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。     

Epithelial transport of Noscapine across cell monolayer and influence of absorption enhancers on in vitro permeation and bioavailability: implications for intestinal absorption

Abstract

The purpose of this study was to investigate the permeation of Noscapine (Nos) across the Caco-2 and Madin–Darby canine kidney (MDCK) cell monolayers and to evaluate the influence of absorption enhancers on in vitro and in vivo absorption of Nos. The bidirectional transport of Nos was studied in Caco-2 and MDCK cell monolayers at pH 5.0–7.8. The effect of 0.5% w/v chitosan (CH) or Captisol (CP) on Nos permeability was investigated at pH 5.0 and 5.8. The effect of 1–5% w/v of CP on oral bioavailability of Nos (150?mg/kg) was evaluated in Sprague–Dawley rats. The effective permeability coefficients (P_eff) of Nos across Caco-2 and MDCK cell monolayers was found to be in the order of pH 5.0?>?5.8?>?6.8?>?7.8. The efflux ratios of P_eff?<?2 demonstrated that active efflux does not limit the absorption of Nos. The use of CH or CP have shown significant (***, p?<?0.001) enhancement in P_eff of Nos across cell monolayer compared with the control group. The CP (1–5% w/v) based Nos formulations resulted in significant (***, p?<?0.001) increase in the bioavailability of Nos compared with Nos solution. The use of CP represents viable approach for enhancing the oral bioavailability of Nos and reducing the required dose.     

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.     

Fenton chemistry and iron chelation under physiologically relevant conditions: Electrochemistry and kinetics

The goal of iron-chelation therapy is to reduce the levels of labile plasma iron, and intravenously administered desferrioxamine is the gold standard of therapeutic agents. Hydroxypyridinones, e.g., CP20 (3-hydroxy-1,2-dimethylpyridin-4(1H)-one), are used or are under investigation as orally administered iron chelators. We determined electrode potentials of CP20, the related hydoxypyridones CP361, CP363, and CP502, and ICL670 (4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic acid) under physiologically relevant conditions to address the question of whether iron in the presence of these chelating agents can carry out Fenton chemistry in vivo. We found that iron(III) but not iron(II) binds tightly to both CP20 and ICL670 at pH 7 and higher, compared to nearly complete binding of 1 microM iron(II) to 10 microM desferrioxamine at pH 7.4 The electrode potentials of the hydroxypyridinones shift to more negative values with decreasing pK(a) values at lower concentrations of iron(III) (0.02 mM) and ligand (0.1 mM). The electrode potential of the iron-CP20 system decreases as a function of increasing pH, with a minimum near pH 10.5. We estimate an electrode potential for the ascorbyl radical/ascorbate couple under physiological conditions of +105 mV, which is higher than the electrode potential of the iron(III) complex of CP20 at all concentrations of iron. The rate of oxidation of iron(II) in the presence of CP20 by hydrogen peroxide increases with the concentrations of both ligand and peroxide. Although iron(II) is oxidized by hydrogen peroxide, the thus-formed Fe(III)(CP20)(3) complex cannot be reduced by ascorbate. Therefore, the tight binding of iron(III) by this class of chelators prevents redox cycling.     

Transport characteristics of L-carnosine and the anticancer derivative 4-toluenesulfonylureido-carnosine in a human epithelial cell line

Purpose. The aim of the present study was to evaluate whether the transepithelial transport of the anticancer compound 4-toluenesulfonylureido-carnosine (Ts-carnosine) and the dipeptide moiety L-carnosine was due to a hPepT1 carrier-mediated flux. Methods. Transport experiments were conducted using Caco-2 cell monolayers and either reversed-phase HPLC-UV or liquid scintillation counting methods for quantification. pK_a, LogD, and LogP were determined using the Sirius GlpK_a meter. Results. L-carnosine was transported across the apical membrane with a K_m,app of 2.48 ± 1.16 mM and a V_max of 2.08 ± 0.34 nmol · cm^–2 · min^–1 and across the basolateral membrane with a K_m,app of 7.21 ± 3.17 mM and a V_max of 0.54 ± 0.10 nmol · cm^–2 · min^–1, and transepithelially with a P_app of 4.46 · 10^–2 ± 6.4 · 10^–6 cm · min^–10. Ts-carnosine had an affinity (K_i) for hPepT1 of 2.33 ± 0.54 mM; however, the transepithelial transport was low as compared to that of L-carnosine. Conclusions. L-carnosine was transported across both the apical and basolateral membrane of Caco-2 cell monolayers in a carrier-mediated manner however, the transepithelial transport followed apparent simple non-saturable kinetics. Ts-carnosine had an affinity for hPepT1 but a relatively low transepithelial transport. This indicates that the transepithelial transport of L-carnosine and Ts-carnosine is not hPepT1 carrier-mediated and that L-carnosine is not a suitable dipeptide moiety for hPepT1-mediated absorption of sulfonamide-type anticancer compounds.     

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.     

Transport of a novel anti-cancer agent,fenretinide across Caco-2 monolayers

Summary Fenretinide is a synthetic retinoid with chemotherapeutic activity against various malignancies. Upon oral administration to animals, fenretinide was found to be incompletely absorbed and excreted primarily in feces. The purpose of this study was to determine the possible reasons for poor oral absorption of fenretinide using Caco-2 cell monolayers. To achieve this purpose, a solid dispersion of fenretinide with Povidone K25 was used. The apparent permeability coefficient (P _app) of fenretinide across Caco-2 monolayers in the presence of bovine serum albumin (BSA) in the receiver was determined. Apical to basolateral (AP-BL) and basolateral to apical (BL-AP) flux studies were performed to determine the role of an efflux mechanism. In the presence of 4% BSA in the receiver, the P _app was found to be (8.8 ± 0.5) × 10⁻⁸ cm/sec. The AP-BL flux increased linearly with an increase in fenretinide concentration (125–640 μM) in the presence of 4% BSA in the receiver. Efflux and paracellular pathways played an insignificant role in the permeability of fenretinide. A significant amount of drug, approximately 13–15% of the initial amount accumulated in the cell membrane. The amount of fenretinide in the donor decreased by 16% over a 3 h period. However, only 0.12% of the initial amount was found in the receiver. Also, the P _app increased with an increase in plasma protein concentration in the receiver. On the basis of these results, the poor permeability of fenretinide can be attributed to its accumulation in the lipophilic cell membrane and poor partitioning into the receiver medium.     

Drug delivery studies in Caco-2 monolayers. III. Intestinal transport of various vasopressin analogues in the presence of lysophosphatidylcholine

The transport of a series of vasopressin and oxytocin analogues with varying lipophilicities was studied in Caco-2 monolayers. Transport was studied across the bare monolayer and after treatment with a phospholipid absorption enhancer, palmitoyl lysophosphatidylcholine. The range in lipophilicity of the analogues, estimated as the capacity factor, was found to be from 0.19 to 3.43. The intrinsic transport of the peptides across Caco-2 monolayers was found to be low. The apparent permeability coefficients, P_app, were in the range of 2 × 10⁻⁸–6 × 10⁷ cm/s. However, peptide transport was significantly greater (P_app in the range of 5 × 10⁻⁶–2 × 10⁻⁵ cm/s) when facilitated by addition of palmitoyllysophosphatidylcholine. The results suggest that polypeptide transport across Caco-2 monolayers does not depend on lipophilicity, but that the facilitated transport does depend on the lipophilicity.     

The Transport and Efflux of Glycosylated Luteinising Hormone-Releasing Hormone Analogues in Caco-2 Cell Model: Contributions of Glucose Transporters and Efflux Systems

Luteinising hormone-releasing hormone (LHRH) analogues have wide therapeutic applications in the treatment of prostate cancers and endocrine disorders. The structure of LHRH was modified using a glycosylation strategy to increase the permeability of the peptide across biological membranes. Lactose, galactose and glucose units were coupled to LHRH peptide, and the impact of glucose transporters, GLUT2 and SGLT1, was investigated in the transport of the analogues. Results showed the contribution of both transporters in the transport of all LHRH analogues. In the presence of glucose transporter inhibitors, reduction in the apparent permeability (P_app) was greatest for compound 6, which contains a glucose unit in the middle of the sequence (P_app = 58.54 ± 4.72 cm/s decreased to P_app = 1.6 ± 0.345 cm/s). The basolateral to apical flux of the glycosylated derivatives and the impact of two efflux pumps was also examined in Caco-2 cell monolayers. The efflux ratios (ERs) of all LHRH analogues in Caco-2 cells were in the range of 0.06–0.2 except for compound 4 (galactose modified, ER = 8.03). We demonstrated that the transport of the glycosylated peptides was facilitated through glucose transporters. The proportion of glucose and lactose derivatives pumped out by efflux pumps did not affect the P_app values of the analogues.     

Permeability Characteristics of Tetragastrins Across Intestinal Membranes Using the Caco-2 Monolayer System: Comparison Between Acylation and Application of Protease Inhibitors

Purpose. Three types of acyl tetragastrin (TG), acetyl-TG (C₂-TG), butyryl-TG (C₄-TG) and caproyl-TG (C₆-TG) were synthesized and their in vitro intestinal permeability characteristics were examined using Caco-2 monolayers. Methods. The disappearance of acyl-TGs from the apical side of Caco-2 monolayers was estimated by analyzing degradation and permeation processes in terms of clearance. Results. The amount of native TG transported to the basolateral side was very low due to its large degradation clearance (CLd) on the apical side. Degradation of TG was reduced by chemical modification with fatty acids, which resulted in an increase in the transport of TG across Caco-2 monolayers. In addition, the permeation clearance (CLp) value of carboxyfluorescein (CF), a paracellular transport and undegradable marker, was increased in the presence of acyl-TGs. Furthermore, we investigated the effects of the protease inhibitors bacitracin and gabexate on the transport of TG across Caco-2 monolayers. In the presence of a low concentration (0.1 mM) of protease inhibitor, the CLd value of TG was reduced, but they did not affect its CLp value. However, a higher concentration (1.0 mM) of bacitracin significantly reduced TG degradation on the apical side, and further increased its CLp value. Conclusions. We demonstrated that acylation of TG made it resistant to intestinal proteases and caused it to enhance absorption of drugs, including itself, across Caco-2 monolayers. Further, bacitracin acted as both a protease inhibitor and an absorption enhancer.     

Isolation and Characterization of Caco-2 Subclones Expressing High Levels of Multidrug Resistance Protein Efflux Transporter

Purpose. The purpose of this study was to isolate Caco-2 subclones that express high levels of multidrug resistance protein (MDR1) and to characterize their kinetics and affinity parameters for MDR1 substrate/inhibitors. Methods. The subclones were selected by a dilution cloning technique. The polarized efflux of [³H]-vinblastine across subclone cell monolayers was quantified by measuring the apparent permeability coefficients (P_app) of [³H]-vinblastine in the basolateral (BL)-to-apical (AP) direction and in the AP-to-BL direction (P_{app BL-to-AP}/P_{app AP-to-BL}) across the cell monolayers. The expression of MDR1 in the Caco-2 subclones compared with the parental Caco-2 cells was confirmed by Western blotting analysis. The kinetics parameters (K _m, V _max) of [³H]-vinblastine and the inhibitory constants (K _I) of several known MDR1 substrates/inhibitors on the transport of [³H]-digoxin determined in the parental Caco-2 cells and Caco-2 subclones were also compared. Results. Three subclones (#1, #20, #21) were selected based on their polarized efflux of [³H]-vinblastine. The P_{app BL-to-AP}/P_{app AP-to-BL} ratios for #1, #20, and #21 were 110, 140, and 112, respectively, and were about 6-fold higher than the ratio observed for the parental Caco-2 cells. In the presence of GF-120918 (2 M), a known MDR1-specific inhibitor, the P_{app BL-to-AP}/P_{app AP-to-BL} ratios were significantly decreased, suggesting that these cells were overexpressing MDR1. The K _m values observed for vinblastine in the Caco-2 subclones were nearly identical to the value observed in the parental Caco-2 cells. In contrast, the V _max values observed in the subclones were approximate 26-69% higher. The K _I values observed for various known MDR1 substrates/inhibitors on [³H]-digoxin transport were nearly identical to those in the parental Caco-2 cells and Caco-2 subclones. The high functional efflux activities of these subclones were stable up to 6 months. Conclusions. Subclones #1, #20, #21 express high levels of MDR1. These Caco-2 subclones may be useful models for profiling drugs for their MDR1 substrate activity and for establishing structure-transport relationships for this efflux transporter.     

Engineering of Dendrimer Surfaces to Enhance Transepithelial Transport and Reduce Cytotoxicity

Purpose. To evaluate the cytotoxicity, permeation, and transport mechanisms of PAMAM dendrimers and surface-modified cationic PAMAM dendrimers using monolayers of the human colon adenocarcinoma cell line, Caco-2. Methods. Cytotoxicity was determined using the MTT assay. The effect of dendrimers on monolayer integrity was determined from measurements of transepithelial electrical resistance (TEER) and [¹⁴C]mannitol apparent permeability coefficient (P_app). The P_app of dendrimers through monolayers was measured in both the apical (A)-to-basolateral (B) and BA directions at 4°C and 37°C and also in the presence and absence of ethylenediamine tetraacetic acid (EDTA) and colchicine. Results. The cytotoxicity and permeation of dendrimers increased with both concentration and generation. The cytotoxicity of cationic dendrimers (G2, G3, G4) was greater than that of anionic dendrimers (G2.5, G3.5) but was reduced by conjugation with lauroyl chloride; the least cytotoxic conjugates were those with six attached lauroyl chains. At 37°C the P_app of cationic dendrimers was higher than that of anionic dendrimers and, in general, increased with the number of attached lipid chains. Cationic dendrimers decreased TEER and significantly increased the P_app of mannitol. Modified dendrimers also reduced TEER and caused a more marked increase in the P_app of mannitol. The P_app values of dendrimers and modified dendrimers were higher in the presence of EDTA, lower in the presence of colchicine, and lower at 4°C than at 37°C. Conclusions. The properties of dendrimers may be significantly modified by surface engineering. Conjugation of cationic PAMAM dendrimers with lauroyl chloride decreased their cytotoxicity and increased their permeation through Caco-2 cell monolayers. Both PAMAM dendrimers and lauroyl-PAMAM dendrimer conjugates can cross epithelial monolayers by paracellular and transcellular pathways.     

Correlation Between Oral Drug Absorption in Humans,and Apparent Drug Permeability in TC-7 Cells,A Human Epithelial Intestinal Cell Line: Comparison with the Parental Caco-2 Cell Line

Purpose. To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (P _app ) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. Methods. Both cell lines were grown for 5–35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. P _app were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. Results. The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The P_app and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and P_app values, allowing to determine a threshold value for P_appof 2 10^–6 cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. Conclusions. On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.     

Rhodamine 123 Requires Carrier-Mediated Influx for Its Activity as a P-Glycoprotein Substrate in Caco-2 Cells

Purpose. The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells. Methods. Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity). Results. R123 secretory P _app (P _app,BA) showed concentration dependence, whereas R123 absorptive P _app (P _app,AB) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P _app,AB, but enhanced R123 and doxorubicin P _app,BA. In calcium-free medium, R123 P _app,AB increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable. Conclusions. R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.     

Effects of dietary flavonoids on the transport of cimetidine via P-glycoprotein and cationic transporters in Caco-2 and LLC-PK1 cell models

1.?The hypotheses tested were to study cimetidine as a substrate of P-glycoprotein (P-gp) and organic cation transport systems and the modulatory effects of eight flavonoid aglycones and glycosides on these transport systems using Caco-2 and LLC-PK1 cells.

2.?Transport and uptake experiments of (20 µM) ³H-cimetidine were performed with and without co-exposure to quercetin, quercetrin, rutin, naringenin, naringin, genistein, genistin, and xanthohumol. Co-treatment decreased basolateral to apical (B to A) permeability (P_app) of cimetidine from 2.02 to 1.24 (quercetin), 1.06 (naringenin), 1.24 (genistein), and 0.96 (xanthohumol) × 10^?6 cm s^?1 in Caco-2 cells and from 10.76 to 1.65 (quercetin), 2.05 (naringenin), 2.88 (genistein), and 1.95 (xanthohumol) × 10^?6 cm s^?1 in LLC-PK1 cells. Genistin significantly reduced B to A P_app of cimetidine to 1.24 × 10^?6 cm s^?1 in Caco-2 cells. Basolateral intracellular uptake rate of cimetidine was enhanced 145–295% when co-treated with flavonoids. Co-treatment with P-glycoprotein and organic cation transporter inhibitors, verapamil and phenoxybenzamine, resulted in reduced B to A permeability and slower basolateral intracellular uptake rate of cimetidine. Intracellular uptake rate of ¹⁴C-tetraethylammonium (TEA) was reduced in the presence of quercetin, naringenin and genistein in LLC-PK1 cells.

3.?In conclusion, quercetin, naringenin, genistein, and xanthohumol reduced P-gp-mediated transport and increased the basolateral uptake rate of cimetidine. Quercetin, naringenin, genistein, but not xanthohumol, reduced intracellular uptake rate of TEA in LLC-PK1 cells. These results suggest that flavonoids may have potential to alter the disposition profile of cimetidine and possibly other therapeutics that are mediated by P-gp and/or cation transport systems.     

Permeability of plumbagin across human intestinal cell in vitro

Plumbagin is the active compound isolated from plants used in traditional medicine for treatment of various diseases such as activities malaria, leishmaniasis, viral infections and cancers. The aim of the study was to investigate the permeability of plumbagin across Caco-2 (human epithelial colorectal adenocarcinoma) cell monolayer and its effects on the expression and function of P-glycoprotein. The integrity of Caco-2 cell monolayer was evaluated by measuring trans-epithelial electrical resistance and permeation (P_app) of Lucifer yellow across the cell monolayer. The effect of plumbagin on P-glycoprotein was detected by measuring its interference with the transport of the P-glycoprotein substrate (R123) and the effect on MDR-1 mRNA expression was detected by RT-PCR. The P_app of plumbagin (2–8 µM) for the apical to basolateral and basolateral to apical directions were 10.29–15.96 × 10^?6 and 7.40–9.02 × 10^?6 cm/s, respectively, with the efflux ratios of 0.57–0.73. Plumbagin is not either a substrate or inhibitor of P-glycoprotein. It did not interfere with the P-glycoprotein-mediated R123 transport across Caco-2 cell monolayer, as well as the function of P-glycoprotein and the expression of MDR-1 mRNA. Results suggest moderate permeability of plumbagin across the Caco-2 cell monolayer in both directions. The transport mechanism is likely to be a passive transport.     

Characterization of the transport of uracil across Caco-2 and LLC-PK1 cell monolayers

Purpose. The purpose of this study was to characterize the transport of uracil, a pyrimidine nucleobase, in Caco-2 and LLC-PK₁ cells. Methods. Caco-2 and LLC-PK₁ cells were grown to confluency on a permeable polycarbonate membrane insert to permit transport and uptake experiments after the loading of uracil on either the apical or basolateral side. Results. The vectorial transport of uracil in both directions was saturable with comparable K_m and V_max in Caco-2 cell monolayers, probably because of a Na⁺-independent transport system located on the basolateral membrane. In LLC-PK₁ cell monolayers, two distinct transport systems, namely a Na⁺-dependent and a Na⁺-independent, were functional in the apical to basolateral (A-B) transport of uracil. The first system was saturable with a K_m value of 3.67 ± 0.40 M, a V_max of 11.31 ± 0.91 pmol/cm²/min, and a Na⁺:uracil coupling stoichiometry of 1.28 ± 0.20. The second system was Na⁺ independent and satuable with a low affinity (K_m, 50.37 ± 9.61 M) and V_max (16.01 ± 4.48 pmol/cm²/min). The two transport systems appeared to be located on the apical membrane. Conclusion. The mechanism of uracil transport differs depending on cell lines; a Na⁺-independent system on the basolateral membrane in Caco-2 cells and both Na⁺-dependent and Na⁺-independent systems on the apical membrane in LLC-PK₁ cells seem to be responsible for the difference.     

Intestinal Absorption Screening of Mixtures from Combinatorial Libraries in the Caco-2 Model

Purpose. Understanding how chemical structures influence transport across the intestinal mucosa will greatly enhance the discovery of orally available drugs. In an attempt to accelerate defining such relationships between structure and transport, six arbitrary mixtures of N-substituted glycine (NSG) peptoids containing 24 physicochemically diverse compounds were evaluated in the Caco-2 model of intestinal absorption. Methods. Samples were analyzed by HPLC and the areas of the peaks representing the components of each mixture were summed to measure 'aggregate' apparent permeability coefficients (P _app) a score of the influence of the common structural element within each mixture towards absorption. Mass spectrometry was used to identify the chemical structure of Caco-2 permeable compounds. Results. Three linear trimeric mixtures were examined and, for each mixture, none of the components was detected in receiver chambers. It was concluded that the components of these mixtures each had a P _app value less than 0.8 × 10^–6 cm/sec, a permeability less than mannitol. Three dimeric mixtures were examined and they exhibited aggregate P _app values of 9.2 × 10^–6 , 14 × 10^–6 and 6.9 × 10^–6 cm/sec. These transport rates reflected the transport of most of the components of each mixture. Furthermore, the components of the dimeric mixtures which were transported at a rate greater than mannitol were apparently transported by passive mechanisms. Conclusions. This study demonstrated that mixtures can be used to study structure-transport relationships in the Caco-2 model. The information obtained from this type of study will be integrated into the design of future chemical libraries. Other potential uses of chemical mixtures with the Caco-2 model are also discussed.     

In Vitro Permeability Across Caco-2 Cells (Colonic) Can Predict In Vivo (Small Intestinal) Absorption in Man—Fact or Myth

Purpose. To evaluate and optimize the use of Caco-2 cell monolayers to predict thein vivo absorption of a broad range of compounds in man. Methods. Caco-2 cells are derived from human adenocarcinoma colon cells and spontaneously differentiate when grown on porous polyethylene terephthalate membranes (PETP) in a 12 well format to form monolayers of polarized cells possessing function similar to intestinal enterocytes. Transport experiments were conducted using 21 day cultured cells in a shaking water bath at 37°C. Radiolabeled mannitol was used to determine monolayer integrity. Apparent permeability coefficient (P_app) was calculated from the appearance of drug in the receiver side. Results. A strong correlation was observed between in vivo human absorption and in vitro P_app for a variety of compounds (R = 0.95, N = 35). For compounds that are substrates of p-glycoprotein (Pgp), use of a Pgp inhibitor resulted in a better estimate of absorption in humans. The results of this study suggest that the overall ranking of compounds with P_app < 1 × 10^–6 cm/sec, between 1–10 × 10^–6 cm/ sec, and > 10 × 10^–6 cm/sec can be classified as poorly (0–20%), moderately (20–70%) and well (70–100%) absorbed compounds, respectively. Conclusions. These data suggest that Caco-2 cells developed under the culturing and transport conditions defined herein can be used to predict in vivo human absorption of compounds regardless of transport mechanism, viz., transcellular, paracellular and carrier-mediated.     

Intestinal absorption mechanisms of berberine,palmatine, jateorhizine,and coptisine: involvement of P-glycoprotein

1. The absorption and transport mechanisms of berberine, palmatine, jateorhizine, and coptisine were studied using a Caco-2 cells uptake and transport model, with the addition of cyclosporin A and verapamil as P-glycoprotein (P-gp) inhibitors and MK-571 as a multidrug resistance-associated protein 2 (MRP₂) inhibitor.

2. In the uptake experiment, berberine, palmatine, jateorhizine, and coptisine were all taken into Caco-2 cells, and their uptakes were increased in the presence of cyclosporin A or verapamil.

3. In the transport experiment, P_app (AP-BL) was between 0.1 and 1.0?×?10⁶ cm/sec for berberine, palmatine, jateorhizine, and coptisine and was lower than P_app (BL-AB). ER values were all >2. Cyclosporin A and verapamil both increased P_app (AP-BL) but decreased P_app (BL-AB) for berberine, palmatine, jateorhizine, and coptisine; ER values were decreased by >50%. MK-571 had no influence on the transmembrane transport of berberine, palmatine, jateorhizine, and coptisine.

4. At a concentration of 1–100 μM, berberine, palmatine, jateorhizine, and coptisine had no significant effects on the bidirection transport of Rho123.

5. Berberine, palmatine, jateorhizine, and coptisine were all P-gp substrates; and at the range of 1–100 μM, berberine, palmatine, jateorhizine, and coptisine had no inhibitory effects on P-gp.