Transport Of FD4 Research Articles

Quaternization of high molecular weight chitosan for increasing intestinal drug absorption using Caco-2 cells as an in vitro intestinal model

Potential use of a quaternized chitosan (MW 600 kDa) with 65% of 3-chloro-2-hydroxypropyltrimethylammonium (600-HPTChC65) as an absorptive enhancer was investigated in Caco-2 monolayers. 600-HPTChC65 (0.005% w/v) quickly reduced transepithelial electrical resistance (TEER) to the maximum level in 40 min with full recovery within 6 h after removal. Its TEER reduction was corresponded to increased FD4 transport across the monolayers and disrupted localization of tight junction proteins ZO-1 and occludin at the cell borders. 600-HPTChC65 was densely localized at the membrane surface and intercellular junctions. This chitosan (0.08–0.32% w/v) reduced the efflux ratio of [3H]-digoxin by 1.7- 2 folds, suggesting an increased [3H]-digoxin transport across the monolayers. Its binding with P-gp on Caco-2 monolayer increased the signal of fluorescence-labeled anti-P-gp (UIC2) reactivity due to conformational change. 600-HPTChC65 (0.32% w/v) had no effect on P-gp expression in the Caco-2 monolayers. These results suggest that 600-HPTChC65 could enhance drug absorption through tight junction opening and decreased P-gp function. Its interaction with the absorptive barrier mainly resulted in disrupting ZO-1 and occludin organization as well as changing in P-gp conformation.

Analysis of absorption-enhancing mechanisms for combinatorial use of spermine with sodium taurocholate in Caco-2 cells

Previously, we reported that the combined use of spermine (SPM) and sodium taurocholate (STC) (SPM–STC) significantly improves the oral absorption of rebamipide (BCS class IV) and pulmonary absorption of interferon-α without any harmful histopathological changes in the gastrointestinal tract and lungs, respectively. In the present study, we examined the effect of SPM–STC on the transport of fluorescein isothiocyanate-labeled dextrans (FDs) across Caco-2 cell monolayers and attempted to clarify the mechanisms underlying the transport enhancement caused by SPM–STC. SPM–STC were found to significantly enhance the transport of FDs, while the treatment with SPM–STC was not harmful, and the decrease in transepithelial electrical resistance was transient and reversible. The voltage-clamp study clearly indicated that the opening of the paracellular route could be mainly responsible for the enhanced transport of FD-4. As for the mechanisms, it was found that SPM–STC caused a significant increase in membrane fluidity, which would lead to the enhanced transport of small-molecule drugs such as rebamipide. Since SPM–STC increased intracellular Ca2+ via Ca2+ uptake through Ca2+ channels and Ca2+ release from the endoplasmic reticulum stimulated by the IP3 pathway, the subsequent possible activation of the MLCK signaling pathway would have led to the contraction of the actin–myosin ring. The rearrangement of tight junction-constituting proteins induced through the MAPK pathway has also been suggested as a possible mechanism for opening tight junctions. Claudin-4, a key protein constituting the tight junction, merged with F-actin along with the plasma membrane, was significantly decreased, which would be at least partial structural evidence for the tight-junction opening.

Effect of double bond in unsaturated long-chain monoglyceride in self-emulsified nanoemulsion on tight junction opening

Unsaturated long chain monoglycerides (LCMs) used to formulate self-emulsified nanoemulsion (SEN) are varied in their degree of unsaturation which may influence their activities on membrane permeability. Therefore, this study highlights the impact of double bond in LCMs on the tight junctions of Madin-Darby Canine Kidney (MDCK) cell monolayer. Two LCMs, Glyceryl monooleate (one double bond) and Glyceryl monolinoleate (two double bonds) were formulated into SENs (SEN2 and SEN3, respectively) with Glyceryl tricaprylate and PEG-40 hydrogenated castor oil (1:1:2), and compared to the SEN (SEN1) consisted of only Glyceryl tricaprylate and PEG-40 hydrogenated castor oil (1:4). Lucifer Yellow (LY, MW 457) or Fluorescein isothiocyanate–Dextran4K (FD4, MW 3000-5000) was added to the aqueous phase of the SENs and tested for their transport across the cell monolayer. The cumulative transport of LY and FD4 achieved by SEN2 and SEN3 were significantly (p < 0.05) higher than those in phosphate buffer, 0.5% PEG-40 hydrogenated castor oil, and SEN1, indicating that the tight junctions were opened by the SENs containing LCMs. The cumulative transport of FD4 achieved by SEN3 was also 2-3 folds higher than SEN2 (p < 0.05). Therefore, LCM with two double bonds have greater potency of opening tight junction than LCM with a single double bond in SEN formulation.

Intestinal Drug Absorption Enhancement by Aloe vera Gel and Whole Leaf Extract: In Vitro Investigations into the Mechanisms of Action.

The co-administration of absorption enhancing agents with macromolecular drugs (e.g., protein and peptide drugs) has been identified as a means to improve the oral bioavailability of these drugs. Absorption-enhancing agents of natural origins have received a great deal of attention due to their sustainable production, in support of green chemistry. In previous studies, certain parts of the Aloe vera leaf (e.g., gel and whole leaf extract) have shown a potential to enhance drug permeation across the intestinal epithelial barrier. The mechanism of the drug-absorption-enhancement action and the capacity for absorption-enhancement of the A. vera gel and whole leaf, were investigated in this study. A clear decrease in transepithelial electrical resistance (TEER) of Caco-2 cell monolayers exposed to A. vera gel and wholeleaf extract, in various concentrations, indicated the opening of tight junctions between the epithelial cells. The transport of Fluorescein isothiocyanate (FITC)-dextran, with a molecular weight of 4 kDa (FD-4), could be enhanced across the Caco-2 cell monolayers, by the A. vera gel and whole-leaf extract, but not the FITC-dextran with larger molecular weights (i.e., 10, 20, and 40 kDa), which indicated a limited drug absorption enhancement capacity, in terms of the molecular size. Accumulation of FD-4 between the Caco-2 cells (and not within the cells), after treatment with the A. vera gel and whole-leaf extract was shown with a confocal laser scanning microscopy (CLSM) imaging, indicating that the paracellular transport of FD-4 occurred after the interaction of the A. vera gel and whole-leaf extract, with the epithelial cell monolayers. Furthermore, changes in the F-actin distribution in the cytoskeleton of the Caco-2 cell monolayers was observed by means of a fluorescence staining, which confirmed tight junction modulation as the mechanism of action for the absorption enhancement effect of the A. vera gel and whole-leaf extract.

Teduglutide, the stable GLP‐2 analog inhibits lipid‐induced LPS transport into the portal vein and intestinal paracellular permeability after systemic inflammation

Lipopolysaccharide (LPS) is a cell wall component of Gram‐negative bacteria that is associated with the metabolic syndrome at one extreme and multiple organ failure at the other, the latter associated with increased intestinal paracellular permeability. We have found that LPS is acutely transported into the portal vein (PV) during lipid absorption via CD36‐ and lipid raft‐mediated transcellular pathways and that GLP‐2 acutely reduces intestinal paracellular permeability during systemic inflammation. We thus hypothesized that a stable GLP‐2 analog teduglutide (TDG) affects LPS transport during lipid absorption and reduces intestinal permeability during systemic inflammation.FITC‐LPS was infused into small intestine from the duodenum with oleic acid (30 mM)/taurocholic acid (10 mM) in anesthetized rats. The appearance of FITC‐LPS was simultaneously monitored in the PV and mesenteric lymph with or without TDG pretreatment (0.5 – 50 μg/kg, iv) 15 min before FITC‐LPS infusion. Intestinal permeability was increased by ip injection of LPS (5 mg/kg). FD4 transport into the PV was measured in vivo at 6 hr after LPS injection with or without TDG treatment (50 μg/kg, ip) at 0, 3, or 6 hr after LPS treatment.LPS was acutely transported into PV at 15 min during lipid absorption, followed by gradual transport into lymph by incorporation to chylomicron at 60 – 90 min. Luminal co‐infusion of the selective agonist for short‐chain fatty acid receptor FFA3, AR420626 (10 μM), which increased GLP‐2 release, inhibited LPS transport into the PV. A DPP4 inhibitor NVP728 (3 μmol/kg, iv) reduced LPS transport into the PV and lymph, suggesting that enhanced endogenous GLP‐2 concentrations reduce LPS transport. TDG iv injection acutely inhibited LPS transport into PV, but enhanced LPS transport into lymph, accompanied by increased lymph output. Luminal L‐NAME (0.1 mM) reversed TDG effects on LPS transport into PV and lymph, suggesting mediation by NO production from myenteric nNOS neurons, which expresses GLP‐2 receptors. Systemic LPS treatment at 6 hr, but not at 1 or 3 hr, increased FD4 movement from the lumen to PV, accompanied by increased expression of the proinflammatory cytokines TNFα, IL‐1β and IL‐6 in the ileal mucosa, suggesting that LPS‐induced subepithelial inflammation increases intestinal epithelial permeability for macromolecules. TDG pretreatment prior to LPS injection aggravated LPS‐induced FD4 permeability into the PV, whereas TDG treatment at 3 – 6 hr after LPS injection inhibited FD4 permeability. Pretreatment with the GLP‐2 receptor antagonist GLP‐2(3‐33) (3 nmol/kg, ip) prior to LPS injection enhanced FD4 permeability.These results suggest that TDG acutely inhibits LPS transport and intestinal permeability, the latter with therapeutic windows at 3 – 6 hr after the induction of systemic inflammation, consistent with half‐life of TDG ~ 2 hr in the circulation. TDG may be a therapeutic candidate for metabolic syndrome such as fatty liver and obesity, and secondary endotoxemia such as occurs in serious illness by inhibiting LPS transport under physiological and pathological conditions.Support or Funding InformationShire Pharmaceuticals, VA Merit Review, NIDDK R01 DK54221This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NUTRIENTS ACUTELY MODULATE INTESTINAL PERMEABILITY INDEPENDENTLY OF THE ENTERIC NERVOUS SYSTEM

BackgroundIt is well known that diet profoundly affects intestinal physiology, with some diets contributing to a “leaky gut”, possibly promoting IBD and metabolic syndrome. Recently, several studies have suggested that the enteric nervous system, composed of neurons and glial cells, could be a potent regulator of intestinal permeability. We hypothesize that nutrients, sensed at the level of the epithelium, could trigger a neuroglial response to regulate intestinal permeability.AimsWe investigated whether nutrients acutely modulate intestinal permeability at the level of the small intestine and if the enteric nervous system plays a role in this effect.MethodsIntact jejunum and Ileum segments from CD1 mice were mounted in Ussing chambers. Transepithelial electrical resistance (TER) and short circuit current (Isc) were recorded for 50 min after stimulation with different nutrients. Two different size probes, 400 Da Fluorescein isothiocyanate (FITC) or 4000 Da FITC‐Dextran (FD4), were used as markers of intestinal permeability. Apical glucose, fructose, glutamine (10 mM) or 5% Intralipid® were used as nutrient stimuli. Enteric neurons were either inhibited with tetrodotoxin (TTX, 0.5 μM) or activated with veratridine (10 μM). Enteric gliotransmission was inhibited with the connexin 43 blocker Gap26 (20 μM).ResultsThe nutrients used in this study were “sensed” by the intestinal epithelium as illustrated by a rapid change in Isc following stimulation (+14, +7, −28 μA/cm2 for glucose, glutamine and Intralipid, respectively, in the jejunum; +132, +42, −14, −56 μA/cm2 for glucose, glutamine, fructose and Intralipid in the ileum). Addition of 5% Intralipid to the luminal side increased TER in both jejunum (+10 Ω/cm2) and Ileum (+10 Ω/cm2). Glutamine triggered a significant (p&lt;0.05) increase of the transport of FITC across the intestine (+600% in jejunum and +60% in ileum after 50 minutes). On the other hand the transport of both FITC and FD4 was significantly reduced in the presence of Intralipid (−73% and −27%, respectively, in the jejunum and −74% and −31%, in the ileum). Results were similar when germ free mice were compared to conventionally raised mice. Neither the activation or inhibition of enteric neurons or the inhibition of gliotransmission affected TER or the transport of the fluorescent probes across the mouse intestine. Additionally, the use of the neuronal blocker, TTX, did not reverse the glutamine‐ or Intralipid‐induced alteration of FITC and FD4 transport across the intestine. Blocking the calcium‐sensing receptor did not alter the response to glutamine.ConclusionsWe have shown glutamine and Intralipid® to be potent regulators of intestinal permeability acting independently of the enteric nervous system, the microbiota, and the calcium‐sensing receptor. We are currently investigating the potential endocrine contribution to these effects.Support or Funding InformationThe work of Dr. Cavin was supported by a postdoctoral fellowship from the Human Frontier Science Program Organization and Alberta innovates.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of sesquiterpenes as permeation enhancers for a model macromolecule across human skin in vitro

The objective of this study was to assess the in vitro transdermal permeation of a model macromolecular compound namely; fluorescein isothiocyanate - dextran 4 kDa (FD-4) across full thickness human skin by means of chemical enhancers. For this purpose, two sesquiterpenes derived from farnesol were synthesized and further evaluated. Transport studies were conducted using vertical Franz diffusion cells followed by tape stripping in an attempt to examine the disposition of FD-4 on and within the skin. The transport of FD-4 across skin was significantly enhanced in the presence of farnesol and two of its derivatives: A 3.02, 2.67 and 3.74-fold increase was observed compared to a control formulation free of sesquiterpenes. Light microscopy studies showed detachment of the superficial layers and in some cases loosening and swelling of cell layers. Differential scanning calorimetry (DSC) studies of stratum corneum (SC) sheets treated with the sesquiteprenes indicated that enhanced permeation of FD-4 was mainly achieved by disruption and fluidization of the SC lipid bilayers whereas infrared spectroscopy (AT-FTIR) data indicated significant decreases in peak intensity. The results showed that the co-administration of selected sesquiterpenes with FD-4 increased its transport across the skin and might be advantageous for transdermal delivery of macromolecules.

Efficacy, safety and mechanism of HP-β-CD-PEI polymers as absorption enhancers on the intestinal absorption of poorly absorbable drugs in rats

Context: Oral bioavailability of some hydrophilic therapeutic macromolecules was very poor, thus leading to their limited application in clinic.Objective: To investigate the efficacy, safety and mechanism of HP-β-CD-PEI polymers on the intestinal absorption of some poorly absorbable drugs in rats.Methods: Effects of HP-β-CD-PEI polymers on the intestinal absorptions of drugs were investigated by an in situ closed loop method in rats. The safety of HP-β-CD-PEI polymer was evaluated by measurement of lactate dehydrogenase (LDH) activity and amount of protein released from rat intestinal perfusate. The absorption enhancing mechanisms were explored by the measurement of zeta potential, transepithelial electrical resistance (TEER) and in vitro transport of FD4 (a paracellular marker) across rat intestinal membranes, respectively.Results: HP-β-CD-PEI polymers, especially HP-β-CD-PEI1800, demonstrated excellent absorption enhancing effects on drug absorption in a concentration-dependent manner and the enhancing effect was more efficient in the small intestine than that in the large intestine. Five percent (w/v) HP-β-CD-PEI1800 obviously decreased the TEER, accompanied with increase in the intestinal transport of FD4, indicating that absorption enhancing actions of HP-β-CD-PEI polymers were possibly performed by loosening tight junctions of intestinal epithelium cells, thereby increasing drug permeation via a paracellular pathway. A good liner relationship between absorption enhancing effects of HP-β-CD-PEI polymers and their zeta potentials suggested the contribution of positive charge on the surface of these polymers to their absorption enhancing effects.Conclusion: HP-β-CD-PEI polymers might be potential and safe absorption enhancers for improving oral delivery of poorly absorbable macromolecules including peptides and proteins.

Characterization of Ocular Iontophoretic Drug Transport of Ionic and Non-ionic Compounds in Isolated Rabbit Cornea and Conjunctiva.

Ocular iontophoresis (IP) in isolated rabbit cornea and conjunctiva was examined in terms of transport enhancement, tissue viability and integrity using electrophysiological parameters by the Ussing-type chamber technique. Lidocaine hydrochloride (LC, a cationic compound), sodium benzoate (BA, anionic compound), and fluorescein isothiocyanate labeled dextran (molecular weight 4400 Da, FD-4, hydrophilic large compound) were used as model permeants. Direct electric current was applied at 0.5-5.0 mA/cm(2) for the cornea and 0.5-20 mA/cm(2) for the conjunctiva for 30 min. LC and BA fluxes across the cornea and conjunctiva were significantly increased by the application of electric current up to 2.3- and 2.5-fold and 4.0- and 3.4-fold, respectively, and returned to their baseline level on stopping the current. Furthermore, a much higher increase by IP application was obtained for the FD-4 transport. The increased FD-4 flux in the conjunctiva returned to baseline on stopping the current, whereas the flux in the cornea was sustained at a higher level after stopping the current. The transepithelial electric resistance of the cornea and conjunctiva was lowered by electric current application but fully recovered after stopping the current up to 2.0 mA/cm(2) for the cornea and 10 mA/cm(2) for the conjunctiva, suggesting that the corneal and conjunctival viability and integrity are maintained even after application of these current densities. These results indicate that ocular IP may be a useful non-invasive technique to achieve drug delivery of hydrophilic large molecules into the eyes.

Thiolated nanocarriers for oral delivery of hydrophilic macromolecular drugs.

It was the aim of this study to investigate the effect of unmodified as well as thiolated anionic poly(acrylic acid) (PAA) and cationic chitosan (CS) utilized in free-soluble form and as nanoparticulate system on the absorption of the hydrophilic compound FD4 across intestinal epithelial cell layer with and without a mucus layer. Modifications of these polymers were achieved by conjugation with cysteine to PAA (PAA-Cys) and thioglycolic acid to CS (CS-TGA). Particles were prepared via ionic gelation and characterized based on their amount of thiol groups, particle size and zeta potential. Effects on the cell layer concerning absorption enhancement, transepithelial electrical resistance (TEER) and cytotoxicity were investigated. Permeation enhancement was evaluated with respect to in vitro transport of FD4 across Caco-2 cells, while mucoadhesion was indirectly examined in terms of adsorption behaviour when cells were covered with a mucus layer. Lyophilized particles displayed around 1000 μmol/g of free thiol groups, particle sizes of less than 300 nm and a zeta potential of 18 mV (CS-TGA) and -14 mV (PAA-Cys). Cytotoxicity studies confirmed that all polymer samples were used at nontoxic concentrations (0.5% m/v). Permeation studies revealed that all thiolated formulations had pronounced effects on the paracellular permeability of mucus-free Caco-2 layers and enhanced the permeation of FD4 3.0- to 5.3-fold. Moreover, polymers administered as particles showed a higher permeation enhancement than their corresponding solutions. However, the absorption-enhancing effect of each thiolated formulation was significantly (p<0.05) reduced when cells were covered with mucus layer. In addition, all formulations were able to decrease the TEER of the cell layer significantly (p<0.05). Therefore, both thiolated polymers as nanoparticulate delivery systems represent a promising tool for the oral administration of hydrophilic macromolecules.

In vitro evaluation of mucoadhesion and permeation enhancement of polymeric amphiphilic nanoparticles

Oleoyl-carboxymethy-chitosan (OCMCS) nanoparticles based on chitosan with various molecular weights were prepared using coacervation process, which demonstrated particle size of 150–350nm, zeta potential of 10–20mV, and high encapsulation efficiency of fluorescein isothiocyanate dextran (FD4). OCMCS nanoparticles were found to be adsorbed onto the excised carp intestinal mucosa, the extent of adsorption increased with increasing chitosan molecular weight. In comparison to FD4 solution, OCMCS nanoparticles promoted FD4 transport through excised carp intestinal mucosa by 3.26–6.52 folds, which were observed via fluorescence microscope. The OCMCS nanoparticulate systems that interacted with the Caco-2 cells decreased the transepithelial electric resistance (TEER) and induced increasing the apparent permeability coefficient (Papp) of FD4 by 3.61–6.32 folds. Cytotoxicity studies in Caco-2 monolayers verified the safety of the delivery system. The improvement of mucoadhesive ability and permeability enable the OCMCS nanosystems suitable carriers for the intestinal absorption of protein drugs.

Immobilization of 2-mercaptoethylamine on oxidized chitosan: a substantially mucoadhesive and permeation enhancing polymer

Thiolated chitosans are mucoadhesive polymers that are able to increase the paracellular permeability and improve the oral bioavailability of hydrophilic and macromolecular compounds. Due to the low hydratability of most thiolated chitosans at pH > 6.5, it was the aim of this study to develop a thiomer as a permeation enhancer for mucosal membranes where a comparatively high pH is prevalent. For this purpose chitosan was oxidized by means of sodium periodate to cleave the polysaccharide structure. The intermediate polymer was coupled with 2-mercaptoethylamine and reduced with sodium cyanoborohydride to obtain the secondary amine moiety. The permeation enhancing effect of 2-mercaptoethylamine-functionalized chitosan conjugate (chitosan-2-MEA) was assessed in Ussing-type chambers using FD4 as a model compound. Further in vitro characterization was conducted regarding water absorbing capacity, cohesive properties, mucoadhesion and cytotoxic effects on Caco-2 cells. By means of fundamental alteration of chitosan's backbone and following thiol immobilization, the paracellular transport of FD4 across rat intestinal mucosa was 5.1-fold improved compared with control (buffer) only. Mucoadhesion could be 20-fold improved likely based on increased chain flexibility and disulfide formation between chitosan-2-MEA and mucus glycoproteins. The novel thiomer showed excellent hydratability in aqueous media even up to pH 11 and was proven to be non-toxic. According to the above-mentioned properties, chitosan-2-MEA seems to be a promising mucoadhesive and permeation enhancing excipient for the development of drug delivery systems.

Sodium hyaluronate as a mucoadhesive component in nasal formulation enhances delivery of molecules to brain tissue

Intranasal administration of molecules has been investigated as a non-invasive way for delivery of drugs to the brain in the last decade. Circumvention of both the blood–brain barrier and the first-pass elimination by the liver and gastrointestinal tract is considered as the main advantages of this method. Because of the rapid mucociliary clearance in the nasal cavity, bioadhesive formulations are needed for effective targeting. Our goal was to develop a formulation containing sodium hyaluronate, a well-known mucoadhesive molecule, in combination with a non-ionic surfactant to enhance the delivery of hydrophilic compounds to the brain via the olfactory route. Fluorescein isothiocyanate-labeled 4 kDa dextran (FD-4), used as a test molecule, was administered nasally in different formulations to Wistar rats, and detected in brain areas by fluorescent spectrophotometry. Hyaluronan increased the viscosity of the vehicles and slowed down the in vitro release of FD-4. Significantly higher FD-4 transport could be measured in the majority of brain areas examined, including olfactory bulb, frontal and parietal cortex, hippocampus, cerebellum, midbrain and pons, when the vehicle contained hyaluronan in combination with absorption enhancer. The highest concentrations of FD-4 could be detected in the olfactory bulbs, frontal and parietal cortex 4 h after nasal administration in the mucoadhesive formulation. Intravenous administration of a hundred times higher dose of FD-4 resulted in a lower brain penetration as compared to nasal formulations. Morphological examination of the olfactory system revealed no toxicity of the vehicles. Hyaluronan, a non-toxic biomolecule used as a mucoadhesive in a nasal formulation, increased the brain penetration of a hydrophilic compound, the size of a peptide, via the nasal route.

Design and Evaluation of a New Gastrointestinal Mucoadhesive Patch System Containing Chitosan-Glutathione

Within this study, a novel gastrointestinal patch system was developed and investigated regarding water-absorbing capacity, adhesive properties, in vitro release, unidirectional release and permeation enhancing effect. Water uptake studies revealed that the weight of patch systems with Ch-GSH increased about 44.5 ± 2.3 mg (127%) after 90 min. This patch system remained even after 180 h on the mucosa and released 49.7 ± 0.7% of FD4 within 8 h. A 2.5-fold higher transport of FD4 can be obtained in contrast to control. In conclusion this patch system could be an interesting possibility for the transport through the intestinal mucosa of macromolecules which will normally be degraded in the intestinal tract.

Poly-L-arginine predominantly increases the paracellular permeability of hydrophilic macromolecules across rabbit nasal epithelium in vitro.

The purpose of this work was to characterize the main transport pathway of hydrophilic macromolecules induced by poly-L-arginine (poly-L-Arg; molecular weight 42.4 kDa) across the excised rabbit nasal epithelium. Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of fluorescein isothiocyanate-labeled dextran (FD-4; molecular weight 4.4 kDa) transport and transepithelial electrical resistance (TEER). The main transport pathway of FD-4 enhanced by poly-L-Arg was evaluated using confocal laser scanning microscopy. Immunolocalization of junction proteins (ZO-1, occludin, and E-cadherin) after treatment with poly-L-Arg was also observed. After apical application of a poly-L-Arg (0.05, 0.5, and 5 mg/mL), the permeability coefficient of FD-4 increased by 1.6-, 2.9-, and 5.2-fold, respectively, compared with the control of 5.2 +/- 1.3 x 10(-7) cm/s. Consistent with the increase in transport, there was a concurrent reduction in TEER. At a concentration of 0.05 mg/mL poly-L-Arg. both FD-4 transport and TEER returned to the control level. A good correlation was obtained between the FD-4 permeability coefficient and 1/TEER. Basolateral application of poly-L-Arg at 5 mg/mL, however, did not increase FD-4 transport. Marked FD-4 fluorescence was located in the paracellular spaces after treatment with apical poly-L-Arg compared with that in the absence of poly-L-Arg. Immunofluorescence of ZO-1, occludin, and E-cadherin in cell-to-cell junctions was reduced and distributed into the cytoplasm by apical application of poly-L-Arg, suggesting that poly-L-Arg regulates the junction proteins to enhance paracellular permeability across the nasal epithelium. After pretreatment with either 2,4-dinitrophenol or ouabain, the enhancing effect of apical poly-L-Arg was abolished, indicating the contribution of metabolic energy (cell viability) to the poly-L-Arg-mediated enhancing effect. In the nasal epithelium, apical poly-L-Arg appears to increase predominantly the paracellular transport of hydrophilic macromolecules via disorganization of tight- and adherens-junction proteins. The regulatory mechanism of the poly-L-Arg effect is likely to be dependent on energy-requiring cellular processes.

Analysis of transient and reversible effects of poly- l-arginine on the in vivo nasal absorption of FITC-dextran in rats

We have investigated whether poly- l-arginine, with a mean molecular weights of 8.9 and 45.5 kDa (poly- l-Arg (10) and poly- l-Arg (50)), can induce transient and reversible effects involving enhancement of the nasal absorption of fluorescein isothiocyanate-labeled dextran (MW 4.4 kDa, FD-4) and determined the main pathway for the increased transport of FD-4 in rats in vivo. Pre-administration and repeated administration studies were conducted involving the selection of different time intervals between intranasal administration of poly- l-Arg and administration of FD-4, with and without poly- l-Arg, to characterize these transient and reversible effects. The degradation of poly- l-Args in a diluted nasal drip was determined from the fluorescence of degraded poly- l-Arg-fluorescamine products. In the pre-administration study, poly- l-Arg exhibited a transient effect on the increased nasal FD-4 absorption depending on its molecular weight, associated with the degradation rate of poly- l-Arg in mucus. In the repeated administration study, additional poly- l-Arg produced similarly enhanced FD-4 absorption. Confocal laser scanning microscopy showed that fluorescence of FD-4 after co-administration of poly- l-Arg (50) was confined mainly to the paracellular spaces. In conclusion, poly- l-Arg exhibited molecular weight-dependent transient and reversible effects on the enhancement of nasal FD-4 absorption paracellularly in rats in vivo. The enzymatic degradation of poly- l-Arg is one of the key determinants of the transient effect on in vivo enhanced absorption of FD-4.

Polarized transport of hydrophilic compounds across rat colonic mucosa from serosa to mucosa is temperature dependent

Background & Aims: In both clinical and experimental studies, intestinal epithelial barrier function is routinely assessed by measuring mucosal permeability to various hydrophilic compounds. We performed experiments to determine whether permeation of several hydrophilic compounds across rat colonic mucosa is polarized. Methods: Sheets of colonic mucosa, stripped of the underlying seromuscular coats, were mounted in Ussing chambers. Results: The rates of permeation across colonic mucosa by numerous hydrophilic compounds (fluorescein isothiocyanate [FITC]-dextrans with molecular weights of 4000 [FD4] and 70,000 [FD70] daltons, fluorescein disulfonic acid [FS], lucifer yellow [LY], lactulose, and mannitol) were several times greater in the serosal-to-mucosal (S→M) direction than in the opposite direction. Increased S→M permeation by FD4, lactulose, and mannitol was evident at 37°C, but not at 4°C. Efflux of FD4 and FS in the S→M direction was dose-dependently inhibited by verapamil, an inhibitor of the P-glycoprotein efflux system. Indomethacin, an anion transporter inhibitor, showed no effect on the S→M permeation of FD4, FD70, FS, or LY. Adding an excess of unlabeled dextran (mol wt, 10,000 daltons) dose-dependently decreased the S→M efflux of FD4, but not FS or LY. Conclusions: The transport across rat colonic mucosa of a number of hydrophilic substances, including some compounds that are commonly used to measure intestinal permeability in clinical practice, is greater in the S→M than in the M→S direction. S→M transport of these hydrophilic solutes is temperature dependent, suggesting that the process is an active one. S→M transport of FD4 may occur via a process that manifests some degree of substrate specificity for polysaccharides. GASTROENTEROLOGY 2000;118:535-543

Absorption Enhancement in Intestinal Epithelial Caco-2 Monolayers by Sodium Caprate: Assessment of Molecular Weight Dependence and Demonstration of Transport Routes

Sodium caprate (CIO), a medium chain fatty acid, is used clinically to enhance rectal absorption of the low molecular weight (MW) drug ampicillin. The main aim of this study was to investigate whether CIO also enhances the permeability of high MW model drugs in a model of the intestinal epithelium. The second aim was to present visual evidence of the route of enhanced transport across the epithelial cell layer. The studies were performed in Caco-2 monolayers cultured on permeable supports. The effects of non-toxic concentrations (≤ 13 mM) of CIO on drug transport across the monolayers was studied using monodisperse 14C-polyethylene glycols (MW 238-502; 14C-PEGs), 125I-Arg8-vasopressin (MW 1,208), 125I-insulin (MW 6,000) and FITC-labelled dextrans (MW 4,400 and 19,600; FD4 and FD20 respectively) as model drugs. Electron and confocal laser scanning microscopy were used to demonstrate transport routes across the epithelium. 10 mM C10 increased the permeability of all 14C-PEGs to approximately the same extent. 13 mM C10 increased the permeability of 125I-Arg8-vasopressin 10-fold. Only small increases in FD4 and FD20 permeabilities were observed. After C10 exposure, both tight junctions with normal morphology and those with dilatations showed an increased permeability to ruthenium red, indicating that C10 enhanced the paracellular transport of molecules with a MW < 1,000. Confocal microscopy showed that C10 increased the transport of FD4 and FD20 by the paracellular route. In conclusion, nontoxic concentrations of C10 can be used to enhance the permeability of drugs of MW up to approximately 1,200. Enhancement of the absorption of molecules larger than 4,000 is quantitatively insignificant. The enhanced permeability occurred via the paracellular pathway.

Ocular absorption of Pz-peptide and its effect on the ocular and systemic pharmacokinetics of topically applied drugs in the rabbit.

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. 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 microl of 20 mM atenolol or propranolol solution to the rabbit eye. 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. 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.

The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro

Two mucoadhesive polymers, chitosan-glutamate and carbomer, were studied in an in vitro model (Caco-2 cell monolayers) with respect to their ability to enhance intestinal peptide drug delivery. Preparations of the polymers at concentrations of 0.5, 1.0, and 1.5% w/v (chitosan), and of 0.5 and 1.0% w/v (carbomer) were applied to the apical side of Caco-2 cell monolayers. The effects on transepithelial electrical resistance (TEER), paracellular transport of a FITC-dextran of a molecular weight of 4400 (FD-4) and [ 14C]mannitol were measured. Paracellular transport of FD-4 was visualized by means of confocal laser scanning microscopy (CLSM). Furthermore, the impact of lowering the pH of the polymer solutions to pH 4 on the integrity of the cell layer was determined. The results show that both polymers were able to decrease TEER of Caco-2 cell layers significantly. In the case of carbomer, CLSM revealed a partial opening of epithelial tight junctions. Lowering of the pH in the control and polymer solutions to pH 4 resulted in every case in the irreversible damage of a large percentage of the cells, as shown by CLSM. Transport studies with [ 14C]mannitol and FD-4 showed only during co-application of carbomer significantly increased fluxes, whereas no difference from the control solution could be detected for chitosan-glutamate. A threshold value of about 50% of TEER reduction has been identified, which allows for transport of hydrophilic compounds across the cell monolayers of the Caco-2 cell model.