In vivo methods for the analysis of the penetration of topically applied substances in and through the skin barrier

The efficacy of a drug is characterized by its action mechanism and its ability to pass the skin barrier. In this article, different methods are discussed, which permit this penetration process to be analysed non‐invasively. Providing qualitative and quantitative information, tape stripping is one of the oldest procedures for penetration studies. Although single cell layers of corneocytes are removed from the skin surface, this procedure is considered as non‐invasive and is applicable exclusively to the stratum corneum. Recently, optical and spectroscopic methods have been used to investigate the penetration process. Fluorescence‐labelled drugs can be easily detected in the skin by laser scanning microscopy. This method has the disadvantage that the dye labelling changes the molecular structures of the drug and consequently might influence the penetration properties. The penetration process of non‐fluorescent substances can be analysed by Raman spectroscopy, electron paramagnetic resonance, CARS and multiphoton microscopic measurements. Using these methods, the concentration of the topically applied formulations in different depths of the stratum corneum can be detected by moving the laser focus from the skin surface deeper into the stratum corneum. The advantages and disadvantages of these methods will be discussed in this article.     

Common cosmetic hydrophilic ingredients as penetration modifiers of flavonoids

Nowadays, flavonoids are present in many cosmetic formulations, mainly in the form of plant extracts. The main reason of still increasing popularity of these substances is their beneficial biochemical activity. The main factor affecting activity of flavonoids in the skin is their skin penetration ability. The studies have evidenced that flavonoids from grape leaf extract as well as flavonoids like quercetin, rutin and catechin can migrate through the model lipophilic membrane from aqueous solution. The influence of common hydrophilic cosmetic additives on the permeation profile of flavonoids has been checked. The partition coefficients of examined flavonoids in the octanol–water extraction system were determined. Correlations between permeation coefficients and log P of particular flavonoids were plotted. To determine the mechanism of influence of hydrophilic substances on the permeation profile of flavonoids, the solubility of these compounds was investigated. Studies suggest that the presence of hydrophilic additives causes the increase in the flavonoid solubility that decreases the activity of flavonoids in the vehicle. In such a situation, the driving force for the penetration is reduced and the decrease of permeation coefficient can be observed.     

The influence of alkane chain length on the skin irritation potential of 1,2-alkanediols

Several studies have reported that 1,2-alkanediols show increasing anti-microbial activity as their alkane chain length increases. However, there are no reports on the influence of alkane chain length on the skin irritation potential of 1,2-alkanediols. To investigate the influence of alkane chain length on the skin irritation potential of 1,2-alkanediols. The objective and subjective (sensory) skin irritation potentials of five 1,2-alkanediols - 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, 1,2-octanediol and 1,2-decanediol - were evaluated. We also estimated percutaneous absorption by measuring in vitro skin penetration using a Franz diffusion cell system. Like anti-microbial activity, sensory irritation potential increased as alkane chain length increased, most likely due to increasing membrane interference and/or intrinsic toxicity of 1,2-alkanediols. 1,2-Hexanediol showed the lowest objective skin irritation potential, which increased when the alkane chain length decreased or increased. Furthermore, percutaneous absorption negatively correlated with the alkane chain length of 1,2-alkanediols. These results show that a lower skin absorption potential is not indicative of a low skin irritation potential. Our results suggest that the factors and processes involved in skin irritation potential are complex and that skin irritation potential is influenced by intrinsic toxicity and the potential for penetration or integration in the lipid bilayer.     

Absorption of materials by or through the living skin

The skin comes into contact with a large range of materials either deliberately or inadvertently. It should be possible to predict the exact transport rates of these materials through the skin as a function of the physicochemical properties of the different compounds. With this sort of knowledge it is possible to predict the exact disposition of compounds and use this in the formulation of new products both in the pharmaceutical and cosmetic field. The information will also be useful from the standpoint of skin toxicology and environmental health. In order to be able to predict this complex process it is necessary to split the overall transport into different component parts. This article will identify these components and provide illustrations. The major areas discussed will be barrier function of the skin, the release properties of different topical formulations and how these may be monitored. Novel means of enhancing the penetration of drugs will be discussed and how some additives that are incorporated into formulations will perhaps alter the barrier function of skin. A mathematical model describing skin penetration has been developed and its use in predicting blood levels will be described. This model has been tested both in animal experiments and in limited human studies and its relevance to these situations will be highlighted.
Absorption cutanée et transcutanée in vivo     

High spatial resolution study of human skin using synchrotron infrared microscopy: application to the penetration of external agents

Human skin sections were studied using synchrotron-powered infrared microscopy. The superior lateral resolution achieved with this technique (diffraction-limited spot sizes) allows the detailed analysis of the biocomposition of the skin layers. It is shown that highly organized lipids are present in the stratum corneum, as evidenced by a slight frequency difference in the symmetric stretch mode of the methylene groups. Biochemical images were generated showing the precise location of the lipids, proteins and collagen across the skin section. More enhanced images were generated using a statistical approach (fuzzy C-means clustering). Penetration of two external agents (cyanophenol and a cosmetic compound, ethylhexyl methoxycinnamate) was studied. The penetration pathway across the stratum corneum is favored at locations of lower lipid concentration. Both compounds penetrated into the epidermis, but neither of them induced a change in the protein secondary structure. It was shown that hair follicle is a favored penetration pathway for these agents.     

Transdermal delivery of two antioxidants from different cosmetic formulations

The efficacy of any cosmetic product containing a functional ingredient is determined by the skin delivery of the active molecule, which is influenced by the type of the vehicle and the molecule itself. This study was designed to compare the percutaneous absorption habits of the antioxidants carcinine and lipoic acid out of various formulations by means of the porcine skin model. Initial evaluation of the in vitro porcine skin model has demonstrated its feasibility for various substances and formulations [1, 2]. Increasing legal requirements for risk assessment in the cosmetic industry have led to the development of this alternative test method. The penetration properties are determined by the OECD Guideline TG 428: Skin Absorption: in vitro Method [3, 4], which allows the use of porcine skin for penetration studies. Porcine skin is used because of its similarity to human skin in terms of its morphology and the essential permeation characteristics [5]. The mass balances for each tested formulation type of the antioxidants show individual penetration behaviours with significant differences. The presented data plainly demonstrate that the lipophilic lipoic acid has a distinct higher penetration potential than the hydrophilic carcinine. The chosen vehicle can enhance or reduce the transdermal delivery of both tested antioxidants. Modern effective cosmetic formulations will work only, if the active ingredients penetrate into the epidermis. In conclusion, the correct selection of a suitable formulation plays an important role during product development.     

Study of surfactant-skin interactions by skin impedance measurements

The stratum corneum (SC) plays a very critical physiological role as skin barrier in regulating water loss through the skin and protects the body from a wide range of physical and chemical exogenous insults. Surfactant-containing formulations can induce skin damage and irritation owing to surfactant absorption and penetration. It is generally accepted that reduction in skin barrier properties occurs only after surfactants have penetrated/permeated into the skin barrier. To mitigate the harshness of surfactant-based cleansing products, penetration/permeation of surfactants should be reduced. Skin impedance measurements have been taken in vitro on porcine skin using vertical Franz diffusion cells to investigate the impact of surfactants, temperature and pH on skin barrier integrity. These skin impedance results demonstrate excellent correlation with other published methods for assessing skin damage and irritation from different surfactant chemistry, concentration, pH, time of exposure and temperature. This study demonstrates that skin impedance can be utilized as a routine approach to screen surfactant-containing formulations for their propensity to compromise the skin barrier and hence likely lead to skin irritation.     

J. Cosmet. Sci., 59, 59–69 (January/February 2008)Biological activities of selected peptides: Skin penetration ability of copper complexes with peptides

This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or γ-glutamylcysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion.     

Sensitive skin: an overview

Sensitive skin is a condition of subjective cutaneous hyper‐reactivity to environmental factors. Subjects experiencing this condition report exaggerated reactions when their skin is in contact with cosmetics, soaps and sun screens, and they often report worsening after exposure to dry and cold climate. Although no sign of irritation is commonly detected, itching, burning, stinging and a tight sensation are constantly present. Generally substances that are not commonly considered irritants are involved in this abnormal response.Sensitive skin and subjective irritation are widespread but still far from being completely defined and understood. A correlation between sensitive skin and constitutional anomalies and/or other triggering factors such as occupational skin diseases or chronic exposure to irritants has been hypothesized. Recent findings suggest that higher sensitivity can be due to different mechanisms. Hyper‐reactors may have a thinner stratum corneum with a reduced corneocyte area causing a higher transcutaneous penetration of water‐soluble chemicals. Alterations in vanilloid receptors and changes in neuronal transmission have been described. Monitoring skin parameters such as barrier function, proclivity to irritation, corneocyte size and sensorial transmission can also be useful to identify regional differences in skin sensitivity.     

Hydration of the stratum corneum

Topically applied water, occlusion and topically applied glycerol were used to investigate and characterize some of the changes which occur in the hydrated stratum corneum. The effects of these treatments were monitored using non-invasive techniques under controlled conditions. The Servomed Evaporimeter was used to determine natural water flux from the skin surface before and after treatment. The performance of the Evaporimeter in this type of study had previously been improved by attaching a paper baffle to the detector. This eliminated the variance in output caused by atmospheric movement. Experiments were carried out at temperatures below the threshold of thermal sweating and emotional sweating was minimized. Skin surface topography was characterized by means of a new type of profilometer. The instrument's design allowed a diamond stylus to traverse the living skin surface without significantly altering its structure. Changes in skin surface roughness were further elucidated using scanning electron microscopy and macrophotography. In vivo penetration of glycerol was assessed by chemical analysis of stratum corneum layers of treated skin. Samples were obtained by sequential stripping of the stratum corneum using adhesive tape. Topically applied water produced only a transient benefit because of rapid evaporation. More prolonged hydration was achieved by suppressing transepidermal water loss with polyethylene film. This occlusive hyperhydration was characterized by a significant reduction in profile roughness and by a smoother macroscopic appearance. Glycerol achieved the same effects by reducing the magnitude of the natural water flux from the skin surface and by reducing the rate of evaporation of water from applied aqueous glycerol solution or cosmetic product. Both effects were seen as the result of lowered water activity in the proximity of glycerol. Smoothing effects of glycerol on the skin surface, and improved appearance, persisted for at least 24 h. This persistence was explained by evidence for diffusion of glycerol into the stratum corneum where it formed a reservoir. Hydration of the skin is known to affect its barrier function and thereby exert a profound effect on penetration of both lipophilic and hydrophilic molecules. Clinically, this effect may be achieved using liberal applications of occlusive petroleum jelly and ointments. The results presented in this paper suggest that the use of humectants could achieve useful hydration using cosmetically acceptable materials.     

冷杉栲胶的制取及鞣革试验

我国冷杉蓄积量丰富,广泛分布于西南、西北广大林区,开发利用冷要用鞣科具有重要意义,本文采用几种不同的浸提方法制取冷杉栲胶：1）水浸提;2）添加亚硫酸盐,包括在浸提过程中和渤提后的溶液中加入两种方法;3）合成单宁改性。用不同方法制取的胶做了靶柑研究,比较其成革性能。试验结果表明,冷杉栲胶经亚硫酸盐和合成单宁处理后,沉淀减少,渗透速度加快,成革丰满坚实,颜色浅淡,鞣革性能得到较大的改善。加亚硫酸盐浸提的栲胶结合靶 和靶要制系数虽有所下降,但渗透速度却快得多,而且栲胶的浸提率大,因而列为首选的方法。浓胶加合成单宁的方法因渗透快和结合力较强,也可供选择。     

Influence des ions sur le pouvoir hydratant de l'ure e: e tude sur peau de porc ex vivo

This study deals with the influence of ions (NaCl and MgSO4) in a W/O emulsion containing 10% urea. Moisturization kinetics are assessed by corneometry on pig skin ex vivo. The formula's influence on urea penetration is measured by infrared spectrometry with an ATR device and the stripping method. Corneometry and spectroscopy were chosen to record simultaneously the hydratation levels and urea localization into superficial cell layers. Urea crystallization after evaporation of emulsions and aqueous solutions is described. Results show that urea does not hydrate nor penetrate when applied to the skin through an aqueous gel. In a W/O emulsion, sodium chloride increases the ability of urea to moisturize without improving penetration. In vitro urea crystallization is disturbed by sodium chloride or magnesium sulphate for solutions and emulsions. This stabilization by ions is correlated with good moisturization values. The stabilization of urea in the solute state provided by ions increases its water epidermal binding capacity without enhancing penetration.     

The effect of cream and ointment bases on the steady state penetration of permeants through intact skin: the reciprocal of the onset time of a pharmacodynamic effect as parameter of response

To characterize cream or ointment bases for cosmetic or pharmaceutical purposes with regard to their effect on permeant penetration through intact healthy skin, the measurement of the pharmacodynamic response of a suitable model drug incorporated in these bases has been shown to be a promising approach. In general, it may be distinguished between thermodynamic vehicle effects owing to different permeant escaping tendencies from the vehicles and penetration-enhancing vehicle effects resulting from a change of the stratum corneum structure, which manifests itself in an increase of the permeant diffusion coefficient and/or its solubility in this barrier. As the latency time of onset of a pharmacodynamic effect, usually used as reciprocal value, represents a suitable response parameter under certain circumstances, this study was done to further evaluate this parameter with regard to the determination of relative bioavailability and penetration enhancement data obtained from simulated dose- and activity-response curves assuming infinite dose conditions, i.e. zero order penetration kinetics and considering varying lag times of drug penetration. The results indicate that bioavailability and enhancement factors may be determined accurately from the horizontal distance between dose- or activity-response curves of a standard and a test preparation as long as the curves are parallel to each other, as it is the case with uniform lag times of permeant penetration. Non-parallel curves observed with varying lag times indicate an influence of the vehicles on the permeant diffusion coefficient in the barrier. Enhancement factors from these curves may be obtained after determination of the lag times from the plateau region of the curves, subsequent subtraction of these values from the measured latency time data, and finally plotting of the reciprocal data as a function of the drug activity. Enhancement factors then correspond to the inverse logarithm of the horizontal distances between the resulting parallel curves.     

In vitro skin permeation and retention of parabens from cosmetic formulations

Parabens are antimicrobial agents widely used in foods, cosmetics and pharmaceutical products. Although non-mutagenic, non-teratogenic and non-carcinogenic, parabens can induce allergic contact dermatitis and posses estrogenic activity. The aim of this work was to assess the skin permeation and retention of methyl- (MP), ethyl- (EP) and propyl- (PP) paraben from three commercial cosmetic creams. The results obtained indicate that parabens are capable of permeating through and accumulating in the skin. The extent of penetration depends more on paraben characteristics (solubility, lipophilicity) than on the composition of the formulation. In particular, the percentage permeated across the skin was independent of the composition of the cream used and decreased in the order MP, EP and PP, in accordance with decreasing solubility. After 8 h of contact with the skin, 60% of MP, 40% of EP and 20% of PP were found across the skin. Concerning skin retention, the percentage remaining in the skin after 8 h depends on both paraben characteristics and on the composition of the formulation used. In conclusion, it appears that only the type of paraben, in particular its water solubility, affects skin penetration whereas the composition of the emulsion, which influences skin retention, plays a secondary role. Finally, excised rabbit ear skin can be considered as a good model for human skin for in vitro experiments.     

冷杉栲胶的制取及鞣革试验

我国冷杉蓄积量丰富，广泛分布于西南、西北广大林区，开发利用冷杉鞣料具有重要意义。本文采用几种不同的浸提方法制取冷杉栲胶：1)水浸提；2)添加亚硫酸盐，包括在浸提过程中和浸提后的溶液中加入两种方法；3)合成单宁改性。用不同方法制取的栲胶做了鞣革研究，比较其成革性能。试验结果表明，冷杉栲胶经亚硫酸盐和合成单宁处理后，沉淀减少，渗透速度加快，成革丰满坚实，颜色浅淡，鞣革性能得到较大的改善。加亚硫酸盐浸提的栲胶结合鞣质和鞣制系数虽有所下降，但渗透速度却快得多，而日.栲胶的浸提率大，因而列为首选的方法。浓胶加合成单宁的方法因渗透快和结合力较强，也可供选择。     

In vitro evaluation of the cutaneous penetration of sprayable sunscreen emulsions with high concentrations of UV filters

The aim of this study was to evaluate the possible penetration through human skin of organic and inorganic filters contained in sunscreen emulsions packaged in aerosol cans, using an in vitro method. Experiments were carried out on two different types of emulsion: W/Si and W/O. This study was conducted using static diffusion cells (Franz cells). The determination of organic UV filters [Methylene Bis Benzotriazolyl Tetramethylbutylphenol (MBBT); Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine (BEMT); Diethylamino Hydroxybenzoyl Hexyl Benzoate (DHHB); Ethylhexyl Methoxycinnamate (EMC); and 2-Ethylhexyl Dimethyl PABA (ED-PABA)] was performed by High Performance Liquid Chromatography (HPLC). Therefore, it was important to develop a single analytical method for the quantification of the five organic filters with the aim of facilitating the experiment. The determination of inorganic filters [titanium dioxide (TiO₂) and zinc oxide (ZnO)] was performed using an emission spectrometric analysis method (ICP-OES). The HPLC and ICP-OES methods were validated. After a penetration test of 24 h duration, the results showed very low penetration only for two of the organic filters (maximum penetration of 1.21 μg cm⁻² h⁻¹ for EMC and 0.14 μg cm⁻² h⁻¹ for MBBT) and no penetration for the inorganic filters. Moreover, more than 50% of each sunscreen agent stayed on the surface on the skin. These results are consistent with those in the literature that presents similar experiments. This study showed that the sprayable sunscreen products developed, which contained high concentrations of UV filters, presented a low level of skin penetration.     

Topically applied KTTKS: a review

Skin ageing is an irreversible process that is caused by both intrinsic and extrinsic factors. The possibility of arresting or delaying skin ageing represents a large research area and has a big potential in the cosmetics sector. Recently, the polypeptide lysine-threonine-threonine-lysine-serine (KTTKS) has attracted a lot of attention and it features in numerous up-market cosmetic products where it has become erroneously associated with the term 'pentapeptide'. In this study, we review in detail KTTKS and its major derivatives, in terms of the limited information in the literature and an appraisal of its physicochemical and theoretical skin permeation properties. There appears to be a sound in vitro basis for its action on fibroblasts due to its stimulatory effect on extracellular matrix synthesis, where the stimulatory effect of KTTKS is specific to collagen types I and III and fibronectin expression. However, there is a surprising absence of in vitro skin penetration data in the literature, and there are relatively few clinical studies using these materials.     

Percutaneous absorption of 2-nitro-p-phenylenediamine

Studies have been done on the absorption and metabolism of the hair colourant 2-nitro-p-phenylenediamine (2-NPPD) in rats with particular reference to skin penetration. When [¹⁴C]2-NPPD was given orally or injected intraperitoneally into male and female rats, excretion was rapid and mainly in the faeces (60%) and urine (35%). No radioactivity was measurable in the expired air. At 3 days after dosing, approximately 2% of the dose was retained in the body. Application of [¹⁴C]2-NPPD in ethanol to the clipped skin of rats gave a high skin penetration. Male rats absorbed less (11.7 μg cm^-2) through the skin than did female rats (24.6 μg cm^-2). Application of [¹⁴C]2-NPPD in a solution of a hair colourant product base to the clipped skin of rats gave a high penetration if the skin was occluded or not rinsed. Penetration was much reduced after rinsing and protection by non-occlusive patches. With these latter conditions, skin penetration in clipped rats treated with a 0.5% (w/v) solution of [¹⁴C]2-NPPD doubled to 6.1 μg cm^-2 when the contact time was increased from 5 to 30 minutes, increased two to three times after two and three repeated applications, increased in proportion to the applied concentration and was reduced by half when applied to unclipped skin. The metabolic pattern in urine from rats after different routes of administration showed only a trace of the parent [¹⁴C]2-NPPD but six other radioactive components. The identity of these components has not yet been fully resolved. Blood levels of radio-activity during 2 days after topical administration were low. Under realistic conditions of use by the consumer the calculated dose through the skin based on the experimental rat data is up to 10 μg/kg body weight per application of a semi-permanent hair colourant product containing 1% of 2-NPPD. L'adsorption transcutanée de la 2-nitro-p-phenylenediamine On a étudié l'adsorption transcutanée et le métabolisme du colorant capillaire 2-nitro-p-phenylènediamine (2-NPPD) sur le rat. Lorsque la [¹⁴C]2-NPPD est administrée par voie orale ou injectée par voie intra-péritonéale sur des rats mâles et femelles, l'excrétion est rapide et principalement dans les fèces (60%) et dans les urines (35%). Il n'y a pas de radioactivité mesurable dans l'air expiré. Trois jours après l'administration, environ 2% de la dose est retenue dans l'organisme.