In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Supersaturation is a very useful method of enhancing the permeation of drugs across membranes such as skin, because unlike other methods, it does not interfere with the ultrastructure of the stratum corneum. Many drugs are able to form inclusion complexes with beta-cyclodextrins (beta-CDs) and this study investigates the anti-nucleating effects of these compounds on supersaturated solutions of diclofenac. The ability of various betaCDs to form inclusion complexes with diclofenac was assessed by measuring their saturated solubilities. Solutions containing hydroxypropyl beta-cyclodextrin (HPbeta-CD, with a molar substitution of 0.9) produced a 7.5-fold increase in the solubility of diclofenac, which suggested that a strong complex was formed between the two compounds. This association was characterized using differential scanning calorimetry. Permeation across silicone membranes of these saturated solutions of diclofenac in the presence of the different betaCDs produced similar flux values suggesting that the overall activity was also similar. The effect of different molar ratios of HPbeta-CD and diclofenac, and the anti-nucleating effect of HPbeta-CD (both on its own and in combination with a known anti-nucleant, hydroxypropylmethyl cellulose (HPMC)) on the diffusion of diclofenac across silicone membranes was investigated. HPbeta-CD appears to have a stabilizing effect on supersaturated solutions of diclofenac as a co-ingredient with HPMC.

For diabetic patients, blood glucose monitoring is an important part in the management of their disease, however the acquisition of blood requires the use of invasive and often painful methods, and the development of a technique that removes these problems would represent a major advance. The uppermost membrane of the skin, the stratum corneum, has been shown to be the main barrier to percutaneous absorption, but there have been claims that polar water-soluble compounds diffuse across it via aqueous pathways. In this study, skin diffusion cells were used to investigate the back diffusion of tritiated water and the convective transport of 3H-glucose across full thickness human skin after the application of a number of different materials to the stratum corneum. Significant amounts of 3H-glucose back diffused only after complete removal of the stratum corneum by tape stripping, and it is likely that any future attempts to monitor blood glucose levels using non-physical techniques will require a certain degree of damage to the stratum corneum. The extraction through the skin of tritiated water and 3H-glucose after the application of solutions with different osmotic pressures were consistent with the theory that solutions with high osmotic pressures dehydrate the stratum corneum which suggests that passive transport of these radiolabelled molecules through porous pathways was insignificant.

Under ideal circumstances, maximum flux is achieved from saturated solutions and, therefore, the diffusion of compounds from supersaturated solutions would provide enhanced penetration. However, due to the inherent lack of stability of supersaturated solutions, they tend to crystallise upon preparation, but in some cases this can be overcome with antinucleant polymers which inhibit or retard crystallisation. Supersaturated solutions of piroxicam for a range of different degrees of saturation up to 5.3 were prepared in a 40:60, v/v, propylene glycol/water cosolvent mixture. Solutions up to 4 degrees of saturation were stable for at least 16 h and their penetration across silicone membranes and full-thickness human skin was investigated using diffusion cells. Relationships between flux and degree of saturation for the supersaturated systems produced linear correlations, but flux values across skin at 0.5 and 1 degrees of saturation were similar. This was partly attributed to differences in the solubility of the drug at different donor phase temperatures. Mechanisms of action for antinucleant polymers were considered, and it was postulated that hydroxypropylmethyl cellulose prevented the formation of a hydrate form of piroxicam, which was less soluble than its anhydrous form in aqueous based solvent systems.

Diffusion cells and attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy have been used to monitor the permeation of a model compound, 4-cyanophenol (CP), across silicone membranes. CP permeation was measured across water saturated silicone membranes and untreated silicone membranes. In all cases the donor phase consisted of a saturated aqueous solution of CP. The receptor phase used in the diffusion cells was distilled water. After calibration of the ATR-FTIR system, it was found that the water saturated membranes yielded permeability co-efficients for CP identical to those found using diffusion cells. In contrast, it was found that the permeability coefficient determined from the ATR-FTIR spectroscopy data for the untreated membrane yielded a value almost two-fold greater than those obtained from both the diffusion cells and the ATR-FTIR spectrometer with the pre-saturated membranes. It is likely that the untreated membrane used in the diffusion cell study contained a substantial amount of water before application of the donor solution due to the inevitable uptake of the water receptor medium. Therefore, as a result of the experimental techniques and procedures used, the only membrane that was not hydrated to some degree before the donor phase was applied was the untreated one analysed by ATR-FTIR spectroscopy. It has been demonstrated that, provided all initial conditions are effectively the same, there is a good correlation between permeability co-efficients measured using diffusion cells and ATR-FTIR spectroscopy. (C) 1997 Elsevier Science B.V.

The flux of a compound across a membrane from any formulation, whether it contains penetration enhancers or not, is limited by its saturated solubility in the vehicle. Under such conditions the concentration of the permeant in the outer layers of the stratum corneum is also saturated. Consequently, when the permeation of a drug from a supersaturated solution leads to enhanced penetration, the concentration of the drug in the outer layers of the membrane is also supersaturated. Therefore, the stratum corneum may possess antinucleant properties which inhibit or retard the crystallisation process. In this study, the enhanced in vitro permeation of supersaturated solutions of piroxicam across human skin in diffusion cells was demonstrated. The amount of permeant in the stratum corneum was determined using a tape stripping technique. Supersaturated solutions up to four degrees of saturation were investigated which produced a linear relationship between the degree of saturation and the amount of piroxicam in the stratum corneum (R2 = 0.970). Furthermore, the amount of piroxicam in the viable layers of the skin also increased with increasing degree of saturation. An analysis of the results suggested that enhanced penetration across human skin from supersaturated solutions of piroxicam may occur as a result of the antinucleating ability of the intercellular lipids of the 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.

Subsaturated, saturated and supersaturated solutions of piroxicam were prepared in a propylene glycol/water cosolvent system. The diffusion of these solutions across a model membrane of Silastic® was investigated. It was shown that in a single cosolvent system the diffusion of the drug was linear with respect to the degree of saturation. However, the results from the flux of supersaturated solutions in different propylene glycol/water vehicles showed that there was an increase in the flux of piroxicam as the proportion of propylene glycol was increased. This was also true with the diffusion of saturated solutions across Silastic®. Further investigations showed that this effect was most likely due to the enhanced partitioning of piroxicam into the membrane.