Parental sex effects in bipolar affective disorder pedigrees

Measurements of transepithelial electrical resistance (TEER), the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) test and monitoring of poly(ethylene glycol) (PEG) transport have been used to study the effects of the non-ionic surfactants Solulan C24 and Solulan 16, either free in solution or as an integral part of niosome bi-layers, on intestinal epithelial cells from man (Caco-2 cell monolayers). The effects on epithelial integrity and on the transport of the hydrophilic drug metformin depend on the concentration of the surfactants. At concentrations above 1% the effect on TEER of the surfactant in niosomal form and free in solution were equivalent whereas cell viability was preserved to a higher concentration of Solulans when the Solulans were present in the niosomal form. It was concluded that the toxic effect of niosomes arises from free surfactant present in the niosome suspension.     

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.     

Inhibition of NK cells by murine CMV-encoded class I MHC homologue m144

In this study the effects of two chitosan salts, namely chitosan hydrochloride and chitosan glutamate (0.5 and 1.5% w/v), on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cell monolayers, using the radioactive marker [14C]-mannitol, were investigated in a slightly acidic (pH 6.2) and neutral (pH 7.4) environment. Both salts are soluble in acidic conditions up to a concentration of 1.5% w/v and solutions of this strength, at a pH of 6.2, caused a pronounced lowering in the TEER of Caco-2 cell monolayers in the order of 70+/-1% (chitosan glutamate) and 77+/-3% (chitosan hydrochloride), 20 min after incubation started. In agreement with the TEER results the transport of the radioactive marker, [14C]-mannitol, was increased 25-fold (chitosan glutamate) and 36-fold (chitosan hydrochloride), respectively, at this pH. However, at a pH of 7.4 both salts are insoluble and prove to be ineffective since no reduction in the TEER values or increase in the transport of [14C]-mannitol were found. The results show that these chitosan salts are potent absorption enhancers in acidic environments. We conclude that there is a need for chitosan derivatives with increased solubility, especially at neutral and basic pH values, for use as absorption enhancers aimed at the delivery of therapeutic compounds in the more basic environment of the large intestine and colon.     

Killing effects of 5-fluorouracil on human biliary tract cancer cell lines

Previous studies have established that a partially quaternized derivative of chitosan, N-trimethyl chitosan chloride (TMC), can be used as an absorption enhancer for large hydrophilic compounds across mucosal surfaces. This study evaluates and compares the effects of the degree of quaternization of TMC, in a neutral environment, on the permeability of intestinal epithelial cells in vitro, where normal chitosan salts are ineffective as absorption enhancers. The effects of TMC-H [61.2% quaternized, (0.05-1.5% w/v)], TMC-L [12.3% quaternized, (0.5-1.5% w/v)], and chitosan hydrochloride [0.5-1.5% w/v] on the transepithelial electrical resistance (TEER) and permeability, for the hydrophilic model compound [14C]mannitol, of intestinal epithelial Caco-2 cell monolayers, were investigated at pH values of 6.20 and 7.40. The viability of the monolayers was checked with the trypan blue exclusion technique. At a pH of 6.20, all the polymers caused a pronounced reduction (37-67% at 0.5% w/v concentrations) in the TEER of Caco-2 cells. On the contrary, at a pH of 7.40, only TMC-H was able to decrease the TEER values, even in a concentration as low as 0.05% w/v (35% reduction). Comparable results were obtained with the permeation of [14C]mannitol. Large increases in the transport rate (18-23-fold at 0.5% w/v concentrations) were found at pH 6.20, whereas only TMC-H was able to increase the permeation of [14C]mannitol at pH 7.40 (31-48-fold at 0.05-1.5% w/v concentrations of TMC-H). For all the polymers studied, no deleterious effects to the cells could be demonstrated with the trypan blue exclusion technique. It is concluded that highly quaternized TMC is a potent absorption enhancer and the potential use of this polymer, especially in neutral and basic environments where normal chitosan salts are not effective, is expected to be an important contribution to the development of effective delivery systems for hydrophilic compounds such as peptide drugs.     

Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes

Selected properties of Caco-2 cells were examined after disparate transepithelial electrical resistance (TEER) measurements were observed in two populations of Caco-2 cells. Comparisons were made between the early passages of Caco-2 cells (Caco-2E, passages 35-47) and the later passages of cells (Caco-2L, passages 87-112). Transmission electron microscopy revealed that regions of Caco-2L cells were composed of multiple cell layers rather than the monolayers observed in Caco-2E cells. Epithelial cell height (or barrier thickness) was not significantly different between the two cell populations. Intercellular and intracellular lumina were observed in the Caco-2L cells, but not in the Caco-2E cells. Results of [3H]thymidine incorporation assays showed significantly higher cell proliferation rates in Caco-2L cells relative to Caco-2E cells. Despite morphological and physiological changes, there were no significant differences in the apparent permeabilities for D-mannitol (paracellular diffusion marker), hydrocortisone (transcellular diffusion marker), or dipeptide, Gly-Sar (carrier-mediated transcellular transport marker) between the two populations of cells. The higher TEER values in Caco-2L cells may be the results of a slight perturbation of tight junctions associated with both the multiple cell layers and the presence of intercellular lumina.     

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.     

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.     

The recording of parenchymal lung changes in smokers by high-resolution computed tomography

Using Caco-2BBe monolayers as a model of the intestinal epithelium, we tested the hypothesis that reactive oxygen metabolites contribute to lactic acid-induced hyperpermeability. Compared to monolayers incubated at normal pH (i.e., 7.4) monolayers incubated in medium titrated to extracellular pH (pHo) 5.0 with 10 mM lactic acid demonstrated increased permeability to both fluorescein sulfonic acid (FS) and fluorescein isothiocyanate-dextran (average molecular mass = 4000 Da; FD4). Lactic acid-induced hyperpermeability to both FS and FD4 was reduced by adding either 30 microM EUK-8, a superoxide dismutase/catalase mimetic, or catalase (10(4) U/mL). Incubation of monolayers with lactic acid increased cellular malondialdehyde content, a measure of lipid peroxidation. EUK-8 (30 microM) completely abrogated this effect. Incubation with ferrous sulfate (100 microM) exacerbated both lactic acid-induced hyperpermeability to FS and lactic acid-induced lipid peroxidation. Iron chelation with 1 mM diethylene triamine pentaacetic acid (DTPA)-trisodium calcium salt attenuated lactic acid-induced hyperpermeability, whereas iron-loaded DTPA (1 mM) was not protective. Treatment with DTPA-trisodium calcium salt also ameliorated lactic acid-induced lipid peroxidation. Incubation with lactic acid (pHo 5.0) for 16 h increased the cellular content of low molecular weight iron species. Incubation with lactic acid (pHo 5.0) for 24 h significantly increased the percentage of oxidized protein-bound thiols in Caco-2BBe cells. We conclude that lactic acidosis induces hyperpermeability in Caco-2BBe monolayers, in part, via an iron-dependent increase in reactive oxygen metabolite-mediated damage.     

Internet resources for orthopaedic surgeons

PURPOSE: Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). METHODS: The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. RESULTS: At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in R123 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. CONCLUSIONS: Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.     

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.     

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.     

Effects of medium-chain fatty acids and their acylglycerols on the transport of penicillin V across Caco-2 cell monolayers

The transport-enhancing effects of medium-chain fatty acids (caproic, caprylic, and capric acids) and their acylglycerols (mono-, di-, and triacylglycerols) were investigated by using Caco-2 cell monolayers as a model of the human intestinal epithelium. Penicillin V was used as a model for a hydrophilic bioactive compound. Among the fatty acids and acylglycerols tested, 1,2-dicaproin, monocaprin, monocaprylin, and capric acid sodium salt effectively enhanced the transport rate, whereas other substances enhanced the rate only slightly or not at all. With each of these four substances, the rate of enhancement was proportional to the concentration at low concentrations, but leveled off at high concentrations. The transport-enhancing effects were well correlated with the reduction in surface tension and with a physico-chemical parameter, denoted by the surface energy-lowering coefficient, characterizing the surface activity of a substance.     

Characterization of P-glycoprotein mediated transport of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor, across MDR1-MDCK and Caco-2 cell monolayers

PURPOSE: Here we characterized the transport properties of morpholine-urea-phenylalanine-homophenylalanine-vinylsulfone-phenyl (K02), a newly developed peptidomimetic cysteine protease inhibitor, across monolayers of P-gp-expressed MDRI transfected MDCK cells (MDR1-MDCK) and Caco-2 cells. METHODS: MDR1-MDCK, MDCK and Caco-2 cells, grown to confluence on Transwell insert membranes, were used to investigate transcellular transport of [14C]-K02. RESULTS: The basolateral to apical (B-A) flux of 10 microM [14C]-K02 across MDR1-MDCK cells was markedly greater than its apical to basolateral (A-B) flux (ratio = 39). This specific B-A transport was temperature dependent and saturable, with an apparent Michaelis-Menten constant and maximum velocity of 69.1 +/- 19.5 microM and 148.9 +/- 16.3 pmol/min/cm2, respectively. This B-A flux was significantly inhibited by cyclosporine (IC50 = 17.1 +/- 0.7 microM), vinblastine (IC50 = 75.9 +/- 13.0 microM) and verapamil (IC50 = 236 +/- 63 microM). In Caco-2 cell monolayers, the B-A flux was reduced about 50% compared to that in MDR1-MDCK and the A-B flux was increased about 8-fold. The apparent Michaelis-Menten constant and maximum velocity values for the B-A transport were 71.8 +/- 45.9 microM and 35.3 +/- 9.0 pmol/min/ cm2. This B-A flux was also significantly inhibited by P-gp substrates/ inhibitors. Western blots showed that the P-gp expression in MDR1-MDCK cells was about 10-fold that in Caco-2 cells. CONCLUSIONS: K02 is transported by P-gp in both MDR1-MDCK and Caco-2 cells, and the in vitro interactions between K02 and various P-gp substrates may provide strategies to overcome the bioavailability barrier by intestinal P-gp.     

In vivo modification of 3'-phosphoadenosine 5'-phosphosulfate and sulfate by infusion of sodium sulfate, cysteine, and methionine

The importance of tissue sulfate concentrations in regulating 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthesis is not known. Therefore, this study was conducted to determine the influence of increased availability of inorganic sulfate on steady-state PAPS concentrations in various tissues. To increase tissue sulfate concentrations, 2-16 mmol/kg of sodium sulfate and sulfur-containing amino acids (cysteine or methionine) were infused intravenously for 2 hr into pentobarbital-anesthetized rats. Serial blood samples were taken during the infusion and analyzed for sulfate concentrations. After 2 hr of infusion, liver, kidney, and brain were removed for quantification of tissue PAPS and sulfate concentrations. Infusion of sodium sulfate, cysteine, and methionine increased serum and tissue sulfate concentrations in a dose- and time-dependent manner. Serum sulfate concentrations increased from 0.8 to 14 mM during the infusion of sodium sulfate, whereas infusions of cysteine and methionine increased serum sulfate concentrations to 4.8 and 2.0 mM, respectively. Tissue sulfate concentrations also increased during sulfate infusion. Liver sulfate concentration increased from 0.8 to 4.8 mM, kidney concentration increased from 0.6 to 31 mM, and brain concentration increased from 0.1 to 0.6 mM. Similar to the serum sulfate concentrations, sulfate infusion was the most effective in increasing tissue sulfate concentrations, cysteine was intermediate, and methionine the least effective. Although sulfate concentrations in liver, kidney, and brain increased 6-, 50-, and 6-fold by infusing sulfate, respectively; tissue PAPS levels were not altered markedly. Hepatic PAPS concentrations increased significantly (30-35%) only when infused with the higher doses (8 or 16 mmol/kg/2 hr) of sodium sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells

Medium chain fatty acids (MCFAs) are used to enhance the permeability of mucosal tissues to hydrophilic drugs, but their mechanism of action is largely unknown. In this study, the absorption-enhancing effects of the sodium salts of two MCFAs, capric acid (C10) and lauric acid (C12), were studied in monolayers of human intestinal epithelial Caco-2 cells. Both MCFAs induced a rapid increase in epithelial permeability to the hydrophilic marker molecule sodium fluorescein. Inhibition of phospholipase C and inhibition or activation of various kinases and buffering of intracellular calcium indicated that the effects on epithelial permeability were mediated through phospholipase C-dependent inositol triphosphate/diacylglycerol pathways. Surprisingly, the inositol triphosphate and diacylglycerol pathways were found to have opposing effects on paracellular permeability. Exposure to the MCFAs also resulted in a concentration dependent reduction of cellular dehydrogenase activity and ATP levels. C10, but not C12, induced redistribution of the tight junction proteins ZO-1 and occludin. These results indicate that the two MCFAs have partially different and more complex mechanisms than previously recognized, which has important implications for their use in vivo.     

Dipeptide transport across rat alveolar epithelial cell monolayers

The transepithelial transport and metabolism of two model peptides, glycyl-D-phenylalanine (Gly-D-Phe) and glycyl-L-phenylalanine (Gly-L-Phe), across primary cultured monolayers of rat alveolar epithelial cells were studied. These tight monolayers (> 2000 omega-cm2) exhibited type I pneumocyte morphological and phenotypic characteristics. A reverse-phase HPLC was used to monitor the appearance of parent dipeptides and their metabolites (D- or L-Phe) in the receiver fluid. The apparent permeability coefficient (Papp) for Gly-D-Phe was about 1.6 x 10(-7) cm/sec at both 1 and 10 mM and in both the apical-to-basolateral (AB) and the basolateral-to-apical (BA) directions. In contrast, the Papp of Gly-L-Phe at 1 mM was about two times higher than that at 10 mM in the AB direction. The Papp of Gly-L-Phe in the BA direction at either concentration was about the same (about 1.4 x 10(-7) cm/sec). Whereas no metabolite was detected during Gly-D-Phe transport, the proportions of a metabolite, L-Phe, observed at 4 hr in the basolateral receiver fluid for 1 and 10 mM apical donor Gly-L-Phe accounted for 83 and 77% of the estimated total Gly-L-Phe (i.e., L-Phe+Gly-L-Phe), respectively. The corresponding values in the BA direction were 40 and 19% of the estimated total Gly-L-Phe in the apical receiver reservoir. Metabolism of Gly-L-Phe was significantly reduced in the presence of 3 microM actinonin (an inhibitor relatively specific for aminopeptides M) in the apical but not the basolateral fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in situ absorption of SDZ-RAD using a human intestinal cell line (Caco-2) and a single pass perfusion model in rats: comparison with rapamycin

PURPOSE: To compare the intestinal absorption and active efflux protein susceptibility of a new immunosuppressive agent (SDZ-RAD) with that of its analog rapamycin. METHODS: Caco-2 cell monolayers were used to examine bidirectional transport of the two compounds at low micromolar concentrations. Single pass rat intestinal perfusion was also used to examine steady state permeability. RESULTS: Rapamycin and SDZ-RAD showed a distinct preference for transport in the basolateral to apical direction of Caco-2 monolayers as efflux was >20 times greater than apical to basolateral transport. Efflux of SDZ-RAD was completely inhibited by verapamil while efflux of rapamycin was mostly inhibited by verapamil and partially inhibited by probenecid. Passive permeability was shown to be 20 x 10(-6) cm/sec for SDZ-RAD and 10 x 10(-6) cm/sec for rapamycin. In situ rat studies also showed the permeability of rapamycin to be half that of SDZ-RAD with permeabilities of 12.6 X 10(-6) for rapamycin and 24.8 x 10(-6) cm/sec for SDZ-RAD. CONCLUSIONS: SDZ-RAD and rapamycin are strong substrates for P-gp-like mediated efflux. Rapamycin is also partially removed from cells by a second efflux system that is not responsive to SDZ-RAD. When these efflux pumps are inhibited SDZ-RAD is likely to be absorbed across the intestine at a faster rate than rapamycin.     

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.     

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.     

DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours

PURPOSE: It has recently been shown that the absorption enhancing and toxic effects of chitosans are dependent on their chemical composition. In this study, the mechanisms underlying these effects were investigated at the cellular level. METHODS: The effects on epithelial cells of chitosans with different chemical composition, absorption enhancing properties and toxicities were studied in Caco-2 monolayers. Chitosan C( 1:31) has a low degree of acetylation (DA) (1%) and a low m.w. (31 kD), and displays dose-dependent absorption enhancement and cytotoxicity; chitosan C(35:170) has a higher DA (35%) and a higher m.w. (170 kD), is less dose-dependent in absorption enhancement, and is not cytotoxic. A third non-toxic chitosan C(49:22) with a high DA (49%), a low m.w. (22 kD), and no influence on epithelial permeability was used as control. RESULTS: C(1:31) and C(35:170) bound tightly to the epithelium. Cellular uptake of the chitosans was not observed. Both chitosans increased apical but not basolateral cell membrane permeability and induced a redistribution of cytoskeletal F-actin and the tight junction protein ZO-1. This resulted in increased paracellular permeability of hydrophilic marker molecules of different molecular weights. Addition of negatively charged heparin inhibited the cellular and the absorption enhancing effects of the chitosans, indicating that these effects are mediated via their positive charges. The onset of the effects of C(35:170) on apical membrane permeability and tight junction structure was much faster than that of C(1:31). C(49:22) did not influence any of the properties of the Caco-2 cell monolayers studied. CONCLUSIONS: The binding and absorption enhancing effects of chitosans on epithelial cells are mediated through their positive charges. The interaction of chitosans with the cell membrane results in a structural reorganisation of tight junction-associated proteins which is followed by enhanced transport through the paracellular pathway.