Stratum Corneum Concentrations Research Articles

Carotenoids in Human SkinIn Vivo: Antioxidant and Photo-Protectant Role against External and Internal Stressors.

The antioxidant system of the human body plays a crucial role in maintaining redox homeostasis and has an important protective function. Carotenoids have pronounced antioxidant properties in the neutralization of free radicals. In human skin, carotenoids have a high concentration in the stratum corneum (SC)—the horny outermost layer of the epidermis, where they accumulate within lipid lamellae. Resonance Raman spectroscopy and diffuse reflectance spectroscopy are optical methods that are used to non-invasively determine the carotenoid concentration in the human SC in vivo. It was shown by electron paramagnetic resonance spectroscopy that carotenoids support the entire antioxidant status of the human SC in vivo by neutralizing free radicals and thus, counteracting the development of oxidative stress. This review is devoted to assembling the kinetics of the carotenoids in the human SC in vivo using non-invasive optical and spectroscopic methods. Factors contributing to the changes of the carotenoid concentration in the human SC and their influence on the antioxidant status of the SC in vivo are summarized. The effect of chemotherapy on the carotenoid concentration of the SC in cancer patients is presented. A potential antioxidant-based pathomechanism of chemotherapy-induced hand-foot syndrome and a method to reduce its frequency and severity are discussed.

Skin Pharmacokinetics of Transdermal Scopolamine: Measurements and Modeling.

Prediction of skin absorption and local bioavailability from topical formulations remains a difficult task. An important challenge in forecasting topical bioavailability is the limited information available about local and systemic drug concentrations post application of topical drug products. Commercially available transdermal patches, such as Scopoderm (Novartis Consumer Health UK), offer an opportunity to test these experimental approaches as systemic pharmacokinetic data are available with which to validate a predictive model. The long-term research aim, therefore, is to develop a physiologically based pharmacokinetic model (PBPK) to predict the dermal absorption and disposition of actives included in complex dermatological products. This work explored whether in vitro release and skin permeation tests (IVRT and IVPT, respectively), and in vitro and in vivo stratum corneum (SC) and viable tissue (VT) sampling data, can provide a satisfactory description of drug "input rate" into the skin and subsequently into the systemic circulation. In vitro release and skin permeation results for scopolamine were consistent with the previously reported performance of the commercial patch investigated. New skin sampling data on the dermatopharmacokinetics (DPK) of scopolamine also accurately reflected the rapid delivery of a "priming" dose from the patch adhesive, superimposed on a slower, rate-controlled input from the drug reservoir. The scopolamine concentration versus time profiles in SC and VT skin compartments, in vitro and in vivo, taken together with IVRT release and IVPT penetration kinetics, reflect the input rate and drug delivery specifications of the Scopoderm transdermal patch and reveal the importance of skin binding with respect to local drug disposition. Further data analysis and skin PK modeling are indicated to further refine and develop the approach outlined.

In vivo Tracking of DNA for Precise Determination of the Stratum Corneum Thickness and Superficial Microbiome Using Confocal Raman Microscopy

The skin barrier function is mostly provided by the stratum corneum (SC), the uppermost layer of the epidermis. To noninvasively analyze the physiological properties of the skin barrier functionin vivo, it is important to determine the SC thickness. Confocal Raman microscopy (CRM) is widely used for this task. In the present in vivo study, a new method based on the determination of the DNA concentration profile using CRM is introduced for determining the SC thickness. The obtained SC thickness values are compared with those obtained using other CRM-based methods determining the water and lipid depth profiles. The obtained results show almost no significant differences in SC thickness for the utilized methods. Therefore, the results indicate that it is possible to calculate the SC thickness by using the DNA profile in the fingerprint region, which is comparable with the SC thickness calculated by the water depth profiles (ANOVA test p = 0.77) and the lipid depth profile (ANOVA test p = 0.74). This provides the possibility to measure the SC thickness by using the DNA profile, in case the water or lipid profile analyses are influenced by a topically applied formulation. The increase in DNA concentration in the superficial SC (0–2 µm) is related to the DNA presence in the microbiome of the skin, which was not present in the SC depth below 4 µm.

Progesterone lipid nanoparticles: Scaling up and in vivo human study.

This investigation describes a scaling up study aimed at producing progesterone containing nanoparticles in a pilot scale. Particularly hot homogenization techniques based on ultrasound homogenization or high pressure homogenization have been employed to produce lipid nanoparticles constituted of tristearin or tristearin in association with caprylic-capric triglyceride. It was found that the high pressure homogenization method enabled to obtain nanoparticles without agglomerates and smaller mean diameters with respect to ultrasound homogenization method. X-ray characterization suggested a lamellar structural organization of both type of nanoparticles. Progesterone encapsulation efficiency was almost 100% in the case of high pressure homogenization method. Shelf life study indicated a double fold stability of progesterone when encapsulated in nanoparticles produced by the high pressure homogenization method. Dialysis and Franz cell methods were performed to mimic subcutaneous and skin administration. Nanoparticles constituted of tristearin in mixture with caprylic/capric triglyceride display a slower release of progesterone with respect to nanoparticles constituted of pure tristearin. Franz cell evidenced a higher progesterone skin uptake in the case of pure tristearin nanoparticles. A human in vivo study, based on tape stripping, was conducted to investigate the performance of nanoparticles as progesterone skin delivery systems. Tape stripping results indicated a decrease of progesterone concentration in stratum corneum within six hours, suggesting an interaction between nanoparticle material and skin lipids.

Effect of potassium present in stratum corneum during non-invasive measurement of potassium in human subjects using reverse iontophoresis.

Reverse iontophoresis (RI) is one of the potential techniques used to monitor the concentration of various analytes in body fluids non-invasively. Transdermal extraction of potassium is investigated using RI. In the present work, the effect of potassium on stratum corneum (SC) during RI, feasibility of RI for continuous monitoring of potassium, and use of potassium as internal standard in RI, are investigated. Tape stripping experiment is carried out to find potassium concentration in SC. RI is carried out continuously for 180 min without passive diffusion and after passive diffusion for 60 min. Skin impedance measurements are done at 20 Hz and 20 kHz. Potassium is found to be in the range 300-650 nmol/cm(2) on SC by tape stripping experiment. Correlation coefficient between blood potassium and extracted potassium through RI after passive diffusion (R(2) = 0.5870) is more than without passive diffusion (R(2) = 0.5117). The skin impedance measurement shows that RI has more effect on SC than superficial layer of SC during RI. The present investigations conclude that it is possible to monitor potassium continuously through RI and using potassium as internal standard in RI.

Possibilities for human skin characterization based on strongly reduced Raman spectroscopic information

Loss‐of‐function mutations in the gene coding for filaggrin are the single most important risk factor for development of atopic dermatitis and associated allergic rhinitis and asthma. Filaggrin is enzymatically degraded to natural moisturizing factor (NMF) in the stratum corneum (SC). In vivo Raman spectra of human skin can be used to quantify the NMF concentration in SC and thereby identify carriers of a loss‐of‐function mutation in the gene coding for filaggrin, which results in decreased NMF content. Here, we demonstrate that strongly reduced Raman spectral information is sufficient to make this differentiation. This is an important step towards development of a dedicated diagnostic device of reduced complexity, size and cost as compared to current state‐of‐the‐art Raman equipment. A genetic algorithm was used to select the spectral regions needed to classify skin based on normal or reduced NMF content in SC. Using the NMF content based on full spectral information as gold standard, only four Raman regions were required to create a linear discriminant analysis model that can differentiate between skin with low NMF and skin with normal NMF with a prediction accuracy of 93 %. Copyright © 2013 John Wiley & Sons, Ltd.

Cutaneous pharmacokinetics of topically applied maxacalcitol ointment and lotion

Maxacalcitol (22-oxacalcitriol), a vitamin D3 analogue, is widely used for the treatment of psoriasis in Japan. The effects of topically applied dermatologic preparations have routinely been assessed by their pharmacodynamic profiles and their concentrations in the skin correlate well with these profiles. Recently, a maxacalcitol lotion formulation (M514102) was developed for the treatment of psoriatic lesions on the face and scalp. To predict the clinical efficacy of the lotion, we investigated the cutaneous bioavailability of topically applied lotion and compared this with that of its ointment formulation in healthy subjects. In the first study, 12 subjects were divided into two groups of 6 each and were treated with the ointment or lotion. Six drug application sites were randomly selected on the left volar forearm. After 0, 2, 4, 6, 8 and 10 h, the formulations were gently removed and tape stripping was performed. Maxacalcitol was extracted from the tape strips and quantified by liquid chromatographic tandem mass spectrometry. In the second study, four drug application sites were randomly selected on the left volar forearm in the 12 subjects. The ointment was applied and spread over two sites and the lotion was applied in the same manner over the remaining two sites. After 8 h, the preparations were gently removed and followed by tape stripping. The average concentrations of maxacalcitol in the stratum corneum (SC) at 2, 4, 6, 8 and 10 h after application were 6.9 +/- 3.3, 12.8 +/- 6.2, 11.8 +/- 4.6, 13.1 +/- 5.2 and 12.3 +/- 3.1 microg/g for the ointment and 3.1 +/- 1.0, 9.1 +/- 3.1, 13.9 +/- 3.4, 13.1 +/- 4.1 and 15.5 +/- 3.1 microg/g for the lotion, respectively. A steady state was observed at approximately 4 and 6 h after application of the ointment and lotion, respectively. In the second study, there was no significant difference between the average of the SC concentrations of the ointment and lotion at 8 h. In conclusion, we observed that assessment of cutaneous bioavailability by using tape stripping was reproducible. Accordingly, the cutaneous bioavailability of the lotion was comparable to that of its ointment. Hence, treatment with the lotion is expected to be as effective as that with the ointment.

Pharmacokinetic investigation of oral itraconazole in stratum corneum level of tinea pedis.

In the present study, the authors administered 100 mg itraconazole (ITCZ) twice daily for a period of 1 week to six patients with hyperkeratotic type tinea pedis, and examined its efficacy, safety profile, and usefulness. ITCZ concentration in stratum corneum was also measured to examine the mobility of the drug into the affected site of planta pedis. ITCZ concentration in the stratum corneum of the affected part was first detected at 1 week after the completion of administration, gradually increased over time, and peaked at 3 weeks, with the sum of ITCZ and hydroxyitraconazole (OH-ITCZ) amounting to 163.7 ng g(-1) on a average. It then gradually decreased to a total sum of 10.3 ng g(-1) on average at 8 weeks following the completion of administration. When compared with the geometric mean minimum inhibitory concentration (MIC) of ITCZ against fresh clinical isolates of dermatophytes (Trichophyton rubrum) (0.06 microg ml(-1)), the stratum corneum ITCZ concentration in this study was 2.1-fold of the geometric mean MIC at 2 weeks following the completion of administration, and 2.4-fold at 4 weeks. Although ITCZ does not produce therapeutic effectiveness (fungistasis) during the period of administration, it starts appearing at 2 weeks after the completion of administration, and after it peaks out at 3-4 weeks, clinical symptoms started improving. These results suggest that satisfying effects can be achieved in a short-term oral ITCZ at a dose of 100 mg twice daily for a period of 1 week in cases of hyperkeratotic type tinea pedis.

Piroxicam delivery into human stratum corneum in vivo: iontophoresis versus passive diffusion

A nonsteroidal anti-inflammatory drug, piroxicam, was administered from a commercially available gel to human volunteers both passively and under the application of an iontophoretic current. The effect of occlusion on the passive delivery of piroxicam was also examined in a separate series of experiments. After treatment, the stratum corneum (SC) at the site of application was progressively tape-stripped and piroxicam transport into the membrane was assessed by UV-analysis of drug extracted from the tape-strips. Analysis of variance did not show any significant difference between passive piroxicam delivery after 30, 60 or 125 min. However, current application enhanced drug uptake into the SC, as indicated by both increased piroxicam concentrations in the horny layer and detectable concentrations at greater depths into the membrane. The total amount of drug recovered in the SC post-iontophoresis was significantly higher than that found following passive diffusion for each application time. The amounts of drug recovered from the tapes after 60 and 125 min of current application were significantly higher than that after 30 min treatment. Finally, the in vivo SC concentration profiles following passive delivery were fitted to the appropriate solution of Fick’s second law of diffusion to determine skin partitioning and diffusivity parameters.

In vivo assessment of enhanced topical delivery of terbinafine to human stratum corneum

Purpose: The objective of this study was to evaluate, using attenuated total reflectance Fourier transform infrared spectroscopy, the stratum corneum (SC) bioavailability of terbinafine (TBF) following topical treatment with four different formulations. Methods: Four skin sites on the ventral forearms of five healthy volunteers were treated for 2 h using one of four formulations based on a vehicle consisting of 50% ethanol and 50% isopropyl myristate. Three of these formulations included a percutaneous penetration enhancer: either 5% oleic acid, 10% 2-pyrrolidone or 1% urea. The SC concentration profile of TBF was measured by repeated infrared spectroscopic measurements while sequentially stripping off the layers of this barrier membrane with adhesive tape. This method was validated by HPLC analysis of TBF extracted from the stripped tapes. Transepidermal water loss (TEWL) measurements were also performed, to permit facile estimation of SC thickness. Results: The SC concentration profiles of TBF were fitted to the appropriate solution of Fick’s second law of diffusion, thereby allowing determination of the characteristic diffusion and partitioning parameters of the permeating drug. This analysis enabled the efficacies of the different formulations tested to be compared to the no-enhancer control. While it was found that the formulation containing 5% oleic acid significantly enhanced the SC availability of TBF, the other formulations did not improve the apparent drug delivery. Conclusions: A facile and minimally invasive methodology to evaluate an important aspect of topical drug bioavailability has been described. The analytical methods used (infrared spectroscopy and HPLC) allow estimates of both relative and absolute drug bioavailability in the SC and may be useful, therefore, in the critical determination of bioequivalence between topical formulations.

Progress in methodologies for evaluating bioequivalence of topical formulations.

Assessment of bioequivalence of topical dermatological formulations is a challenge. Currently, comparative clinical studies are used to establish bioequivalence for most formulations (except corticosteroids). This article reviews different in vivo methodologies for determining bioequivalence, and the progress made in this area. Dermatopharmacokinetics is the term used to describe the pharmacokinetics of topically applied drugs in the stratum corneum. A tape stripping procedure used in dermatopharmacokinetic methodology measures the drug concentration in stratum corneum at the site of application. Various studies have shown dermatopharmacokinetics to be a reliable and reproducible method for determining bioequivalence, and have indicated that it is applicable for all topical dermatological drug products. However, confidence in this methodology needs to be established, particularly regarding its relevance to clinical drug efficacy.

Assessment and prediction of the cutaneous bioavailability of topical terbinafine, in vivo, in man.

OBJECTIVE : The in vivo effectiveness of a topical, dermatological formulation depends on the bioa-vailability of the drug within the skin at the site of action. Unlike oral drugs, or those delivered trans-dermally for systemic effect, the amount of active agent reaching the general circulation after topical application is a measurement of questionable relevance with respect to its local bioavailability. In ad-dition, the level of a dermatological drug, which can be found in the blood is invariably very small and difficult, if not impossible, to quantify easily. While local, ‘skin' concentrations of topical drugs can be obtained from biopsies of the site of application, this approach is very invasive and is unacceptable for routine use. Other practical alternatives have not been abundantly proposed, and the vasoconstriction assay for corticosteroids remains the only method (albeit imperfect itself) with significant credibility. Recently, the U.S. Food and Drug Administration has suggested a new, so-called dermatopharma-cokinetic (DPK), approach. METHODS: The idea is to evaluate topically applied drug levels in the stratum corneum (SC) in vivo as a function of time post-application and post-removal of the formulation, and to generate a SC con-centration versus time profile from which such “classical” measures as maximum drug level, time to reach this maximum, and the area under the curve can be obtained (i.e., in an analogous fashion to blood level measurements for an orally administered drug). The DPK method assumes that: (a) in normal circumstances, the SC is the rate-determining barrier to percutaneous absorption, (b) the SC concentration of drug is directly related to that which diffuses into the underlying viable epidermis, and (c) SC drug levels are more useful and relevant for assessing local, dermatological efficacy than plasma concentrations. Whereas methodological and validation issues for the DPK technique remain to be answered, it is clear that there is an attractive logic behind this idea. However, examination of the draft guidance reveals that this methodology will be labor-intensive even for relatively simple evaluations of bioequivalence. Our objective, therefore, is to begin an examination of whether experiments (following, in general, a DPK methodology) of relatively short duration can be analyzed to produce physicochemical parame-ters that describe drug transport in the SC and which can therefore be used to predict drug uptake as a function of time. RESULTS : Using the antimycotic drug, terbinafine (molecular weight = 291 Da; log(octanol/water partition coefficient) = 3.3), we have measured, in vivo, in man, the SC concentration versus depth profile following a 0.5-h exposure to a simple formulation. The data have been analyzed mathematicaly to yield the SC/vehicle partition coefficient (K)of the drug and its characteristic diffu-sion parameter (D/L2, where D is the drug's diffusivity across the SC of thickness L). CONCLUSION: With these values, the mathematical model has been used to predict the integrated quantity of drug in the SC following treatment periods of 2 and 4 h, and the predictions have then been compared to experiment. In addition, the longer-time data have been evaluated independently to determine whether K and D/L2 are sensitive to the duration of drug treatment.

LC analysis of benzophenone-3: II application to determination of ‘in vitro’ and ‘in vivo’ skin penetration from solvents, coarse and submicron emulsions

The aim of this study was to determine the skin penetration of benzophenone-3 in vitro and in vivo in order to investigate a possible influence of formulation. Six different vehicles, three solvents and three different emulsion types were evaluated in vitro and in vivo. Each vehicle was applied to the skin model at 2 mg cm −2. First, histological studies on ear pigskin and human skin were evaluated. In vitro measurements were performed with static diffusion cells using pigskin at 1, 2, 4, and 8-h. In vivo, benzophenone-3 concentration in stratum corneum was evaluated by the stripping method after 30-min application on forearm of volunteers. It was shown that ear pigskin and human skin appear similar and in both experiments significant differences between vehicles were noticed. The six vehicles could be ranked in the same order of benzophenone-3 skin concentration. At 8-h, the highest concentration of benzophenone-3 in skin was obtained with propylene glycol, and O/W submicron emulsion. On the contrary, the two oily solvents, W/O emulsion and O/W coarse emulsion restrain the concentration of this UV-filter in the skin. At each time, permeability in vitro and in vivo were well correlated. Low concentrations were measured in the receptor fluid suggesting that percutaneous absorption of this UV-filter across the skin would be minimal. The in vitro and in vivo skin penetration capacity of benzophenone-3 from six vehicles was confirmed and quantified. A satisfactory relationship between binary in vitro and in vivo was established.

Effect of application time of betamethasone-17-valerate 0.1% cream on skin blanching and stratum corneum drug concentration

The effect of application time on skin blanching response and stratum corneum concentration after topical application of 0.1% betamethasone-17-valerate cream on healthy volunteers was assessed. Total corticosteroid content in the stratum corneum (SC) was determined at different application times (0.5, 8, 10 and 24 h) from five subjects in whom blanching was also evaluated at the same application times by visual score, colorimetry ( L-, a- and b-values) and Laser Doppler flowmetry. No significant differences between corticosteroid concentration in the SC at 8, 10 and 24 h was observed when compared using ANOVA and t-test ( P > 0.05) while drug content at 0.5 h was significantly lower ( P < 0.05). Significant differences between the blanching response at 0.5 h and the other time points (8, 10 and 24 h) were observed by visual assessment and a-value readings from a chromameter. However, at 24 h visual score and †a- values were lower than those measured at 8 and 10 h. but this difference was significant only for †a- values . This findings suggest that skin blanching may not be related to drug concentration in the stratum corneum and that †a- readings may be more sensitive and accurate than visual score in evaluating skin blanching. L-values were not significantly different at all the time points while b-values at 0.5 h were significantly different only from those at 8 and 10 h. Skin blanching was not observed when laser Doppler flow was measured while the concentration parameter was capable of detecting blanching; however, the concentration values were not significantly different at all the application times. The results of this study suggest that the duration of application should be carefully chosen to assess betamethasone-17- valerate topical bioavailability since after long application time skin blanching response may not be related to drug concentration in the stratum corneum.

Levels of terbinafine in plasma, stratum corneum, dermis-epidermis (without stratum corneum), sebum, hair and nails during and after 250 mg terbinafine orally once daily for 7 and 14 days.

In earlier skin pharmacokinetic studies we have shown that terbinafine is rapidly delivered to the stratum corneum, nails and hair both through sebum and by direct diffusion through dermis-epidermis. In the present study the skin pharmacokinetic profile of terbinafine was studied in two groups of eight human male volunteers during and after 250 mg orally once daily for 7 and 14 days. In the 7-day study high terbinafine levels were found in sebum (19.0 micrograms/g) and stratum corneum (2.5 micrograms/g), and a concentration in stratum corneum above the minimal inhibitory concentration for most dermatophytes was still found 48 days after the last day of medication. Terbinafine was found in peripheral nail clippings after 7 days of medication and the concentration was, in the 7-day study, 0.5 microgram/g 1 day after stopping medication; it was still 0.2 microgram/g 90 days after stopping treatment. The results in the 14-day study were in parallel with, but higher than, in the 7-day study. The elimination of terbinafine from several compartments is biphasic, with a faster initial elimination followed by a slower secondary elimination. For nails, the elimination is slower compared with the other compartments. The results indicate that terbinafine may be effective in short-term treatment of several dermatophytoses. The concentration of 0.2 microgram/g of terbinafine found in nails 90 days after stopping medication, following 7 days of treatment, indicates that the duration of therapy, even in tinea ungium, may be shorter than is currently the case.

Oleic acid concentration and effect in human stratum corneum: non-invasive determination by attenuated total reflectance infrared spectroscopy in vivo

Attenuated total reflectance infrared (ATR-IR) spectroscopy was used to study, in vivo, the mechanism of action of a putative skin penetration enhancer, oleic acid. Specifically, the spectroscopic technique monitored structural changes and enhancer concentration in the stratum corneum (SC) (skin's outermost and least permeable layer) following treatment with oleic acid in ethanol. As previously observed in vitro, oleic acid increased the hydrocarbon chain disorder of the inter-cellular lipid domains of the SC in vivo. The effects of the enhancer were prolonged but reversible. It also appeared that the treatment of the skin with oleic acid for 0.5 hour caused a gradient of effect (i.e. increased lipid disorder) that decreased from superficial to inner stratum corneum layers. However, this gradient was normalized by increasing the time of exposure of the skin to the enhancer. Although the oleic acid-induced lipid hydrocarbon chain disorder in vivo maximized at a concentration of 1% oleic acid in ethanol, the in vitro uptake of radiolabelied enhancer into excised SC increased linearly with concentration up to 10%. Further experiments showed that the saturation of response in vivo was not solubility limited. Precise analysis of the IR spectra enabled an absorption band to be assigned mainly to oleic acid and to be used, therefore, to quantity SC uptake of enhancer in vivo. A quite linear correlation between (radiochemical) in vitro and (spectroscopic) in vivo assessments of oleic acid in SC was found. Overall, then, the results reported support the conclusions: (a) that ATR-IR can monitor the effects of penetration enhancers on skin barrier function in vivo, and (b) that, in the case of oleic acid, the technique can estimate the level of enhancer within the SC as a function of time and, possibly, position.

Griseofulvin Levels in Stratum Corneum

Griseofulvin concentration in stratum corneum is determined by using a gasliquid, chromatographic procedure and electron capture detector. A scraping technique is used for obtaining three distinct levels from stratum corneum, and histological evidence shows these levels. Concentration of griseofulvin is found to be 16.4 ng/mg of palmar skin in level 1, 9.7 ng/mg in level 2, and 4.5 ng/mg in level 3. There is a more rapid buildup of drug after oral administration than thought previously. The drug can be detected in stratum corneum in eight hours. After discontinuing use of griseofulvin, concentration falls off more rapidly in skin than in blood. However, buildup is somewhat dependent on climate, the concentration being higher in warm weather than in cool. These findings emphasize that stratum corneum can act as a compartment for drugs such as griseofulvin.