矢车菊黄素在Caco-2细胞模型中的吸收机制研究

目的 研究矢车菊黄素（centaureidin）在Caco-2细胞单层模型中的吸收机制。方法 以HPLC分析矢车菊黄素浓度,用Caco-2 细胞单层模型评价吸收时间、药物浓度、介质pH值、抑制剂等对矢车菊黄素吸收的影响,研究矢车菊黄素的吸收机制,计算表观渗透系数（apparent permeability coefficient, Papp）。结果 药物的吸收与药物浓度和吸收时间正相关;弱酸性介质条件下有利于药物的吸收;2,4-二硝基酚（DNP）对药物吸收无影响,但异博定（verapamil）可增加药物的吸收;从肠腔侧到基底侧的转运小于基底侧到肠腔侧的转运。结论 矢车菊黄素在Caco-2细胞模型中的吸收主要是被动转运,受P-糖蛋白的外排作用。     

Transepithelial Transport of Diphenhydramine Across Monolayers of the Human Intestinal Epithelial Cell Line Caco-2

Purpose. The transepithelial transport characteristics of theantihistamine, diphenhydramine, were studied in human intestinal Caco-2 cellmonolayers to elucidate the mechanisms of its intestinal absorption. Methods. The transepithelial transport and the cellular accumulationof diphenhydramine were measured using Caco-2 cell monolayersgrown in Transwell chambers. Results. The transepithelial transport of diphenhydramine from theapical to basolateral side was saturable, and the flux and cellularaccumulation of diphenhydramine were dependent on the apicalextracellular pH (pH 7.4 > 6.5 > 5.5). Transport and accumulation ofdiphenhydramine from the apical side were inhibited by anotherantihistamine, chlorpheniramine, while typical substrates for the renal organiccation transport system such as tetraethylammonium, cimetidine andguanidine had no effect. The transepithelial transport and cellularaccumulation of diphenhydramine from the basolateral side were alsopH-dependent and inhibited by chlorpheniramine. In addition, intracellulardiphenhydramine preloaded was preferentially effluxed to the apicalside, suggesting the involvement of the secretory pathway indiphenhydramine transport. Furthermore, diphenhydramine uptake from boththe apical and basolateral sides was stimulated by preloadingmonolayers with chlorpheniramine (trans-stimulation effect). Conclusions. Transepithelial transport of diphenhydramine acrossCaco-2 cells is mediated by pH-dependent, specific transport systemsthat exist in both the apical and basolateral membranes.     

Binding and Transport of Some Bioadhesive Plant Lectins Across Caco-2 Cell Monolayers

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Stereoselective and Carrier-Mediated Transport of Monocarboxylic Acids Across Caco-2 Cells

Purpose. To characterize the transport mechanism of monocarboxylic acids across intestinal epithelial cells by examining the stereoselectivity of the transcellular transport of several chiral monocarboxylic acids. Methods. The transport of monocarboxylic acids was examined using monolayers of human adenocarcinoma cell line, Caco-2 cells. Results. The permeability of L-[¹⁴C]lactic acid at a tracer concentration (1 µM) exhibited pH- and concentration-dependencies and was significantly greater than that of the D-isomer. The permeabilities of both L-/ D-[¹⁴C]lactic acids involve saturable and nonsaturable processes; the saturable process showed a higher affinity and a lower capacity for L-lactic acid compared with the D-isomer, while no difference between the isomers was seen for the nonsaturable process. The transport of L-lactic acid was inhibited by chiral monocarboxylic acids such as (R)/(S)-mandelic acids and (R)/(S)-ibuprofen in a stereoselective manner. Mutually competitive inhibition was observed between L-lactic acid and (S)-mandelic acid. Conclusions. Some chiral monocarboxylic acids are transported across the intestinal epithelial cells in a stereoselective manner by the specific carrier-mediated transport mechanism.     

Transport of Proteolytic Enzymes Across Caco-2 Cell Monolayers

Purpose. To investigate the mechanisms by which proteolytic enzymes, such as trypsin, chymotrypsin, papain, and bromelain, are able to cross the intestinal mucosal barrier after oral administration to man. Methods. Filter-grown Caco-2 cell monolayers were incubated with proteolytic enzymes and then the transepithelial electrical resistance (TEER) and the transport of the paracellular marker fluorescein were monitored. The effects of the enzymes on the cells were investigated by light microscopy and by biochemical assays. Transport of intact proteases across the cells was verified by monitoring the proteolytic activity and MALDI-TOF mass spectroscopic identification of undegraded trypsin. Results. Depending on time, concentration, and side of exposure to Caco-2 cell monolayers, all proteases decreased the TEER and increased the transport of fluorescein. Some morphological and metabolic changes were observed. The effects were reversible, but until 24 hours after removal of the proteases. Under the conditions of this in-vitro model, approximately 10% of the apically applied dose reached the basolateral compartment as biologically active, non-degraded molecules. Conclusions. Proteolytic enzymes were found to exert considerable effects on the barrier function of Caco-2 monolayers, facilitating the transport of normally non-absorbable compounds. This suggests the also reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may occur by self-enhanced paracellular transport.     

Transport Characteristics of Peptidomimetics. Effect of the Pyrrolinone Bioisostere on Transport Across Caco-2 Cell Monolayers

Purpose. To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. Methods. Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. Results. In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (P_app) calculated for the BL-to-AP and AP-to-BL transport (P_BLAP/P_APBL) varied between 1.7 and 36.2. When individual pairs were compared, P_BLAP/P_APBL ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 M cyclosporin A to the transport buffer reduced the P_BLAP /P_APBL ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 M cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. Conclusions. The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.     

Caco-2 Cell Monolayers as a Model for Drug Transport Across the Intestinal Mucosa

Human colon adenocarcinoma (Caco-2) cells, when grown on semipermeable filters, spontaneously differentiate in culture to form confluent monolayers which both structurally and functionally resemble the small intestinal epithelium. Because of this property they show promise as a simple, in vitro model for the study of drug absorption and metabolism during absorption in the intestinal mucosa. In the present study, the transport of several model solutes across Caco-2 cell monolayers grown in the Transwell diffusion cell system was examined. Maximum transport rates were found for the actively transported substance glucose and the lipophilic solutes testosterone and salicylic acid. Slower rates were observed for urea, hippurate, and salicylate anions and were correlated with the apparent partition coefficient of the solute. These results are similar to what is found with the same compounds in other, in vivo absorption model systems. It is concluded that the Caco-2 cell system may give useful predictions concerning the oral absorption potential of new drug substances.     

Mechanism of Intestinal Absorption of Ranitidine and Ondansetron: Transport Across Caco-2 Cell Monolayers

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability coefficients (P _app) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. P _app values for ranitidine and ondansetron (bioavailability of 50 and 100% in humans, respectively) were 1.03 ± 0.17 × 10^–7 and 1.83 ± 0.055 × 10^–5 cm/sec, respectively. The P _app value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.     

Carrier-Mediated Transport of Macrolide Antimicrobial Agents Across Caco-2 Cell Monolayers

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Increased Transport of Resveratrol Across Monolayers of the Human Intestinal Caco-2 Cells is Mediated by Inhibition and Saturation of Metabolites

Purpose The study's aim was to investigate the dose-dependent effect of sulfation and glucuronidation on intestinal absorption of resveratrol, a dietary constituent found in grapes and various medical plants.Materials and Methods The intestinal epithelial membrane transport kinetics and metabolism of resveratrol (10–200 μM) was studied using Caco-2 monolayers cultured in Transwells.Results Along with resveratrol it was possible to identify three metabolites, namely, resveratrol-4′-O-glucuronide (M1), resveratrol 3-O-gucuronide (M2), and resveratrol-3-O-sulfate (M3) by LC/MS and NMR. Efflux of the glucuronides M1 and M2 followed Michaelis–Menten kinetics significantly favouring basolateral efflux. The predominant metabolite was the monosulfate M3, however, its formation was strongly inhibited at higher resveratrol concentrations. As biotransformation was either inhibited or saturated, total amount of resveratrol transported across the Caco-2 monolayers increased as much as 3.5-fold at 200 μM resveratrol. This value might be even higher when taking into account the high intracellular concentration of resveratrol, which accounted for up to 61% of the applied dose.Conclusions Our data demonstrate a concentration-dependent biotransformation of resveratrol in Caco-2 cells, which may also apply to human enterocytes affecting oral bioavailability.     

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

Purpose. The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl --aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA). Methods. Pregabalin transport was studied in rat intestine and Caco-2 monolayers. An in vitro Ussing/diffusion chamber model and an in situ single-pass perfusion model were used to study rat intestinal transport. An in vitro diffusion chamber model was used to evaluate Caco-2 transport. Results. In rat ileum, pregabalin transport was saturable and inhibited by substrates of intestinal LNAA carriers including neurontin (gabapentin), phenylalanine, and proline. Weak substrates of intestinal LNAA carriers (-alanine, --aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 mono-layers that showed a high capacity for phenylalanine transport, pregabalin transport was concentration- and direction-independent and equivalent in magnitude to the paracellular marker, mannitol. The in vitro and in situ rat ileal permeabilities of the LNAA carrier-mediated compounds neurontin, pregabalin, and phenylalanine correlated well with the corresponding in vivo human oral absorption. Conclusions. The transport of pregabalin was mediated by LNAA carriers in rat ileum but not in Caco-2 monolayers. Caco-2 was not an appropriate model for evaluating the in vivo human oral absorption of pregabalin and neurontin.     

Transport of Peptidomimetic Renin Inhibitors Across Monolayers of a Human Intestinal Cell Line (Caco-2): Evidence for Self-Enhancement of Paracellular Transport Route

Pharmaceutical Research -     

Comparison of Bidirectional Cephalexin Transport Across MDCK and Caco-2 Cell Monolayers: Interactions with Peptide Transporters

Purpose. Bidirectional transport studies were conducted to determine whether Madin-Darby canine kidney (MDCK) cell monolayers could be used as an alternative to the traditional Caco-2 assay as a fast-growingin vitro model of peptide transport. Methods. Transport of cephalexin and glycylsarcosine across MDCK and Caco-2 cell monolayers was quantified using LC-LC/MS. Glycylsarcosine, p-aminohippuric acid (PAH), and tetraethylammonium chloride (TEA) were tested as inhibitors of cephalexin transport. Results. The ratio of apparent cephalexin permeabilities (apical to basolateral/basolateral to apical) obtained from MDCK monolayers was almost 5-fold greater than that obtained from Caco-2 monolayers. The opposite trend was observed for glycylsarcosine. When MDCK monolayers were used, glycylsarcosine reduced the cephalexin/apparent permeability ratio almost 90%. PAH and TEA did not inhibit cephalexin transport across MDCK or Caco-2 cell monolayers. Conclusion. MDCK cell monolayers may be a promising, fast-growing alternative to Caco-2 cells for identifying peptide transporter substrates. However, differences in the apical-to-basolateral transport of cephalexin and glycylsarcosine suggest that the basolateral transport mechanisms for these compounds are different in the two cell lines. Additionally, because the activity of the peptide transporter in MDCK cells was low, scaling factors may be required when using this cell line to predict in vivo drug absorption.     

Transcellular Transport of Benzole Acid Across Caco-2 Cells by a pH-Dependent and Carrier-Mediated Transport Mechanism

The pH-dependent transcellular transport of [¹⁴ C]benzoic acid across a Caco-2 cell monolayer is shown to be mediated by a monocarboxylic acid-specific carrier-mediated transport system, localized on the apical membrane. Evidence for the carrier-mediated transport of benzoic acid includes (a) the significant temperature and concentration dependence, (b) the metabolic energy dependence, (c) the inhibition by unlabeled benzoic acid and other monocarboxylic acids, (d) countertransport effects on the uptake of [¹⁴C]benzoic acid, and (e) effects of a proteinase (papain) and amino acid-modifying reagents. Furthermore, since carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin significantly inhibited the transport of [¹⁴C] benzoic acid, the direct driving force for benzoic acid transport is suggested to be the inwardly directed proton gradient. From these results, together with previous observations using intestinal brush border membrane vesicles, the pH dependence of the transcellular transport of certain organic weak acids across Caco-2 cells is considered to result mainly from a proton gradient-dependent, carrier-mediated transport mechanism, rather than passive diffusion according to the pH-partition theory.     

Quantification and Imaging of Mannitol Transport Through Caco-2 Cell Monolayers Using a Positron-Emitting Tracer

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Dipeptide Transport Across Rat Alveolar Epithelial Cell Monolayers

The transepithelial transport and metabolism of two model peptides, glycyl-D-phenylalanine (Gly-D-Phe) and glycyl-L-phenylalanine (Gly-L-Phe), across primary cultured monolayers of rat alveolar epithelial cells were studied. These tight monolayers (>2000 -cm²) exhibited type I pneumocyte morphological and phenotypic characteristics. A reverse-phase HPLC was used to monitor the appearance of parent dipeptides and their metabolites (D- or L-Phe) in the receiver fluid. The apparent permeability coefficient (P_app) for Gly-D-Phe was about 1.6 × 10^–7 cm/sec at both 1 and 10 mM and in both the apical-to-basolateral (AB) and the basolateral-to-apical (BA) directions. In contrast, the P _app of Gly-L-Phe at 1 mM was about two times higher than that at 10 mM in the AB direction. The P _app of Gly-L-Phe in the BA direction at either concentration was about the same (about 1.4 × 10^–7 cm/sec). Whereas no metabolite was detected during Gly-D-Phe transport, the proportions of a metabolite, L-Phe, observed at 4 hr in the basolateral receiver fluid for 1 and 10 mM apical donor Gly-L-Phe accounted for 83 and 77% of the estimated total Gly-L-Phe (i.e., L-Phe + Gly-L-Phe), respectively. The corresponding values in the BA direction were 40 and 19% of the estimated total Gly-L-Phe in the apical receiver reservoir. Metabolism of Gly-L-Phe was significantly reduced in the presence of 3 µM actinonin (an inhibitor relatively specific for aminopeptidase M) in the apical but not the basolateral fluid. Under all experimental conditions, the monolayers remained intact, as indicated by no appreciable changes in the bioelectric parameters of transepithelial potential difference and electrical resistance. The above data provide evidence for cellular metabolism of Gly-L-Phe as well as paracellular restricted diffusional transport of intact Gly-D-Phe and Gly-L-Phe and comparatively lower transcellular transport of Gly-L-Phe across the rat alveolar epithelial cell monolayer.     

Transport of Poly(Amidoamine) Dendrimers across Caco-2 Cell Monolayers: Influence of Size, Charge and Fluorescent Labeling

Purpose To investigate the transport of poly(amidoamine) (PAMAM) dendrimers with positive, neutral and negatively charged surface groups across Caco-2 cell monolayers. Methods Cationic PAMAM-NH₂ (G2 and G4), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5–G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC). The permeability of fluorescently labeled PAMAM dendrimers was measured in the apical-to-basolateral direction. ¹⁴C-Mannitol permeability was measured in the presence of unlabeled and FITC labeled PAMAM dendrimers. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin or rhodamine phalloidin, and visualized using confocal laser scanning microscopy to examine tight junction integrity. Results The overall rank order of PAMAM permeability was G3.5COOH > G2NH₂ > G2.5COOH > G1.5COOH > G2OH. ¹⁴C-Mannitol permeability significantly increased in the presence of cationic and anionic PAMAM dendrimers with significantly greater permeability in the presence of labeled dendrimers compared to unlabeled. PAMAM dendrimers had a significant influence on tight junction proteins occludin and actin, which was microscopically evidenced by disruption in the occludin and rhodamine phalloidin staining patterns. Conclusions These studies demonstrate that enhanced PAMAM permeability is in part due to opening of tight junctions, and that by appropriate engineering of PAMAM surface chemistry it is possible to increase polymer transepithelial transport for oral drug delivery applications.     

Efflux Ratio Cannot Assess P-Glycoprotein-Mediated Attenuation of Absorptive Transport: Asymmetric Effect of P-Glycoprotein on Absorptive and Secretory Transport Across Caco-2 Cell Monolayers

Purpose. The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. Methods. Studies were performed in Caco-2 cell monolayers. Apparent permeability (P _app) in absorptive (P _app,AB) and secretory (P _app,BA) directions as well as efflux ratios (P _app,BA / P _app,AB) were determined for substrates as a function of concentration. Transport of these compounds (10 M) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 M), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. Results. Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent K _m for P-gp-mediated efflux of digoxin was >6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent K _m was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent J _max was somewhat similar in both transport directions. Conclusions. P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent K _m of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.     

Mechanisms of Transport and Structure-Permeability Relationship of Sulfasalazine and Its Analogs in Caco-2 Cell Monolayers

Purpose. To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells. Methods. Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters. Results. Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BLAP) than from apical to basolateral direction (APBL) at 100, 200, and 500 M, respectively. Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4×10^–5 cm/s, 1.9×10^–8 cm/s, and 369 M, respectively. The efflux of sulfasalazine was completely blocked at 4°C and in the presence of an uncoupler of oxidative phosphorylation. Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms. Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group. Conclusions. The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters. This may partially explain its low absorption in vivo.