Model Permeant Research Articles

Evaluation of Fluorescent Probe Surface Intensities as an Indicator of Transdermal Permeant Distributions Using Wide-Area Two-Photon Fluorescence Microscopy

The application of high-speed two-photon fluorescence microscopy (HTPM) to examine transdermal transport processes has enabled the noninvasive visualization of permeant spatial distributions over a larger, more clinically relevant wide area of the skin. Earlier studies demonstrated that the transdermal fluorescent probe distribution over a 2 × 2mm skin area was well represented by a significantly reduced sampling of the 400 microscale skin sites (100 × 100 μm) constituting the wide area. In the present study, the 400 microscale skin sites are considered individually, and the site-to-site variability in permeant distributions is used as a model to reflect the range in experimentally measured skin permeabilities resulting from the inherent stratum corneum structural heterogeneity. The correlation established between the permeant surface intensity and the corresponding permeant intensity gradient at each skin site provides an indication of the potential for screening transdermal permeant distributions solely based on the evaluation of microscale permeant surface intensities. The strong linear correlation between the intensity gradient and the surface intensity for the hydrophilic model permeant, sulforhodamine B, demonstrated that surface intensities provide a robust indicator of the corresponding transdermal probe distributions at the microscale. For the hydrophobic model permeant, rhodamine B hexyl ester, however, weak correlations were observed between these two parameters. This result suggests that the stratum corneum microscale surface intensity does not validly capture the corresponding intensity gradients for the entire range of skin permeabilities typically encountered as a result of the inherent stratum corneum heterogeneity. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2354–2365, 2003

The synergic effects of various electrolytes and electroporation on the in vitro skin permeation of calcein

Various electrolytes in test solutions applied to the skin were evaluated with regard to their effects on enhanced skin permeation of calcein as a model permeant by electroporation (EP), which is a physical means to increase skin penetration by applying a high voltage pulse on the skin surface. Calcein solution (1.0 mM) containing different electrolytes at a concentration of 150 mM was applied to excised hairless rat skin, and a 10-ms electric pulse of 300 V was applied to the skin surface ten times (one pulse every second) at the beginning of the in vitro permeation experiments. The following results were obtained: (i) addition of several electrolytes, such as CaCl 2 and NaCl, further increased the EP-enhanced skin permeation of calcein when compared to treatment without these electrolytes; (ii) Ca 2+ and Mg 2+ exerted a greater effect than other cations (Na +, Zn 2+, Cu 2+, Fe 3+ and Al 3+); (iii) with simultaneous application of CaCl 2 and EP, the effect of anodal EP was much greater than that of cathodal EP; (iv) the penetration-enhancing effects of CaCl 2 were also obtained with EP pretreatment followed by calcein addition; and (v) skin permeation was greatly increased particularly with simultaneous application of EP and Ca 2+. These substantial combined synergic effects of EP and electrolytes, particularly those containing CaCl 2, MgCl 2 and CaBr 2, may be related to the disruptive and retrievable functions of the biggest barrier of skin, the stratum corneum, of these electrolytes.

Structure–Activity Relationship for Chemical Skin Permeation Enhancers: Probing the Chemical Microenvironment of the Site of Action

Studies were previously conducted in our laboratory on the influence of n‐alkanols, 1‐alkyl‐2‐pyrrolidones, N,N‐dimethlyalkanamides, and 1,2‐alkanediols as skin permeation enhancers on the transport of a model permeant, corticosterone (CS). The experiments were conducted with hairless mouse skin (HMS) in a side‐by‐side, two‐chamber diffusion cell, with enhancer present in an aqueous buffer in both chambers. The purpose of the present study was to extend these studies and investigate in greater detail the hypothesis that a suitable semipolar organic phase may mimic the microenvironment of the site of enhancer action, and that the enhancer partitioning tendency into this organic phase may be used to predict the enhancer potency. CS flux enhancement along the lipoidal pathway of HMS stratum corneum was determined with the 1‐alkyl‐2‐azacycloheptanones, 1‐alkyl‐2‐piperidinones, 1,2‐dihydroxypropyl decanoate, 1,2‐dihydroxypropyl octanoate, n‐alkyl‐β‐D‐glucopyranosides, 2‐(1‐alkyl)‐2‐methyl‐1,3‐dioxolanes, 1,2,3‐nonanetriol, and trans‐hydroxyproline‐N‐decanamide‐C‐ethylamide as enhancers. Enhancement factors (E values) were calculated from the permeability coefficient and solubility data over a range of E values. Comparisons of the enhancer potencies for all studied homologous series and the carbon number of the n‐alkyl group revealed a nearly semilogarithmic linear relationship with a slope of ∼0.55, which is consistent with the hydrophobic effect. Moreover, comparisons of the enhancer potencies of all the enhancers with the n‐hexanol–phosphate buffered saline (PBS), n‐octanol–PBS, n‐decanol–PBS, and n‐hexane–PBS partition coefficients showed very good correlations for the n‐alkanol solvents but not for n‐hexane. This result supports the interpretation that the enhancer potency is directly related to the ability of the enhancer molecule to translocate to a site of action via its free energy of transfer from the bulk aqueous phase to a semipolar microenvironment in the stratum corneum lipid lamella that is well mimicked by water‐saturated n‐alkanols. © 2003 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1305–1322, 2003

Mechanistic studies of the effect of hydroxypropyl-β-cyclodextrin on in vitro transdermal permeation of corticosterone through hairless mouse skin

Literature reports reveal that the issue of whether cyclodextrins may act as skin permeation enhancers has not been resolved. Accordingly, in vitro skin transport studies were conducted to address this question. Corticosterone ( 3 H -CS and/or non-radiolabeled CS) was chosen as the model permeant for transport experiments with hairless mouse skin (HMS) and with a synthetic cellulose membrane of 500 molecular weight cut off (MWCO), the latter to help establish baseline behavior. Hydroxypropyl-β-cyclodextrin (HPβCD) was selected as the representative cyclodextrin. The CS/HPβCD complexation constant was determined both from solubility data (saturation conditions) in phosphate buffered saline (PBS), pH 7.4 and with data obtained from PBS/silicone polymer partitioning experiments, the latter experiments permitting the determination of the complexation constant at low CS concentrations. These results were used in the calculations of the free CS concentrations in the donor chamber of the transport experiments. The CS transport experiments were conducted at CS solubility saturation and under supersaturation (resulting from autoclaving at 121 °C) conditions as well at very low (tracer level) concentrations. The effect of polyvinylpyrrolidone as a solution additive was also evaluated. The following were the key outcomes of this study. Contrary to literature reports, there was no evidence that HPβCD is an enhancer for CS transport through HMS. The CS permeability coefficient values obtained with HMS in all of the experiments were found to be the same within experimental error when calculated on the basis of the free CS concentration as the driving force for permeation. The constancy of the permeability coefficient in the presence and absence of HPβCD is interpreted to mean that, in these experiments, HPβCD did not alter the barrier properties of HMS stratum corneum to any significant extent nor did it enhance CS transport in any other manner such as by a carrier mechanism involving the aqueous boundary layer or by a carrier mechanism within the stratum corneum.

Theoretical Description of Transdermal Transport of Hydrophilic Permeants: Application to Low‐Frequency Sonophoresis

Application of ultrasound enhances transdermal transport of drugs (sonophoresis). The enhancement may result from enhanced diffusion due to ultrasound‐induced skin alteration and/or from forced convection. To understand the relative roles played by these two mechanisms in low‐frequency sonophoresis (LFS, 20 kHz), a theory describing the transdermal transport of hydrophilic permeants in both the absence and the presence of ultrasound was developed using fundamental equations of membrane transport, hindered‐transport theory, and electrochemistry principles. With mannitol as the model permeant, the role of convection in LFS was evaluated experimentally with two commonly used in vitro skin models— human cadaver heat‐stripped skin (HSS) and pig full‐thickness skin (FTS). Our results suggest that convection plays an important role during LFS of HSS, whereas its effect is negligible when FTS is utilized. The theory developed was utilized to characterize the transport pathways of hydrophilic permeants during both passive diffusion and LFS with mannitol and sucrose as two probe molecules. Our results show that the porous pathway theory can adequately describe the transdermal transport of hydrophilic permeants in both the presence and the absence of ultrasound. Ultrasound alters the skin porous pathways by two mechanisms: (1) enlarging the skin effective pore radii, or (2) creating more pores and/or making the pores less tortuous. During passive diffusion, both HSS and FTS exhibit the same skin effective pore radii (r = 28 ± 13 Å). In contrast, during LFS, r within HSS is greatly enlarged (r > 125 Å), whereas r within FTS does not change significantly (23 ± 10 Å). The observed different roles of convection during LFS across HSS and FTS can be attributed to the different degrees of structural alteration that these two types of skin undergo during LFS. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:545–568, 2001

Penetration of compounds through human stratum corneum as studied by Fourier transform infrared photoacoustic spectroscopy

The penetration of the lipophilic model permeant, 1-cyanodecane, into isolated human stratum corneum (SC) was followed nondestructively by step-scan Fourier transform infrared (FTIR) photoacoustic spectroscopy (PAS) with phase modulation technique. The uptake of the compound in the SC was quantified by monitoring the alterations in the spectra in the course of the penetration using multivariate analysis. Step-scan technique in conjunction with phase modulation offers the possibility for controllable depth profiling (sampling depth up to 30 μm) during the penetration process. Based on Fick’s second law and assuming a virtually layered structure of the membrane, depth-dependent diffusion coefficients were derived by numerical fitting of the spectroscopic data. For 1-cyanodecane, the diffusion coefficient in the inner region of the SC is 1.6-fold that measured in the outer region.

Effect of preparation conditions on properties and permeability of chitosan–sodium hexametaphosphate capsules

Capsules were obtained by interpolymer complexation between chitosan (polycation) and sodium hexametaphosphate (SMP, oligoanion). The effect of the preparation conditions on the capsule characteristics was evaluated. Specifically, the influence of variables such as pH, ionic strength, reagent concentration, and additives on the capsule permeability properties was investigated using dextran as a model permeant. The capsule membrane permeability was found to increase by decreasing the chitosan/SMP ratio as well as adding mannitol to the oligoanion recipient bath. Increasing the ionic strength or the pH of the initial chitosan solution was also found to enhance the membrane permeability, moving the membrane exclusion limit to higher values. Generally, the capsules prepared under all tested conditions had a relatively low permeability which rarely exceeded a molecular cut-off of 40 kD based on dextran standards. Furthermore, the diffusion rate showed a strong temporal dependence, indicating that the capsules prepared under various conditions exhibit different apparent pore size densities on the surface. The results indicated that, in order to obtain the desired capsule mass-transfer properties, the preparation conditions should be carefully considered and adjusted. Adding a polyol as well as low salt amount (less than 0.15%) is preferable as a means of modulating the diffusion characteristics, without disturbing the capsule mechanical stability.

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.

Pore induction in human epidermal membrane during low to moderate voltage iontophoresis: A study using AC iontophoresis

The present study aimed to investigate new pore induction as a flux-enhancing mechanism in human epidermal membrane (HEM) with low to moderate voltage electric fields. The extent of pore induction and the effective pore sizes of these induced pores were to be assessed using a low frequency (12.5 Hz) low to moderate voltage (2.0 to 4.0 V) square-wave alternating current (ac) “passive” permeation method (ac iontophoresis). This ac approach was to allow for inducing and sustaining a state of pore induction in HEM while permitting no significant transport enhancement via electroosmosis; thus, transport enhancement entirely due to new pore induction (enhanced passive permeation) was to be assessed without any contributions from electroosmosis. Good proportionality between the increase in HEM permeability and its electrical conductance was found with the “passive” transport data obtained during square-wave ac iontophoresis using urea as the model permeant. Typically, at 3.0 to 4.0 V, HEM conductance increases (and permeability increases) ranged from around 3- to 30-fold. These results appear to be the first direct evidence that new pore induction in HEM is a significant flux enhancing mechanism under moderate voltage conditions. The extents of pore induction in HEM under low frequency moderate voltage (2.0 to 3.0 V) ac, pulsed direct current (dc), and continuous dc were also compared. The extents of pore induction from square-wave ac and pulsed dc were generally of the same order of magnitude but somewhat less than that observed during continuous dc iontophoresis at the same applied voltage and duration, suggesting less extent of pore induction with reversing polarity or when a brief delay is provided between pulses to allow for membrane depolarization. The average effective pore sizes calculated for the induced pores from the experimental data with urea and mannitol as probe permeants and the hindered transport theory were 12 ± 2 Å, which are of the same order of magnitude as those of preexisting pores determined from conventional passive diffusion experiments.

SKIN PERMEATION ENHANCEMENT BY FATTY ACIDS

The present study elucidates the skin permeation enhancement effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA). The effects were studied using human stratum corneum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. PUFA — linoleic (LA), α-linolenic (ALA) and arachidonic acids — enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids. Based on this and other studies, a set of mechanisms of action is proposed for fatty acids.

Influence of the Permeation Enhancers 1-Alkyl-2-pyrrolidones on Permeant Partitioning into the Stratum Corneum

□ In a previous study, the enhancing effects of a series of 1-alkyl-2-pyrrolidones (APs; 1-ethyl, 1-butyl, 1-hexyl, and 1-octyl-2-pyrrolidone) on the transport of steroidal permeants across hairless mouse skin were investigated via a parallel pathway skin model. Isoenhancement concentration conditions were deduced under which different APs induce essentially the same transport enhancement for the lipoidal pathway of the stratum corneum (SC). As a continuing effort to understand the mechanism of action of permeation enhancers, the influence of the APs on permeant partitioning into hairless mouse SC was investigated under the isoenhancement concentration conditions using β-estradiol (E2β) as the model permeant. The amount of E2β uptake into SC was found to be essentially the same for all the APs under these isoenhancement conditions. This result suggests that inducing a higher partitioning tendency for E2β into the lipoidal pathway of hairless mouse SC is a principal mechanism of action of the APs in enhancing transdermal transport. The uptake of the APs into SC lipoidal domains was also determined, and the results show only a modest (approximately 2-fold) increase in the uptake of the APs in going from 1-ethyl- to 1-octyl-2-pyrrolidone under isoenhancement conditions. This indicates the potency of the APs as permeation enhancers is only very modestly dependent upon the alkyl chain length in this chain length region when compared at concentrations in the microenvironment where the action occurs in the lipid domains.

Comparison of permeability data from traditional diffusion cells and ATR-FTIR spectroscopy. Part II. Determination of diffusional pathlengths in synthetic membranes and human stratum corneum

In this study, the morphological structure of the inner and outer regions of human stratum corneum (SC) were investigated using Attenuated Total Reflectance Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. Furthermore, diffusional pathlengths in silicone membranes and human SC were determined using ATR-FTIR spectroscopic data and regular skin diffusion cell data. SC membranes were fully hydrated throughout the experiments. It was shown that diffusion coefficients for a model permeant, 4-cyanophenol (CP), were lower in the more compact regions of the inner layers of the SC when compared to diffusion coefficients in the outer layers. Partition coefficients between SC and aqueous vehicles were higher in the outer layers than the inner layers. These data demonstrate a 4-fold lower permeability of skin to CP in the inner layers relative to the outer layers of the SC. The combination of diffusion cell data and ATR-FTIR spectroscopic data was also used to determine diffusional pathlengths across synthetic silicone membranes and human SC. In all cases, the pathlengths were similar to the thickness of the membranes. For SC, this appears to contrast the commonly held theory that diffusion occurs via a tortuous route within the intercellular lipids, and may therefore imply a transcellular route. Alternatively, the calculated pathlengths may be a reflection of the total length of rate limiting steps in the diffusional process rather than overall diffusional distance. This implies that lateral molecular diffusion within the head groups or lipid tails (depending on the lipophilicity of the permeant) of the lipid bilayers may be a relatively rapid process. These results have demonstrated that the previously observed morphological differences between the inner and outer regions of the SC are reflected in variations in permeability, and that the diffusional route through fully hydrated human SC may indeed be via a direct pathway.

In vitro human skin barrier modulation by fatty acids: skin permeation and thermal analysis studies.

This study aims to elucidate the skin permeation enhancement and the skin perturbation effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA). The skin permeation enhancement effects were studied using human stratum comeum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. The perturbation effects were studied using differential thermal analysis (DTA) on SC after pretreatment with FA/PG. SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). Nonanoic and decanoic acids exert barely noticeable effects on the thermal behaviour of SC, suggesting that they easily mix with the skin lipids. All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. DTA revealed that the cis-9- and 13-isomers form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70 degrees/80 degrees C. PUFA--linoleic (LA), alpha-linolenic (ALA) and arachidonic acids--enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. LA and ALA form separate domains but do not completely suppress the SC lipid transitions at 70 degrees/80 degrees C. Increase in the enthalpy changes of 70 degrees/80 degrees transitions linearly correlates to the decrease in the permeability coefficients, suggesting that an increased perturbation of the skin lipids not necessarily has to yield an increased PABA permeation. The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids.

Skin permeation model of phenylalkylcarboxylic homologous acids and their enhancer effect on percutaneous penetration of 5-Fluorouracil

This study was conducted primarily to establish the rat skin penetration model of acidic homologous series with a wide range of lipophilicity (from phenylpropionic acid to phenylcaprylic acid), and to compare it with other homologous series (alkylanilines) by means of adequate permeability-partition correlations, in order to find out if there could be a common model of penetration for all the compounds. The influence of pH on acid penetration through the skin was also analyzed. Standard in vitro skin permeation methods using rat skin were used to determine the permeability coefficients of phenylalkylcarboxylic acids in the conditions established. Membrane/water partition coefficients were also assessed, and the correlations between permeability and partition values were established. A linear relationship between the logarithms of permeabilities of penetrants and the corresponding membrane/water partition coefficients was found. The apparent discordance between this type of correlation and the probabilistic (i.e. parabolic) model previously established with other homologous series (alkylanilines) is attributed to a self-enhancing effect on penetration in the case of the more lipophilic compounds of the series. In fact, when 5-FU was used as a polar model permeant, pretreatment of the membrane with phenyloenanthic acid gave it an enhancer ratio of 4.5, thus confirming its enhancer effect.

A mechanistic investigation of the in vitro human skin permeation enhancing effect of Azone®

In order to clarify the mechanism of the enhancing action of Laurocapram (Azone) on the skin permeation of drugs, the following experiments were conducted. Partitioning of an homologous series of alkyl anilines into n-octanol and isopropyl myristate (IPM) in the absence and presence of Azone (1%, 5%, and 10%, w/w) was determined. The addition of 1% and 5% of enhancer to n-octanol and 1% of enhancer to IPM slightly increased the partition coefficients of the alkyl anilines assayed. This effect was more pronounced for 10% Azone in both cases. The percutaneous penetration-enhancing effect of Azone for similar concentrations through the human skin was investigated using 5-fluorouracil as a hydrophilic model permeant, four compounds selected from the alkyl anilines (aniline, 4-ethylaniline, 4- n-butylaniline and 4- n-pentylaniline) with a wide range of lipophilicity values (log PC Oct from −0.87 to 4.2 where PC Oct is the octanol/water partition coefficient) and two compounds which belong to the phenyl alkanols series, 2-phenylethanol and 4-phenylbutanol with lipophilicity values of log PC Oct of 1.18 and 2.34, respectively. The enhancer effect of Azone depended predominantly on two parameters, the concentration of the enhancer used and the lipophilicity of the compound assayed. When the concentration of the Azone employed was 1% (w/w), it only acted on the compounds with a lipophilicity value of less than log PC Oct of 1 but if the enhancer concentration was increased to 5% (w/w) this lipophilicity threshold increased to a log PC Oct value of 2.69. Therefore the number of drugs whose penetration may be increased by the use of Azone will be greater at the higher concentration. The less the lipophilicity value of the penetrant, the greater the enhancer effect observed. In the case of 10% Azone the penetration of the compounds tested could be also increased for 5-FU, aniline, 4-ethylaniline, 2-phenylethanol and 4-phenylbutanol, but for this last compound there is a reduction in the degree of penetration if it is compared with the permeability coefficients obtained with 5% of Azone. Finally, the Azone cannot enhance the skin permeability for the highest lipophilic compounds (log PC Oct ≥ 3).

A protocol for the assessment of receiver solution additive-induced skin permeability changes. An example with γ-cyclodextrin

In the in vitro evaluation of transdermal drug delivery, the receiver solution composition can affect the experimental outcome. In particular, receiver solution accumulation of permeants having low aqueous solubility may give misleading results. Additives can be used to increase the solubility in the receiver solution, but have the potential of altering the skin barrier function. We offer a protocol for the evaluation of receiver solution additives with respect to potential detrimental effects on skin permeation, using bovine serum albumin as a control additive and examining the effect of additive concentration. A test receiver solution additive, y-cyclodextrin, has been effectively used in vitro to address limited receiver solution solubility in the evaluation of transdermal delivery of a model permeant, progesterone. At the cyclodextrin concentrations used, flux experiments detected no alterations in the barrier function of skin.

Enhancement of in vitro permeability of porcine buccal mucosa by bile salts: kinetic and histological studies

In this study the enhancing effects of di-and tri-hydroxy bile salts on buccal penetration were investigated using fluorescein isothiocyanate (FITC) as a model permeant. The permeability of the porcine buccal mucosa to this compound in the presence and absence of bile salts was determined in vitro. In conjunction with kinetic studies, histological studies were carried out to investigate the interaction between the bile salts and the buccal epithelium at the light and electron microscopic level. In the presence of the bile salts at 0.l M concentration, the permeability of buccal mucosa to FITC increased by a 100–200 fold compared to FITC alone. Calculation of enhancement ratios provided normalization of the permeability data for the enhancer treated tissue with respect to its own control. No significant difference was observed between the enhancing effects of di-hydroxy and tri-hydroxy bile salts. After the bile salt treatment significant morphological and ultrastructural changes occurred. At LM level, partial loss of superficial cells was observed as well as separation of the epithelium from the basal lamina. Upon a 4 hour bile salt treatment, two important changes were observed with FFEM: (1) the fracture plane preferentially ran across the cytoplasmic proteins and through the cytoplasmic space domains; (2) abnormally structured deposits were found in the cytoplasm. Similar to the kinetic studies, no differences were found between the histological effects of di- and tri-hydroxy bile salts.

Enhanced delivery of 5-fluorouracil through shed snake skin by two new transdermal penetration enhancers

The effectiveness of a new biodegradable penetration enhancer, dodecyl N, N-dimethylamino isopropionate (DDAIP) was compared to dodecyl N, N-dimethylamino acetate (DDAA), another biodegradable penetration enhancer, and to Azone®, lauryl alcohol (LA), and oleic acid (OA) in vitro using shed snake skin in modified Franz-type diffusion cells. 5-Fluorouracil (5FU), a hydrophilic drug with poor skin permeability, was used as a model permeant. Skin samples were pretreated with pure liquid enhancers. 5FU flux through the control and enhancer treated skin increased linearly with its concentration in the donor compartment. DDAIP and DDAA interacted with the skin rapidly (< 2 h), and the duration of action is at least 24 h. The transdermal flux of 5FU increased substantially (> 40-fold) with the dose of DDAIP up to 10 μ1. Thereafter the flux did not increase significantly. With DDAA no plateau effect was observed with the applied volumes up to 50 μ1. Skin pretreatment with 5 μ1 of DDAIP, DDAA, Azone®, LA, and OA increased the permeability of 5FU 69-, 24-, 23-, 5-, and 2-times, respectively. Substitution of a methyl group for a hydrogen atom in the acetate moiety of DDAA was observed to markedly increase transdermal penetration enhancement.

Transport methods for probing the barrier domain of lipid bilayer membranes

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.

Shed Snake Skin and Hairless Mouse Skin as Model Membranes for Human Skin During Permeation Studies

Difficulties in obtaining and using human skin have tempted many workers to employ animal membranes for percutaneous absorption studies. We have investigated the suitability of two species of snake (Elaphe obsoleta, Python molurus) for this purpose and compared our in vitro experimental results for human skin and for hairless mouse, a currently popular model. The effects of long-term hydration on the membranes were investigated over 8 d using tritiated water as a model permeant. The initial permeability coefficients of all the membranes were similar (0.74-2.2 X 10(-3) cm 2h-1). Although the human and squamate skins did not change significantly over the test period, the permeability of hairless mouse skin increased 37 times. The actions of typical enhancers on the permeabilities of the membranes to a model penetrant 5-fluorouracil (5-FU) were tested using 3% Azone in Tween 20/saline, propylene glycol (PG), 2% Azone in PG, and 5% oleic acid in PG. While the data from snake membranes tended to underestimate the enhancer effects, those from hairless mouse skin greatly overestimated the changes. None of the membranes was a completely reliable model for assessing human percutaneous absorption as modified by accelerants. Pretreatment with acetone did not significantly change the permeability of human or squamate skins to 5-FU, although that of hairless mouse increased twentyfold. An overall conclusion is that, wherever possible, human skin should be used in absorption studies and not hairless mouse or snake skin; otherwise, misleading results may be obtained.