remaining after a 2-hr incubation), affording Trp as the major metabolite and Trp-Ala as a minor metabolite. When DSIP was added to the basolateral (BL) side of the monolayer, the same metabolites were detected, but the peptide was more stable (70.6 +/- 3.0% remaining after a 2-hr incubation). Inclusion of bestatin, an inhibitor of aminopeptidases, at concentrations up to 0.29 mM with DSIP on the AP side of the Caco-2 cell monolayer increased the stability of the peptide only slightly but dramatically altered the distribution of the metabolites (Trp-Ala became the major metabolite, and Trp became the minor metabolite). Inclusion of other aminopeptidase inhibitors (e.g. amastatin, puromycin) alone, dipeptidylpeptidase IV inhibitors (e.g. diprotin A, Gly-Pro) alone, inhibitors of proteases that require heavy metals for proper activity (e.g. EDTA, 1,10-phenanthroline) alone, or cysteine protease inhibitors (e.g. leupeptin) alone did not lead to significant stabilization of the peptide. However, inclusion of a combination of 0.29 mM bestatin and 1 mM diprotin A with DSIP on the AP side of the monolayers resulted in a substantial increase in the stability of the peptide (83.2 +/- 3.7% remaining after a 2-hr incubation). However, under these conditions, a new metabolite (Trp-Ala-Gly-Gly-Asp-Ala-Ser) was observed with a formation that could be inhibited by inclusion of 1 mM captopril, an inhibitor of peptidyl dipeptidase A. Therefore, the stability of DSIP could be further increased (95.1 +/- 1.6% remaining after a 2-hr incubation) by incubating the peptide with 0.29 mM bestatin, 1 mM diprotin A, and 1 mM captopril. However, even when the major metabolic pathways were inhibited on the AP side of the cell monolayer, no DSIP was detected on the BL side of a Caco-2 cell monolayer. These results suggest that a yet unidentified metabolic pathway is preventing the AP-to-BL flux of DSIP or that DSIP has lower "intrinsic" ability to permeate across cultured intestinal epithelial cells than across cultured brain endothelial cells, a cell culture model of the blood-brain barrier.
Asunto(s)
Péptido Inductor del Sueño Delta/metabolismo , Mucosa Intestinal/metabolismo , Secuencia de Aminoácidos , Antimetabolitos/farmacología , Transporte Biológico , Células CACO-2 , Cromatografía Líquida de Alta Presión , Células Epiteliales , Epitelio/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Intestinos/citología , Leucina/análogos & derivados , Leucina/farmacología , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , TemperaturaRESUMEN
Previous studies have shown that the metabolism of delta sleep-inducing peptide (DSIP) in the blood-brain barrier (BBB) is catalyzed by amino-peptidases. In this study, we have shown that peptidyl dipeptidase A in cultured bovine brain microvessel endothelial cells (BBMEC), a model of the BBB, and a purified form of this enzyme can also metabolize DSIP by sequential hydrolyses of dipeptides or tripeptides from the carboxyl terminus of this nonapeptide. Both the dipeptidase and tripeptidase activity associated with peptidyl dipeptidase A can be inhibited by captopril. Total stabilization of DSIP to metabolism in BBMEC could be achieved by inclusion of an inhibitor of peptidyl dipeptidase A (e.g., captopril) and an inhibitor of aminopeptidases (e.g., bestatin).
Asunto(s)
Barrera Hematoencefálica , Circulación Cerebrovascular , Péptido Inductor del Sueño Delta/metabolismo , Endotelio Vascular/metabolismo , Microcirculación , Peptidil-Dipeptidasa A/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Modelos Cardiovasculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Peptidil-Dipeptidasa A/aislamiento & purificación , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The characteristics of cyclosporin A (CsA) transport across Caco-2 monolayers were investigated. CsA (0.25-5.0 microM) was transported in a time and concentration dependent manner. The total amount of apical (AP) to basolateral (BL) transport was non-linearly related to CsA concentration from 0.25 to 1 microM and was linear from about 1 to 5 microM. Average permeability coefficient (Papp) values obtained in the AP to BL direction showed CsA concentration (0.5 and 5.0 microM) dependence, whereas those of the reverse (BL to AP) process did not. Papp values for the AP to BL direction were also markedly lower. When the P-glycoprotein pump inhibitors, chlorpromazine and progesterone, were included in the transport medium we observed a significant increase in CsA (0.5 and 5.0 microM) transport from the AP to BL direction; transport was decreased in the reverse direction. This study suggests that CsA is transported across Caco-2 cells by passive diffusion, but that a polarized efflux system (presumably a P-glycoprotein pump) located at the apical membrane can attenuate the net AP to BL transport.