Skin Penetration and Mechanisms of Action in the Delivery of the D2-Agonist Rotigotine from Surfactant-Based Elastic Vesicle Formulations

Purpose. This study was performed to investigate the effect of elastic and rigid vesicles on the penetration of the D₂ dopamine agonist rotigotine across human skin and to further elucidate the mechanisms of action of the elastic vesicles. Methods. A series of rotigotine-loaded vesicles were prepared, ranging from very elastic to very rigid. The drug penetration from these vesicles across human skin was studied in vitro using flow-through diffusion cells. Micelle and buffer solutions were investigated as controls. For the most elastic vesicle composition, two additional variables were investigated. Coapplication of drug and vesicles was compared to pretreatment, and the effect of the drug entrapment efficiency was investigated. Results. The very elastic vesicle formulation L-595/PEG-8-L (50/50) gave steady-state fluxes of 214.4 ± 27.8 ng/(h · cm²). This formulation was the most effective formulation and significantly better than the rigid vesicle formulations as well as the micelle and buffer controls. However, coapplication and a high drug entrapment efficiency were essential factors for an optimal drug delivery from elastic vesicle formulations. Conclusions. Elastic vesicles are promising vehicles for transdermal drug delivery. It is essential that drug molecules are applied together with and entrapped within the vesicles themselves, suggesting that elastic vesicles act as drug carrier systems and not solely as penetration enhancers.     

Comparison of HT29-18-C1 and Caco-2 Cell Lines as Models for Studying Intestinal Paracellular Drug Absorption

Purpose. To compare the permeability characteristics of HT29-18-C₁ colonic epithelial cell line with Caco-2, an established model of intestinal drug transport. Methods. Cell lines were grown as epithelial monolayers. Permeability was measured over a range of transepithelial electrical resistance (R_t) using a group of drug compounds. Results. HT29-18-C₁ develop R_t slowly when grown in culture, allowing permeability to be measured over a wide range (80–600 ·cm²). In contrast, Caco-2 monolayers rapidly develop R_t of 300 ·cm² and require Ca²⁺-chelation to generate R_t equivalent to human intestine (60–120 ·cm²). Permeability of atenolol, ranitidine, cimetidine, hydrochlorothiazide and mannitol across HT29-18-C₁ decreased 4–5 fold as R_t developed from 100–300 ·cm² indicating they permeate via the paracellular route. In contrast, ondansetron showed no difference in permeability with changing R_t consistent with transcellular permeation. Permeability profiles across low R_t HT29-18C₁ and pulse EGTA-treated Caco-2 monolayers were the same for all 5 paracellular drugs suggesting that transient Ca²⁺ removal does not alter selectivity of the tight junctions. Permeabilities of cimetidine, hydrochlorothiazide and atenolol across 100 ·cm² HT29-18-C₁ monolayers reflect more closely those reported for the human ileum in vivo than did mature Caco-2 monolayers. Conclusions. HT29-18-C₁ monolayers can be used to study drug permeability at R_t values similar to human intestine without the need for Ca²⁺ chelation. As such, they offer a useful alternative to Caco-2 for modelling intestinal drug absorption.     

Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.     

Radioligand-Binding Assay Employing P-Glycoprotein-Overexpressing Cells: Testing Drug Affinities to the Secretory Intestinal Multidrug Transporter

Purpose. To develop a rapid and reliable system for affinity determination of conventional as well as newly synthesized compounds to P-gp. Methods. The principles of radioligand-binding assay were adapted to the human intestinal P-gp. Acceptor protein was obtained from the human carcinoma cell line Caco-2, where overexpression of P-gp was induced by growing cells in the presence of the cytostatic drug vinblastine. ³H-Verapamil was chosen as radioligand. Results. The saturability and specificity of ³H-verapamil as the radioligand for the binding to P-gp was demonstrated. From concentration dependence of displacement of the radioligand by various non-labeled ligands for P-gp, affinity constants to P-gp binding sites were calculated. The binding results obtained were in agreement with those published earlier where influx and efflux experiments with cell monolayers had been conducted in order to functionally characterize the P-gp -drug interaction. Conclusions. A radioligand-binding assay on the basis of P-gp overexpressing Caco-2 cells has been developed. The method might be suitable for high-throughput screening of drug interaction with human P-gp. It will allow modeling of the interaction of drugs with the human multi-drug transporter and has also the potential to serve as a high-throughput screening tool to detect compounds prone to P-gp mediated intestinal secretion and potential P-gp related drug/drug interactions in drug discovery and early development.     

Influence of Morphometric Factors on Quantitation of Paracellular Permeability of Intestinal Epithelia In Vitro

Purpose. The relative contribution of the small and large intestine to paracellular absorption is a subject of some controversy. Direct comparison of paracellular permeability in different epithelia is complicated by variations in junctional density and/or the absorptive surface area. Methods. This study used a combination of morphometric analyses and in vitro absorption studies to define permeability characteristics in relation to the amount of paracellular pathway present in rat ileum, colon and the model epithelium, Caco-2. Results. Mucosal to serosal amplification was higher in ileum (3.9) than colon (1.9) or Caco-2 (1). Tight junctional density (1p) of ileal crypts was 3 fold greater (91 m/cm²) than that measured in ileal villi, colonic surface and crypt cells or Caco-2 monolayers (34–37 m/ cm²). However, when the relative contributions of the crypts and villi was taken into account there was no significant difference in the mean 1p per mucosal area for the three epithelia studied. Using these data to correct for morphometric differences the permeabilities of a range of small hydrophilic molecules (atenolol, D-PheAsp and PEG oligomers MW 282-634) was measured. Permeability of rat ileum and colon were virtually identical for all compounds studied. In contrast, Caco-2 monolayers showed a significantly lower permeability than intestinal tissues with the difference increasing markedly with molecular size. Conclusions. These studies suggest the importance of accounting for morphological variation when comparing the permeability characteristics of different epithelial systems.     

Autophagy-dependent removal of α-synuclein: a novel mechanism of GM1 ganglioside neuroprotection against Parkinson’s disease

GM1 ganglioside is particularly abundant in the mammalian central nervous system and has shown beneficial effects on neurodegenerative diseases.In this study,we investigated the therapeutic effect of GM1 ganglioside in experimental models of Parkinson’s disease(PD)in vivo and in vitro.Mice were injected with MPTP(30 mg·kg-1·d?1,i.p.)for 5 days,resulting in a subacute model of PD.PD mice were treated with GM1 ganglioside(25,50 mg·kg^?1·d^?1,i.p.)for 2 weeks.We showed that GM1 ganglioside administration substantially improved the MPTP-induced behavioral disturbance and increased the levels of dopamine and its metabolites in the striatal tissues.In the MPP⁺-treated SH-SY5Y cells andα-synuclein(α-Syn)A53T-overexpressing PC12(PC12^(α-Syn A53T))cells,treatment with GM1 ganglioside(40μM)significantly decreasedα-Syn accumulation and alleviated mitochondrial dysfunction and oxidative stress.We further revealed that treatment with GM1 ganglioside promoted autophagy,evidenced by the autophagosomes that appeared in the substantia nigra of PD mice as well as the changes of autophagy-related proteins(LC3-II and p62)in the MPP⁺-treated SH-SY5Y cells.Cotreatment with the autophagy inhibitor 3-MA or bafilomycin A1 abrogated the in vivo and in vitro neuroprotective effects of GM1 ganglioside.Using GM1 ganglioside labeled with FITC fluorescent,we observed apparent colocalization of GM1-FITC andα-Syn as well as GM1-FITC and LC3 in PC12^(α-Syn A53T)cells.GM1 ganglioside significantly increased the phosphorylation of autophagy regulatory proteins ATG13 and ULK1 in doxycycline-treated PC12^(α-Syn A53T)cells and the MPP⁺-treated SH-SY5Y cells,which was inhibited by 3-MA.Taken together,this study demonstrates that the anti-PD role of GM1 ganglioside resulted from activation of autophagy-dependentα-Syn clearance.     

Influence of Chitosan Microspheres on the Transport of Prednisolone Sodium Phosphate Across HT-29 Cell Monolayers

Purpose. The present study was performed to investigate the influence of chitosan microspheres on transport of the hydrophilic, antiinflammatory drug prednisolone sodium phosphate (PSP) across the epithelial barrier. Methods. Microspheres were prepared using a precipitation method and loaded with PSP. Transport studies were performed in a diffusion cell chamber using the polarized human cell line HT-29_B6. Porcine small intestine and fluorescence-labeled microspheres were used to investigate penetration ability of microspheres. Results. It was shown that transport of PSP drug solution was not saturable across the cell monolayers (P = 8.68 ± 8.24 × 10^–6 cm sec^–1) and no sodium dependency could be established. EGTA treatment resulted in an increased permeability (P = 18.69 ± 1.09 × 10^–6 cm sec^–1). After binding of prednisolone to chitosan microspheres its permeability was enhanced drastically compared with the drug solution (P = 35.37 ± 3.21 ×10^–6 cm sec^–1). This effect was prevented by EGTA treatment (P = 15.11 ± 2.57 × 10^–6 cm sec^–1). Furthermore the supporting effect of chitosan microspheres was impaired by pH and ion composition of the medium, whereas the effect remained unchanged in cells treated with bacterial lipopolysaccharides. In vitro incubation of fluorescence-labeled microspheres in the lumen of freshly excised intestine revealed a significant amount of the spheres in the submucosa. Conclusions. Chitosan microspheres are a useful tool to improve the uptake of hydrophilic substances like PSP across epithelial layers. The effect is dependent on the integrity of the intercellular cell contact zones and the microparticles are able to pass the epithelial layer. Their potential benefit under inflammatory conditions like in inflammatory bowel disease, in order to establish high drug doses at the region of interest, remains to be shown.     

Transdermal Delivery of Pergolide from Surfactant-Based Elastic and Rigid Vesicles: Characterization and in Vitro Transport Studies

Purpose. The aim of this study was to investigate the effect of elastic and rigid vesicles on the penetration of pergolide across human skin. Methods. Vesicles used consisted of the bilayer-forming surfactant L-595 (sucrose laurate ester) and the micelle-forming surfactant PEG-8-L (octaoxyethylene laurate ester), together with the stabilizer sulfosuccinate. A series of L-595/PEG-8-L/sulfosuccinate vesicles were investigated, ranging from very rigid to very elastic. Pergolide-loaded elastic and rigid vesicles were visualized using Cryo-TEM and characterized for size and stability. Transdermal penetration of pergolide from different vesicle compositions was studied in vitro using flow-through Franz diffusion cells. A saturated buffer solution served as the control. Results. Vesicle composition had a major effect on the physico-chemical characteristics, morphology and drug solubility of the vesicular system. L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles gave the best balance between vesicle stability and elasticity, as well as the highest drug solubility. Transport studies clearly showed that elastic vesicles were superior to rigid vesicles. Elastic vesicles enhanced the drug transport compared to the buffer control, although rigid vesicles decreased the drug transport. The best drug transport was achieved from L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles, resulting in a steady-state flux of 13.6 ± 2.3 ng/(h*cm²). This was a 6.2-fold increase compared to the most rigid vesicles. Conclusions. This study supports the hypothesis that elastic vesicles are superior to rigid vesicles as vehicles for transdermal drug delivery.     

Wheat Germ Agglutinin Binds to the Epidermal Growth Factor Receptor of Artificial Caco-2 Membranes as Detected by Silver Nanoparticle Enhanced Fluorescence

Purpose. The purpose of this study was to identify one of the ligands that mediate carbohydrate-specific cytoadhesion and cytoinvasion of wheat germ agglutinin (WGA)-containing drug delivery systems. Methods. The receptor-ligand studies were performed with isolated epidermal growth factor (EGF) receptors as well as biomimetic membranes prepared from Caco-2 and A-431 cells. The binding of fluorescent labeled WGA was detected by the silver nanoparticle enhanced fluorescence technique. Results. The binding of WGA to isolated EGF receptors is saturable and the equilibrium is reached within 1 min. The interaction between WGA and isolated EGF receptors is fully inhibited by the complementary carbohydrate and at least 85% of WGA binding to artificial Caco-2 membranes is caused by protein-carbohydrate interactions involving the tetrasialo-binding motif. The integrity and the presence of EGF-receptors in artificial Caco-2 membranes as well as their WGA-binding capacity were confirmed by immunoblot detection. Conclusions. The glycosylated extracellular domain III of the EGF receptor is involved in the WGA-Caco-2 cell interaction. Accordingly, receptor mediated endocytosis is the basic mechanism for internalization of WGA. As the EGF receptor is overexpressed in a high number of tumors but also occurs in non-malignant tissue at considerable density, WGA-mediated drug delivery opens exciting possibilities for specific binding and uptake of poorly absorbable drugs.     

Phospholipid vesicle–bound lysozyme to enhance permeability in human intestinal cells

Background: Oral peptide and protein drug delivery still remain the area of challenges for pharmaceutical scientists due to their low stability and permeability in gastrointestinal (GI) tract. In this study phospholipid vesicle–bound lysozyme were prepared and assessed for their physicochemical properties, secondary structure, and permeation across Caco-2 cells.

Results: Lysozyme was found to be substantially bound onto negatively charged vesicles via electrostatic interaction as evidenced by zeta potential measurements regardless of cholesterol content. In contrast, the size of phospholipid vesicle–bound lysozyme became larger with the increasing cholesterol content. The secondary structure of vesicle-bound lysozyme examined by FTIR was unchanged compared to that in buffer solution. The apparent permeability of vesicle-bound lysozyme across Caco-2 cells monolayer was significantly enhanced with a size dependent manner compared to that of solution.

Conclusion: The permeation across Caco-2 cell monolayers of phospholipid vesicle–bound lysozyme was demonstrated to be significantly enhanced with a size-dependent manner.     

Targeting Efficiency of Galactosylated Liposomes to Hepatocytes in Vivo: Effect of Lipid Composition

Purpose. To investigate the effects of the lipid composition of galactosylated liposomes on their targeted delivery to hepatocytes. Methods. Several types of liposomes with a particle size of about 90 nm were prepared using distearoyl-L-phosphatidylcholine (DSPC), cholesterol (Chol) and cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol), and labeled with [³H]cholesterol hexadecyl ether. Their tissue disposition was investigated in mice following intravenous injection. The binding and internalization characteristics were also studied in HepG2 cells. Results. Compared with [³H]DSPC/Chol (60:40) liposomes, [³H]D-SPC/Chol/Gal-C4-Chol (60:35:5) liposomes exhibit extensive hepatic uptake. Separation of the liver cells showed that galactosylated liposomes are preferentially taken up by hepatocytes, whereas those lacking Gal-C4-Chol distribute equally to hepatocytes and nonparenchymal cells (NPC). Increasing the molar ratio of DSPC to 90% resulted in enhanced NPC uptake of both liposomes, suggesting their uptake via a mechanism other than asialoglycoprotein receptors. DSPC/Chol/Gal-C4-Chol (60:35:5) and DSPC/Chol/Gal-C4-Chol (90:5:5) liposomes exhibited similar binding to the surface of HepG2 cells, but the former were taken up faster by the cells. Conclusions. The recognition of galactosylated liposomes by the asialoglycoprotein receptors is dependent on the lipid composition. Cholesterol-rich galactosylated liposomes, exhibiting less non-specific interaction and greater receptor-mediated uptake, are better for targeting drugs to hepatocytes in vivo.     

N-Trimethyl Chitosan Chloride as a Potential Absorption Enhancer Across Mucosal Surfaces: In Vitro Evaluation in Intestinal Epithelial Cells (Caco-2)

Purpose. Previous studies have established that chitosan hydrochloride and glutamate are potent absorption enhancers for large hydrophilic compounds across mucosal surfaces. However, these compounds lack solubility at neutral pH values. A partially quaternized and well-soluble derivative of chitosan, N-trimethyl chitosan chloride, was synthesized and the effects of this polymer on the transepithelial electrical resistance and permeability of intestinal epithelial cells were investigated in vitro. Methods. N-trimethyl chitosan chloride was synthesized by reductive methylation and characterized with NMR. The effect of this polymer (1.0–2.5% w/v) on the transepithelial electrical resistance of intestinal epithelial cells, using Caco-2 cell monolayers, was investigated. Permeation of the hydrophilic model compounds [^l4C]-mannitol (MW 182.2), FITC-Dextran (MW 4400) and the peptide drug buserelin (MW 1299.5), in the presence of N-trimethyl chitosan chloride (1.5–2.5% w/v), was followed for 3 hours. The transport process of the fluorescent marker, FITC-Dextran 4400, across the cell monolayers was visualised with confocal laser scanning microscopy. Viability of the cells was checked with the trypan blue exclusion technique. Results. N-trimethyl chitosan chloride was found to be a perfectly water-soluble, partially quaternized (about 12%) derivative of chitosan. This polymer (1.5–2.5% w/v) caused a pronounced and immediate reduction (25–85%) in the transepithelial electrical resistance of Caco-2 cells. Large increases in the transport rate of [^!4C]-mannitol (32–60 fold), FITC-Dextran 4400 (167–373 fold) and buserelin (28–73 fold) were demonstrated. Confocal laser scanning microscopy confirmed that N-trimethyl chitosan chloride opens the tight junctions of intestinal epithelial cells to allow increased transport of hydrophilic compounds through the paracellular transport pathway. No deleterious effects to the cells could be demonstrated with trypan blue. Conclusions. The potential use of N-trimethyl chitosan chloride as an absorption enhancer across mucosal surfaces could be an important contribution towards the development of effective delivery systems for hydrophilic drugs.     

Are MDCK Cells Transfected with the Human MDR1 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K _M,V _max) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K _I) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [³H]-digoxin than did Caco-2 and MDCK-WT cells. K _M values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 M, respectively, whereas V _max values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm^–2s^–1, respectively. Known P-gp substrates/inhibitors showed, in general, lower K _I values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.     

Metabolism,Uptake, and Transepithelial Transport of the Stereoisomers of Val-Val-Val in the Human Intestinal Cell Line,Caco-2

Purpose. The purpose of this study was to determine the stereospecificity of the apical oligopeptide transporter(s) for the stereoisomers of Val-Val-Val and to determine whether the interaction of these molecules with this transporter(s) could be correlated with their cellular uptake and/or transepithelial transport. Methods. The interactions of these stereoisomers with this transporter(s) were evaluated by determining their ability to inhibit [³H]cephalexin uptake into Caco-2 cells. The metabolism of these stereoisomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of these stereoisomers were studied using the Caco-2 cell monolayers. Results. The L-L-L tripeptide was totally degraded within 1 h in the Caco-2 cell homogenate and within 2 h when applied to the apical side of a Caco-2 cell monolayer. In contrast, 36.7 ± 1.3% and 69.7 ± 0.9% of L-Val-L-Val-D-Val remained after 2 h in the cell homogenate and in the apical bathing solution, respectively. The other six stereoisomers of Val-Val-Val were completely stable in the Caco-2 cell homogenate. Five of the stereoisomers (L-L-L, L-L-D, L-D-L, D-L-L, D-D-L) significantly inhibited the cellular uptake of [³H]cephalexin (91%, 62%, 14%, 45%, 16%, respectively). The other stereoisomers had no effect on the [³H]cephalexin uptake. When the cellular uptake of the stereoisomers was determined, the D-L-L and L-D-L tripeptides showed the highest intracellular concentrations (1.32 ± 0.25 and 0.62 ± 0.20 nmol/mg protein after a 2-h incubation, respectively). In contrast, the intracellular concentrations of the other stereoisomers were less than 0.1 nmol/mg protein. Moreover, the cellular uptake of the D-L-L and L-D-L tripeptides was inhibited by Gly-Pro by 82% and 68%, respectively, whereas Gly-Pro showed moderate to no inhibitory effect on the cellular uptake of the other stereoisomers. The permeability coefficients of the stereoisomers across the Caco-2 cell monolayers were very low (1.8 to 3.1 × 10^–7 cm/sec) and almost identical. Gly-Pro had no effect on their transepithelial transport. Conclusions. These results suggest that the interaction of the Val-Val-Val stereoisomers with the apical oligopeptide transporter(s) could be a good predictor of their cellular uptake. However, since the major transepithelial transport mechanism of Val-Val-Val stereoisomers is passive diffusion via the paracellular route, the binding of these molecules to the oligopeptide transporter(s) is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates permeation of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.     

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.     

Messenger RNA Expression of Transporter and Ion Channel Genes in Undifferentiated and Differentiated Caco-2 Cells Compared to Human Intestines

Purpose. The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. Method. Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. Results. Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. Conclusions. Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.     

In Vitro and In Situ Permeability of a 'Second Generation' Hydroxypyridinone Oral Iron Chelator: Correlation with Physico-Chemical Properties and Oral Activity

Purpose. The in vitro and in situ transport of CGP 65015 ((+)-3-hydroxy-1-(2-hydroxyethyl) -2-hydroxyphenyl-methyl-1 H-pyridin-4-one), a novel oral iron chelator, is described. The predictive power of these data in assessing intestinal absorption in man is described. Methods. Caco-2 epithelial monolayer and in situ rat jejunum perfusion intestinal permeability models were utilized. In vivo iron excretion and preliminary animal pharmacokinetic experiments were described, lonization constants and octanol/aqueous partition coefficients were measured potentiometrically. Solubilities and intrinsic dissolution rates were determined using standard procedures. Results. Caco-2 cell (P_app 0.25 X 10^–6 cm.s^–1) and rat jejunum (P_w 0.4) permeabilities of CGP 65015 were determined. The log D(pH 7.4) of CGP 65015 was 0.58 and its aqueous solubility was > 0.5 mg.ml^–1 (pH 3–9). The intrinsic dissolution rate of CGP 65015 in USP simulated intestinal fluid was 0.012 mg.min^–1.cm^–2. CGP 65015 promotes iron excretion effectively and dose dependently in animals. Conclusions. Caco-2 and rat intestinal permeabilities predict incomplete oral absorption of CGP 65015 in man. Preliminary rat pharmacokinetics support this. Physico-chemical data are, also, in line and suggest that CGP 65015 may, in addition, be solubility/dissolution rate limited in vivo. Nevertheless, early animal pharmacological data demonstrate that CGP 65015 is a viable oral iron chelator candidate.     

Metabolism,Uptake, and Transepithelial Transport of the Diastereomers of Val-Val in the Human Intestinal Cell Line,Caco-2

Purpose. The purpose of this study was to determine whether the binding of the diastereomers of Val-Val to the apical oligopeptide transporter(s) could be correlated with their cellular uptake and transepithelial transport. Methods. The Caco-2 cell culture system was used for all experiments. The binding of the diastereomers of Val-Val was evaluated by determining their ability to inhibit [³H]cephalexin uptake. The stability of the diastereomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of the individual diastereomers were studied using Caco-2 cell monolayers. Results. 10 mM concentrations of L-Val-L-Val, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val inhibited cellular uptake of [³H]cephalexin (0.1 mM) by 92%, 37%, 70%, and 18%, respectively. When the cellular uptake of Val-Val diastereomers (1 mM) were evaluated, the intracellular concentrations of L-Val-D-Val and D-Val-L-Val were 15 and 50 times higher, respectively, than that of D-Val-D-Val. The cellular uptake of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) (>95%), whereas Gly-Pro had no effect on the cellular uptake of D-Val-D-Val. L-Val-L-Val was not detected in the Caco-2 cells, probably due to its metabolic lability. When the transepithelial transport of the Val-Val diastereomers (1 mM) was determined, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val transport rates were similar. The transepithelial transport of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) 36% and 30%, respectively, while Gly-Pro inhibited carnosine (1 mM) transepithelial transport by 65%. Gly-Pro had no effect on the transepithelial transport of D-Val-D-Val. Conclusions. These results suggest that the major transepithelial transport route of L-Val-D-Val, D-Val-L-Val and D-Val-D-Val is passive diffusion via the paracellular route. The binding of Val-Val diastereomers to the oligopeptide transporter(s) is a good predictor of their cellular uptake, however, the binding is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates efflux of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.     

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.     

Ciprofloxacin decreases survival in HT-29 cells via the induction of TGF-��1 secretion and enhances the anti-proliferative effect of 5-fluorouracil

Background and purpose:

Fluoroquinolones are potent anti-microbial agents with multiple effects on host cells and tissues. Previous studies have highlighted their pro-apoptotic effect on human cancer cells and an immunoregulatory role in animal models of inflammatory bowel disease. We examined the effect of ciprofloxacin on proliferation, cell cycle and apoptosis of HT-29 cells, a human colonic epithelial cell line sensitive to transforming growth factor (TGF)-β1-mediated growth inhibition and its role in TGF-β1 production. We also examined the effect of ciprofloxacin on proliferation of HT-29 cells in combination with 5-fluorouracil (5-FU), a well-established pro-apoptotic agent.

Experimental approach:

Using subconfluent cultures of HT-29 and Caco-2 cells, we studied the effect of ciprofloxacin, TGF-β1 and 5-FU on proliferation, apoptosis, necrosis and cell cycle. The effect of ciprofloxacin on TGF-β1 mRNA expression and production was studied in RNA extracts and cell culture supernatants respectively, using confluent cultures.

Key results:

Ciprofloxacin decreased proliferation of HT-29 cells in a concentration- and time-dependent manner. This was mediated by accumulation of HT-29 cells into the S-phase but without any effect on apoptosis or necrosis. Additionally, ciprofloxacin enhanced the antiproliferative effect of 5-FU. Interestingly, ciprofloxacin was found to up-regulate TGF-β1 production by HT-29 cells and its anti-proliferative effect was abolished when TGF-β1 was blocked. Confirming this mechanism further, ciprofloxacin had no effect on Caco-2, a human colonic epithelial cell line that lacks functional TGF-β1 receptors.

Conclusions and implications:

We demonstrate a novel anti-proliferative and immunoregulatory effect of ciprofloxacin on human intestinal epithelial cells mediated via TGF-β1.