Lipid bilayer permeation by neutral aluminum citrate and by three alpha-hydroxy carboxylic acids

Several groups have proposed that aluminum (Al) may permeate biological membranes as a neutral complex with citrate. We tested this hypothesis by measuring aluminum citrate flux across unilamellar phospholipid vesicles (liposomes). Results from two independent procedures show that lipid bilayer permeation by the neutral aluminum-citrate complex is slow (P approximately equal to 1 x 10(-11) cm.s-1). We then compared aluminum-citrate permeation with permeation by a series of alpha-hydroxy carboxylic acids and by trimethylcitrate. This comparison showed that the aluminum-citrate flux is limited by diffusion across the water/lipid interface. This is due to hydrogen bonding between water and the citrate carboxyl groups, and by hydration of the bound metal in the aqueous phase. By analogy with citric acid, steric hindrance of diffusion within the bilayer does not affect the permeation rate of aluminum citrate. Elevated tissue levels of Al in subjects fed a diet supplemented with citric acid and Al(OH)3 cannot be explained by lipid bilayer permeation of the neutral complex.     

Defining the factors that affect solute permeation of gap junction channels

This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Differential permeation of propranolol enantiomers across human skin in vitro from formulations containing an enantioselective excipient.

This work tested the hypothesis that a stereospecific topical formulation could be used to engineer differential permeation rates for each enantiomer of an applied racemate across human skin in vitro. Racemic and enantiomerically pure R or S propranolol HCI were formulated with cellulose tris(3,5-dimethyl phenyl carbamate) (CDMPC) and applied to excised human skin using side-by-side Franz-type diffusion cells. When the pure enantiomers were used, there was a marked difference between the penetration rates of R and S propranolol (flux ratio: 2.06; P = 0.04). When racemic propranolol was used, the difference was reduced, although still statistically significant (flux ratio: 1.2; P = 0.08), particularly in view of the differential activities of the two enantiomers. Control experiments, in which no CDMPC was present, produced equal permeation rates. The results can be rationalised in terms of differential adsorption onto CDMPC within the vehicle, whereby S-propranolol is preferentially bound relative to R-propranolol. This causes an imbalance in the apparent donor phase concentrations that (in accordance with Fickian diffusion laws and thermodynamic activity) gives rise to differences in permeation rates. The diminished differential observed when the racemate was used, rather than individual enantiomers, is less easily rationalised. In this work, it was the permeation of the eutomer (S-propranolol) that was retarded, although the general principle of stereoselectively retarded skin permeation has been established.     

Transbuccal delivery of 5-aza-2 -deoxycytidine: effects of drug concentration, buffer solution, and bile salts on permeation

Delivery of 5-aza-2 -deoxycytidine (decitabine) across porcine buccal mucosa was evaluated as an alternative to the complex intravenous infusion regimen currently used to administer the drug. A reproducible high-performance liquid chromatography method was developed and optimized for the quantitative determination of this drug. Decitabine showed a concentration-dependent passive diffusion process across porcine buccal mucosa. An increase in the ionic strength of the phosphate buffer from 100 to 400 mM decreased the flux from 3.57 +/- 0.65 to 1.89 +/- 0.61 microg/h/cm2. Trihydroxy bile salts significantly enhanced the flux of decitabine at a 100 mM concentration (P > .05). The steady-state flux of decitabine in the presence of 100 mM of sodium taurocholate and sodium glycocholate was 52.65 +/- 9.48 and 85.22 +/- 7.61 microg/cm2/h, respectively. Two dihydroxy bile salts, sodium deoxytaurocholate and sodium deoxyglycocholate, showed better enhancement effect than did trihydroxy bile salts. A 38-fold enhancement in flux was achieved with 10 mM of sodium deoxyglycocholate.     

Intercellular permeation and cyclosis-mediated transport of a fluorescent probe in Characeae

Intercellular communication and transport is the essential prerequisite for the function of multicellular organisms. Simple diffusion as a transport mechanism is often inefficient in sustaining the effective exchange of metabolites, and other active transport mechanisms become involved. In this paper, we use the giant cells of characean algae as a model system to explore the role of advection and diffusion in intercellular transport. Using fluorescent dye as a tracer, we study the kinetics of the permeation of the fluorophore through the plasmodesmata complex in the node of tandem cells and its further distribution across the cell. To explore the role of cytoplasmic streaming and the nodal cell complex in the transport mechanism, we modulate the cytoplasmic streaming using action potential to separate the diffusive permeation from the advective contribution. The results imply that the plasmodesmal transport of fluorescent probe through the central and peripheral cells of the nodal complex is differentially regulated by a physiological signal, the action potential. The passage of the probe through the central cells of the nodal complex ceases transiently after elicitation of the action potential in the internodal cell, whereas the passage through the peripheral cells of the node was retained. A diffusion-advection model is developed to describe the transport kinetics and extract the permeability of the node-internode cell wall from experimental data.     

Screening of Venlafaxine Hydrochloride for Transdermal Delivery: Passive Diffusion and Iontophoresis

The objective of the study was to investigate in vitro transdermal delivery of venlafaxine hydrochloride across the pigskin by passive diffusion and iontophoresis. For passive diffusion, experiments were carried out in Franz diffusion cell whereas for iontophoretic permeation, the diffusion cell was modified to contain both the donor and return electrode on the same side of skin. Anodal iontophoresis was carried out using a current density of 0.5 mA/cm². Donor concentrations used were 585.5 mg/ml (saturated solution) and 100 mg/ml. Experiments initially performed to determine the transport efficiency of venlafaxine ions showed promising results. Iontophoresis increased the permeation rate at both concentration levels over their passive counterparts (P < 0.01), but surprisingly higher steady-state flux was obtained from lower donor drug load (P < 0.01). The favorable pH of the unsaturated solutions is suggested to be the cause for this effect. Mild synergistic effect was observed when iontophoresis was carried out incorporating peppermint oil in the donor but the same was not found in passive diffusion. Highest steady-state flux obtained in the experiment was 3.279 μmol/cm²/h when peppermint oil (0.1%) was included in the donor. As the maintenance requirement of venlafaxine hydrochloride is approximately 9.956 μmol/h, the results suggested that the drug is a promising candidate for iontophoretic delivery.     

Analysis of the permeation of cryoprotectants in cartilage

Some tissues, such as cartilage and cornea, carry an internal fixed negative charge, leading to a swelling pressure that is balanced by tensile stress in the tissue matrix. During the addition and removal of cryoprotectants the changes in osmotic pressure will cause the tissue to deform. Because of the fixed charge and osmotic deformation, the permeation process in such tissues differs from ordinary diffusion processes. In this paper a biomechanical multi-solute theory is introduced to describe this process in cartilage tissue. Typical values for the physiological and biomechanical properties are used in the simulation. Several parameters - the aggregate modulus, the fixed charge density and the frictional parameter - are analyzed to show their impact on the process. It is shown that friction between water and cryoprotectant has the greatest influence but the fixed charge density is also important. The aggregate modulus and the frictional parameter between the cryoprotectant and the solid matrix have the least influence. Both the new biomechanical model and the conventional diffusion model were fitted to published experimental data concerning the time course of mean tissue cryoprotectant concentration when cartilage is immersed in solutions of dimethyl sulphoxide or propylene glycol: in all cases and with both models a good fit was obtained only when a substantial amount of non-solvent water was assumed.     

Validation of a Static Franz Diffusion Cell System for <Emphasis Type="Italic">In Vitro</Emphasis> Permeation Studies

Over the years, in vitro Franz diffusion experiments have evolved into one of the most important methods for researching transdermal drug administration. Unfortunately, this type of testing often yields permeation data that suffer from poor reproducibility. Moreover, this feature frequently occurs when synthetic membranes are used as barriers, in which case biological tissue-associated variability has been removed as an artefact of total variation. The objective of the current study was to evaluate the influence of a full-validation protocol on the performance of a tailor-made array of Franz diffusion cells (GlaxoSmithKline, Harlow, UK) available in our laboratory. To this end, ibuprofen was used as a model hydrophobic drug while synthetic membranes were used as barriers. The parameters investigated included Franz cell dimensions, stirring conditions, membrane type, membrane treatment, temperature regulation and sampling frequency. It was determined that validation dramatically reduced derived data variability as the coefficient of variation for steady-state ibuprofen permeation from a gel formulation was reduced from 25.7% to 5.3% (n = 6). Thus, validation and refinement of the protocol combined with improved operator training can greatly enhance reproducibility in Franz cell experimentation.     

A theoretical analysis for determining the nonlinear hydraulic permeability of a soft tissue from a permeation experiment

The nonlinear nature of the hydraulic permeability, as well as the corresponding pressure and displacement fields, in a soft tissue are studied for steady-state permeation. From a two-phase continuum model analytical expressions are derived that can be used with the results from a permeation experiment to determine the dependence of the permeability on the strain. In the process it is found that, because of the compaction of the tissue arising from fluid flow, it is necessary to distinguish between the apparent and intrinsic permeability. The former, which is an averaged quantity, is the permeability usually obtained in permeation studies. However, as shown from the analysis, it can differ substantially from the latter, which is the local permeability in the tissue.     

Enzyme-modified cheese exerts inhibitory effects on allergen permeation in rats suffering from indomethacin-induced intestinal inflammation

We have found that an enzyme-modified cheese (EMC) inhibited the permeation of allergens such as ovalbumin (OVA), using Caco-2 cells as an in vitro intestinal epithelial model. In addition, NPWDQ (Asn-Pro-Trp-Asp-Gln, aa 107-111 of alphas(2)-casein) was isolated from EMC and identified as one of the responsible peptides for this inhibitory activity (Tanabe et al., J. Agric. Food Chem., (2007)). In this study, we aimed to clarify the mechanism by which NPWDQ inhibited allergen permeation in vitro, and also to evaluate the effects of EMC on allergen permeation in vivo. Intestinal permeability for both fluorescein isothiocyanate conjugated dextran and horseradish peroxidase was decreased in Caco-2 cells by the addition of NPWDQ, indicating that NPWDQ might inhibit both paracellular and transcellular transports. Next, intestinal inflammation was induced by subcutaneous injections of indomethacin to rats. When OVA was injected into the jejunal and ileac loops of indomethacin-administered rats with and without NPWDQ, it was found that the addition of NPWDQ effectively diminished OVA permeation from both loops. Although the plasma OVA concentration of indomethacin-administered rats after oral OVA challenge was markedly elevated over that of normal rats, supplemental administration of EMC to the rats effectively suppressed OVA permeation. These results suggest that EMC is useful for the prevention of food allergy by inhibiting allergen permeation probably by enforcing the intestinal barrier.     

Probing the skin permeation of fish oil/EPA and ketoprofen-3. Effects on epidermal COX-2 and LOX

This work employed immunocytochemistry (ICC) techniques to study the effect of topically applied fish oil and ketoprofen on cyclooxygenase (COX-2) and lipoxygenase (LOX) within freshly excised porcine ear skin. Maintained in Hanks buffer immediately post excision, full thickness membranes were mounted in Franz diffusion cells and dosed with 1 ml of individual formulations containing ketoprofen, fish oil or both. At different timepoints, the diffused areas were recovered and relative activities of COX-2 and LOX determined. It was found that the fish oil formulation qualitatively inhibited the expression of both COX-2 and LOX enzymes. As expected, ketoprofen had no effect upon LOX expression but a significant decrease on COX-2 expression was observed. The formulation containing both fish oil and ketoprofen proved to be the most effective at inhibiting the expression of both COX-2 and LOX. Considered together with data from earlier papers, a mechanism of EPA permeation enhancement by ketoprofen may be elucidated and also show the ability of such a formulation to inhibit these enzymes and thus indicate the efficacy of such a formulation.     

Properties of permeation pathways induced in the human red cell membrane by malaria parasites

The intracellular development of malaria parasites in mature erythrocytes imposes on the host cell a major demand for supply of nutrients and disposal of waste products. So as to cope with these demands, the erythrocyte membrane undergoes profound alterations in its basic permeability properties. A few hours after being invaded by Plasmodium falciparum parasites, and before any structural changes are apparent on the surface, the molecular traffic across the red cell membrane changes both in intensity and in composition of permeating substances. The changes are of a gradual nature, developmentally related and dependent on de novo protein synthesis, but do not occur concurrently for all the classes of permeants. Molecules which permeate very poorly into uninfected cells, such as hexitols (e.g., sorbitol and myoinositol), amino acids (e.g., glutamine, threonine, and histidine), a variety of organic acids and metal ions show a marked increase in their permeation rates across the host cell membrane. Likewise, substances whose normal permeation pathways conform with those of facilitated diffusion (e.g., hexoses, nucleosides, choline, and some amino acids), gain access into the host cytosol either by modified or additional permeation pathways. It has been proposed that three major new pathways are induced in the membrane of infected cells: (1) one of pore-like properties, which can accommodate most of the water soluble permeants, including anionic substances; (2) a protein-lipid interface, which can accommodate compounds of relatively higher hydrophobic character; and (3) modified constitutive transporters or modified lipid surroundings with altered transport activities. The pores are blocked by permeant bioflavonoid glycosides whose sites of binding are endofacial, and amount to less than a thousand per cell. In addition to serving as specific targets for transport blockers, the new sites of permeation can also serve as routes for enhanced delivery of cytotoxic agents into parasitized cells.     

The in vitro use of the hair follicle closure technique to study the follicular and percutaneous permeation of topically applied drugs

Recent studies on follicular permeation emphasise the importance of hair follicles as diffusion pathways, but only a limited amount of data are available about the follicular permeation of topically applied drugs. This study examines the use of a hair follicle closure technique in vitro, to determine the participation of hair follicles in transdermal drug penetration. Various substances, with different lipophilicities, were tested: caffeine, diclofenac, flufenamic acid, ibuprofen, paracetamol, salicylic acid and testosterone. Diffusion experiments were conducted with porcine skin, the most common replacement material for human skin, in Franz-type diffusion cells over 28 hours. Different experimental settings allowed the differentiation between interfollicular and follicular permeation after topical application of the test compounds. A comparison of the apparent permeability coefficients of the drugs demonstrates that the percutaneous permeations of caffeine and flufenamic acid were significantly higher along the hair follicles. In the cases of paracetamol and testosterone, the follicular pathway appears to be of importance, while no difference was found between interfollicular and follicular permeation for diclofenac, ibuprofen and salicylic acid. Thus, the hair follicle closure technique represents an adequate in vitro method for gaining information about follicular or percutaneous permeation, and can replace in vivo testing in animals or humans.     

Crossflow filtration of baker's yeast with periodical stopping of permeation flow and bubbling

The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 x 10(-5) m(3) /m(2) s (290 L/m(2) h) from 2 x 10(-5) m(3)/m(2) s (72 L/m(2) h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux.(c) John Wiley & Sons, Inc.     

Preparation and <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation of a New Fentanyl Patch Based on Functional and Non-functional Pressure Sensitive Adhesives

In this study, some single-layer and double-layer transdermal drug delivery systems (TDDSs) with different functional and non-functional acrylic pressure-sensitive adhesives (PSAs) were prepared. For this purpose, fentanyl as a drug was used. The effects of PSAs type, single-layer and double-layer TDDSs on skin permeation and in vitro drug release from devices were evaluated using a hydrodynamically well-characterized Chien permeation system fitted with excised rat abdominal skin. The adhesion properties of devices such as peel strength and tack values were obtained as well. It was found that TDDS with –COOH functional PSA showed the lowest steady-state flux. Double-layer TDDS displayed a constant flux up to 72 h. In double- and single-layer devices after 1 and 3 h, respectively, drug release followed Higuchi’s kinetic model. Formulations with the highest percentage of –COOH functional PSA have displayed the lowest flux. The double-layer TDDSs with non-functional PSA demonstrated the suitable skin permeation rate close to Duragesic® TDDS and suitable adhesion properties.     

L-malic-acid permeation in resting cells of anaerobically grown Saccharomyces cerevisiae

The study of permeation of L-malic acid in cells of Saccharomyces cerevisiae at pH 3.0 was carried out with (U-14C)-labelled L-malic acid. Resting cells were used in these experiments. They were previously anaerobically grown on glucose. This study showed that this transport is the result of two competitive mechanisms, one for the uptake and one for the efflux. The uptake mechanism seems to be a simple diffusion of the L-malic acid in a non-dissociated form. The efflux mechanism seems to be an active transport of L-malic acid that is very dependent on the temperature. At the steady state, the result of uptake and efflux mechanisms leads to an intracellular concentration which is twice or three times the extracellular concentration.