Noninvasive measurement of phenylalanine by iontophoretic extraction in patients with phenylketonuria.

Phenylketonuria is an autosomal recessive disorder characterized by elevated concentrations of phenylalanine. Elevated phenylalanine concentrations can impair intellectual functions and the disease is treated with a lifelong diet and frequent monitoring of plasma phenylalanine concentrations. Previous in vitro studies have demonstrated the feasibility of iontophoretically enhanced transdermal transport of phenylalanine. Here we evaluate the feasibility of transdermal iontophoretic extraction of phenylalanine in vivo. Phenylalanine was iontophoretically extracted from the skin of healthy volunteers and of patients with phenylketonuria for up to 6 h and concentrations were compared with those measured in plasma. The amount of phenylalanine iontophoretically extracted from the skin declined over time, suggesting contribution of phenylalanine from the skin in the initial extraction. Phenylalanine iontophoretically extracted from skin correlated with plasma phenylalanine levels at plasma levels above 300 micromol/L. This correlation supports the feasibility of iontophoretic phenylalanine extraction for monitoring phenylketonuria.

Correlation of in vivo topical efficacies with in vitro predictions using acyclovir formulations in the treatment of cutaneous HSV-1 infections in hairless mice: an evaluation of the predictive value of the C* concept.

The purpose of this study was to carry out an extensive examination of the C* concept for prediction of the topical antiviral efficacies of acyclovir (ACV) formulations in a hairless mouse model for the treatment of cutaneous herpes simplex virus type-1 (HSV-1) infections. This method is based on estimation of the free drug concentration at the target site (C*), which is presumed to be the basal cell layer of the epidermis. Five different formulations (containing 5% ACV) were examined in a finite dose multiple dosing regimen (twice a day application) to simulate the clinical situation. For determination of C*, in vitro ACV fluxes across the hairless mouse skin were measured in an in vivo-in vitro experimental design that approximated the in vivo antiviral treatment protocol. Then, the in vivo antiviral efficacies were measured using a 1-day delayed (after HSV-1 virus inoculation) 4-day treatment protocol. 10 microL/cm2 dose of ACV formulation was applied every 12 h for 4 days after which the lesions were scored and efficacies were calculated. Our results indicate that, over a wide range of efficacies, the predictions based on C* (estimated from the experimental fluxes) are in good agreement with the in vivo antiviral efficacies. These studies, therefore, support the validity of the C* concept for various ACV formulations and suggest that the C* approach has potential for future practical situations.

Mechanistic aspects of iontophoresis in human epidermal membrane.

A large number of factors are involved in the movement of ions and molecules across human epidermal membrane (HEM) under the influence of an electric field. These factors and their interplay need to be understood if our knowledge of iontophoretic transport of drugs across HEM is to reach a point where physical models and strategies may be employed for useful quantitative predictions. In a typical in vitro experiment, the fully hydrated HEM is positioned between aqueous compartments of a two-chamber diffusion cell. When a low electric field is applied across the HEM under these conditions, the transport enhancement of ions in the pre-existing pores of the stratum corneum is the result of, (a) the direct interaction of the electric field with the charge of the ion in question, and (b) convective solvent flow (electroosmosis); in the case where the permeant is non-ionic under these circumstances, transport enhancement is by convective solvent flow only. At moderate-to-high voltage iontophoresis (> or = around 1.0 V applied across a single HEM), in addition to the direct field effect and convective solvent flow in the pre-existing pores, there can generally be a significant (e.g. 10- to 100-fold enhancement) contribution to transport enhancement arising from new pore induction (electroporation). Much of the recent work in our laboratory has been devoted to defining and quantifying HEM electroporation, and an especially difficult aspect has been that of dealing with the large HEM membrane-to-membrane variabilities with regard to, (a) the extent of new pore induction, and (b) the characteristics of the newly induced pores. Recently we discovered that the extent of relevant (i.e. permeant accessible) pore induction may be correlated to the change in HEM electrical conductance (and quantifiable) if an appropriate matching background electrolyte can be selected having ion sizes comparable to that of the permeant. For example, employing tetraethylammonium (TEA) pivalate (PIV) for which the ion sizes are approximately 3.5 A, but not KCl (ion sizes approximately 1.9 A), as the background electrolyte for TEA (as the permeant) gave very good results; in this example, the sizable contribution of pore induction to iontophoresis was quantitatively factored out from the total iontophoretic enhancement. Experiments with a large number of HEM samples gave good agreement with the Nernst-Planck (N-P) predictions of the direct field effect when TEA-PIV was used as the background electrolyte for TEA transport, but large variations (up to 300%) between N-P predictions and experimental results were observed with KCl as the background electrolyte. Another area of recent effort has been HEM pore size determinations, both at low voltages (i.e. for pre-existing pores) and at voltages where the newly induced pores dominate HEM permeability. The sizes of pre-existing pores of HEM have been determined with the hindered diffusion theory (using experimental fluxes of several probe permeants of different known molecular sizes) to be generally in the range, 10-20 A, by a number of investigators in our laboratory for a large number HEM samples. Deducing pore sizes of electric field induced pores under steady electroporation conditions has been a more challenging task. We succeeded recently in developing a novel method for 'passively' determining pore sizes (i.e. by passive diffusion with hindered diffusion theory) under steady electroporation conditions: by using low frequency (12.5 Hz) a.c. at 2-5 V. We have been able to sustain electroporation at a nearly constant state of electroporation long enough to carry out a set of 'passive' diffusion experiments with relatively good precision to obtain the sizes of the newly induced pores. Studies to date have revealed that the sizes of pores induced with 2-5 V are of the same order of magnitude as those of the pre-existing pores (i.e. 10-20 A). Finally, another research question of interest has been that of pore charge

Quantitative in vivo iontophoretic studies.

An experimental methodology was developed to evaluate the physicochemical basis for in vitro-in vivo correlations in iontophoretic delivery situations. This experimental methodology can be used to quantitatively evaluate the extent of interaction between chemical permeation enhancers and iontophoresis for drug delivery. The inherent advantages of using Ag/AgCl electrodes are fully exploited to control pH and minimize depletion of permeant during prolonged periods of iontophoresis.

Pore charge distribution considerations in human epidermal membrane electroosmosis.

The aim of this study was to assess the extent to which a model with pores having only net negative charges would adequately describe transdermal electroosmosis in human epidermal membrane (HEM) at neutral pH. Such information would enhance the predictive value of the modified Nernst-Planck model for transdermal iontophoresis, in addition to providing insights regarding the likelihood of significant pore charge distribution in HEM. Baseline results (the control) obtained from 0.1 to 0.4 V anodal and cathodal electroosmosis experiments with synthetic polycarbonate membranes (Nuclepore membranes), using radiolabeled urea and mannitol as the model permeants, demonstrated that such a membrane system can be modeled by the electrokinetic (electroosmosis) theory with the assumption of the pores possessing only negative charges. The studies with HEM were carried out at low voltage (/approximately = 1.0 V) where significant field-induced pore formation in HEM occurred. In both the low and high voltage studies, radiolabeled urea, mannitol, and water were employed as permeants in cathodal and anodal iontophoresis experiments. The results of the low voltage iontophoresis experiments suggest significant pore charge distribution in HEM (a significant deviation between the predictions from the single pore charge type assumption and the experimental data). Under the higher applied voltage conditions (>/approximately = 1.0 V), results from anodal and cathodal electroosmosis studies were consistent with the model in which the HEM has only pores that are net negatively charged.

The effect of temperature upon the permeation of polar and ionic solutes through human epidermal membrane.

The temperature dependence of in vitro permeation through human epidermal membrane (HEM) was determined for urea, mannitol, tetraethylammonium ion (TEA), and corticosterone. The effect of temperature upon HEM electrical resistance was also measured. The majority of the experiments involved measuring the permeability coefficients of a specific permeant at 27 degrees C and 39 degrees C for a given HEM sample, the electrical resistance was also measured at each temperature. Similar experiments were also conducted with a model synthetic porous membrane. The effect of temperature was quantitated as the ratio of the permeability at 39 degrees C to the permeability at 27 degrees C for each permeant. These ratios observed for HEM with urea, mannitol, and TEA as the permeants were 1.66 +/- 0.05, 1.76 +/- 0.14, and 1.71 +/- 0.11, respectively. The change in temperature was shown to have a similar effect upon the electrical conductance of the HEM samples. The observed ratio for corticosterone permeation was 4.5 +/- 0.4. The experimental ratios observed for the three polar/ionic permeants were shown to approach those obtained from the model porous membrane and differed greatly from the ratio observed for the more lipophilic corticosterone, indicating differences in the effective transport mechanism/pathway for these classes of permeants. The permeability of urea was also observed to be inversely proportional to the electrical resistance of the HEM samples; this relationship was shown to be independent of temperature over the temperature range studied. The temperature dependence data and the observed relationship between urea permeability and electrical resistance strongly support the existence of a porous permeation pathway through the HEM as an operative diffusional route for polar-ionic permeants.

Mechanistic studies of the 1-alkyl-2-pyrrolidones as skin permeation enhancers.

The influences of 1-ethyl-, 1-butyl-, 1-hexyl-, and 1-octyl-2-pyrrolidone in their saline solutions on the transport of beta-estradiol, corticosterone, and hydrocortisone across hairless mouse skin under in vitro conditions have been investigated by the physical model approach. The experimental data were interpreted with a physical model that treats the stratum corneum as a diffusional barrier with a lipoidal pathway and a pore pathway. Enhancement factors (E values) for the lipoidal pathway were calculated from the permeability coefficients and solubility data as a function of the 1-alkyl-2-pyrrolidone concentration for all three permeants. A pattern of increasing E values with increasing 1-alkyl-2-pyrrolidone chain length was found, and the results were essentially the same for all three steroidal permeants. A nearly semilogarithmic linear relationship was also obtained between the enhancement potency and the carbon number of the alkyl chain; there was about an approximately 3.5-fold increase in the enhancement potency per 1-alkyl-2-pyrrolidone methylene group. An important outcome of this research is that the enhancement potencies of the 1-alkyl-2-pyrrolidones were essentially the same as those for the previously studied n-alkanols when compared at the same carbon numbers of the alkyl groups. This result is somewhat surprising as it suggests that the enhancer action resides (in its entirety) in the alkyl group, and the nature of the polar head group may not be intrinsically important in transdermal enhancement of the lipoidal pathway within a class of permeation enhancers.

Iontophoretic transport across a synthetic membrane and human epidermal membrane: a study of the effects of permeant charge.

The effects of permeant charge (z) on iontophoretic-enhanced transport were investigated with synthetic Nucleopore membranes and with human epidermal membranes using a four-electrode potentiostat with side-by-side diffusion cells. The modified Nernst-Planck model (Nernst-Planck theory with an additional transport term to correct for the effect of the convective solvent flow due to electroosmosis) was first examined in a Nuclepore membrane system with model permeants calcein (z = -4), salicylate (z = -1), and a series of polystyrene sulfonates (from monomer to molecular weight of approximately 8000 with a z range of -1 to approximately -40). The flux enhancement (E) for each permeant was determined at 470 mV. Mannitol (a neutral molecule) was used as a probe to determine a correction for convective solvent flow under the same applied voltage conditions. Good agreement between the experimental results and the predictions from the modified Nernst-Planck model was found for calcein, salicylate, and polystyrene sulfonates up to molecular weight of approximately 1800 (z approximately -8). The flux enhancements for the higher molecular weight polystyrene sulfonates with greater z values were more than a factor of three lower than theoretical predictions; the electrophoretic effect and counterion binding to the permeants are proposed as possible explanations for these discrepancies between experiment and the modified Nernst-Planck theory. In the studies with human epidermal membranes, iontophoretic flux enhancements for calcein, salicylate, and taurocholate were determined at 250 and/or 470 mV. The flux enhancements were generally consistent with the results calculated from the modified Nernst-Planck model.

Relationship of skin target site free drug concentration (C*) to the in vivo efficacy: an extensive evaluation of the predictive value of the C* concept using acyclovir as a model drug.

For the past few years, our laboratory has been involved in the development of a novel approach for predicting topical in vivo efficacy based on the estimation of skin target site free drug concentration (C*) from in vitro flux data. We have used acyclovir (ACV) as a model drug in the treatment of cutaneous herpes simplex virus type 1 infections in hairless mice. The goal of this study was to rigorously evaluate the applicability of this approach over the entire range of topical efficacy (i.e., from 0 to 100%). We employed a variety of ACV formulations differing in solvent compositions, enhancers, and excipients (and therefore in their efficacies) to achieve this goal. The C* values were estimated from the in vitro flux data obtained in an in vivo-in vitro experimental design that closely approximated the in vivo treatment protocol. For the in vivo antiviral efficacy studies, a finite dose of ACV formulation was applied twice a day, beginning the day after virus inoculation, for 4 days. The lesions were scored on the fifth day, and the efficacies were calculated as described earlier. Our results indicate that, for a variety of formulations over a wide range of efficacies, the predictions based on C* are in good agreement with the observed in vivo efficacies. These findings strongly demonstrate the predictive value of C* over the entire range of topical efficacy, thereby further strengthening its potential for future studies. The findings also indicate that although the excipients in a formulation may alter the rate and extent of available drug at the target site, in these cases, they do not seem to have any effect on the in vivo potency of the drug.

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 A, which are of the same order of magnitude as those of preexisting pores determined from conventional passive diffusion experiments.

A mechanistic study of the effects of the 1-alkyl-2-pyrrolidones on bilayer permeability of stratum corneum lipid liposomes: a comparison with hairless mouse skin studies.

The influence of a series of 1-alkyl-2-pyrrolidones (C2-C8) on the transport behavior of lipophilic and polar/ionic permeants across hairless mouse skin was recently investigated by employing a physical model approach that treats the stratum corneum barrier as a diffusional system of parallel lipoidal and pore pathways. In this previous study, the transport enhancement effects (enhancement factor, EHMS) on the lipoidal pathway of the stratum corneum were found to be essentially the same for all steroidal probe permeants investigated at various concentrations of these 1-alkyl-2-pyrrolidones. In the present research, the relationship between solute transport enhancement in the lipoidal pathway of hairless mouse skin and the transport enhancement in the stratum corneum lipid liposome bilayer was studied by comparing the enhancement factor for the lipoidal pathway in the hairless mouse skin, EHMS, with that for the stratum corneum lipid liposome, ESCLL, at equal solution concentrations of the 1-alkyl-2-pyrrolidones. The release rates of D-mannitol, D-glucose, 3-O-methyl-D-glucose, sucrose, and raffinose from stratum corneum lipid liposomes were determined, and the ESCLL values for these permeants were compared with the EHMS values obtained with hairless mouse skin using the steroidal permeants. An important finding in this study was a semiquantitative correlation between the enhancement effects induced by the 1-alkyl-2-pyrrolidones, except 1-ethyl-2-pyrrolidone, with the liposome bilayer using sugar molecules as permeants and those found with the lipoidal pathway in hairless mouse skin using steroid molecules as permeants.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative description of the effect of molecular size upon electroosmotic flux enhancement during iontophoresis for a synthetic membrane and human epidermal membrane.

This study focused upon quantitatively determining the influence of permeant molecular size upon flux enhancement which results from electroosmosis. The first phase of the study involved validation of a fundamental model describing the molecular size dependence of flux enhancement which results from convective solvent flow. This was accomplished using a model synthetic membrane (stack of 50 Nuclepore membranes) and four model permeants with a molecular weight range of 60-504 (urea, mannitol, sucrose, and raffinose). The steady-state flux of each permeant was determined under passive conditions and applied voltages of 125, 250, 500, and 1000 mV using side-by-side diffusion cells and a four-electrode potentiostat system. On the basis of the permeability enhancement for each permeant at each applied voltage (relative to the passive permeability) it was possible to calculate the effective solvent flow velocity from each permeant at each field strength. An important finding was that the flux enhancement due to electroosmosis was strongly molecular weight dependent (i.e., the flux enhancement ratio was around 4 times greater for raffinose than for urea, with mannitol and sucrose yielding intermediate values), while the calculated effective flow velocity at each voltage was independent of the molecular weight of the permeant. This coupled with a linear correlation between flow velocity and applied voltage served to establish the validity of the method and model. The second phase of the study was an extension of the model to human epidermal membrane (HEM). These experiments involved simultaneously measuring the fluxes of [14C]urea and [3H]sucrose across HEM samples under passive, 250 mV, and 500 mV conditions. Similar to the Nuclepore system, the observed flux enhancement ratios with HEM were approximately 3 times greater for sucrose than for urea. A detailed analysis of the HEM data showed semiquantitative agreement between predictions of the model and experimental results.

Iontophoretic transport of oligonucleotides across human epidermal membrane: a study of the Nernst-Planck model.

The objective of this study was to investigate the transport behavior of a series of oligonucleotides with human epidermal membrane (HEM) and to examine the applicability of the modified NERNST-PLANCK model to transdermal iontophoresis of these macromolecules. Iontophoretic transport experiments were first carried out in a synthetic model membrane system (Nuclepore membranes) with a four-electrode potentiostat to examine the baseline modified NERNST-PLANCK model. The modified NERNST-PLANCK model derived from the Einstein relation and the Stokes-Einstein equation taken from previous work did not hold for the oligonucleotides. Results obtained in the Nuclepore studies were, however, consistent with predictions of the modified NERNST-PLANCK model using the experimentally determined electromobilities and diffusion coefficients. The electromobilities of the oligonucleotides (determined by capillary electrophoresis) were found to be more than a factor of two smaller than expected from the Einstein relation between electromobilities and diffusion coefficients (the latter determined in diffusion cell experiments). A correlation between these electromobilities and the theoretical electromobilities estimated by considering the effects of counterion binding and the effects of mobility reduction according to colloid theory was also observed. These results suggest that the modified NERNST-PLANCK model predictions are satisfactory only when the electromobilities and the effective molecular size of the oligonucleotides are known and are used directly to predict the iontophoretically enhanced transport. Results with the HEM experiments generally agreed with model predictions based on the experimental electromobilities. The oligonucleotide HEM flux data also suggest the existence of pores with effective pore radii greater than the effective radii estimated in previous studies with small molecular weight model permeants.

Quantification of pore induction in human epidermal membrane during iontophoresis: the importance of background electrolyte selection.

It has been shown that significant pore induction (electroporation) occurs in human epidermal membrane (HEM) during iontophoresis even at moderate applied voltages (1-10 V). Recent efforts in our laboratory have been aimed at quantifying HEM electroporation by examining the proportionality between flux enhancement due to electroporation and electrical conductance changes during iontophoresis. The specific purpose of the present study was to test the hypothesis that by matching the background electrolyte ion sizes with the permeant ion sizes, the flux enhancement due to electroporation can be quantified by the change in HEM electrical conductance. In this study, radiolabeled tetraethylammonium (TEA(+)), methylammonium (MA(+)), and mannitol were the permeants. Potassium chloride (KCl), tetraethylammonium bromide (TEAB), tetraethylammonium pivalate (TEAP), and sodium fluoride (NaF) were the background electrolytes. Iontophoresis experiments were carried out over an applied voltage range of 1 to 3 V. The experimental flux enhancement results were compared with the theoretical predictions from the Nernst-Planck model after corrections were made: (a) for HEM pore induction during iontophoresis based on electrical conductance changes and (b) for electroosmosis employing mannitol as the neutral probe permeant. In experiments where the ion sizes of the background electrolyte and permeant were closely matched (e.g., TEA(+) as the permeant and TEAP as the background electrolyte), there was excellent agreement between experimental results and theoretical predictions of the modified Nernst-Planck model, with only modest data scatter. When the electrolyte and permeant sizes were quite different (e.g., TEA(+)/KCl and MA(+)/TEAP), the experimental flux data were inconsistent with model predictions and there were large variations in the experimental results. The results of the present study illustrate that permeant flux enhancement can be predicted by the modified Nernst-Planck model even during moderate voltage iontophoresis when electroporation is operative.

Characterization of the transport pathways induced during low to moderate voltage iontophoresis in human epidermal membrane.

This report describes the results of iontophoresis experiments involving the transport of polar nonelectrolytes across human epidermal membrane (HEM) at a moderate applied voltage of 2.0 V and where the data are interpreted via a convective transport model and hindered transport theory. A principal finding is that although HEM iontophoresis at 2.0 V resulted in a large increase in HEM porosity, the pore radii of the newly induced pores in HEM as calculated from the iontophoresis data using the hindered transport theory were found to be in the range of 6-12 A. This supports the view that electroporation at these modest applied voltages results in pores with sizes the same order of magnitude but somewhat smaller than those estimated for the preexisting pores in HEM prior to electroporation. This outcome is also important from a practical standpoint, as flux enhancement for large molecules (such as oligonucleotides and polypeptides) arising from electroporation under these conditions would be expected to be significantly less than if the resulting pore sizes were much greater. Providing a "prepulse" of 4.0, 8.0, and 15 V prior to the 2.0 V iontophoresis generally gave greater increases in HEM conductance (and, therefore, in porosity) but did not significantly change the deduced effective pore radii (around 5-9 A). The alteration during and the recovery of HEM after iontophoresis was also investigated. The recovery behavior was found to be dependent upon both the duration of the applied voltage and the magnitude of its effects: the recovery for a HEM sample that experienced a large increase in electrical conductance during iontophoresis was generally poorer than that for a sample that was more resistant to the electric field. Incomplete recovery was generally observed in experiments with long iontophoresis duration (50 min) and with the higher voltages (4.0, 8.0 V, and 15 V). In these cases, the barrier properties of HEM were more greatly altered as indicated by larger increases in the electrical conductance and passive permeability of HEM after iontophoresis.

Transdermal iontophoretic drug delivery: mechanistic analysis and application to polypeptide delivery.

Three factors are of primary importance in determining the iontophoretic flux of a charged solute: the electrochemical potential gradient across the skin, an increase in skin permeability to passive transport due to iontophoresis (loosely defined as skin damage), and a current-induced water flux. The latter two factors can also affect the transport of uncharged solutes during iontophoresis. A method of correcting for the skin damage effect is introduced. The contributions of the water transport effect relative to that of the applied voltage drop for charged solutes is estimated. It is shown that the water transport contribution is generally lower than the contribution due to the applied voltage drop. The observed iontophonetic flux of the enhancement factors due to the applied voltage drop alone are compared with the theoretical predictions based on the constant field assumption. It is shown that the theoretical predictions are higher than the experimental observations. This work also examines, for the first time, a synergism of iontophoresis and pretreatment with a chemical penetration enhancer as a means for delivering high molecular weight polypeptides. It is shown that a 2-h pretreatment with absolute ethanol followed by iontophoresis dramatically increases the permeability coefficient of insulin through human skin.

Flux enhancement effects of ionic surfactants upon passive and electroosmotic transdermal transport.

This study focused upon the enhancement effects of ionic surfactants upon passive and electroosmotic transdermal flux. The first phase of the study involved validating theories relating surface properties of a membrane to electroosmotic solvent flow under appropriate experimental conditions using a synthetic model membrane (stack of 50 Nuclepore membranes). Numerical solutions to the Poisson-Boltzmann equation and the equations of fluid motion served as the theoretical basis for the experimental studies. Important outcomes of the model membrane studies were that electroosmotic solvent flow velocity was enhanced by the addition of an anionic surfactant, sodium dodecyl sulfate, and reversed by the addition of a cationic surfactant, dodecyltrimethylammonium bromide. The effective membrane pore wall surface charge densities were determined under a variety of experimental conditions. Adsorption of dodecyl sulfate to the pore wall increased the net negative charge on the pore wall. A reversal of the net pore wall surface charge density resulted from the adsorption of dodecyltrimethylammonium. The interrelationship between electroosmosis, surfactant adsorption, and ionic strength was also evaluated. The second phase of the study was an investigation of the effects of sodium dodecyl sulfate upon the transport of neutral polar permeants through human epidermal membrane (HEM). Fluxes of [14C]urea and [3H]sucrose were simultaneously measured across HEM samples under passive and 250 mV conditions; flux measurements were made before, during, and after HEM exposure to sodium dodecyl sulfate. A systematic analysis of the experimental data made it possible to elucidate the specific contributions of sodium dodecyl sulfate and the applied electric potential to the overall flux enhancement. Sodium dodecyl sulfate enhanced the intrinsic passive permeability of the HEM, and it also enhanced the contribution of electroosmosis to the flux during iontophoresis.

Influence of the treatment protocol upon the in vivo efficacy of cidofovir (HPMPC) and of acyclovir (ACV) formulations in topical treatment of cutaneous HSV-1 infection in hairless mice.

In recent studies we found that the topical effectiveness of acyclovir (ACV) formulations was a single-valued function of C-the target site free drug concentration. The topical efficacy was the same when the therapy was initiated 0, 1, or 2 days after intracutaneous herpes simplex virus type-1 (HSV-1) inoculation in hairless mice. The purpose of the present study was to examine the hypothesis that the topical effectiveness of cidofovir (HPMPC) would not be a single valued function of C and that it would be dependent upon when the therapy was initiated relative to the time of viral infection. Formulations of HPMPC and ACV in 95% DMSO as a vehicle were used. Hairless mice intracutaneously infected with HSV-1 were used, and 20 microL of the test formulation was topically applied twice a day. In protocol A, the treatment was continued until the fourth day after virus inoculation, whereas in protocol B the treatment was terminated on the day of virus inoculation. Treatment was initiated on various days ranging from day -6 to day 4, and the lesions were scored on day 5. Treatment of ACV according to protocol A proved efficacious whether started as early as 6 days before virus inoculation or later, whereas the efficacy of ACV was annihilated if applied following protocol B. For HPMPC, on the other hand, the in vivo efficacies were found to be strongly dependent on how early the therapy was initiated, and significant efficacy was observed even when the treatment was terminated on the day of virus inoculation. This difference was attributed to the virus-independent intracellular phosphorylation of HPMPC and slow clearance of its metabolites from the cell. It was also noted that, similar to ACV, for HPMPC the topical efficacy is likely to be a function of C for a fixed protocol. However, unlike for ACV, for HPMPC the efficacy was not a single-valued function of C.

Assessment of correlation between skin target site free drug concentration and the in vivo topical antiviral efficacy in hairless mice for (E)-5-(2-bromovinyl)-2'-deoxyuridine and acyclovir formulations.

Recently, we reported that the in vivo efficacy of acyclovir (ACV) formulations was a single valued function of skin target site free drug concentration (C) irrespective of the formulation compositions. A long-term objective of this research has been to generalize the C concept using model drugs which are similar to as well as different from ACV in their mechanism of actions. (Bromovinyl)deoxyuridine (BVDU) was selected as a model drug based on the reported similarity in its mechanism of action with ACV. The relationship between the C predictions and the in vivo efficacies for some topical formulations containing different concentrations (0.05-10%) of either ACV or BVDU in 95% DMSO as a vehicle with or without 5% Azone as skin permeation enhancer was examined. Hairless mice infected cutaneously with HSV-1 were used to quantitatively estimate the in vivo topical antiviral efficacy. A finite dose of the test antiviral formulation was applied twice a day for 4 days, starting the day after virus inoculation. On the fifth day, the lesions were scored and the efficacy values were calculated. For each formulation, in vitro flux experiments were performed in an in vivo-in vitro experimental design that closely approximated the in vivo study protocol. As was previously shown, with all ACV formulations, a good correlation was found between the C predictions and the in vivo topical efficacy. With the BVDU formulations, on the other hand, this was found not to be the case. BVDU formulations with 5% Azone were generally much more effective than those without Azone at comparable C values. This finding is believed to be the first of its kind showing that skin "permeation enhancers" may enhance efficacy by more than simply increasing skin permeation rates.

Mechanistic studies of the effect of hydroxypropyl-beta-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.