Intestinal permeability of ophthalmic beta-blockers for predicting ocular permeability

The purpose of this study was to investigate the intestinal permeability of ophthalmic beta-blockers and evaluate the utility of intestinal membrane for predicting the ocular permeability. The penetrations of beta-blockers were measured across the isolated jejunum and colon of the albino rabbit using a two-chamber glass diffusion cell. beta-Blockers tested include atenolol, carteolol, tilisolol, timolol, and befunolol. Colonic membrane showed lower permeability of hydrophilic drugs than jejunal membrane. Scraping the entire cell monolayer of jejunum increased the drug permeability. There was a significant correlation between colonic permeability coefficients and lipophilicities of beta-blockers. The permeability coefficients through jejunum and scraped jejunum were not susceptible to drug lipophilicities. Jejunum, scraped jejunum, and colon showed permeability coefficients almost equal to those of sclera, conjunctiva, and cornea, respectively. There was a significant correlation between permeability coefficients through colon and cornea. These results indicate that the steady-state permeability of ophthalmic beta-blockers through ocular membranes may be predicted by measuring the permeability through certain intestinal membranes. However, the analyses of intestinal permeability using Fick's equation showed the functional difference of intestinal permeability from ocular permeability of ophthalmic beta-blockers.     

Novel approaches for oral delivery of macromolecules

Traditional forms of administrations of nonabsorbable drugs and peptides often rely on their parenteral injection, since the intestinal epithelium is poorly permeable to these therapeutical agents. A number of innovative drug delivery approaches have been recently developed, including the drug entrapment within small vesicles or their passage through the intestinal paracellular pathway. Zonula occludens toxin, a recently discovered protein elaborated by Vibrio cholerae, provided tools to gain more insights on the pathophysiology of the regulation of intestinal permeability through the paracellular pathway and to develop alternative approaches for the oral delivery of drugs and macromolecules normally not absorbed through the intestine.     

Castor-oil induced diarrhoea in ponies: a model for acute colitis

A reproducible, reversible model of colitis induced in ponies by administering castor oil (2.5 ml/kg bodyweight [bwt] per os) was characterised by abdominal pain, fever, watery diarrhoea, dehydration, hypovolaemia, toxaemia, leucopenia, decreased serum Cl, Na and K levels and metabolic acidosis. The signs were most severe between 24 and 48 h post induction, stabilisation was frequently observed after 72 h, although diarrhoea could persist beyond 96 h. Morphological and in vitro transport studies (right ventral colon) were conducted on tissues from animals destroyed at 24, 48 and 72 h. In the caecum and colon, surface epithelial disruption and exfoliation from the basement membrane occurred between 24 and 48 h. Early signs of recovery were evident by replenishment of denuded areas with columnar epithelium at 72 h. The crypt epithelium was unaffected throughout the intestinal tract. In vitro transport studies were consistent with the morphological findings. Decreased Na-Cl absorption and normal Cl secretion indicated an impaired surface epithelium coincident with an undamaged cryptal epithelium. Increased mucosal permeability was demonstrated by high ionic conductance and large unidirectional isotopic fluxes. Tissue conductance improved during in vitro incubation suggesting epithelial repair after removal of castor oil. Changes in the population and proportion of bacteria in the faeces as diarrhoea ensued were confirmed at necropsy with a predominance of E. coli and Enterobacter/Klebsiella sp in the large bowel. The experimental induction of castor oil colitis showed many similarities to intestinal endotoxaemia and the secretory type diarrhoea encountered in naturally occurring acute colitis syndromes in horses. The model could prove applicable in studying the pathophysiological mechanisms precipitating such life-threatening disorders.     

Effects of different absorption enhancers on the permeation of ebiratide, an ACTH analogue, across intestinal membranes

The permeation of ebiratide (H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2), a novel ACTH analogue, across the intestinal mucosae has been examined by use of isolated intestinal membranes from rats in a modified Ussing chamber. Regional differences were observed in the permeation of ebiratide across intestinal membranes; the order of membrane permeability was jejunum > ileum > duodenum > colon. Overall, the permeation of ebiratide was relatively poor. The effects of various absorption enhancers were examined to increase the intestinal permeability to ebiratide. Sodium glycocholate and sodium caprate had no significant enhancing effect on the permeability of the jejunal membrane, but significantly enhanced the permeation of ebiratide through the colonic membrane. On the other hand, N-dodecyl-beta-D-maltopyramoside (LM) significantly enhanced the permeation of ebiratide through both jejunal and colonic membranes. In general, the absorption-enhancing effects of these agents were more predominant in the colon than in the jejunum. Membrane damage by the absorption enhancers was evaluated by measuring the amount of protein released from the intestinal membrane. It was found that all the absorption enhancers slightly increased the amount of protein released, but that the amounts of protein released in the presence of these enhancers were much less than in the presence of ethylenediaminetetraacetic acid (EDTA), used as a positive control. These findings suggest that the absorption enhancers, especially LM might be useful adjuvants for improving the intestinal absorption of peptide and protein drugs, including ebiratide.     

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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.     

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.     

Insulin modulates cellular proliferation in developing human jejunum and colon

Several lines of evidence suggest an important role for insulin in the regulatory mechanism of rodent small intestinal development. To investigate its potential implication in human gut, the immunofluorescent localization of insulin receptors (IR) and the influence of insulin (30 microU or 3 mU/ml) on [3H]-thymidine incorporation and on lactase and alkaline phosphatase activities were studied in fetal jejunum and colon (14-19 weeks). We demonstrate the early presence of IR, mainly detected in the basolateral portion of enterocytes and colonocytes along the crypt-villus axis. Insulin increased [3H]-thymidine incorporation as well as epithelial labeling indices in cultured explants from jejunum and colon without affecting enzymic activities. This study establishes, for the first time, that insulin stimulates proliferation of epithelial cells expressing IR in both segments without affecting brush border hydrolases in the developing human gut.     

Bile regulates the expression of major histocompatibility complex class II molecules on rat intestinal epithelium

BACKGROUND & AIMS: Major histocompatibility complex (MHC) class II molecules are expressed on intestinal epithelial cells, and the intensity of this expression is regulated. The aim of this study was to test the hypothesis that bile regulates the expression of MHC class II molecules on intestinal epithelium. METHODS: Rats were deprived of intestinal bile by external drainage for 24 or 48 hours, and their intestines were collected, sectioned, and stained with the anti-MHC class II monoclonal antibodies OX4 and OX6. For one group of rats, bile flow was deviated from its usual entry point to the ileum. RESULTS: Compared with intact animals, MHC class II expression was observed to be diminished within 24 hours and totally absent after 48 hours of bile drainage. For the group in which bile flow was deviated to the ileum, staining was only observed in the region distal to the entry point. Analysis by bioassay and enzyme-linked immunosorbent assay of bile showed the presence of tumor necrosis factor and interferon gamma, respectively. CONCLUSIONS: It is concluded that the presence of bile is required for the expression of MHC class II molecules on gut epithelium and that the cytokine components of bile may be the inducing agents.     

Calcium channel blockers modify jejunal uptake of D-galactose in rabbits

Calcium channel blockers modify the intestinal uptake of lipids. This study was undertaken to test the hypothesis that two different types of calcium channel blockers influence the uptake of D-galactose, a sugar absorbed by the sodium-dependent glucose transporter (SGLT1) in the intestinal brush border membrane. Nisoldipine (1 mg/kg/day) or verapamil (4 mg/kg/day) were given by mouth to New Zealand white rabbits for three weeks, and then the rates of uptake of varying concentrations (2-64 mM) of galactose were examined in an in vitro preparation of jejunum using the incorporation of 14C-labeled substrate into intact tissue segments. The maximal transport capacities (Vmax) for D-galactose were increased in animals given nisoldipine or verapamil, as compared to controls. The value of the apparent Michaelis constant Km* for D-galactose was higher with nisoldipine group and lower with verapamil, than in controls. The apparent passive permeability (Pd*) of D-galactose was estimated from the uptake of L-glucose: Pd* was lower with nisoldipine and higher with verapamil, as compared to controls. The effect of these drugs on sugar uptake is not due to differences in the animals' food intake, body weight gain, or mucosal surface area. Thus, the two different classes of calcium channel blockers, the dihydropyridine nisoldipine and the phenylalkylamine verapamil, have different effects on the K(m)* and Pd*, but not on the Vmax of D-galactose uptake.     

Biochemical and morphological correlations in human gallbladder with reference to membrane permeability

There is good evidence that gallbladder epithelium is permeable to a diverse range of molecules which move into the epithelial cell from the lumen or the basement membrane. Morphological investigations have shown both secretory mucous droplets, components of the endocytosis pathway together with evidence of a system allowing passage of molecules across the basement membrane. This indicates that the gallbladder epithelium may be influenced by molecules presented via the apical and basal membranes, complicating our understanding of gallbladder function, particularly in disease. Gallbladder disease increases the proteoglycan content of the basement membrane, but the implication of this in terms of permeability remains to be defined. Indeed, it remains unknown whether this precedes disease or is a manifestation of the disease process. The removal of water from hepatic bile by gallbladder involves two counter ion transport systems. Autoradiography shows that ion transport occurs into the lateral intracellular spaces but it remains unclear whether this leads to a hypertonic solution in these spaces causing an osmotically driven water absorption or if the process involves an osmotically linked isotonic secretion. These ion pumps are reversible, for water is absorbed during the interdigestive phase but fluid is secreted into the lumen during digestion or in the presence of disease. Appropriate neural stimulation can increase or decrease fluid absorption from the lumen while vasoactive intestinal peptide or secretin promote fluid secretion, probably mediated by prostaglandins leading to raised cyclic AMP acting at the cellular level. Immediate control may depend on intracellular Ca2+ which activates a calmodulin-protein kinase, phosphorylating the counter ion transporters to downregulate their activity. Failure of this regulatory process may explain the initial increase in bile concentrating potential seen in the development of gallstones although the mechanism of such failure remains unknown. More concentrated bile increases movement of biliary compounds into gallbladder epithelial cells which alter gallbladder function in a complex manner. Secondary bile acids are raised in gallstone disease and increase permeability of the gallbladder epithelium to molecules including cholesterol. This cholesterol absorbed from the lumen may have paramount importance to gallbladder function. Raised biliary cholesterol reduces gallbladder motility, possibly by increasing the amount of cholesterol in gallbladder muscle membranes and reducing contraction in response to cholecystokinin. However, increased secondary bile acids are also associated with an alteration in phospholipid acyl groups which may alter ion transport activity and/or cholesterol solubility within the micelle/vesicle. As the acyl groups show increased arachidonate levels the production of prostaglandins could be raised, although currently it is not known if this phospholipid arachidonate enters the epithelial cells. In addition, gallbladder inflammation is associated with raised phospholipase A2 activity, leading to formation of fatty acids and lysophospholipid which causes membrane damage. The fatty acids are likely to displace cholesterol from the micelle but may also act directly on the epithelium, possibly increasing prostaglandin production and thus stimulating mucin secretion. Increased mucin secretion is seen early in gallstone disease but the evidence presently available cannot determine if this is a causative factor.     

Intraepithelial gamma delta T cells are closely associated with apoptotic enterocytes in the bovine intestine

The T cell population in the intestinal epithelium, comparable in size to the T cell pool in the spleen, is characterized by the predominant distribution of T cells bearing gamma delta T cell receptors. To determine the functional significance of the intraepithelial lymphocytes, we observed gamma delta T cells present in the jejunal epithelium in cattle, in which there is predominance of gamma delta T cells. Immunohistochemistry of frozen sections demonstrated that gamma delta T lymphocytes were densely distributed in the villous epithelium but there were fewer in the lamina propria and they were not present in the crypt epithelium. Ultrastructurally, intraepithelial gamma delta T cells were characterized by possessing electron-dense granules and interdigitating with enterocyte cytoplasm. Enterocytes, which were inserted by processes of intraepithelial lymphocytes or contacted by their cell bodies, showed morphologic changes seen in apoptotic cell death, such as elevated electron density of the cytoplasm and condensation of the chromatin. Apoptotic cells and cell debris were found in macrophages, which gathered in the subepithelial region of villus tips. These findings suggest that in the small intestine of cattle, gamma delta T cells are involved in the renewal of epithelial cells by inducing apoptosis of epithelial cells.     

The contribution of the distal gastrointestinal tract to glucagon-like immunoreactivity secretion in the rat

Radioimmunoassay of acid ethanol extracts of the rat intestinal tract showed the presence of glucagon-like immunoreactivity (GLI) from the duodenum to the colon with maximal concentrations in the ileum and colon. Twenty percent glucose instilled in the duodenum at a dose of 2g/kg body weight stimulated a twofold increase in peripheral plasma GLI concentration. When the instilled glucose load was restricted to only the duodenum and jejunum, or to only the ileum and colon, the increase in peripheral plasma GLI was approximately half that seen when the entire gut was exposed to the glucose. It is concluded that the distal gut as well as the proximal gut releases GLI in the rat.     

Role of phosphodiesterases in the regulation of gonadotropin- releasing hormone secretion in GT1 cells

The development of the bovine small intestine was examined in 39 embryos and fetuses by light microscopic and transmission electron microscopic methods. Special reference was paid to the histogenesis of the ephithelium. In contrast to the duodenum the epithelium of jejunum and ileum undergoes a degeneration by vacuolation of its villous epithelial cells. The demonstration of the acid phosphatase activity of these vacuoles shows their lysosomal character. This degenerative process of the small intestinal epithelium is also known in large intestine where it leads to the destruction of the intestinal villi. Both seem to be part of a 'principle of construction of the intestine of the vertebrates' (Wille, 1984).     

Use of protease inhibitors to improve calcitonin absorption from the small and large intestine in rats

The objective of this study was to examine the effects of protease inhibitors on the absorption of calcitonin from different regions of the intestine in rats. The absorption experiments were investigated by in-situ use of closed intestinal loops in rats and stability of calcitonin was examined in mucosal homogenates and intestinal fluids. The intestinal absorption of calcitonin was evaluated by measurement of its hypocalcaemic effect. No substantial hypocalcaemic response was observed when calcitonin was administered into the jejunum or colon. A slight hypocalcaemic effect was observed after administration of calcitonin into the ileum. Of the co-administered protease inhibitors, bacitracin (20mM) strongly promoted calcitonin absorption from the jejunum, ileum and colon. A significant hypocalcaemic effect was also obtained after intestinal administration of calcitonin with soybean trypsin inhibitor (10mgmL(-1)), camostat mesylate (20mM) or aprotinin (2mgmL(-1)). In the stability experiment, bacitracin reduced the degradation of calcitonin in the different intestinal homogenates. Soybean trypsin inhibitor significantly reduced the degradation of calcitonin in the fluids of the small intestine. We also examined the different endopeptidases in gut luminal fluids and the different exopeptidases in gut mucosal homogenates of rats. The ranking order for the total endopeptidase activity of the intestinal fluids was jejunum > ileum > colon. That for total exopeptidase activity of the intestinal mucosa was jejunum > ileum > colon. These results suggest that endo- and exopeptidases might be responsible for the hydrolysis of calcitonin and that protease inhibitors might usefully improve absorption of calcitonin to the systemic circulation from the large intestine.     

Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin

In contrast to sessile cell types, lymphocytes migrate through the vasculature to become diffusely distributed in tissues or organized in lymphoid structures. A complex array of adhesion molecules including selectins, integrins and their counter-receptors mediate lymphocyte homing and migration into tissues and may be constitutively expressed or induced. However, the molecules that mediate the tissue-specific retention of lymphocytes within the parenchyma have not been identified. Along the epithelium at the basolateral surface of enterocytes, intestinal intraepithelial lymphocytes are found. These T cells of the mucosal immune system serve as a model for the tissue-specific compartmentalization of lymphocytes. We investigated whether the localization of these intestinal intraepithelial lymphocytes could be mediated by specific interactions between adhesion molecules expressed selectively on this subpopulation of T cells and tissue-restricted adhesion molecules on epithelial cells. Here we show that heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes in vitro are mediated by E-cadherin and the alpha E beta 7 integrin.     

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.