Dynamics of glycerine and water transport across human skin from binary mixtures.

OBJECTIVE: Skin transport properties of glycerine and water from binary mixtures contacting human skin were determined to better understand the mechanism of skin moisturization by aqueous glycerine formulations. METHODS: Steady-state permeation for 3 H2 O and 14 C-glycerine across split-thickness human skin in vitro and desorption dynamics of the same permeants in isolated human stratum corneum (HSC) were experimentally determined under near equilibrium conditions. These data were compared to a priori values developed in the context of a thermodynamic model for binary mixtures of glycerine and water and a previously determined water sorption isotherm for HSC. This allowed the estimation of diffusion and partition coefficients for each permeant in the HSC, as well as HSC thickness, as a function of composition of the contacting solution. These data may be used to estimate water retention and associated HSC swelling related to the absorption and slow release of glycerine from the skin. RESULTS: It took 6+ days for glycerine to completely desorb from HSC immersed in glycerine/water binary solutions. Desorption of both 3 H2 O and 14 C-glycerine from HSC was slower in pure water than from binary mixtures, a result that is largely explained by the greater swelling of HSC in water. Parametric relationships were developed for water and glycerine intradiffusivities in HSC as functions of HSC water content, and a mutual diffusion coefficient was estimated by analogy with glycerine/water binary solutions. The intradiffusivity of 14 C-glycerine in HSC as inferred from sorption/desorption experiments was shown to be approximately 10-fold less than that inferred from permeation experiments, whereas the corresponding values for 3 H2 O were comparable. CONCLUSION: These studies confirm that glycerine enters HSC in substantial quantities and has a long residence time therein. The coupling between bulk water and glycerine transport projected from binary solution data suggests the net effect of glycerine is to slow water loss from the skin. The data support the concept of glycerine as a humectant with an excellent balance of skin penetration and retention characteristics; however, they do not rule out the possibility of an additional biological effect on skin barrier homoeostasis.

Lipid solubility and molecular weight: whose idea was that.

Gene Cooper was a bright theoretician, a skilled product developer, and a motivational leader who applied his talents to the skin science area early in his career. His work led to the development of finite dose skin absorption models, chemical penetration enhancer technologies and quantitative structure-penetration relationships for chemicals contacting human skin. His ideas regarding the impact of molecular weight and lipid solubility on skin transport catalyzed the later development by Potts and Guy of the first successful skin permeability model. But Gene's most important contribution to the field was as a scientific role model and an inspirational leader who launched the careers of several young scientists, including the author of this article.

Hydroxyisohexyl 3-cyclohexene carboxaldehyde allergy: relationship between patch test and repeated open application test thresholds.

BACKGROUND: Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) is a synthetic fragrance ingredient. Case reports of allergy to HICC appeared in the 1980s, and HICC has recently been included in the European baseline series. Human elicitation dose-response studies performed with different allergens have shown a significant relationship between the patch-test threshold and the repeated open application test (ROAT) threshold, which mimics some real-life exposure situations. Fragrance ingredients are special as significant amounts of allergen may evaporate from the skin. OBJECTIVES: The study aimed to investigate the relationship between elicitation threshold doses at the patch test and the ROAT, using HICC as the allergen. The expected evaporation rate was calculated. MATERIALS AND METHODS: Seventeen HICC-allergic persons were tested with a dilution series of HICC in a patch test and a ROAT (duration up to 21 days). Seventeen persons with no HICC allergy were included as control group for the ROAT. Results The response frequency to the ROAT (in microg HICC cm(-2) per application) was significantly higher than the response frequency to the patch test at one of the tested doses. Furthermore the response rate to the accumulated ROAT dose was significantly lower at half of the doses compared with the patch test. The evaporation rate of HICC was calculated to be 72% over a 24-h period. CONCLUSIONS: The ROAT threshold in dose per area per application is lower than the patch test threshold; furthermore the accumulated ROAT threshold is higher than the patch test threshold, which can probably be explained by the evaporation of HICC from the skin in the open test.

Dermal capillary clearance: physiology and modeling.

Substances applied to the skin surface may permeate deeper tissue layers and pass into the body's systemic circulation by entering blood or lymphatic vessels in the dermis. The purpose of this review is an in-depth analysis of the dermal clearance/exchange process and its constituents: transport through the interstitium, permeability of the microvascular barrier and removal via the circulation. We adapt an 'engineering' viewpoint with emphasis on quantifying the dermal microcirculatory physiology, providing the theoretical framework for the physics of key transport processes and reviewing the available computational clearance models in a comparative manner. Selected experimental data which may serve as valuable input to modeling attempts are also reported.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

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.

Two-stage kinetic analysis of fragrance evaporation and absorption from skin.

Human in vivo fragrance evaporation data from a previously published study are reanalysed in terms of compartmental pharmacokinetic models in which the microscopic rate constants are functions of the physicochemical properties of the fragrance components. According to the proposed analysis, which is restricted to low doses, absorption and evaporation of each component are first-order processes occurring from either the skin (one-compartment model) or the skin and a more rapidly depleted vehicle layer (two-compartment models). Evaporation rates of ingredients from a 12-component mixture containing a musk fixative followed single exponential decays that were well described by the one-compartment model. An otherwise identical mixture without fixative yielded evaporation rates that could be characterized as biexponential decays associated with loss from two compartments. This result shows that ingredient interactions qualitatively and quantitatively change evaporation rate profiles of fragrance components; however, an attempt to account for these interactions explicitly by means of activity coefficients inserted as multipliers for the microscopic rate constants was unsuccessful. Re-examination of this approach in the context of a diffusion/evaporation model is suggested. The developed models have potential utility for dermal risk assessment and for prediction of aroma evolution following topical application of complex fragrances.

Visualization of the lipid barrier and measurement of lipid pathlength in human stratum corneum.

Detailed models of solute transport through the stratum corneum (SC) require an interpretation of apparent bulk diffusion coefficients in terms of microscopic transport properties. Modern microscopy techniques provide a tool for evaluating one key property-lipid pathway tortuosity-in more detail than previously possible. Microscopic lipid pathway measurements on alkali expanded human SC stained with the lipid-soluble dyes methylene blue, Nile red, and oil red O are described. Brightfield, differential interference contrast, fluorescence, and laser scanning confocal optics were employed to obtain 2-dimensional (2-D) and 3-dimensional (3-D) images. The 2-D techniques clearly outlined the corneocytes. Confocal microscopy using Nile red yielded a well-delineated 3-D structure of expanded SC. Quantitative assessment of the 2-D images from a small number of expanded SC samples led to an average value of 3.7 for the ratio of the shortest lipid-continuous pathway to the width of the membrane. This was corrected for the effect of alkaline expansion to arrive at an average value of 12.7 for the same ratio prior to swelling.

Kinetics of finite dose absorption through skin 1. Vanillylnonanamide.

Despite the considerable success in predicting the steady-state dermal absorption rates of chemical compounds from large reservoirs applied to skin, correspondingly little progress has been made in predicting the absorption rate and extent for small doses of topically applied compounds. In the latter case, steady-state absorption rates are generally not obtained, and rapid evaporation or penetration of the dose solvent makes application of permeability coefficient models problematic. This report presents a new analysis of the finite dose problem in terms of a diffusion model with three parameters-a characteristic time for diffusion, h2/D; a skin solubility factor, S(m)h; and a capacity factor for absorption of the dose during the dry down period, M*. These parameters can be related to the molecular weight and oil and water solubilities of the permeant in a manner similar to models describing steady-state absorption from saturated solutions. Some variation of the parameter values based on the chemical nature and volume of the dose solvent is anticipated. The applicability of the model is demonstrated by analyzing the in vitro absorption rates of varying doses of vanillylnonamide (VN, synthetic capsaicin) applied to excised human skin from propylene glycol. The analysis shows that a three-parameter model that assigns all of the resistance to transport to diffusion through the stratum corneum is able to explain most of the significant features of VN absorption through skin.

A physico-chemical properties based model for estimating evaporation and absorption rates of perfumes from skin.

Because of their potential for inducing allergic contact dermatitis (ACD) if used improperly, perfumes are carefully assessed for dermal safety prior to incorporation into cosmetic products. Exposure assessment for these materials often involves the conservative assumption of 100% absorption of each component. This report describes an improved method to estimate the absorption and evaporation of perfume ingredients from skin, based on their physico-chemical properties. The effect of environmental variables such as temperature and wind velocity can be accounted for in a logical way. This was accomplished using a first-order kinetic approach expected to be applicable for small doses applied to skin. Skin penetration rate was calculated as a fraction of the maximum flux estimated from the compound's lipid solubility, S(lip) (represented by the product of octanol/water partition coefficient, K(octt), and water solubility, S(w)), and molecular weight, MW. Evaporation rates were estimated from a modified Henry's Law approach with a stagnant boundary layer whose thickness is a function of surface airflow, v. At a given value of v, evaporation rate was assumed proportional to the ratio P(vp)/S(lip), where P(vp) is the vapour pressure of the ingredient at skin temperature, T. The model predicts a relationship for total evaporation from skin of the form %evap = 100x/(k+x) where x = P(vp)MW(2.7)/(K(oct)S(w)) and k is a parameter which depends only on v and T. Comparison with published data on perfume evaporation from human skin in vivo showed good agreement between theory and experiment for two closely related perfume mixtures (r(2) = 0.52-0.74, s = 12-14%, n = 10). Thus, the method would seem to have a good prospect of providing skin absorption estimates suitable for use in exposure assessment and improved understanding of dose-related contact allergy.

Percutaneous absorption of vanilloids: in vivo and in vitro studies.

The percutaneous absorption of three highly lipophilic analogs of capsaicin--vanillylnonanamide (VN), olvanil, and NE-21610--was measured in vivo in the CD:VAF rat, and in vitro through excised CD: VAF and SkH:Fz rat skin and human cadaver skin. Absorption and skin metabolism were monitored by radiolabel techniques. The rank order of penetration in all species was VN > olvanil > NE-21610, in accordance with that expected from their physical properties. Rat skin was more permeable than human skin by factors ranging from 4 to 8 for VN, 10 to 20 for olvanil, and approximately 10 to 100 for NE-21610. All three compounds were extensively metabolized during passage through fresh SkH:Fz rat skin, with the primary route of degradation for at least two of the compounds involving hydrolysis of the amide bond (the metabolites of NE-21610 were not identified). For the in vitro studies a range of receptor solutions was employed to determine a set of conditions that best mimicked in vivo absorption. The results with phosphate-buffered saline containing a preservative and 1-6% polyoxyethylene-20 oleyl ether (Oleth-20) were in good agreement with in vivo results for all three compounds for periods up to 24 h post-dose; after this time, in vivo absorption rates declined but in vitro rates remained relatively constant. Buffered saline or saline containing 0.5% bovine serum albumin led to marked underestimates of in vivo penetration for olvanil and NE-21610, whereas a 1:1 ethanol: water solution led to gross overestimates of the in vivo absorption rates for all three compounds.

Improving the sensitivity of in vitro skin penetration experiments.

The institution of a readily-implemented sample screening and data handling procedure for in vitro skin penetration studies yields substantial improvements in sensitivity for distinguishing between formulations, treatments, penetrants, etc. The procedure involves four steps: 1) prescreen the tissue samples to determine their intrinsic permeability; 2) apply treatments using a randomized complete block (RCB) design, with blocking by tissue permeability; 3) apply a variance-stabilizing transformation to the penetration data, followed by outlier testing; and 4) analyze the transformed data according to an RCB analysis of variance, using tissue permeability as the blocking variable. For penetration studies in which high sample variability is a concern, the above procedure commonly yields a sensitivity advantage of several-fold versus alternative methods of comparison.

Vanilloids. 1. Analogs of capsaicin with antinociceptive and antiinflammatory activity.

As part of a program to establish structure-activity relationships for vanilloids, analogs of the pungent principle capsaicin, the alkyl chain portion of the parent structure (and related compounds derived from homovanillic acid) was varied. In antinociceptive and antiinflammatory assays (rat and mouse hot plate and croton oil-inflamed mouse ear), compounds with widely varying alkyl chain structures were active. Short-chain compounds were active by systemic administration in the assays mentioned above but they retained the high pungency and acute toxicity characteristic of capsaicin. In contrast, the long chain cis-unsaturates, NE-19550 (vanillyloleamide) and NE-28345 (oleylhomovanillamide), were orally active, less pungent, and less acutely toxic than capsaicin. The potential of these compounds as antiinflammatory/analgesic agents is discussed in light of recent data on the mechanism of action of vanilloids on sensory nerve fibers.

Retention of etidronate in human, dog, and rat.

The retention of radioactivity in human, rat, and dog following a single injected dose of radiolabeled etidronate disodium (EHDP) is shown to follow power-law decay curves with similar slopes for times up to 4, 60, and 80 days, respectively. During this period retention declines with time according to a weak inverse power of the time since dosing, with an exponent ranging from -0.05 (dog) to -0.09 (human and rat). Direct analyses of dog bones either 90 days after a single dose or 365 days after cessation of chronic dosing indicate a more rapid bone clearance of EHDP than predicted by the initial power law. Direct skeletal analysis also shows a more rapid loss of radioactivity in the rat between 60 and 365 days, indicative of either a second power law or a terminal exponential phase in the retention function occurring after 60 days. These data are used to estimate the minimum and maximum amounts of drug that would remain in the body following long-term treatment in humans. For the intermittent cyclic EHDP treatment (ICT) regimen for osteoporosis (repeated cycles of 14 daily doses of 400 mg orally followed by 76 days drug free), the projected retention of EHDP after 3 years of treatment is 25-50 times the daily absorbed dose. Thus, for a 60 kg woman with a daily absorbed dose of 12 mg, the retained mass of EHDP would be about 300-600 mg.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical analysis of fresh, excised human skin: a comparison with frozen skin.

Samples of human allograft skin prepared without freezing ("fresh skin") were found to have electrical and sodium ion transport properties which differed only slightly from those of skin which had been similarly treated but stored frozen ("frozen skin"). The fresh skin samples were less permeable to sodium ions during passive diffusion and less conductive than frozen skin at low current levels. They were more permselective for sodium versus chloride during constant-current iontophoresis and showed slightly more asymmetry in their current-voltage properties. Overall, the electrical behavior of the two tissues was similar enough to support the use of frozen tissue in iontophoresis studies. However, caution should be exercised when considering the use of frozen skin for applications, such as those based on electroosmosis, where the observed differences could have a major impact on the results.

DC electrical properties of frozen, excised human skin.

DC current-voltage relationships and sodium ion transport measurements for human allograft skin immersed in saline buffers have been determined using a four terminal potentiometric method and diffusion cells of our own design. About three-fourths of the skin samples were deemed suitable for study on the basis of their high resistivities and similar j-V characteristics. Most of these samples yielded sodium ion permeability coefficients less than or equal to those reported for human skin in vivo. The current-voltage relationship in these tissues was time dependent, highly nonlinear, and slightly asymmetric with respect to the sign of the applied potential. Skin resistance decreased as current or voltage increased. For current densities less than 15 microA/cm2 and exposure times of 10-20 min, this decrease was almost completely reversible; at higher current densities, both reversible and irreversible effects were observed. The overall dependence of current on voltage was nearly exponential and was satisfactorily described by an equation of the form j approximately sinh V. Diffusion potentials, sodium ion membrane transference numbers, and sodium ion flux enhancement factors during iontophoresis were measured for skin immersed both in normal saline solutions and in saline solutions of differing concentrations. The sign of the diffusion potentials and the value of the sodium ion transference number (0.51 in normal saline at pH 7.4) indicated a weak permselectivity of the skin for transport of sodium ion versus chloride. At a current density of 71 microA/cm2 and transmembrane potentials in the range of 1.1-1.6 V, the flux enhancement for sodium ion was three to five times greater than that predicted for an uncharged homogeneous membrane according to electrodiffusion theory. For transmembrane potentials less than 0.17 V, agreement of this theory with the data was better but still incomplete.

Application of liquid chromatography with on-line radiochemical detection to metabolism studies on a novel class of analgesics.

Vanilloids are a class of compounds structurally related to capsaicin, the pungent principle of hot peppers, which are under development as a novel class of analgesics. Vanilloids undergo extensive first-pass metabolism when dosed orally to rats and mice. These compounds, as well as capsaicin, would be anticipated to be susceptible to three major routes of metabolism: (omega, beta)-oxidation of the alkyl side chain, hydrolysis of the amide bond and conjugation of the phenolic group. Olvanil [N-(3-methoxy-4-hydroxybenzyl)oleamide], radiolabelled with either 14C at the benzylic carbon or 3H in the oleyl side chain, was studied in various in vitro, in situ and in vivo metabolism models to determine the major route(s) of intestinal and hepatic metabolism in rats for this new class of compounds. Models used in metabolism studies included isolated hydrolytic enzymes, cell-free intestinal and liver supernatants, hepatocytes, enterocytes, perfused intestine and whole animal studies. Reversed-phase liquid chromatography (LC) with on-line radiochemical detection was used to examine the metabolic profiles from the different models. The major metabolic route for olvanil in both the intestine and the liver was found to be hydrolysis of the amide bond. The benefits of selective 14C and 3H labels in conjunction with LC with on-line radiochemical detection are discussed.

A nonparametric method for evaluating results from laboratory antinociceptive tests.

A method is presented in which Cox's proportional hazards model, a survival analysis technique, is used to assess the results of hot-plate antinociceptive testing. The method appropriately handles censored data values and variable pretest latency times without making arbitrary assumptions about the distribution of the data. It may be used to characterize and compare dose-response curves or to examine the effect of agent or other treatment variables on the response. The technique is easily implemented using the SAS statistical software package. Due to the similar way in which data is obtained, we believe the method to be applicable to several other laboratory models of pain, including the tail-flick, tail-immersion, and paw-pressure (Randall-Selitto) assays.