Caco-2 cell permeability of a new (hydroxybenzyl)ethylenediamine oral iron chelator: correlation with physicochemical properties and oral activity

This study describes the transport of CGP 75254A, a novel oral iron chelator, across Caco-2 cells in an attempt to model intestinal epithelial cell permeability in man. CGP 75254A was dosed to the apical side of Caco-2 cell monolayers, together with [14C]mannitol as an internal permeability standard. The apparent permeability (Papp) was calculated from the cumulative appearance of drug in the basolateral fluid with time. The [14C]mannitol Papp indicated that the Caco-2 monolayers remained intact and that the iron chelator was not toxic to the cells. Permeabilities of CGP 75254A were compared with the Caco-2 permeabilities of compounds of known absorption in man. The results predict that absorption of CGP 75254A is likely to be virtually complete at pH values between 5.5 and 7.0. However, at pH 8.0 permeability is predicted as negligible. Cell permeability data are in full accordance with key physicochemical properties of CGP 75254A and suggest that the drug is passively absorbed. The results, which suggest likely quantitative absorption in vivo, are supported by preliminary pharmacological experiments in marmosets.     

Absorption enhancement, structural changes in tight junctions and cytotoxicity caused by palmitoyl carnitine in Caco-2 and IEC-18 cells

Palmitoyl carnitine chloride (PCC) has been shown to be an effective enhancer of intestinal transport of hydrophilic molecules. The exact mechanism by which the epithelial barrier function is decreased is not clear. In an attempt to elucidate the mechanism of action of PCC, we studied the relationship among absorption enhancement, cell viability and tight junction protein localization in the human colonic Caco-2 cell line and the rat small intestinal cell line IEC-18. Filter-grown cells were exposed to 0 to 1 mM PCC for 30 min, and the efficacy of PCC treatment was determined by assessing the transepithelial electrical resistance and the apparent permeability for mannitol and PEG-4000. Membrane lysis and cytotoxicity were assessed by measurement of lactate dehydrogenase leakage and uptake of propidium iodide and neutral red. The immunolocalization of the tight junctional protein ZO-1 was quantified using CSLM and image-processing software. In both cell lines, PCC caused a dose-dependent decrease in transepithelial electrical resistance and a concomitant increase in the permeability for mannitol and PEG-4000. The transport enhancement was accompanied by an increase in apical membrane permeability and a reduction in cell viability. At higher PCC concentrations (>/=0.4 mM), the distribution of the tight junctional protein ZO-1 was changed and cells were unable to recover viability. PCC is effective as an absorption enhancer for hydrophilic macromolecules. However, lytic effects on the cell membrane and reduced cell viability were concomitant with transport enhancement.     

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 co-efficients (Papp) 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. Papp values for ranitidine and ondansetron (bioavailability of 50 and approximately 100% in humans, respectively) were 1.03 +/- 0.17 x 10(-7) and 1.83 +/- 0.055 x 10(-5) cm/sec, respectively. The Papp 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.     

Paracellular calcium transport across Caco-2 and HT29 cell monolayers

Intestinal calcium absorption has been shown to include two processes, a saturable transcellular movement and a non-saturable paracellular pathway. The potential utility of cell monolayers for studying transepithelial intestinal calcium transport has already been demonstrated; however, simultaneous evaluation of the contribution of the saturable transcellular and of the non-saturable paracellular processes to the total transepithelial transport has not yet been attempted. The aim of this study was to investigate the contribution both of transcellular and paracellular transport processes to the total transepithelial calcium transport in two cell culture monolayers. Caco-2 cells and a clone derived from HT-29 cells (HT29-Cl.19A), two cell lines derived from colon adenocarcinomas which are known to be able to exhibit typical enterocytic differentiation, were used. Cell monolayers were grown on a permeable support and used after 15 days of culture when these cells express enterocytic differentiation and high transepithelial resistance. Isotopic transport rate measurements were performed in the absence of a chemical gradient. The paracellular route was evaluated using [3H]mannitol. Calcium and [3H]mannitol transport rates across cell monolayers were not significantly different. Augmentation of calcium uptake by 200 mM sorbitol did not significantly increase calcium or mannitol transepithelial transport; however, calcium accumulation in the cells was increased by about 200%. Modulation of the monolayer permeability by addition of 10 nM vasoactive intestinal polypeptide (VIP) or 0.5 mM carbachol treatment, which respectively increased and decreased the transepithelial resistance, consequently modified calcium and mannitol transport in a parallel manner. Our results show that Caco-2 and HT29-Cl.19A cell monolayers are good models for studying the calcium paracellular transport pathway.     

Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers

PURPOSE: To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. METHODS: Caco-2 cell monolayers were grown for 3-27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. RESULTS: The apparent permeability coefficient (Papp) of CsA (0.5 microM) in the basolateral-to-apical (B-->A) direction increased with culture age and was higher than the apical-to-basolateral (A-->B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 microM) significantly inhibited CsA transport in the B-->A direction from day 17 to 27, while elevating CsA transport in the A-->B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 microM) in the B-->A direction was significantly higher than in the A-->B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks approximately 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. CONCLUSIONS: P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.     

Determinants of transmission success of individual clones from mixed-clone infections of the rodent malaria parasite, Plasmodium chabaudi

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 (Papp) calculated for the BL-to-AP and AP-to-BL transport (P(BL-->AP)/P(AP-->BL)) varied between 1.7 and 36.2. When individual pairs were ompared, P(BL-->AP)/P(AP-BL) 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 microM cyclosporin A to the transport buffer reduced the P(BL-->AP)/P(AP-->BL) 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 microM 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.     

Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells

There is mounting evidence from human epidemiological, animal in vivo, and in vitro studies to suggest beneficial effects related to the consumption of quercetin and its glucosides. However, there is limited knowledge on the oral bioavailability of these natural products. This study examined the intestinal epithelial membrane transport of quercetin, quercetin 4'-glucoside, and quercetin 3,4'-diglucoside, using the Caco-2 human colonic cell line, a model of human intestinal absorption. The apparent permeability (Papp) of each agent was measured in both apical to basal and basal to apical directions. The apical to basolateral flux of quercetin, Papp 5.8 +/- 1.1 x 10(-6) cm x sec(-1) (mean +/- SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.48 +/- 0.09 x 10(-6) cm x sec(-1) (P < 0.01). Under identical conditions, the Papp for the transcellular marker propranolol was about 5-fold higher than for quercetin (P < 0.001). Interestingly, the reverse, basolateral to apical, flux of quercetin (Papp 11.1 +/- 1.2 x 10(-6) cm x sec(-1)) was almost 2-fold higher than the apical to basolateral flux (P < 0.001). In similar experiments, quercetin 4'-glucoside demonstrated no absorption, Papp < 0.02 x 10(-6) cm x sec(-1) in the apical to basal direction, but did demonstrate basal to apical flux, Papp 1.6 +/- 0.2 x 10(-6) cm x sec(-1). Quercetin 3,4'-diglucoside showed a low apical to basolateral transport (Papp 0.09 +/- 0.03 x 10(-6) cm x sec(-1)); its reverse, basolateral to apical, transport was, however, 4-fold higher (P < 0.05). In these cells, glucose was actively transported with an apical to basolateral Papp of 36.8 +/- 1.1 x 10(-6) cm x sec(-1). These observations suggest facile absorption of quercetin through the human intestinal epithelium, but contrary to a previous proposal, they do not support an active transport process for quercetin glucosides.     

Preparation, characterization, and in vitro release of ibuprofen from AI2O3/PLA/PMMA composites

PURPOSE: To investigate in vitro the mechanisms involved in the gastro-intestinal absorption of the HIV protease inhibitor, saquinavir mesylate (Invirase), whose oral bioavailability is low, variable, and significantly increased by co-administration with ritonavir, also an HIV protease inhibitor but with higher oral bioavailability. METHODS: Confluent epithelial layers of human Caco-2 cells mimicking the intestinal barrier. RESULTS: Both saquinavir and ritonavir showed polarized transport through Caco-2 cell monolayers in the basolateral to apical direction (secretory pathway), exceeding apical to basolateral transport (absorptive pathway) by factors of 50-70 and 15-25, respectively. Active efflux was temperature dependent, saturable and inhibited by verapamil and cyclosporin A. Saquinavir and ritonavir decreased each other's secretory permeability and hence elevated their net transport by the absorptive pathway. CONCLUSIONS: Saquinavir and ritonavir are both substrates for an efflux mechanism in the gut, most likely P-glycoprotein, which acts as a counter-transporter for both drugs. Together with sensitivity to gutwall metabolism by cytochrome P-450 3A, this may partially account for the low and variable oral bioavailability of saquinavir in clinical studies and for its increased bioavailability after co-administration with ritonavir.     

Microsurgical management of craniopharyngiomas--outcomes in 56 patients

PURPOSE: The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H2-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H2-antagonists on the function of the intestinal tight junctions. METHODS: Effect of the H2-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. RESULTS: Four different H2-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H2-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. CONCLUSIONS: We hypothesize that the H2-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.     

Transport of L-valine-acyclovir via the oligopeptide transporter in the human intestinal cell line, Caco-2

It has been reported that conjugating acyclovir, a potent antiviral with low oral bioavailability, to L-valine increases its urinary excretion in rats. However, it was also reported that this increase is not found for the D-valine ester, suggesting that a carrier-mediated mechanism is involved in its intestinal absorption. Therefore, mechanisms involved in the transepithelial transport of L-valine-acyclovir were investigated using the intestinal cell line, Caco-2, as a model system for the intestinal epithelium. Only the mucosal-to-serosal transport of acyclovir was increased by conjugation with L-valine (approximately 7-fold), suggesting the involvement of a carrier-mediated mechanism. This conclusion was supported by the finding that this increase was saturable. The mucosal-to-serosal transport of L-valine-acyclovir could be inhibited by L-glycylsarcosine, but not by L-valine, suggesting the involvement of the dipeptide carrier. Also it was found that L-valine-acyclovir inhibits the uptake of cephalexin, a substrate for the oligopeptide transporter. Stability of the esters in either the mucosal or serosal bathing solution is more than 90% after completion of the transport study. However, after transport, the receiver solution contained approximately 90% of acyclovir. Based on these findings it was concluded that absorption of the L-valine ester of acyclovir occurs as a result of uptake by the oligopeptide transporter at the apical cell membrane followed by intracellular hydrolysis of the ester and efflux of acyclovir.     

Epithelial transport of drugs in cell culture. VIII: Effects of sodium dodecyl sulfate on cell membrane and tight junction permeability in human intestinal epithelial (Caco-2) cells

This study demonstrates how the common pharmaceutical wetting agent sodium dodecyl sulfate (SDS) increases the absorption of drugs and peptides across the human intestinal epithelium. First, an assay that could follow the reversible and irreversible time-dependent effects of SDS on the permeability of Caco-2 cell monolayers with high reproducibility was developed. SDS (0.40 mM) exposure for 20 min resulted in reversible absorption enhancement of mannitol (M(r), 182 g/mol), 1-deamino-8-D-arginine-vasopressin (M(r), 1071 g/mol), and polyethylene glycol (M(r), 4000 g/mol). A longer (2 h) exposure to SDS resulted in irreversible absorption enhancement. Second, transepithelial electrical resistance measurements (TEER) together with fluorescence and transmission electron microscopy were used to study the effects of SDS on epithelial integrity, cell membranes, intracellular calcium concentration, cytoskeleton, and tight junctions. The effect of SDS (0.40 mM) on epithelial integrity was immediate. A significant decrease in transepithelial electrical resistance measurements was obtained with 1 min after exposure to SDS that was concomitant with increases in the permeability of the apical cell membranes and intracellular calcium concentration. SDS shortened the microvilli of the cells and produced apical (but not basolateral) membrane wounds, actin disbandment, disorganization of the terminal web, and structural separation of the tight junctions. The absorption enhancement was not reduced after repair of the apical cell membranes, indicating that SDS enhances drug and peptide absorption across the intestinal epithelium by the paracellular pathway.     

The multidrug-resistant human lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems

Drug accumulation studies with the anticancer agents adriamycin and vincristine were carried out on the MDR variant of the human lung cell lines DLKP, DLKP-A10 which overexpresses the MDR associated P-glycoprotein efflux pump. Reduced cellular accumulation of both agents was observed in the resistant variant. The subsequent addition of verapamil and cyclosporin A resulted in partial restoration of cellular accumulation of both drugs in the DLKP-A10 resistant variant while complete restoration of cellular drug levels was observed in the SKMES-1/ADR cell line. These results suggested that the accumulation defect observed in the SKMES-1/ADR cell line was P-glycoprotein mediated and that accordingly, the cells exhibited characteristics consistent with the classical MDR phenotype. In contrast, while P-glycoprotein also appears to mediate a reduction in cellular drug accumulation in the DLKP-A10 cells, an alternative transport mechanism may also be present. No significant increase in the expression of either the MRP or LRP transport proteins was observed in the resistant cells. Metabolic inhibition by antimycin A (but not sodium azide or 2-deoxy-D-glucose) resulted in complete restoration of drug accumulation suggesting the presence of an alternative energy dependent transport mechanism. Fluorescent microscopy studies indicated different cellular localisation of the drug within the parental and resistant cells despite equivalent intracellular concentrations. These studies also revealed the presence of an ATP-dependent, vesicular sequestration mechanism which may be involved in the reduction of nuclear adriamycin accumulation in the DLKP-A10 cell line. This was indicated by observation of the disruption of cytoplasmic vesicles by antimycin A and also inhibition of cytoplasmic drug sequestration by the carboxylic ionophores, monensin and nigericin, accompanied by increased adriamycin accumulation and redistribution of the drug from the cytoplasm to the nucleus.     

A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model

Caco-2 cell monolayers mimic intestinal absorptive epithelium and represent a very useful tool for studying transepithelial transport. The literature on Caco-2 cells is controversial regarding transepithelial resistance and permeabilities of different marker compounds across monolayers. This paper discusses probable causes for these discrepancies. First, we present the role of culture conditions, such as the nature of the support or the passage number, on cell biology and transport properties. Further, we compare the presence of transport proteins in Caco-2 cells to mammalian intestinal tissue and discuss their implication for drug absorption. We also examine the advantages and disadvantages of systems such as Transwell and side-by-side diffusion chambers. A summary of comparisons between permeabilities across Caco-2 monolayers and mammalian intestinal tissues is provided. We conclude that the origin of Caco-2 cells and the culture conditions are in part responsible for the discrepancies encountered in the literature.     

Estimation of oral drug absorption in man based on intestine permeability in rats

Predicting the fraction of an oral dose absorbed in humans is of considerable interest at an early stage of a research program in the pharmaceutical industry. Models described in the literature to predict the oral absorption in man include: the permeability in Caco-2 cells, absorption from a perfused segment of rat intestinal lumen and uptake into everted rings. The present study used an isolated and vascularly perfused rat small intestine to determine the permeability values of eleven compounds across the intestinal epithelium. A good correlation was obtained between the permeability values determined in this model and the proportion of an oral dose absorbed in humans. Compared to the other models, the present one could allow the appearance in the artificial bloodstream and the intestinal metabolism of a compound to be studied simultaneously.     

Demonstration of interleukin-1 (IL-1) as an autocrine growth inhibitor in renal cancer cell line, TC-1

In this report, we demonstrated that Interleukin-6 (IL-6) production could be induced by stimulating renal cell carcinoma cell lines, namely ACHN, Caki-1 and TC-1 cells with Interleukin-1 beta (IL-1 beta). IL-1 beta had no effects on cell proliferation in ACHN cells. However, IL-1 beta could suppress cell proliferation in Caki-1 and TC-1 cells. Flow cytometric cell cycle analysis by double staining method with propidium iodide and Proliferating cell nuclear antigen (PCNA) disclosed IL-1 beta caused cell accumulation at G1 phase. Fine granules were visualized in perinuclear area of TC-1 cells treated with IL-1 beta under microscopy. High electron density granules and spherically dilated rough endoplasmic reticula were observed by electron microscopic examinations. In TC-1 cell culture, IL-1 beta excretion into the supernatant was demonstrated by bioassay and ELISA. These results suggest that IL-1 beta functions as an "autocrine growth inhibitor" against TC-1 cells. Half-maximal inhibition of IL-1 beta and IFN-alpha was 6.5 pg/ml, and 720 U/ml, respectively for TC-1 proliferation and combination of these cytokines showed enhanced activity in cell growth inhibition.     

Predicting drug absorption from molecular surface properties based on molecular dynamics simulations

PURPOSE: To develop an efficient method for generating representative conformations for calculation of the conformationally dependent molecular surface area, and to investigate the relation between this parameter and the permeability in Caco-2 cells. METHODS: High temperature molecular dynamics (MD) simulations were used to obtain 1000 conformations of six beta-blocking agents and their prodrugs. The Boltzmann averaged (B.a.) polar surface area of the 1000 conformations was correlated to the apparent permeability coefficients (Papp) for transport across filter-grown Caco-2 cells. RESULTS: Sampling of 1000 conformations during the MD simulations was sufficient for obtaining a representative set of conformations. The B.a. polar water accessible surface area (PWASA) yielded an excellent linear correlation with Papp for both series of compounds under study (R2 = 0.98). Thus, the improved permeability of the prodrugs could be explained by a reduced PWASA. The improvement of permeability after derivatization correlated positively with the size of the non-polar water accessible surface area-suggesting a synergistic effect of the cyclopropyl and the non-polar parts of the molecule to shield the polar parts from contact with water. CONCLUSIONS: An efficient method for generating the representative conformations for calculation of the B.a. polar surface area has been established. An excellent linear correlation explaining the improved permeability of the prodrugs was obtained.     

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.     

Permeability characteristics of novel mydriatic agents using an in vitro cell culture model that utilizes SIRC rabbit corneal cells

The purpose of this study was to evaluate the permeability characteristics of a previously reported in vitro corneal model that utilizes SIRC rabbbit corneal cells and to investigate the permeability of three novel esters of phenylephrone chemical delivery systems (CDS) under different pH conditions using this in vitro model. The SIRC rabbit corneal cell line was grown on transwell polycarbonate membranes, and the barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a voltohmmeter. The permeabilities of esters of phenylephrone CDS across the SIRC cell layers were measured over a pH range 4.0-7. 4. The esters tested include phenylacetyl (1), isovaleryl (2), and pivalyl (3). The SIRC rabbit corneal cell line, when grown on permeable filters, formed tight monolayers of high electrical resistance with TEER values increasing from 71.6 +/- 20.8 Omega.cm2 at day 3 in culture to 2233.42 +/- 15.2 Omega.cm2 at day 8 in culture and remained constant through day 14 in culture. The transepithelial permeability coefficients (Papp) at pH 7.4 ranged from 0.58 x 10(-6) cm/s for the hydrophilic marker, mannitol, to 43. 5 x 10(-6) cm/s for the most lipophilic molecule, testosterone. The Papp at pH 7.4 for phenylephrine was 4.21 x 10(-6) cm/s. The Papp values and the lag times of the three esters of phenylephrone were pH dependent. The Papp for 1, 2, and 3 at pH 7.4 were 14.76 x 10(-6), 13.19 x 10(-6), and 12.86 x 10(-6) cm/s, respectively and the permeabilities decreased at conditions below pH 7.4. The lag times at pH 7.4 were 0.10, 0.17, and 0.12 h for 1, 2, and 3, respectively, and the values increased at lower pH conditions. The TEER values of SIRC cell line observed at day 8 to day 14 in the present investigation are similar to the resistance value reported for rabbit cornea (2 kOmega.cm2). All the esters showed significantly (p < 0.05) higher permeabilities than phenylephrine at pH 7.4. The rate and extent of transport of the drugs across the cell layers were influenced by the fraction of ionized and un-ionized species and the intrinsic partition coefficient of the drug. The results indicate that the permeability of ophthalmic drugs through ocular membranes may be predicted by measuring the permeability through the new in vitro cell culture model.     

The effect of apotransferrin on iron release from Caco-2 cells, an intestinal epithelial cell line

The Caco-2 cell line grown in bicameral chambers was used to study the effect of transferrin in the basal chamber on the transepithelial transport of iron. We have shown that when iron was offered as 59Fe on the apical surface of the Caco-2 cells, transport of 59Fe into the basal chamber was stimulated by 50 micromol/L apotransferrin. Here, we examined the effect on 59Fe transport of lower concentrations of apotransferrin, as well as the effects on transport of ovo-, cobalt-, and ferri-transferrin and of iron chelators with an affinity for iron greater than that of transferrin. The stimulation of 59Fe transport was more sensitive to the presence of apotransferrin with a Km of 0.078 +/- 0.008 micromol/L compared with ferri-transferrin with a Km of 1.24 +/- 0.39 micromol/L (P < .006). 59Fe transport was less sensitive to diethylenetriaminopenta-acetic acid (DTPA) than apotransferrin with Kms of 1.52 +/- 0.70. The chelator nitrilotriacetic acid (NTA) exhibited no stimulation of 59Fe transport. Analysis of laser scanning confocal micrographs showed that apotransferrin labeled with Texas Red is internalized by Caco-2 cells from the basal side and localizes in distinct vesicles above the nucleus. The sensitivity of apotransferrin in stimulating Fe transport suggests a unique interaction of apotransferrin with the basal surface of the intestinal epithelium.