Regulation of Tight Junction Permeability by Calcium Mediators and Cell Cytoskeleton in Rabbit Tracheal Epithelium

The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, ¹⁴C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of ¹⁴C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance. These results suggest that high extracellular calcium levels and low intracellular calcium levels are required for the normal maintenance of the junctional permeability in the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tight junction permeability may exert their action through the calcium and cytoskeleton mechanisms.     

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.     

Role of Paracellular Pathway in Nonelectrolyte Permeation Across Rat Colon Epithelium Enhanced by Sodium Caprate and Sodium Caprylate

The enhancing effects of 0.25% sodium caprate (C10) and sodium caprylate (C8) on the paracellular permeation of seven water-soluble nonelectrolytes (inulin, polyethylene glycol 900, mannitol, erythritol, glycerol, thiourea, and urea) across the isolated rat colonic epithelium were examined using the Ussing-type chamber technique. The paracellular changes were also measured by impedance analysis. In both the presence and the absence of enhancers, the permeation clearances (Pm) for inulin (12–15 in molecular radius) to erythritol (3.2 ) increased linearly with the increase in their free diffusion coefficients (Dfr), showing the existence of a paracellular shunt pathway unrestricted to any molecular size. Glycerol (2.9 ), thiourea (2.6 ), and urea (2.3 ) had higher clearances than the expected linear values, showing the existence of a restricted paracellular or transcellular pathway. Both C10 and C8 increased the permeabilities in the two pathways, but C10 was more effective than C8. The increase in the permeabilities via the shunt pathway caused by the enhancers was greater than that via the restricted pathway, and thus, the two-phase pattern in the relationship of Pm and Dfr was similar to that in the absence of enhancers. The transcellular permeabilities for urea and thiourea, which were obtained from the efflux experiments, were increased by the enhancers. However, the relative increase caused by C10 was smaller than that of the paracellular-restricted permeabilities. The paracellular changes probably were due to the increase in pore area per unit diffusive path length. A decrease in the resistance of the intercellular junctions involving a simultaneous increase of membrane capacitance was observed in the presence of C10, corresponding to an increase of pore area per unit path length. The effect of C10 on the paracellular permeability was reversible, and the junctional resistance, membrane capacitance, and Pm of mannitol returned to the control level following the removal of C10.     

Does Fluid Flow Across the Intestinal Mucosa Affect Quantitative Oral Drug Absorption? Is It Time for a Reevaluation?

Purpose. Hydrophilic and charged solutes have a lower membrane permeability which is due to a lower partition into the lipid membrane (low solubility in the membrane phase) and/or a slower transcellular diffusion coefficient. They are therefore anticipated to be absorbed through the paracellular route, which is a consequence of diffusion and a convective volume flow through the water-filled intercellular space. Methods. Two approaches have been used to investigate the mechanisms underlying the paracellular drug transport across the intestinal mucosa: (a) including water transport by exposing the apical side of the epithelium with a hypotonic solution, and (b) stimulated paracellular transport by widening of tight junction and increased water absorption as a consequence of the sodium-coupled transport of nutrients. Results. Among the first studies that recognized this fluid flux dependent transmucosal transport of drugs, was one published by Oschenfahrt & Winne in 1973 and the one by Kitazawa et al. in 1975. During the last two decades the importance of this paracellular route for drug delivery have been explored in vitro and in situ. Conclusions. The limits concerning molecular weight, shape, ionization and the effect of physiological stimulants, such as luminal concentrations of nutrients, osmolality and motility, are currently under investigation. However, recently published in vivo human data by ourselves and others indicate that the promising results obtained in vitro and in situ for various hydrophilic compounds might not be valid in quantitative aspects in humans, especially not for drugs with a molecular weight over 200.     

Selective Paracellular Permeability in Two Models of Intestinal Absorption: Cultured Monolayers of Human Intestinal Epithelial Cells and Rat Intestinal Segments

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.     

Riboflavin-Enhanced Transport of Serum Albumin Across the Distal Pulmonary Epithelium

Purpose. Conjugation of bovine serum albumin (BSA) with riboflavin (BSA-riboflavin) increases its uptake into cultured epithelial cells. Our purpose was to determine whether transport of BSA-riboflavin across the intact distal pulmonary epithelium is also increased, and whether transcytosis plays a role. Methods. In anesthetized rats, we instilled ³H-BSA-riboflavin or ³H-BSA into the trachea and measured their appearance in blood. In isolated, perfused rat lungs we measured the distal pulmonary epithelium permeability-surface area product (PS) for FITC-BSA or FITC-BSA-riboflavin. Results. In intact rats we found 2.1 times more ³H-BSA-riboflavin than ³H-BSA appeared in blood 60 min after intratracheal instillation of the protein. In isolated, perfused rat lungs we found that BSA-riboflavin had double the PS of BSA (2.63 vs. 1.46 × 10^–5 cm³/sec). The addition of transcytosis inhibitors monensin or nocodazole (both 3 × 10^–5 M) reduced the BSA-riboflavin PS to that of BSA and had no effect on the PS of unconjugated BSA. Simultaneous measurements of ³H-sucrose PS showed no differences in paracellular transport among any of the experimental groups. Conclusions. Conjugation with riboflavin increases the flux of BSA across the distal pulmonary epithelium. The increased transport appears to be due to transcytosis, which apparently does not play a significant role in the movement of unconjugated BSA across the distal pulmonary epithelium.     

Poly-l-Arginine Enhances Paracellular Permeability via Serine/Threonine Phosphorylation of ZO-1 and Tyrosine Dephosphorylation of Occludin in Rabbit Nasal Epithelium

PURPOSE: The purpose of the present study is to explore whether a poly-L-arginine (poly-L-Arg)-induced increase in tight junctions (TJ) permeability of fluorescein isothiocyanate-labeled dextran (MW 4.4 kDa, FD-4) is associated with the Ca2+-dependent signaling and occurs following the phosphorylation/dephosphorylation of TJ proteins. METHODS: Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of FD-4 transport and membrane conductance (Gt) in the presence of various inhibitors that are involved in the Ca2+-dependent pathway and the phosphorylation/dephosphorylation of TJ proteins. The resultant distribution of TJ proteins was observed using confocal laser scanning microscopy (CLSM) in an immunostaining. RESULTS: The increase in TJ permeability of FD4 induced by 0.2 mg/ml poly-L-Arg was not altered by treatment with inhibitors (of possible Ca2+ mobilization pathways followed by exposure of poly-L-Arg, suggesting that the promoting effect of poly-L-Arg is independent of Ca2+-related signaling. On the other hand, the protein kinase C (PKC) and tyrosine phosphatase inhibitors suppress the increase in TJ permeability by poly-L-Arg, indicating that serine/threonine phosphorylation by way of Ca2+-independent PKC and tyrosine dephosphorylation of junction proteins may have occurred. Furthermore, immunofluorescent monitoring of ZO-1, a TJ associated protein, and occludin, an integral membrane protein localizing at TJ, after preincubation with PKC and tyrosine phosphatase inhibitors followed by poly-L-Arg treatment has shown that the internalization of ZO-1 and occludin occurred by way of serine/threonine phosphorylation by PKC activation and by way of tyrosine dephosphorylation, respectively, providing TJ disassembly. CONCLUSIONS: We conclude that poly-L-Arg enhances the paracellular permeability of FD-4 (i.e., macromolecules), at least, by way of both serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium.     

Cyclodextrins in Nasal Delivery of Low-Molecular-Weight Heparins: In Vivo and in Vitro Studies

PURPOSE: To test the hypothesis that cyclodextrins reversibly enhance nasal absorption of low-molecular-weight heparins (LMWHs) and to investigate the mechanisms by which cyclodextrins enhance LMWH absorption via the nose. METHODS: Absorption of LMWHs was studied by measuring plasma anti-factor Xa activity after nasal administration of various LMWH formulations to anesthetized rats. In vivo reversibility studies were performed to investigate if the effects of cyclodextrins are reversible and diminish with time. The absorption-enhancing mechanisms of cyclodextrins were investigated in cell culture model. The transport of enoxaparin and mannitol, changes in transepithelial electrical resistance (TEER), and distribution of tight junction protein ZO-1 were investigated. RESULTS: Formulations containing 5% dimethyl-beta-cyclodextrin (DMbetaCD) produced the highest increase in the bioavailability of LMWH preparations tested. In vivo reversibility studies with 5% DMbetaCD showed that the effect of the absorption enhancer at the site of administration diminished with time. Transport studies using 16HBE14o(-) cells demonstrated that the increase in the permeability of enoxaparin and mannitol, reduction in TEER, and the changes in the tight junction protein ZO-1 distribution produced by 5% DMbetaCD were much greater than those produced by beta-cyclodextrin (betaCD) or hydroxyl-propyl-beta-cyclodextrin (HPbetaCD). CONCLUSIONS: Of the cyclodextrins tested, DMbetaCD was the most efficacious in enhancing absorption of LMWHs both in vivo and in vitro. The study also suggests that cyclodextrins enhance nasal drug absorption by opening of cell-cell tight junctions.     

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

Purpose Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2. Materials and Methods The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors. Results Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4–70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling. Conclusions Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.     

常用实验动物鼻上皮毒性病理学研究简介

目的 介绍常用实验动物鼻上皮毒性病理学研究进展,为吸入途径给药及非吸入途径药物临床前安全性评价毒性病理学研究提供参考.方法 系统介绍了常见实验动物鼻解剖学与组织学及其种属差异、常用实验动物组织病理学检查鼻腔上皮的取材方法、吸入药物引起的鼻上皮损伤的特点、使用吸入药物的动物数据来评估人类风险以及非吸入途径给药实验引起的鼻上皮病变特点和机制.结果 不同种属实验动物鼻大体和显微解剖学及组织学差别显著;常用实验动物例如大鼠、猴、比格犬及兔的组织病理学检查鼻腔上皮的取材方法各有其特点;吸入药物引起的鼻上皮损伤及非吸入途径给药实验引起的鼻上皮病变特点和机制有所不同.结论 本文可帮助提高国内常用实验动物鼻上皮的制片和诊断水平,为吸入药物及非吸入途径给药药物的临床前安全性评价提供更加客观、全面、准确的形态学数据.     

Correction of Permeability with Pore Radius of Tight Junctions in Caco-2 Monolayers Improves the Prediction of the Dose Fraction of Hydrophilic Drugs Absorbed by Humans

PURPOSE: To improve predictions of fraction dose absorbed (Fa) for hydrophilic drugs, a correction of paracellular permeability using the pore radius of tight junctions (TJs) in Caco-2 monolayers was performed. METHODS: The apparent permeability coefficient (P9app)) of drugs was measured using the Caco-2 assay and the parallel artificial membrane permeation assay (PAMPA), and values were corrected with the pore radius of TJs. RESULTS: An equation for calculating the pore radius of TJs from the P(app) of lucifer yellow was obtained. The optimal pore radius of TJs in Caco-2 monolayers for predicting human Fa was calculated to be 7 A. The correlation between the actual and predicted Fa was improved by using the P(app) corrected with the pore radius of TJs. Permeability in the PAMPA, which was corrected using the pore radius and membrane potential, was well correlated with that in the Caco-2 assay. Most of the hydrophilic drugs tested in this study were absorbed mainly through the paracellular pathway. CONCLUSIONS: The results suggest the necessity of optimizing paracellular permeation for the prediction of Fa, and also the importance of the paracellular pathway to the absorption of hydrophilic drugs. This method might contribute to the setting of appropriate dosages and the development of hydrophilic drugs.     

Estimation of the Relative Contribution of the Transcellular and Paracellular Pathway to the Transport of Passively Absorbed Drugs in the Caco-2 Cell Culture Model

Purpose. The objective of this investigation was to determine, using the Caco-2 cell culture model, the extent to which the paracellular and transcellular routes contributed to the transport of passively absorbed drugs. An effort was also made to determine the controlling factors in this process. Methods. We selected a heterologous series of drugs with varying physicochemical parameters for the investigation. Effective permeability coefficients of the model drugs (naproxen, phenytoin, salicylic acid, chlorothiazide, furosemide, propranolol, diltiazem, ephedrine, and cimetidine), at pH 7.2 and pH 5.4, were estimated using confluent monolayers of Caco-2 cells. The biophysical model approach, based on molecular size restricted diffusion within an electrostatic field of force, used by Adson et al. (1 ,2), was employed to estimate the permeability coefficients of the ionized and unionized forms of the drugs for the paracellular and transcellular route. Results and Conclusions. The permeability coefficients of the acidic drugs was greater at pH 5.4, whereas that of the basic drugs was greater at pH 7.2 and the transcellular pathway was the favored pathway for most drugs, probably due to its larger accessible surface area. The paracellular permeability of the drugs was size and charge dependent. The permeability of the drugs through the tight junctions decreased with increasing molecular size. Further, the pathway also appeared to be cation-selective, with the positively charged cations of weak bases permeating the aqueous pores of the paracellular pathway at a faster rate than the negatively charged anions of weak acids. Thus, the extent to which the paracellular and transcellular routes are utilized in drug transport is influenced by the fraction of ionized and unionized species (which in turn depends upon the pKa of the drug and the pH of the solution), the intrinsic partition coefficient of the drug, the size of the molecule and its charge.     

Effect of Chitosan on the Permeability of Monolayers of Intestinal Epithelial Cells (Caco-2)

Pharmaceutical Research -     

Effect of Medium-Chain Glycerides on Physiological Properties of Rabbit Intestinal Epithelium in Vitro

Medium chain glycerides (MCGs) have been reported to enhance intestinal absorption of hydrophilic drugs. However, the mechanisms involved in absorption enhancement are not well understood. The effects of MCGs (CapMul MCM) on physiological properties of rabbit ileum and distal colon, including active ion transport, transepithelial resistance (R_t) and passive permeability, have been investigated in vitro. CapMul MCM inhibited active ion transport (measured as a decrease in short-circuit current, I_sc) in both intestinal segments in a concentration-dependent manner. The inhibition of I_sc was rapidly reversible (within 100 min) upon removal of CapMul MCM. The data indicate that CapMul MCM preferentially affected ion transport by villus cells in the ileum and surface cells in the distal colon. Ion transport in crypt cells in both segments was not significantly altered. R_t of the ileum was not significantly affected by 5% CapMul MCM, while mannitol transport was 6 fold enhanced. Treatment of distal colon with 1% CapMul MCM reduced R_t by 95%, while mannitol transport was 100 fold enhanced. In a parallel experiment, mucosal(m)-to-serosal(s) transport of cephalexin, a -lactam antibiotic, in the ileum was about 40% reduced in the presence of 5% CapMul MCM, whereas transport in the s-to-m direction was 2.5 fold enhanced. Treatment of the distal colon with 1% CapMul MCM resulted in 25 fold enhancement of cephalexin transport in either direction. These results suggest that absorption enhancement by MCGs results from an increased permeability of the intestine confined to the villus or surface epithelium.     

Ocular Absorption of Pz-Peptide and Its Effect on the Ocular and Systemic Pharmacokinetics of Topically Applied Drugs in the Rabbit

Purpose. To determine the corneal and conjunctival penetration of 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz-peptide) and to evaluate its effect on the corneal and conjunctival penetration of hydrophilic solutes as well as on the ocular and systemic absorption of topically applied atenolol and propranolol in the rabbit. The hydrophilic solutes were mannitol, fluorescein, FITC-dextran 4,000, and FITC-dextran 10,000. Methods. Drug penetration across the rabbit cornea and conjunctiva was evaluated using the modified Ussing chamber. Ocular and systemic absorption of topically applied atenolol and propranolol was evaluated by analyzing the drug concentration in various anterior segment tissues at 45 min and in the blood over 240 min, respectively, following topical instillation of 25 l of 20 mM atenolol or propranolol solution to the rabbit eye. Results. The conjunctiva was 29 times more permeable than the cornea to 3 mM Pz-peptide. Conjunctival Pz-peptide transport was 1.7 times more extensive in the mucosal-to-serosal than in the opposite direction, whereas corneal Pz-peptide transport showed no directionality. The apparent permeability coefficient of Pz-peptide across the cornea and the conjunctiva increased over the 1–5 mM range, suggesting that Pz-peptide enhanced its own transport across both epithelial tissues. The cornea appeared to be more sensitive than the conjunctiva to the penetration enhancement effect of Pz-peptide. Thus, whereas Pz-peptide elevated the corneal transport of mannitol, fluorescein, and FD4 by 50%, 57%, and 106%, respectively, it did not affect the conjunctival transport of mannitol and fluorescein, while enhancing FD4 transport by only 46%. Moreover, while Pz-peptide enhanced the ocular absorption of topically applied hydrophilic atenolol, it did not affect the ocular absorption of lipophilic propranolol. Interestingly, Pz-peptide did not affect the systemic absorption of either beta adrenergic antagonist. Conclusions. Pz-peptide appears to facilitate its own penetration across the cornea and the conjunctiva. Pz-peptide appears to increase the ocular absorption of topically applied hydrophilic but not lipophilic drugs, while not affecting the systemic absorption of either type of drugs.     

In Vitro Nasal Transport Across Ovine Mucosa: Effects of Ammonium Glycyrrhizinate on Electrical Properties and Permeability of Growth Hormone Releasing Peptide,Mannitol, and Lucifer Yellow

Transport of growth hormone releasing peptide across ovine nasal mucosa in the absence or presence of ammonium glycyrrhizinate (AMGZ) was studied in vitro. Ovine nasal mucosa was stripped from underlying cartilage and mounted in Ussing chambers. Transepithelial conductance (G _t) and short-circuit current (I _sc) were monitored during experiments to assess tissue viability and integrity. Radiolabeled mannitol (Man; MW 182) and growth hormone releasing peptide (GHRP, SK&F 110679; MW 873) were employed to measure transport rates across the epithelium, and fluorescence spectroscopy was employed to measure rates of lucifer yellow (LY; MW 521) transport. Effects of AMGZ on ovine nasal mucosal viability and transport were determined from changes in electrical properties or fluxes of [³H]GHRP, [³H]Man, and LY. Results demonstrate that electrical properties of ovine nasal mucosa are stable over the time course of the experiments (G _t = 8.3 ± 0.5 mS/cm² and I _sc = 3.7 ± 0.2 µEq/hr · cm²; n = 21). Man fluxes were comparable in the mucosal (m)-to-serosal (s) and s-to-m directions [0.10 ± 0.01 (n = 17) and 0.10 ± 0.01 (n = 4) %/hr · cm², respectively]. Transport of GHRP and LY in the m-s direction was similar to that of Man [0.08 ± 0.01 (n = 11) and 0.09 ± 0.01 (n = 3) %/hr · cm², respectively]. GHRP flux was equivalent in the m-s and s-m directions. GHRP did not significantly alter ion transport processes as indicated by the lack of any change in G _t or I _sc. Luminal addition of AMGZ (2%, 24 mM) increases G _t and transport of both LY and [³H]Man approximately fourfold without altering transport of [³H]GHRP. No changes in transport or G _t were seen with luminal addition of AMGZ (1%, 12 mM). These studies suggest that transport of the hexapeptide GHRP occurs by a passive process and that AMGZ selectively increases the permeability of the mucosa to the low molecular weight molecules, Man and LY, but not to GHRP in vitro.     

Concentration-Dependent Effects of Polyethylene Glycol 400 on Gastrointestinal Transit and Drug Absorption

Purpose. The aim of the study was to investigate the effect of different concentrations of polyethylene glycol 400 (PEG 400) on liquid transit through, and ranitidine absorption from, the gastrointestinal tract. Methods. Six healthy male volunteers received, on four separate occasions, 150 mL water containing 150 mg ranitidine and either 0 (control), 1, 2.5, or 5 g PEG 400. The solutions were radiolabeled with technetium-99m to allow their gastrointestinal transit to be followed using a gamma camera. Urine samples were collected over a 24-h period to assess the amount of ranitidine excreted and hence absorbed. Results. No significant differences in gastric emptying were noted between the four solutions. In contrast, the presence of 1, 2.5, and 5 g PEG 400 reduced the mean small intestinal transit times of the solutions by 9, 20, and 23%, respectively, against the control. In terms of drug absorption, the mean cumulative amount of ranitidine excreted was reduced by 38% in the presence of both 2.5 and 5 g PEG 400, although it was significantly increased by 41% in the presence of 1 g PEG 400. Conclusions. The results show that low concentrations of PEG 400 enhance the absorption of ranitidine possibly via modulation of intestinal permeability, while high concentrations have a detrimental effect on ranitidine absorption presumably via a reduction in the small intestinal transit time.     

Development of a Human Nasal Epithelial Cell Culture Model and Its Suitability for Transport and Metabolism Studies Under in Vitro Conditions

A human nasal epithelial cell culture model has been adapted to observe transport and metabolism of drugs, e.g., peptides. Human nasal epithelial cells, isolated by protease treatment of human nasal conchae, grew to confluency after 6-8 days using DMEM supplemented with 1% nonessential amino acids, 1% glutamine, 10% FCS and 1% antibiotics. These cultures expressed microvilli and actively beating cilia as documented by light microscopy and scanning electron microscopy (SEM). Tight junctions were confirmed by dome formation and positive actin staining using FITC-labelled phalloidin. Preliminary transport studies, carried out with FITC-labelled Dex-tran (FD 4, MW 4400) and Sulforhodamine (SR 101, MW 607), demonstrated the intact barrier function of the cultured monolayer, grown on filter membranes. In addition, the cultured cells metabolized Leu-Enkephalin to Des-Tyr-Leu-Enkephalin demonstrating the presence of aminopeptidase, a naturally occurring enzyme in the human nasal mucosa.