Non-invasive raman spectroscopic detection of carotenoids in human skin.

Carotenoids are thought to play a significant part in the skin's anti-oxidant defense system, and may help prevent malignancy. Inability to measure skin carotenoid content readily has, however, made it difficult to establish the relationship between carotenoid concentration and the occurrence of cutaneous malignancy. We have measured in vivo carotenoid concentration using a noninvasive optical method, Raman spectroscopy. To validate our instrumentation, abdominoplasty skin was evaluated by both Raman spectroscopy and high-performance liquid chromatography determination for carotenoid content. Evaluation of the Raman signal in specific carotenoid solutions was also performed. Precision of Raman measurements within skin sites, within subjects, and between subjects was measured. Sensitivity of the method was evaluated as a function of anatomical region and the distribution of carotenoids within the stratum corneum. Lastly, we evaluated the Raman signal in actinic keratosis and basal cell carcinoma lesions and perilesional skin and compared this with region-matched sites in healthy subjects. Our results indicate that the Raman scattering method reflects the presence of carotenoids in human skin and is highly reproducible. Evaluation of five anatomical regions demonstrated significant differences in carotenoid concentration by body region with the highest carotenoid concentration noted in the palm. Comparison of carotenoid concentrations in basal cell carcinomas, actinic keratosis, and their perilesional skin demonstrate a significantly lower carotenoid concentration than in region-matched skin of healthy subjects. These results represent the first evidence that carotenoid concentration in the skin correlate with the presence or absence of skin cancer and precancerous lesions.

Development of in vivo bioequivalence methodology for dermatologic corticosteroids based on pharmacodynamic modeling.

BACKGROUND: Dermatologic corticosteroid products produce skin blanching that is related to clinical potency and dose. (For application of the vasoconstrictor assay to bioavailability and bioequivalence assessment, dose is defined in terms of duration of treatment exposure [dose duration], so the terms dose and dose duration have been used interchangeably). The vasoconstrictor assay is the method of choice to assess dermatologic corticosteroid products bioequivalence if dose-response is validated. This article examines dose-response validation to meet objectives of US Food and Drug Administration (FDA) bioequivalence guidance for dermatologic corticosteroid products. METHODS: An exploratory dose-response study was conducted to determine applicability of the empirical maximum effect (Emax) model to the individual subject and population dose-response relationships of six dermatologic corticosteroid product creams that varied from the most to the least potent classes. Products were applied to the skin of 10 healthy subjects in each of two dosing periods for dose durations of 0.5, 1, 2, and 6 hours. Skin blanching was measured by reflectance colorimeter through 24 hours after application. Area under the effect curve (AUEC) was determined for each dose duration. An Emax model was fitted to the AUEC versus dose duration data. A similar analysis was conducted for a bioequivalence study on two formulations of a dermatologic corticosteroid product in 40 healthy subjects. RESULTS: In the exploratory study, the number of individual subject data sets for which the Emax model provided an acceptable fit generally increased with the potency of the dermatologic corticosteroid product. On the basis of population modeling, dose-response data of all products, except the lowest potency cream, were adequately described by the Emax model. Values for population ED50 (the dose duration required to achieve 50% of the fitted AUECmax value) decreased with increase in dermatologic corticosteroid product potency. CONCLUSIONS: Acceptable model fits to all individual subject dose-response data were not achieved for any dermatologic corticosteroid product. However, population dose-responses were adequately described by the Emax model. On the basis of these data, the optimal dose duration used for comparison of multisource dermatologic corticosteroid products is recommended to be equal to the ED50 based on population modeling of pilot dose-response study data.

Differentiation of involved and uninvolved psoriatic skin from healthy skin using noninvasive visual, colorimeter and evaporimeter methods.

BACKGROUND/AIMS: Uninvolved skin of psoriasis may not be entirely normal. The object was to characterize healthy, uninvolved psoriatic skin and lesional skin by biophysical methods. METHODS: Involved and uninvolved psoriatic and age-gender matched healthy skin was measured objectively with a colorimeter and evaporimeter and subjectively with visual assessment in 14 subjects. METHODS: Visual assessment of erythema (E), scaling (S) and induration (I) as well as the target lesion score at the involved psoriatic skin sites were significantly elevated (p<0.05) above uninvolved psoriatic or healthy skin sites. No difference between uninvolved psoriatic and healthy skin was measured visually. Transepidermal water loss at involved psoriatic skin >uninvolved psoriatic skin >healthy skin (p<0.05). Objective assessment of skin color in 3 color scales, L*, a*, and b*, differentiated involved and uninvolved psoriatic skin from healthy skin sites. Involved psoriatic skin demonstrated higher (p<0.01) a-scale values and lower (p<0.01) L* and b* scale values than uninvolved psoriatic skin. Further, colorimeter L* and a* scale values at uninvolved psoriatic skin sites were lower and higher (p<0.05), respectively, than healthy skin. The individual chromameter parameters (L*, a*, b*) correlated well with the visual parameters (E, S and I). Composite colorimeter description (L*× b*)/a* significantly differentiated healthy skin from both involved and uninvolved psoriatic skin. CONCLUSIONS: These collective data highlight that even visually appearing uninvolved psoriatic skin is compromised compared with healthy skin. These objective, noninvasive but differential capabilities of the colorimeter and evaporimeter will aid in the mechanistic quantification of new psoriatic drug therapies and in conjuction with biochemical studies, add to understanding of the multifactorial pathogenesis of psoriasis.

Topical 0.050% betamethasone dipropionate. Pharmacokinetic and pharmacodynamic dose-response studies in humans.

BACKGROUND AND DESIGN: Effective topical drug therapy requires drug delivery into the skin to produce the desired pharmacodynamic response. For topical corticosteroids, the visual skin-blanching assay has been used to rank the potency of the corticosteroids and their overall efficacy. While vehicles have been shown to influence the resulting blanching response, the dose of drug applied has not always produced proportional differences in the blanching assay. The mechanism of the nonproportional pharmacodynamic response to the corticosteroid dose is unclear. We describe four methods for assessing the dose-response relationship of topical betamethasone dipropionate on the ventral forearm of six human subjects: duration, concentration, film thickness, and surface area. Drug uptake analysis in human stratum corneum and the resulting pharmacodynamic response, measured visually and with a chromameter, were performed with each method to quantify the dose-response relationship. RESULTS: Only the concentration and duration methods demonstrated an increase in mean drug uptake with increasing dose. The maximal mean pharmacodynamic response reflected the mean drug uptake with all four methods. Application conditions for maximal pharmacodynamic activity of topical betamethasone dipropionate in the skin include short duration of treatment (< or = 2 hours), a lower concentration than commercially marketed, and thin film thicknesses (1 to 5 microns). CONCLUSION: A dose response can be produced by increasing the drug concentration or the duration of application time. Achievement of steady-state betamethasone dipropionate uptake into the stratum corneum was not commensurate with the maximal pharmacodynamic response. Very small amounts of this potent corticosteroid within the skin appear to maximize the receptor response to drug.

Circadian activity of topical 0.05% betamethasone dipropionate in human skin in vivo.

The influence of treatment duration, vehicle, and time of day of application on topical 0.05% betamethasone dipropionate uptake into human stratum corneum and the resulting skin-blanching response was investigated in human subjects. Drug uptake into stratum corneum and the resulting skin color changes measured with a chromameter demonstrate an equilibrium delay. Maximal drug uptake occurred at 2 h, whereas maximal skin color changes occurred 6 h after a single application. Extent of decreased skin color was dependent on vehicle, treatment duration, and time of day of application. Time of maximal decreased skin color occurred at midnight independent of vehicle, treatment duration, or time of day of application. This time of maximal drug activity coincides with the well-known time period of lowest circulating cortisol concentrations (2000-0400 h). Application of a single 2- or 6-h dose of the 0.05% cream at 1600 h produced more extensive and prolonged changes in skin color over 24 h than a 0900-h application in the same subject. These data demonstrate that the extent and duration of topical corticosteroid activity in human skin is influenced by vehicle, treatment duration, and time of day of application. The prolonged changes in skin color measured with a single dose applied at 1600 h suggest that a once-a-day dosing regimen in the late afternoon may be sufficient for dermatologic therapy. Elucidation of these circadian responses with topical corticosteroids may provide a rational basis for the future re-evaluation of the appropriate therapeutic regimen with this class of drugs in dermatologic medicine.

In vivo pharmacokinetics and pharmacodynamics of topical ketoconazole and miconazole in human stratum corneum.

A direct study evaluating whether differential drug uptake of topical 2% miconazole and 2% ketoconazole from cream formulations into human stratum corneum correlated with differential pharmacological activity against Candida albicans was investigated in healthy human subjects. A single 24-h topical dose of 2% ketoconazole cream or 2% miconazole cream was applied unoccluded, at the same dose (2.6 mg of formulation per cm2 of surface area), at four skin sites on both ventral forearms of six human subjects. At the end of the treatment, residual drug was removed with a tissue from all sites and the treated site was tape stripped 11 times, either 1, 4, 8, or 24 h later. The first tape disc was discarded. The remaining tape discs, 2 through 11, were combined and extracted for drug quantification by high-performance liquid chromatography and bioactivity against C. albicans growth in vitro. Topical 2% ketoconazole produced 14-, 10-, and 7-fold greater drug concentrations in stratum corneum than 2% miconazole at 1, 4, and 8 h after a single topical dose. Ketoconazole and miconazole concentrations in the stratum corneum were similar 24 h after drug removal. Tape disc extracts from 2% ketoconazole-treated skin sites demonstrated significantly greater bioactivity in the bioassay than 2% miconazole. The increased efficacy of 2% ketoconazole compared with that of 2% miconazole in vitro reflects their differential uptake into the stratum corneum and inherent pharmacological activity. Tape stripping the drug-treated site in conjunction with a bioassay is therefore a useful approach in the determination of bioavailability of topical antifungal agents.

Disparity of in vitro and in vivo oleic acid-enhanced beta-estradiol percutaneous absorption across human skin.

The permeation enhancing property of 5% oleic acid in ethanol on beta-estradiol was investigated in vitro and in vivo using symmetrical and asymmetrical side-by-side diffusion cells and the human skin sandwich flap, respectively. beta-Estradiol permeability in vitro and in vivo was similar in 75% ethanol (ETOH). Oleic acid (5%) did not alter beta-estradiol permeability in vivo but increased permeability six-fold in vitro in symmetrical diffusion cells. beta-Estradiol permeability in oleic acid was not different from that in ETOH, however, using asymmetrical diffusion cells. Stratum corneum-to-vehicle partition coefficients of beta-estradiol in the vehicles were similar, yet fourfold more steroid was detected in skin biopsies from the in vitro symmetrical diffusion cells. Thus, oleic acid increased beta-estradiol permeability in vitro only when skin was equilibrated with fatty acid. Attention to in vitro diffusion cell design and its relevance in vivo is critical to defining the mechanisms of enhanced solute permeation.

New approaches to assess topical corticosteroid bioequivalence: pharmacokinetic evaluation.

An ideal method for measuring the bioavailability of topical corticosteroids should be simple, accurate, and adaptable to a variety of settings and should not require extensive special training to perform. Drug uptake into the stratum corneum, measured by tapestripping, is correlated with the pharmacodynamic response of skin blanching, observed in the vasoconstrictor assay. Differences in stratum corneum drug uptake can be objectively quantitated as a function of time, occlusion, dose applied, and vehicle. Tapestripping measurements are reproducible within individual subjects, but large interindividual variabilities may exist. The chromameter, a new technology, objectively quantitates color numerically and can be used to measure skin blanching as part of the pharmacodynamic response to topical corticosteroids. The chromameter offers an easy, objective method with which to quantitate the pharmacodynamic response of topical corticosteroids. Both methods allow a more mechanistic approach than currently used methods to investigate topical drug pharmacokinetics and pharmacodynamics.

Acyclovir bioavailability in human skin.

Clinical experience demonstrates that oral acyclovir (ACV) is superior to topical ACV in treating recurrent cutaneous herpes simplex virus type 1 (HSV-1) infections. Cutaneous HSV-1 infections are complex in their pathology, affecting the basal epidermis in skin as well as establishing a latency phase in sensory ganglia. In vitro and in vivo human skin model systems were used in the present study to quantitate ACV disposition and absorption in skin and blood following two routes of administration and to investigate whether bioavailability differences were the result of insufficient drug delivery. Physiochemical and physiologic parameters determined from these experiments were used to develop a mathematical model to predict ACV disposition and absorption in human subjects. Model predictions and in vivo data agree; topical administration of commercial 5% ACV ointment and cream result in a 48 times greater total epidermal ACV concentration than after oral administration. Mathematical modeling of the ACV concentration gradient through the epidermis revealed, however, that the drug concentration in the target site of HSV-1 infections, the basal epidermis, is 2-3 times less after topical administration than after oral administration. Thus, the observed lack of clinical efficacy with topical ACV therapy in the recurring HSV-1 infection likely reflects the insufficient delivery of the drug to the target site of the HSV-1 infection, the basal epidermis.

Feasibility of measuring the bioavailability of topical betamethasone dipropionate in commercial formulations using drug content in skin and a skin blanching bioassay.

An in vivo technique has been developed which simultaneously compares a skin blanching bioassay with drug content in human stratum corneum following topical application of four 0.05% betamethasone dipropionate formulations. Bioavailability of drug from commercial cream and ointment formulations was assessed by quantification of drug content in tape-stripped stratum corneum and skin blanching in the treated skin site under occluded conditions. Tape-stripping removed stratum corneum to a varying degree between individuals but was consistent (35%) within an individual with all formulations, day to day. A correlation (r = 0.9935) between the amount of drug in the treated stratum corneum normalized for surface area and the corresponding skin blanching score was observed with four 0.05% betamethasone dipropionate formulations. Increasing the amount of drug in the tape-stripped stratum corneum correlated with an increased skin blanching score. Ointment formulations delivered more drug to the skin and produced greater blanching scores than the cream formulations. Topical corticosteroid content in the treated skin site can therefore be quantified and correlates well with the resulting pharmacodynamic activity.

Percutaneous absorption of benzoic acid across human skin. II. Prediction of an in vivo, skin-flap system using in vitro parameters.

The possibility of predicting the behavior of in vivo systems based on physical and chemical parameters determined by in vitro experiments is examined using benzoic acid. The physical and chemical parameters governing percutaneous absorption of benzoic acid--permeability, partition coefficient, and skin thickness--were determined by in vitro experiments as described in Ref. 1. These parameters were used, in combination with benzoic acid elimination kinetics, to predict the results of in vivo experiments using a comprehensive mathematical model. The in vivo system consists of a congenitally athymic (nude) rat with a surgically constructed human skin sandwich (HSSF) flap on which a donor cell is placed. To apply the in vitro parameters to an in vivo system requires a suitable pharmacokinetic model describing distribution and elimination for benzoic acid in the nude rat. Blood concentrations of benzoic acid following a bolus intravenous injection are closely described by a two-compartment open pharmacokinetic model with elimination occurring from only one compartment. The mathematical model of the rat-donor cell system combines this two-compartment model of the rat with a percutaneous absorption model to provide useful estimates of the measured in vivo blood levels. Comparisons of predicted and measured results suggest that the parameters determined by in vitro experimentation can be used to predict the behavior of complex in vivo systems, if a suitable mathematical model is available.

Percutaneous absorption of benzoic acid across human skin. I. In vitro experiments and mathematical modeling.

The percutaneous absorption of benzoic acid across human skin in vitro was experimentally and mathematically modeled. Skin partition coefficients were measured over a range of benzoic acid concentrations in both saline and distilled water. The permeation of benzoic acid was measured across isolated stratum corneum, stratum corneum and epidermis, and split-thickness skin. These experiments demonstrated that the stratum corneum was the rate-limiting barrier and that the flux is proportional to the concentration of the undissociated species. The permeation data were analyzed with a comprehensive non-steady-state mathematical model of diffusion across skin. Two adjustable parameters, the effective skin thickness and diffusivity, were fit to the permeation data by nonlinear regression.

Mechanism of ethanol-enhanced estradiol permeation across human skin in vivo.

The influence of ethanol on the permeation of 17 beta-estradiol (estradiol) across viable human skin in vivo was investigated with the human skin sandwich flap model. Maintaining continuous delivery of a constant concentration of the solute in phosphate-buffered saline, pH 7.4 (PBS), or mixtures of ethanol in PBS to the skin surface revealed that steady-state flux of estradiol was achieved within 30-60 min and maintained throughout 4 hr. The 10-fold decrease in in vivo flux and permeability coefficient (Kp) of tracer estradiol solutions in ethanol or ethanol solutions compared with PBS vehicle reflected the 10-fold difference in the apparent partition coefficients (Km) of estradiol from the respective vehicles into isolated human stratum corneum. Neither the stratum corneum thickness nor the diffusion coefficient of estradiol was significantly different among the vehicles tested. In vivo flux of estradiol in ethanol or ethanol solutions across viable human skin was increased with saturated solutions of estradiol. Further, in vivo flux of estradiol from vehicles such as PBS, ethanol, and ethanol mixtures, which minimally alter the rate-limiting barrier, can be successfully predicted with knowledge of only two physicochemical parameters, the estradiol concentration in the vehicle and the Km of estradiol from the vehicle into isolated human stratum corneum.

Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for epidermal function and gene therapy.

In the present study, human apolipoprotein E (apoE) was monitored in the circulation of athymic mice and rats bearing human epidermal grafts. Human apoE was detected in the systemic circulation of graft-bearing animals as long as the graft remained on the animal. Within 24 hr of graft removal, human apoE was not detectable in plasma, indicating that apoE resulted from continuous production of the protein by grafted keratinocytes. These results show that proteins as large as apoE (299 amino acids) traverse the epidermal-dermal barrier and achieve systemic distribution where they may produce effects on distal tissues. The feasibility of using grafts of genetically-altered keratinocytes for the delivery of secreted proteins is clearly reinforced by the demonstration that an epidermally derived protein exhibits systemic distribution. Finally, by virtue of its systemic distribution, apoE produced in a peripheral tissue such as skin, may function in the reverse transport of cholesterol from peripheral tissues to the liver.

Cutaneous blood flow and percutaneous absorption: a quantitative analysis using a laser Doppler velocimeter and a blood flow meter.

Cutaneous blood flow has been directly quantitated in vivo for the first time without animal death utilizing the rat skin sandwich flap. This was accomplished by conducting experiments that made a direct correlation between two instruments: a laser Doppler velocimeter and an electromagnetic blood flow meter. Data demonstrate that the correlation between these two instruments is high and reproducible (r = 0.96) with a small (1.3%) coefficient of variation. Blood flow to skin in the unmanipulated state varies from 0.7 to 1.2 mls/min in an anesthetized rat. Application of the blood flow correlation to the determination of percutaneous absorption of caffeine across human skin and benzoic acid across rat skin demonstrates that assuming cutaneous blood flow is a particular value day to day in any skin type results in an apparent wide range of total compound absorbed across that skin on independent occasions. Utilizing actual blood flow measurements to calculate the amount of chemical absorbed reduces the range of variability in the total amount of chemical absorbed and provides a more accurate knowledge of events occurring during a particular time of the absorption process. Quantitation of cutaneous blood flow will be useful in physiologic and pharmacologic studies where actual cutaneous blood flow is likely to be important to the processes studied, e.g., delivery of drug to skin, metabolism within the skin, and disposition of drug to blood and skin following topical drug application.

An experimental skin sandwich flap on an independent vascular supply for the study of percutaneous absorption.

Further insights into the composite interactive processes of topically applied agents and percutaneous absorption and metabolism by functional skin in vivo have been hampered by the lack of a model system wherein the blood flow to and from the skin is independent but experimentally accessible. Utilizing microsurgical techniques, split-thickness skin grafting with syngeneic skin grafts, and the congenitally athymic (nude) rat, a skin sandwich flap system has been generated that has an independent but accessible vasculature and thus fills this void. We describe the methodology that has been developed to create the flap and present experiments that: demonstrate a lack of significant collateral circulation; quantify the microcirculation of the skin sandwich flap, host side, and graft side at various times during and after the flap has been generated, and note that blood flow to the flap is basically unchanged from host skin; demonstrate the utility of the system in measuring the amount of [14C]benzoic acid that appears in the flap when deposited on the surface in volatile and nonvolatile vehicles as a function of time; and demonstrate the fact that the flap can be reused, and that the total amount of [14C]benzoic acid absorbed across skin does not change in a substantial way as the flap ages.

The development of a rat/human skin flap served by a defined and accessible vasculature on a congenitally athymic (nude) rat.

Experience in microvascular surgery on rats and availability of athymic (nude) rats led us to believe that a long-term functional rat/human skin sandwich flap could be generated on a defined and experimentally accessible vasculature on nude rats. Such a system has been developed and validated. Microvasculature has been assessed. The volume of blood to the flap ranges from 1 to 2 ml/min, collateral circulation to the flap exists, but is negligible, and there is little change in the capillary blood flow as the flap ages. The flap can be utilized to study absorption of compounds from a half-cell diffusion chamber or from direct deposition on the skin, and can be utilized to study various parameters of percutaneous absorption, e.g., the effect of hydration on the stratum corneum. Transdermal flux can be determined. Altering the microcirculation directly affects the percutaneous absorption of compounds that are rapidly absorbed. The absorption of benzoic acid through an experimentally vasoconstricted area (iontophoresis of phenylephrine) significantly alters the time to peak absorption, with values being 14 times that of the control site. The system has been utilized to assess metabolic activity of skin in situ using [3H]adenine arabinoside and studying the appearance of its major metabolite, [3H]Ara-H, in flap blood, as well as the back diffusion of this compound into the donor chamber. Recently the human/rat skin sandwich flap component has been developed. With this system, it has been demonstrated that benzoic acid, when applied to the human skin component of the flap has an absorption profile which is quite different from that when benzoic acid is applied to rat skin, peak flux occurred 2 hr after application. This contrasts with 10 min to peak flux when the same experiment is carried out on the rat/rat skin sandwich flap. To our knowledge, the human/rat skin sandwich flap is the first example of a viable, functional human organ that is chronically maintained by a biologic support system which has the added distinction of being on an independent but accessible vasculature. The validation experiments strongly suggest that this system will be important in gaining insights into the more sophisticated in vivo components of skin, relative to toxicology and pharmacology.