Abdel-Halim Ghanem

Hindered Diffusion of Polar Molecules Through and Effective Pore Radii Estimates of Intact and Ethanol Treated Human Epidermal Membrane

The in vitro passive transport of urea, mannitol, sucrose and raffinose across intact and ethanol treated human epidermal membrane was investigated. The intent of this study was to characterize the barrier properties and permeation pathways of these membranes for polar permeants under passive conditions. Based upon the relative permeabilities of these four solutes and hindered diffusion theory, the experimental data was adequately modeled for both membrane systems according to permeation through a porous membrane. Effective pore radii estimates for intact human epidermal membrane fell between 15 Å to 25 Å while similar estimates fell compactly between 15 Å to 20 Å for ethanol treated human epidermal membrane. Similarities between the relative permeabilities of human epidermal membrane for the four permeants studied and the relative permeabilities of these same permeants through ethanol pretreated human epidermal membrane indicate that significant similarities exist between the permeation pathways for both membrane systems. The results of this study have important implications for transdermal drug delivery in general and more specifically for strategies of designing effective chemical permeation enhancement systems.

The effects of ethanol on the transport of β-estradiol and other permeants in hairless mouse skin. II. A new quantitative approach☆

The influence of ethanol on the transport behavior of β-estradiol and other permeants in hairless mouse skin was investigated over a 0–100% ethanol/saline concentration range. At high ethanol levels (> 50%), there were significant increases in new pore formation in the stratum corneum component of the skin. With pure ethanol, pore pathway transport dominated the permeation for all solutes, irrespective of polarity. At low ethanol levels (<25%), ethanol had little or no effect on the pore pathway. However, the transport of β-estradiol and hydrocortisone via the lipid pathway of the stratum corneum was greatly enhanced. The enhancement factor, E, for β-estradiol and for hydrocortison was calculated from the experimental permeation data by correcting for the dermis /epidermis permeability coefficient, the stratum corneum pore pathway permeability coefficient, and by using solubility data to correct for chemical potential changes with solvent composition. The E-values for β-estradiol and for hydrocortisone were found to be of the same magnitude (7 to 9 at 25% ethanol and of the order of 100 at 50% ethanol). It is proposed that the ethanol enhancement effects at low ethanol levels may be interpreted in terms of fluidity increases in the transport rate-limiting lipid domains.

Studies on the Effects of Applied Voltage and Duration on Human Epidermal Membrane Alteration/Recovery and the Resultant Effects upon Iontophoresis

The effects of applied voltage and the duration of application upon human epidermal membrane (HEM) alterations and recovery were investigated. All experiments were conducted using a two-chamber diffusion cell with constant DC voltage (250–4000 mV) applied over a predetermined period, and HEM changes were monitored by measuring the electrical resistance before and after voltage termination. The key findings were that the rate of decrease in resistance was strongly dependent upon the applied voltage, the reversible recovery times were dependent upon both the magnitude and the duration of the applied field (frequently were several orders of magnitude greater than times for attaining significant resistance reduction), and reversible recovery times were much longer when lower voltages were applied for longer times to attain the same decrease in electrical resistance than for higher voltages at short times. These findings closely parallel those obtained on electrical breakdown/recovery of bilayer membranes (electroporation). The second part of this work examined the hypothesis that decreases in HEM electrical resistance induced by the applied voltage are accompanied by proportional increases in HEM permeability. A study was designed to test this hypothesis involving a four-stage protocol with HEM: passive transport, 250-mV iontophoresis, 2000-mV iontophoresis for 10 min, then back to 250-mV iontophoresis. The data obtained strongly support the view that the HEM alterations induced by the electric field result in pore formation and in the expected changes in HEM permeability.

The effects of ethanol on the transport of lipophilic and polar permeants across hairless mouse skin: Methods/validation of a novel approach

The influence of ethanol on the in vitro transport behavior of some lipophilic and polar/ionic permeants in hairless mouse skin has been investigated over a 0–100% ethanol/saline concentration range in a two-chamber diffusion cell. The lipophilic permeants were employed in probing the transport enhancing effects of ethanol upon the lipoidal pathway of the stratum corneum, and the polar/ionic permeants were used to quantify the influence of ethanol on thepore pathway of the stratum corneum over the entire range of ethanol concentrations. The following were the important interpretations of the data. The lipophilic permeants (estrone, s-estradiol, and hydrocortisone) were mainly transported via the lipoidal pathway up to around 50% ethanol. The permeation enhancement factor, E, for the lipoidal pathway was calcuklated from the transport data for the three lipophilic permeants. In order to calculate the E values, it was first necessary to establish the validity of Henrys law by comparing the ratios of permeant solubilities (in different ethanol/saline solutions) to ratios of permeant partition coefficients (in hexadecane/ethanol-saline systems). The calculated E values were found to be about the same for all three permeants: E = 7.0 ± 2.0 at 25% ethanol and E = 112 ± 19 at 50% ethanol. These large enhancing effects of ethanol upon the lipoidal pathway were somewhat surprising, and it is suggested that ethanol (< 50%) may work as an effective ‘fluidizing’ agent at some locus in the stratum corneum lipid bilayer at or near the polar head plane, but not in the bilayer hydrocarbon interiors. The polar/ionic permeants (tetraethylammonium bromide, mannitol, estrone ammonium sulfate, and vidarabine) all were transported via the pore pathway at all ethanol concentrations. Ethanol up to around 25% had little effect upon the pore pathway; however, at higher concentrations (∼ 50%), ethanol greatly enhanced pore transport and, at very high ethanol levels (t~ 75%), the pore pathway appeared to dominate the transport of all permeants including the lipophilic permeants.

Short chain alkanols as transport enhancers for lipophilic and polar/ionic permeants in hairless mouse skin : mechanism(s) of action

The influences of short chain n-alkanols (from C1 to C5) and isopropanol on the transport of lipophilic (β-estradiol and hydrocortisone) and polar/ionic (tetraethylammonium ion) permeants across hairless mouse skin have been investigated. Permeability studies employing a two-chamber diffusion cell were carried out over wide ranges of alkanol (in saline) concentrations with an aim toward quantifying the reversible enhancement effects of the added alkanol upon the lipoidal pathway of the stratum corneum. An enhancement factor, E (for the lipoidal pathway of the stratum corneum), was calculated from permeability coefficient and solubility data, and the E values for β-estradiol and for hydrocortisone were found to be nearly always the same in all instances. A pattern of increasing E values with increasing alkanol chain length up to C5 with these two permeants was found. A nearly semi-logarithmic linear relationship was also obtained between the enhancement potency and the carbon number of the n-alkanols; there was about 4-fold increase in the enhancement potency per n-alkanol methylene group. Pretreatment studies showed that the n-alkanol effetcts at low concentrations were reversible as far the lipoidal pathway of the stratum corneum was concerned. These results demonstrate the general usefulness of this approach for evaluating the action of enhancers on the barrier function of the stratum corneum. It is suggested that the short chain alkanols may work at low concentrations as effective ‘fluidizing’ agents at some locus in the stratum corneum lipid bilayer at or near the polar head plane, but not in the deep bilayer hydrocarbon interiors.

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 (3H-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-beta-cyclodextrin (HPbetaCD) was selected as the representative cyclodextrin. The CS/HPbetaCD 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 degrees 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 HPbetaCD 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 HPbetaCD is interpreted to mean that, in these experiments, HPbetaCD 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.

Transport of β-Estradiol in Freshly Excised Human Skin in Vitro: Diffusion and Metabolism in Each Skin Layer

This paper describes an experimental and theoretical evaluation of β-estradiol (E2) transport in post-surgery fresh human skin in vitro. Necessary auxiliary experimental methods were newly developed for these studies. The experimental fluxes of E2 and the metabolite, estrone (El), using the dermis, stripped skin, and split-thickness skin were consistent with a model considering the human skin as a three-layer (stratum corneum, viable epidermis, and dermis) membrane with the enzyme activity mainly residing in the basal layer of the viable epidermis. The diffusion and metabolism parameters for each skin layer were determined in the overall transdermal transport of E2. Compared to fresh hairless mouse skin, fresh human skin appears more resistant to the stratum corneum diffusion of E2 and is much less capable of metabolizing E2 to El. These in vitro results have been extrapolated to the possible in vivo human skin situation with blood vessels directly beneath the viable epidermis providing “sink” conditions a short distance from the dermo-epidermal junction. The model analysis has demonstrated that there would be less metabolism and that a much smaller amount of the transdermal metabolite (El) would be taken up by the blood capillary due to the shorter dermis path length for permeants in vivo than in the in vitro case using dermatomed split-thickness skin.

Improved stability of the human epidermal membrane during successive permeability experiments

Abstract In many cases it is instructive to use a single human epidermal membrane (HEM) sample to perform successive in vitro permeability experiments under varied experimental conditions. This study focused upon the feasibility of such successive permeability experiments in side-by-side, two-chamber diffusion cells. It was shown that for permeability experimental protocols which involved performing one permeability experiment per day and extensive washing between permeability experiments, the barrier properties of HEM samples were altered significantly within the first 72 h of the protocol. However, if the HEM is supported in the diffusion cell with a porous synthetic membrane, a single HEM sample remains essentially unaltered with respect to mannitol permeability and electrical resistance for up to 5 days. This suggests that protecting the HEM from physical stress is an essential element in performing successive permeability experiments.

Macromolecule transport in and effective pore size of ethanol pretreated human epidermal membrane

Abstract This study has examined the transport behavior of macromolecules of up to about 18 000 molecular weight (Mol. Wt) and the feasibility of using the theory of restricted diffusion of molecules through cylindrical pores to predict/characterize the pore sizes of synthetic membranes and ethanol pretreated human epidermal membrane (HEM). To minimize membrane and skin variabilities, experiments were conducted consecutively with each membrane using a two-chamber diffusion cell. Reference permeants or electrical resistance measurements were used to monitor possible membrane changes during a given set of runs. All HEM specimens exhibited the same pattern of significantly decreasing permeability with increasing polystyrene sulfonate (PSS) molecular weight. Previously obtained permeability data for ethanol pretreated HEM with polypeptides (leuprolide, CCK-8 and insulin) were found to be consistent with those for PSS; comparable permeability coefficients were observed for polypeptides and PSSs of comparable sizes. An analysis based on the theory of restricted diffusion for PSS transport across a synthetic Nuclepore® membrane yielded results consistent with the nomical pore size ( ∼ 75 A ) of this membrane. A similar analysis of the PSS data obtained with ethanol pretreated HEM yielded estimates of effective pore size for this membrane in the range, 22–54 A.

Quantitation of simultaneous diffusion and metabolism of β-estradiol in hairless mouse skin: Enzyme distribution and intrinsic diffusion/metabolism parameters

Abstract This paper describes a systematic experimental and theoretical study of the simultaneous diffusion and metabolism of β-estradiol (E 2β ) in hairless mouse skin (in vitro). The strategy involved (a) considering a general three-layer skin model (stratum corneum, epidermis, and dermis), (b) considering three possible enzyme distributions (Model A: homogeneous enzyme distribution across both epidermis and dermis; Model B: homogeneous enzyme distribution in the epidermis; and Model C: homogeneous enzyme distribution in the ‘basal cell layer’ only of the epidermis), and (c) carrying out a wide range of independent diffusion experiments so that a ‘best’ model may be deduced in which all of the experimental data are consistent with the model and a single set of transport and metabolism parameters. The various diffusion/metabolism experiments included using three skin membranes (dermis, stripped skin, and full-thickness skin), two membrane configurations (transport of permeants in the direction: stratum corneum → epidermis → dermis, and in the reverse direction), two permeants (E 2β and estrone, E 1 , the principal metabolite), and measuring three fluxes (forward fluxes of E 2β and E 1 and the back flux of E 1 ). Analysis of all of the experimental data demonstrated that Model C was superior to Models B and A; within the uncertainties of the experiments and model fitting, Model C agreed well with the data in all instances while the predictions of Models B and A exhibited significant deviations from the experimental data.