Basic & Clinical Pharmacology & Toxicology, 2014, 115, 101–109
Doi: 10.1111/bcpt.12188
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Percutaneous Penetration - Methodological Considerations Rikke Holmgaard1, Eva Benfeldt2 and Jesper B. Nielsen3 Department of Orthopedic Surgery, Køge Sygehus, Køge, Denmark, 2Department of Dermatology, University of Copenhagen, Roskilde Hospital, Roskilde, Denmark and 3Institute of Public Health, University of Southern Denmark, Odense, Denmark
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(Received 4 October 2013; Accepted 18 December 2013) Abstract: Studies on percutaneous penetration are needed to assess the hazards after unintended occupational skin exposures to industrial products as well as the efficacy after intended consumer exposure to topically applied medicinal or cosmetic products. During recent decades, a number of methods have been developed to replace methods involving experimental animals. The results obtained from these methods are decided not only by the chemical or product tested, but to a significant degree also by the experimental set-up and decisions made by the investigator during the planning phase. The present MiniReview discusses some of the existing and well-known experimental in vitro and in vivo methods for studies of percutaneous penetration together with some more recent and promising methods. After this, some considerations and recommendations about advantages and limitations of the different methods and their relevance for the prediction of percutaneous penetration are given. Which method to prefer will depend on the product to be tested and the question asked. Regulatory guidelines exist for studies on percutaneous penetration, but researchers as well as regulatory bodies need to pay specific attention to the vehicles and solvents used in donor and sampling fluids so that it reflects in-use conditions as closely as possible. Based on available experimental data, mathematical models have been developed to aid predictions of skin penetration. The authors question the general use of the present mathematical models in hazard assessment, as they seem to ignore outliers among chemicals as well as the heterogeneity of skin barrier properties and skin conditions within the exposed populations.
This MiniReview will discuss some of the existing and wellknown experimental in vitro and in vivo models together with some more recent and promising models and give some considerations and recommendations about advantages and limitations of the various models and their relevance for predictions of percutaneous penetration. An overview of the methodologies discussed can be found in table 1. Dermal exposure may be unintentional after environmental or occupational exposure or intentional after the use of topically applied medication or use of cosmetic products. In all cases, the assessment of the potential for percutaneous penetration or temporary deposition within the skin is an essential element in assessing risk as well as efficacy after dermal exposures. Decades of preventive efforts have decreased the inhalational exposures at work places. At the same time, topically applied pharmaceuticals are being used ever more often. Together, this has increased the relative importance of dermal exposures. Knowledge concerning percutaneous penetration and the potential to reach target sites closes the gap from exposure assessment and hazard identification to risk assessment. If a substance is unable to penetrate the stratum corneum (SC) or Author for correspondence: Jesper Bo Nielsen, Institute of Public Health, University of Southern Denmark, J.B. Winsløws Vej 9b, 2nd floor, 5000 Odense C, Denmark (fax +45 6550 3682, e-mail [email protected]).
affect the skin barrier function in any way, then the need for further assessment of risk becomes less evident. If a substance penetrates the skin or reaches targets within the skin, specific information is needed to qualify the hazard and risk assessment. Thus, information on penetration kinetics, including rate of penetration (flux), Lag-time and temporary deposition in different skin layers, will be needed. The ultimate goal of skin penetration research is to assess the risk to human beings after dermal exposure to hazardous chemicals. Consequently, results from in vivo studies in human beings, including skin sampling, will remain the gold standard in skin penetration studies. In the present MiniReview, a short presentation of three more recent experimental human methodologies (microdialysis, open-flow microperfusion and spectroscopy) is included together with a more traditional approach based on tape stripping. For ethical, logistical and financial reasons, in vivo studies in human beings will not be able to cover the increasing need for data on percutaneous penetration. Animal studies have been used extensively and are still being used. However, acknowledging both physiological and structural differences between species, which may jeopardize the extrapolation of results from animals to human beings, and the implementation of the European REACH program followed by the more general political urge to reduce, refine and replace studies in experimental animals, the need for validated in vitro or in silico models to study percutaneous penetration has increased.
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Table 1. Advantages and limitations related to different methods for studying percutaneous penetration. The + and tioned defining feature, and + to +++ indicates if it is more or less characteristic for the method.
indicates if the method has the men-
Method Advantages/limitations
Static cells
Flow through
Tape stripping
Raman Spectroscopy
DMD
OFM
In vitro In vivo Full-thickness skin Dermatomed skin Limited time consumption Minimally invasive Continuous sampling Multiple application sites Simple design Low cost Good reproducibility
+
+
+ + +
+ + +
+ + +
+ + +
+ + +
+ + + + ++
+ + + + ++
+ + + + + ++
+ +++ + + +
+ + +
+ + +
?
+
+
Furthermore, due to the increasing number of chemicals to which human beings are exposed, data from clinical and experimental studies will not be able to satisfy the need for quantitative information, and mathematical models for predicting penetration will therefore be necessary. The validity of these in silico models will, however, still depend on the continued input of relevant data from experimental studies on percutaneous penetration. For all the above reasons, studies on percutaneous penetration are highly relevant. In these cases, the OECD guidance is to combine knowledge from different experimental models (multimodel approach) to mitigate the drawbacks from relying on a single model. Skin penetration research can be considered very versatile due to the different areas of interests mentioned previously. Over time, different models have been used to gain knowledge of kinetics related to pharmaceuticals, pesticides and other industrial chemicals and products. All models have their advantages and limitations. The model of choice will depend on the research question to be answered, because different models may provide the researcher with different types of information. When planning experimental studies, awareness of the advantages, limitations and model-specific challenges makes it possible to design an experimental study in agreement with the scientific question to be answered and the penetrant of choice. Skin Structure The skin is the largest single organ of the body, accounting for more than 5.5% of the body mass; in average about 4 kg covering 1.7 m² depending on the height and weight of the individual [1]. The human skin surface is continually exposed to chemicals, mechanical injury, microorganisms, UV-light, temperature variations and water, and the most important function of the skin is to act as a barrier against these exposures. Besides barrier properties against exogenous exposures, the skin helps maintaining homoeostasis. The skin can be divided into the upper epidermis, underneath is the more vascular dermis and below the subcutaneous layers (fig. 1). Topically applied substances have to penetrate the avascular and
+ + + ++
Fig. 1. Schematic illustration of the skin structure.
lipophilic SC and continue through the more aqueous lower epidermis and dermis to reach the systemic circulation. Lipophilic substances will easily cross the SC, but the penetration rate will decrease as it reaches the hydrophilic epidermis and dermis – leading to increased deposition and giving the appearance of a reservoir – the reservoir effect. Determinants of Percutaneous Penetration Percutaneous penetration will – for the vast majority of exogenous chemicals – occur via passive diffusion. The exception to this generalization concerns larger or protein-bound molecules, which may use carrier-mediated transportation besides passive diffusion. The passive diffusion process follows Fick’s law, which defines flux as the multiplication of the concentration differences across the membrane and a concentrationindependent constant, the chemical-specific permeability coefficient Kp. Thus, the penetration rate (flux) will be proportional to the experimentally chosen concentration gradient across the membrane and if the concentration gradient diminishes during the experimental period, the flux will also be reduced. This is evident in experiments with finite doses applied on the skin and/or if the concentration of the chemical
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is allowed to become significant on the lower side of the skin. The latter can be practically avoided by having a sufficiently large volume of receptor fluid or by continuously replacing the receptor fluid with fresh receptor fluid. The permeability coefficient (Kp) has been described to depend mainly on the molecular weight (MW), the octanol/water partition coefficient (Kow) and molecular size (stereochemistry), whereas other chemical characteristics such as melting point, hydrogen bonding acceptor capability [H(a)] only marginally affect penetration rates [2,3]. Besides these determinants, vapour pressure, ionization (which is pH-dependent) and susceptibility to protein binding will affect the concentration of unbound and uncharged chemical available for percutaneous penetration at the surface of the skin at any time during the experimental period. Thus, if a chemical has a sufficient vapour pressure, a substantial part of the applied chemical may evaporate and thereby significantly reduce the concentration gradient and the observed flux. Besides the chemical-specific characteristics, the passive diffusion will also depend on experimental conditions related to the physicochemical environment at the experimental setting. In the in vivo situation, the absorbed chemical will reach the lymph or blood circulation after penetrating the upper layers of the skin. To mimic this situation, the sampling fluid applied experimentally for sampling the penetrant should have relevant physico-chemical characteristics related to solubility and pH; otherwise, experiments may under- or overestimate the true in vivo flux. Likewise, if a chemical is not applied as neat chemical, it will be dissolved in a solvent, which may in turn affect the penetration characteristics. This is why it needs to be well argued if the chemical is not applied under user-relevant conditions. The above-mentioned determinants are general to all in vivo and in vitro experimental models, but model-specific characteristics posing challenges to the transferal of experimentally observed penetration rates or time lag also exist. To facilitate a harmonized interpretation of in vitro data from different experimental models, a number of guidance documents have been published [4–7]. In the following sections, some of these models will be discussed under each type of experimental model. Skin Penetration Models for in vitro Studies The first in vitro model to study skin penetration was developed in the 1940s in response to the threat of World War II chemical warfare agents intended for inhalational as well as dermal exposure [8]. The original model resembles the presently used static and flow-through diffusion cells. The static diffusion cell, also known as the Franz diffusion cell (fig. 2), has been one of the most used in vitro models in skin penetration research since 1975 [9]. In the mid-eighties, the flow-through system was developed (fig. 3), and it is used for the same purposes as the static model. Both static and flow-through diffusion cells will work with full thickness as well as epidermal skin barriers [10,11]. Various guidelines indicate that the skin samples that may be used during in vitro studies are split thickness [6] or when justified, full thickness
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Fig. 2. Illustration of the static diffusion cell. The penetrating substance is diffusing from the donor chamber through the skin to the receptor chamber from where it is collected through the collection pipe. The ‘○’ symbolizes the penetrating substance.
Fig. 3. Illustration of the flow-through system. The penetrating substance symbolised by ‘○’ is sampled in the passing receptor fluid and collected in vials from the outlet tube.
up to 1 mm. Full thickness means that the upper approximately 1 mm of the skin including SC, epidermis and part of dermis is mounted in the diffusion cell, whereas epidermal barriers mean that the upper 200–400 lm skin has been separated (e.g. through techniques such as dermatome or heat separation) before being mounted in the diffusion cell. In both models, a skin sample is mounted between a donor chamber and a receptor chamber with the SC side towards the donor chamber. Skin from both human donors and experimental animals may be used, but human skin samples are preferred as it will avoid the use of experimental animals and consequently also avoid extrapolation between species when data are being used. Irrespective of the donor source, the barrier integrity should be checked before application of test substances. The basic procedure is that the penetrant of choice is applied on the donor side, and percutaneous penetration can be measured after sampling from the receptor side [6] – figs 2 and 3. Specific OECD guidelines have been developed [6] for both models, and for regulatory purposes both models are acceptable, though each of them have their advantages and limitations [12,13].
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In vitro techniques are ideal for screening percutaneous penetration of large numbers of topically applied substances, as experimental cost and time consumption are low. Ethical considerations are fewer than for in vivo studies and relates to the use of experimental animals or the consent to use of human skin samples obtained from operative procedures, that is, typically surgical waste. Many studies would be hazardous if conducted in vivo, for example, studies regarding chemical warfare agents [14]. When comparing data between studies, the in vitro methods have the advantage of a simple design, great predictability and little variability. Consequently, these methods have been easy to standardize. Another advantage can be the possibility of working with radiolabelled substances, which is associated with easy quantification of penetration of the substance. However, the researcher needs to take notice that the radiolabel may be separated from the substance itself due to enzymatic degradation/metabolism during the penetration pathway, and quantification of penetration by counting the radiolabel will – in that case – be erroneous. Due to a lack of biochemical, physiological and immunological systems (missing skin metabolism, lack of blood flow, etc.) in vitro studies do not reproduce real physiological conditions. However, in vitro studies have – when thoughtfully designed – been shown to be able to predict skin penetration in vivo [15–17]. As most substances penetrate the skin by passive diffusion, an in vitro method may be an excellent method for initial studies or, as mentioned previously, for high-throughput screening of topically applied drugs and other substances. The two in vitro methods mentioned previously – the static diffusion cell and the flow-through system – both have advantages and challenges [12] and the choice of cell needs to be carefully considered in relation to the research question posed. The flow-through system mimics the microcirculation in the skin as the continuous flow in the receptor chamber removes the substance once it has permeated the skin. This is an advantage when dealing with substances with a low solubility in the receptor fluid, and sink conditions are also easier maintained in studies running for longer time periods compared with the method based on static diffusion cells, where the concentration of the penetrant in the receptor chamber slowly increases during the experimental period. In the flow-through system, the length of the outlet tubing will affect the time lag, especially at low flow rates, and the complexity of the method – compared with the simpler and less expensive version based on the static cells – is technically more challenging. Both in vitro models will work with finite and infinite dosing. Use of infinite doses will allow estimation of time lag, and subsequently, the maximal flux from which the apparent permeability coefficient, Kp, can be calculated. Infinite dosing will, however, also very often keep the topical side of the skin covered with donor fluid throughout the experimental period, which is oftentimes different form the in vivo exposure situation. However, in transdermal therapeutic delivery systems (TTS), the occlusive environment underlying the patch will make the underlying skin moist, and in this case, the resemblance to a live situation may be closer. This is also the case in occupational toxicology where risk assessment in occlusive/
occluded environments is relevant, for example, when employees use gloves when handling toxic substances, which may – due to incorrect choice or use of gloves or damage to the glove membrane – become trapped between glove and skin. Use of finite dosing will more closely resemble most in vivo situations in relation to infrequent short-term occupational exposures (e.g. splashes) or the use of cosmetic or medicinal products. Use of finite doses will allow an estimate of the fractional permeation of the applied dose. The amount of a substance permeating the skin depends on the area of skin available for penetration, and calculation of different measures for permeation is therefore adjusted to that area. If the entire area of the donor cell is not covered, the adjustment is flawed, and the calculations will overestimate the actual permeation rate. The present OECD guideline [6] suggests adding 25 lL/cm2 to the donor chambers in flowthrough as well as static cells, which corresponds to a thickness of the donor fluid of 250 lm. If the natural surface tension in the donor solution is not reduced by adding detergents or solvents, this volume is (at least in our hands) too small to assure full coverage of the entire area, especially as the skin surface, besides having a natural uneven surface, has a tendency to be slightly thicker in the middle of the donor chamber. Our suggestion is to increase the volume applied to the cells to 50 lL/cm2. Both methods are also ideal for studies exploring the reservoir effect of the skin [18]. Furthermore, the effect of different penetration enhancers, which are often applied to a solution in order to change the skin permeability, can easily and inexpensively be studied in different applications. Enhancers or detergents present in the receptor solution may interact with the SC by causing either disruption of the SC lipids, structural protein changes or improved partitioning of the drug [19]. Ethanol, as an example, makes reversible changes to the skin barrier described in different ways as intercellular lipid removal [20], lipid fluidization [21] lipid disordering or lipid extraction and modulation of the lipid barrier [22]. The effect of any enhancer or enhancer-added solution can be studied in vitro without ethical considerations. Skin Penetration Models for in vivo Studies In the development of drugs for human topical use or in risk assessment in occupational settings, the most directly transferrable kinetic research is conducted by in vivo human studies, because there is no need for extrapolation to human conditions as is the case after animal or in vitro studies. Some methods can be used both in vitro and in vivo. These methods can be used in, for example, the development of new drugs from the laboratory concept to the final product tested in clinical settings. By avoiding a change in methodology during the path of development, an intermethodological variability can be avoided. Among the presently developed in vivo methods, a few also have the advantage of chronological real-time continuous sampling – dermal microdialysis and open-flow microperfusion are mentioned below – which is a relevant feature in pharmaco- and toxicokinetic research.
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Tape Stripping Tape stripping (TS) has become one of the traditional investigative methods in in vivo skin penetration research and is known as a non-invasive method, which can be used to determine the kinetics of penetrants. By repeated application and removal of adhesive tape to the same site on the skin, it is possible to remove and sample successively layers of the SC and determine the absorption profile and potential temporary deposit within the upper SC of the penetrant at the time of sampling (fig. 4). After stripping a specified skin area, the penetrant is extracted from the tape and subsequently analysed with traditional analytical methods. The sampling method has some manual steps, which requires trained personnel to avoid significant interpersonal and interlaboratory variability [23]. Spectroscopy In the early 1990s, spectroscopy was developed for skin penetration studies as a method for SC research. Spectroscopic methods are optical methods based on light scattering and generally have until recently had only but a limited depth range confined to the SC or just below the SC. More recently, imaging techniques based on confocal fluorescence microscopy have been developed that provide a pictorial description of skin structures including visualization of the different skin strata, which enables optical sectioning of the specimen to be performed in a non-invasive manner. Two major confocal fluorescence microscopy techniques have been used in skinrelated experiments: laser scanning confocal fluorescence microscopy (LSCM) and two-photon excitation fluorescence microscopy (TPEFM). Both techniques are suitable to explore the epidermis; however, TPEFM has particular advantages over LSCM. Two-photon excitation is a non-linear process in which a fluorophore absorbs two photons simultaneously. These photons are generally of low energy compared with the one-photon absorption process in conventional fluorescence microscopy techniques, resulting in an overall low extent of photobleaching and photodamage to the specimens [24]. In addition, as infrared light is used as the excitation source in TPEFM, the penetration depth in thick specimens can be up to 800 lm, allowing in-depth, three-dimensional examination of biological specimens. This penetration depth is far superior to
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that obtained using LCSM, where a maximum depth of approximately 150–200 lm is generally obtained [25]. The first publication showing concordance between drug penetration investigated by Confocal Raman spectroscopy (Confocal microscopy in combination with Raman spectroscopy) and the TS methodology was recently published [26]. Other spectroscopic methods such as Near IR and Terahertz spectroscopy are currently being developed [27,28]. A challenge for all these methods is that they are not quantitative by nature, though analytical ways to semi-quantitative measurements are being developed. The methods are fully non-invasive and can provide detailed information about the layers studied. Given that a test molecule can act as a fluorophore, it is expected to become possible to study specific depositional patterns during the absorption process for such molecules. As the methods are applicable in vivo as well as in in vitro settings, it should be possible to use these imaging techniques to study experimentally induced structural artefacts in experimental settings. Microdialysis Dermal MD (DMD) was first described by Anderson et al. in 1991 in a human study concerning percutaneous absorption of solvents, using ethanol as penetrating substance and MD sampling in the dermis [29]. Since then, the DMD method (fig. 5) has been used for sampling of a large number of topically applied drugs and other skin penetrants [30] in both healthy and damaged/diseased skin in human beings as well as in animals. DMD can sample endogenous and exogenous substances in all types of tissue by use of a thin catheter with a semi-permeable membrane imitating a small blood vessel. This vessel (the microdialysis probe) is connected to an inlet and an outlet tube perfused with a tissue compatible fluid (perfusate). The exchange of molecules across the membrane occurs by passive diffusion driven by the concentration gradient. The microdialysis probes have specific pore sizes, which set upper limits (cut-off value) for the molecules that can be sampled, but also excludes larger molecules and proteins from entering the sampling fluid. Thus, unless protein has been added to the perfusate, which is only done if it enhances recovery of the substance of interest, then DMD sampling delivers protein and enzyme-free samples, which makes the pre-analytical steps relatively uncomplicated. The method requires prior considerations of the suitability of the substance of interest for microdialysis sampling, as the typical combination seen in many topical medical treatments, that is, a high or very high lipophilicity of the drug and a low drug concentration in the topical product, both make DMD sampling challenging as the resulting samples may be of very low (below LOQ) concentration [31]. Open-flow Microperfusion:
Fig. 4. Tape stripping. Illustration of the sampling of the upper layers of the skin – the stratum corneum (SC) - by repeated tape application and removal of successive layers of SC cells.
In 1997, a similar technique – OFM – was introduced by Trajanoski et al. [32].. OFM is also designed as a continuous tissue-specific sampling method. Where the DMD probe has a
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enzymes and some cells will be included in the sample fluid collected by the catheter. Thus, the resulting sample requires technically more demanding pre-analytical steps before analysis of the sample fluid. The use of in vivo methods has significant ethical implications. In the case of TS, the skin trauma invoked when removing the SC layers is most often negligible leaving only a small temporarily sore area. For DMD and OFM, the considerations concern the necessary skin trauma inflicted when inserting the probe/catheter horizontally in the dermis (figs 5 and 6). Lasting tissue damage is not seen, but the insertion trauma is followed by a histamine release, causing a reversible wheal and flare reaction [35]. Fig. 5. Illustration of the microdialysis probe placed in the dermis, which is sampling increasing dermal drug concentrations after topical drug penetration (modified from Benfeldt and Serup 1999). The ‘○’ symbolizes the penetrating substance.
semi-permeable membrane, the OFM sampling catheter has a membrane-free macroscopically perforated area with unrestricted access to and exchange of solutes in the peri-catheter tissue. Therefore, the OFM technique does not have the same limitations regarding sampling efficacy towards large and/or protein-bound penetrants as the MD technique. Since 2006, the OFM method has been utilized for dermal sampling [33,34] (fig. 6). The method is, due to the open exchange area, relevant for sampling of large and/or lipophilic penetrants, which is where the DMD sampling methodology is often challenged. However, OFM is a more demanding method both technically and labourwise, as the method needs a push- as well as a pull-pump function connected to the sampling catheters to counteract the tendency to induce oedema in the tissue surrounding the probe due to the open exchange area. As a consequence of the open exchange area, proteins,
Fig. 6. Illustration of the open-flow microperfusion catheter placed in the dermis. The topically added substance symbolized by ‘○’ is penetrating through the skin and sampled in the dermis. The push/pull system is connected to the probe and the samples are collected in an exchangeable glass capillary. Markings on the probe make a correct positioning in the skin easy.
From Experimental Observations to the Real World When conducting experimental studies, in vitro as well as in vivo, researchers often take pride in following specified guidelines and standard procedures in the laboratory. This approach increases repeatability and reduces variability in results. However, the human population is most often better characterized by heterogeneity than homogeneity, and a significant fraction of the population suffers from a diseased or otherwise damaged skin barrier for exogenous or endogenous (including genetic) reasons. When we study skin penetration, it is therefore important not only to create reliable results in relation to a normal skin profile but also to adjust the experimental studies to real-life conditions, which are often not as straightforward as in the ‘standard operation procedures’ applied in the laboratory. The use of an experimental model that can be adapted to a real-life situation would therefore be most relevant for the study of situations that may deviate from the average. Known exceptions are damaged or diseased skin [36]. Irrespective of whether the affected skin barrier is caused by blunt trauma, wet work, delipidization of the SC or the presence of dermatitis, a decrease in skin barrier integrity has proven to increase percutaneous penetration [37–40]. A mutation in the filaggrin gene, which is carried by approximately 9% of the European population, is associated with a decrease in natural moisturizing factor of the skin, a key parameter for skin barrier function and a risk factor for the development of dermatitis [41–43]. Patients suffering from dermatitis have previously been shown to have impaired skin barrier function in lesional [44] as well as non-lesional skin [45]. The impact of an impaired skin barrier function may be seen in both the occupational setting but also in penetration and/or reaction to transdermally administered medical products, topical formulations as well as cosmetic products. In some situations, the experimental focus is on percutaneous penetration of the active ingredient and not on the commercial product that consumers are exposed to, which is often a mixture of different substances. The tested individual ingredients are then used to formulate non-tested mixtures. These mixtures have often been demonstrated to have a higher penetration rate than the tested active ingredient [46,47]. The research results from testing pure chemicals are in these
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situations potentially misleading and may affect the risk assessment in occupational settings where an added detergent/ enhancer affects the skin integrity and thereby increase skin permeability [48,49]. This is an area that still lacks relevant experimental data to support mathematical modelling, which will allow valid predictions of the percutaneous penetration of formulated products including if the formulations of already approved products are changed. Often individuals are exposed to several products at the same time. Interactions between two or more unintended dermal exposures in occupational settings have been described, causing increased absorption just as well as an absence of any interaction of practical importance [50,51]. In the occupational setting, prevention of dermal absorption to single chemicals has been handled through the implementation of hazard indicators raising awareness for individuals handling/using chemicals with a potential for dermal absorption. Following description of clear inconsistencies [52], much work has been done internationally to develop better and more consistent hazard indicators [53], but still only taking into account the single substances and not addressing the potential for interaction. Models to handle known occupational co-exposures to several chemicals need to be developed and handled preventively through regulations. However, one difficulty in obtaining this goal is if one of the exposures is not perceived as a dermal exposure to a chemical. Such an exposure could be through the use of sunscreens including nanomaterials or other cosmetic products. On the topic on interaction between unintentional occupational exposures and intentional use of skin care products, much more experimental evidence is needed as most people at work places are not aware of the potential interactions, and because the hazard indicators used at work sites (i.e. skin notations) do not take this potential enhancement into account. Use of topical creams has been demonstrated to change the lipid composition in the skin, and thereby potentially also the percutaneous penetration of other substances [54]. How much this may quantitatively affect penetration rate and time lag is presently not known. Likewise, it is not known for how long after exposure to the cosmetic product that the lipid composition will be affected. Given the very frequent and often long-term use of topical cosmetics, this is an issue that as previously mentioned deserves more attention. The examples described previously demonstrate the continuing need for more experimental data. The challenge will not be to generate in vivo or in vitro experimental data on all imaginable combinations, but to generate sufficient data to allow valid predictions and modelling. The use of mathematical models is, however, not without caveats. Several models have been developed and refined over the last decades to predict steady-state flux or the permeability coefficient (Kp). These models are most often based on physicochemical properties related to partition coefficients between octanol and water, MW, and different measures of molecular volume and steric structures [55–57]. The original algorithm from 1992 based on the Flynn database has been revisited several times, but the overall r2 values for the quantitative structure–activity relationship (QSAR) models related to
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percutaneous penetration seldom exceed 0.7 except when excluding specific outliers, in which case the r2 value may approach 0.9. However, from a preventive perspective, it seems equally important to be able to identify the outliers than to create sophisticated models. Basically, regulatory guidelines based solely on QSAR models may miss the outliers and potentially produce a significant underestimation of the true penetration potential of a chemical. However, for specific groups of chemicals, predictive models may be used to predict Kp or maximal flux for other chemicals belonging to the same group. Prediction of Kp or maximal flux is relevant, but from a preventive perspective, the time lag is almost equally important, as a high Kp not always correlates with a short lag time. Thus, a short time lag and a medium flux may produce higher total absorption during a short exposure period than a higher maximal flux but a long time lag. Further, lack of knowledge on lag time may jeopardize biological monitoring based on blood sampling shortly after dermal exposures to a chemical with long time lag by producing significant underestimation of the actual exposure. Moreover, most modelling is presently performed based on well-known physiochemical determinants predicting the percutaneous penetration of single chemicals [3,55], but efforts are needed to allow also for valid predictions on absorption into and through compromised skin after exposure to combinations of chemicals. It can be concluded that we continue to be in need of data on percutaneous penetration from well-validated experimental models to help refine in silico models and to find and describe those chemicals and/or exposure situations, where reality deviates from the predictive mathematical models. A range of experimental in vitro and in vivo approaches exist, and each model has strengths and limitations. Before planning and conducting the experimental work, some initial and important considerations and questions therefore need to be addressed. What are the reasons for conducting this particular study? What question is the study expected to answer, and more importantly, what can we use it for? Is it possible to relate the experimental set-up to a real-life scenario? Does the study method chosen meet the required conditions for extrapolations and predictions - and will our results reflect the real world scenarios or can they potentially mislead us, thereby allowing unacceptable exposure to individuals who are not aware of an existing hazard? Acknowledgement Supported by COST Action BM0903 (SKINBAD). References 1 Goldsmith LA. My organ is bigger than your organ. Arch Dermatol 1990;126:301–2. 2 Magnusson BM, Anissimov YG, Cross SE, Roberts MS. Molecular size as the main determinant of solute maximum flux across the skin. J Invest Dermatol 2004;122:993–9. 3 Nielsen JB, Sorensen JA, Nielsen F. The usual suspects - influence of physicochemical properties on lag time, skin deposition, and percutaneous penetration of nine model compounds. J Toxicol Environ Health A 2009;72:315–23. 4 EFSA. EFSA guidance on dermal absorption. EFSA J 2012;10:10.
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5 OECD. Guidance notes for the estimation of dermal absorption values. Series on testing and assessment no 156. 2011. 6 OECD-428. OECD guidelines for testing of chemicals. Skin absorption: In vitro method, 2004. 7 WHO. Dermal Absorption. Environmental Health Criteria 235. World Health Organization, Geneva, 2006. 8 Pendlington R. In vitro percutaneous absorption measurements. In: Chilcott RP, Price S (eds). Principles and Practice of Skin Toxicology. Wiley & Sons, Chichester, UK, 2008;129–48. 9 Franz TJ. Percutaneous absorption on the relevance of in vitro data. J Invest Dermatol 1975;64:190–5. 10 van de Sandt JJ, van Burgsteden JA, Cage S, Carmichael PL, Dick I, Kenyon S et al. In vitro predictions of skin absorption of caffeine, testosterone, and benzoic acid: a multi-centre comparison study. Regul Toxicol Pharmacol 2004;39:271–81. 11 Wilkinson SC, Maas WJM, Nielsen JB, Greaves LC, van de Sandt JJM, Williams FM. Interactions of skin thickness and physicochemical properties of test compounds in percutaneous penetration studies. Int Arch Occup Environ Health 2006;79:405–13. 12 Holmgaard R, Nielsen JB. Dermal Absorption of Pesticides Evaluation and Prevention. Danish Ministry of Environment, Copenhagen, 2009. 13 Holmgaard R. Skin penetration studied by static diffusion cells, microdialysis and open-flow micrpperfusion - sampling of fentanyl and benzoic acid in human skin ex vivo. University of Southern Denmark, PhD Thesis, 2012. 14 Vallet V, Cruz C, Josse D, Bazire A, Lallement G, Boudry I. In vitro percutaneous penetration of organophosphorus compounds using full-thickness and split-thickness pig and human skin. Toxicol Vitro 2007;21:1182–90. 15 Bronaugh RL, Stewart RF, Simon M. Methods for in vitro percutaneous.absorption studies. 7. Use of excised human-skin. J Pharm Sci 1986;75:1094–7. 16 Hotchkiss SAM, Hewitt P, Caldwell J, Chen WL, Rowe RR. Percutaneous.absorption of nicotine-acid, phenol, benzoic acid and triclopyr butoxyethyl ester through rat and human skin in vitro further validation of an in vitro model by comparison with in vivo data. Food Chem Toxicol 1992;30:891–9. 17 van de Sandt JJM, Meuling WJA, Elliott GR, Cnubben NHP, Hakkert BC. Comparative in vitro-in vivo percutaneous absorption of the pesticide propoxur. Toxicol Sci 2000;58:15–22. 18 Bronaugh RL, Stewart RF. Methods for in vitro percutaneous absorption studies IV: the flow-through diffusion cell. J Pharm Sci 1985;74:64–7. 19 Williams AC, Barry BW. Penetration enhancers. Adv Drug Deliv Rev 2004;56:603–18. 20 Bommannan D, Potts RO, Guy RH. Examination of the effect of ethanol on human stratum-corneum in vivo using infrared-spectroscopy. J Controlled Release 1991;16:299–304. 21 Ghanem AH, Mahmoud H, Higuchi WI, Liu PC, Good WR. The effects of ethanol on the transport of lipophilic and polar permeants across hairless mouse skin - methods validation of a novelapproach. Int J Pharm 1992;78:137–56. 22 Dias M, Naik A, Guy RH, Hadgraft J, Lane ME. In vivo infrared spectroscopy studies of alkanol effects on human skin. Eur J Pharm Biopharm 2008;69:1171–5. 23 Pershing LK, Bakhtian S, Poncelet CE, Corlett JL, Shah VP. Comparison of skin stripping, in vitro release, and skin blanching response methods to measure dose response and similarity of triamcinolone acetonide cream strengths from two manufactured sources. J Pharm Sci 2002;91:1312–23. 24 Zipfel WR, Williams RM, Webb WW. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 2003;21:1368– 76. 25 Laiho LH, Pelet S, Hancewicz TM, Kaplan PD, So PTC. Two-photon 3-D mapping of ex vivo human skin endogenous
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fluorescence species based on fluorescence emission spectra. J Biomed Opt 2005;10:024016. Mateus R, Abdalghafor H, Oliveira G, Hadgraft J, Lane ME. A new paradigm in dermatopharmacokinetics - Confocal Raman spectroscopy. Int J Pharm 2013;444:106–8. Stamatas GN, Kollias N. In vivo documentation of cutaneous inflammation using spectral imaging. J Biomed Opt 2007;12:051603. Ney M, Abdulhalim I. Modeling of reflectometric and ellipsometric spectra from the skin in the terahertz and submillimeter waves region. J Biomed Opt 2011;16:067006. Anderson C, Andersson T, Molander M. Ethanol absorption across human skin measured by in vivo microdialysis technique. Acta Derm Venereol 1991;71:389–93. Chaurasia CS, Muller M, Bashaw ED, Benfeldt E, Bolinder J, Bullock R et al. AAPS-FDA workshop white paper: microdialysis principles, application and regulatory perspectives. Pharm Res 2007;24:1014–25. Holmgaard R, Nielsen JB, Benfeldt E. Microdialysis Sampling for Investigations of Bioavailability and Bioequivalence of Topically Administered Drugs: current State and Future Perspectives. Skin Pharmacol Physiol 2010;23:225–43. Trajanoski Z, Brunner GA, Schaupp L, Ellmerer M, Wach P, Pieber TR et al. Open-flow microperfusion of subcutaneous adipose tissue for on-line continuous ex vivo measurement of glucose concentration. Diabetes Care 1997;20:1114–21. Holmgaard R, Benfeldt E, Nielsen JB, Gatschelhofer C, Sorensen JA, H€offerer C et al. Comparison of Open-Flow Microperfusion and Microdialysis Methodologies When Sampling Topically Applied Fentanyl and Benzoic Acid in Human Dermis Ex Vivo. Pharm Res 2012;29:1808–20. Bodenlenz M, Hofferer C, Magnes C, Schaller-Ammann R, Schaupp L, Feichtner F et al. Dermal PK/PD of a lipophilic topical drug in psoriatic patients by continuous intradermal membrane-free sampling. Eur J Pharm Biopharm 2012;81:635–41. Groth L, Serup J. Cutaneous microdialysis in man: effects of needle insertion trauma and anaesthesia on skin perfusion, erythema and skin thickness. Acta Derm Venereol 1998;78:5–9. Kezic S, Nielsen JB. Absorption of chemicals through compromised skin. Int Arch Occup Environ Health 2009;82:677–88. Grandjean P. Skin penetration - hazardous chemicals at work: Taylor and Francis, 1990. Benfeldt E, Serup J, Menne T. Effect of barrier perturbation on cutaneous salicylic acid penetration in human skins: in vivo pharmacokinetics using microdialysis and non-invasive quantification of barrier function. Br J Dermatol 1999;140:739–48. Nielsen JB. Percutaneous penetration through slightly damaged skin. Arch Dermatol Res 2005;296:560–7. Ortiz PG, Hansen SH, Shah VP, Menne T, Benfeldt E. The effect of irritant dermatitis on cutaneous bioavailability of a metronidazole formulation, investigated by microdialysis and dermatopharmacokinetic method. Contact Derm 2008;59:23–30. Palmer CNA, Irvine AD, Terron-Kwiatkowski A, Zhao YW, Liao HH, Lee SP et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 2006;38:441–6. Kezic S, Kemperman PMJH, Koster ES, de Jongh CM, Thio HB, Campbell LE et al. Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum. J Invest Dermatol 2008;128:2117–9. O’Regan GM, Sandilands A, McLean WHI, Irvine AD. Filaggrin in atopic dermatitis (Reprinted from J Allergy Clin Immunol vol 122, pg 689-93, 2008). J Allergy Clin Immunol 2009; 124:R2–6. Ortiz PG, Hansen SH, Shah VP, Menne T, Benfeldt E. Impact of adult atopic dermatitis on topical drug penetration: assessment by
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cutaneous microdialysis and tape stripping. Acta Derm Venereol 2009;89:33–8. Jakasa I, Koster ES, Calkoen F, McLean WHI, Campbell LE, Bos JD et al. Skin barrier function in healthy subjects and patients with atopic dermatitis in relation to filaggrin loss-of-function mutations. J Invest Dermatol 2011;131:540–2. Liu KH, Kim JH. In vitro dermal penetration study of carbofuran, carbosulfan, and furathiocarb. Arch Toxicol 2003;77:255–60. Buist HE, van de Sandt JJM, van Burgsteden JA, de Heer C. Effects of single and repeated exposure to biocidal active substances on the barrier function of the skin in vitro. Regul Toxicol Pharmacol 2005;43:76–84. Nielsen JB, Nielsen F, Sorensen JA. In vitro percutaneous penetration of five pesticides-effects of molecular weight and solubility characteristics. Ann Occup Hyg 2004;48:697–705. Mills PC, Magnusson BM, Cross SE. Penetration of a topically applied nonsteroidal anti-inflammatory drug into local tissues and synovial fluid of dogs. Am J Vet Res 2005;66:1128–32. Korinth G, Geh S, Schaller KH, Drexler H. In vitro evaluation of the efficacy of skin barrier creams and protective gloves on percu-
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taneous absorption of industrial solvents. Int Arch Occup Environ Health 2003;76:382–6. Nielsen JB. Natural oils affect the human skin integrity and the percutaneous penetration of benzoic acid dose-dependently. Basic Clin Pharmacol Toxicol 2006;98:575–81. Nielsen JB, Grandjean P. Criteria for skin notation in different countries. Am J Ind Med 2004;45:275–80. Sartorelli P, Ahlers HW, Alanko K, Chen-Peng C, Cherrie JW, Drexler H et al. How to improve skin notation. Position paper from a workshop. Regul Toxicol Pharmacol 2007;49:301–7. Monteiro-Riviere NA, Inman AO, Mak V, Wertz P, Riviere JE. Effect of selective lipid extraction from different body regions on epidermal barrier function. Pharm Res 2001;18:992–8. Potts RO, Guy RH. Predicting Skin Permeability. Pharm Res 1992;9:663–9. Moss GP, Dearden JC, Patel H, Cronin MTD. Quantitative structure-permeability relationships (QSPRs) for percutaneous absorption. Toxicol Vitro 2002;16:299–317. Fitzpatrick D, Corish J, Hayes B. Modelling skin permeability in risk assessment - the future. Chemosphere 2004;55:1309–14.
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Percutaneous Penetration - Methodological Considerations Rikke Holmgaard1, Eva Benfeldt2 and Jesper B. Nielsen3 Department of Orthopedic Surgery, Køge Sygehus, Køge, Denmark, 2Department of Dermatology, University of Copenhagen, Roskilde Hospital, Roskilde, Denmark and 3Institute of Public Health, University of Southern Denmark, Odense, Denmark
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(Received 4 October 2013; Accepted 18 December 2013) Abstract: Studies on percutaneous penetration are needed to assess the hazards after unintended occupational skin exposures to industrial products as well as the efficacy after intended consumer exposure to topically applied medicinal or cosmetic products. During recent decades, a number of methods have been developed to replace methods involving experimental animals. The results obtained from these methods are decided not only by the chemical or product tested, but to a significant degree also by the experimental set-up and decisions made by the investigator during the planning phase. The present MiniReview discusses some of the existing and well-known experimental in vitro and in vivo methods for studies of percutaneous penetration together with some more recent and promising methods. After this, some considerations and recommendations about advantages and limitations of the different methods and their relevance for the prediction of percutaneous penetration are given. Which method to prefer will depend on the product to be tested and the question asked. Regulatory guidelines exist for studies on percutaneous penetration, but researchers as well as regulatory bodies need to pay specific attention to the vehicles and solvents used in donor and sampling fluids so that it reflects in-use conditions as closely as possible. Based on available experimental data, mathematical models have been developed to aid predictions of skin penetration. The authors question the general use of the present mathematical models in hazard assessment, as they seem to ignore outliers among chemicals as well as the heterogeneity of skin barrier properties and skin conditions within the exposed populations.
This MiniReview will discuss some of the existing and wellknown experimental in vitro and in vivo models together with some more recent and promising models and give some considerations and recommendations about advantages and limitations of the various models and their relevance for predictions of percutaneous penetration. An overview of the methodologies discussed can be found in table 1. Dermal exposure may be unintentional after environmental or occupational exposure or intentional after the use of topically applied medication or use of cosmetic products. In all cases, the assessment of the potential for percutaneous penetration or temporary deposition within the skin is an essential element in assessing risk as well as efficacy after dermal exposures. Decades of preventive efforts have decreased the inhalational exposures at work places. At the same time, topically applied pharmaceuticals are being used ever more often. Together, this has increased the relative importance of dermal exposures. Knowledge concerning percutaneous penetration and the potential to reach target sites closes the gap from exposure assessment and hazard identification to risk assessment. If a substance is unable to penetrate the stratum corneum (SC) or Author for correspondence: Jesper Bo Nielsen, Institute of Public Health, University of Southern Denmark, J.B. Winsløws Vej 9b, 2nd floor, 5000 Odense C, Denmark (fax +45 6550 3682, e-mail [email protected]).
affect the skin barrier function in any way, then the need for further assessment of risk becomes less evident. If a substance penetrates the skin or reaches targets within the skin, specific information is needed to qualify the hazard and risk assessment. Thus, information on penetration kinetics, including rate of penetration (flux), Lag-time and temporary deposition in different skin layers, will be needed. The ultimate goal of skin penetration research is to assess the risk to human beings after dermal exposure to hazardous chemicals. Consequently, results from in vivo studies in human beings, including skin sampling, will remain the gold standard in skin penetration studies. In the present MiniReview, a short presentation of three more recent experimental human methodologies (microdialysis, open-flow microperfusion and spectroscopy) is included together with a more traditional approach based on tape stripping. For ethical, logistical and financial reasons, in vivo studies in human beings will not be able to cover the increasing need for data on percutaneous penetration. Animal studies have been used extensively and are still being used. However, acknowledging both physiological and structural differences between species, which may jeopardize the extrapolation of results from animals to human beings, and the implementation of the European REACH program followed by the more general political urge to reduce, refine and replace studies in experimental animals, the need for validated in vitro or in silico models to study percutaneous penetration has increased.
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Table 1. Advantages and limitations related to different methods for studying percutaneous penetration. The + and tioned defining feature, and + to +++ indicates if it is more or less characteristic for the method.
indicates if the method has the men-
Method Advantages/limitations
Static cells
Flow through
Tape stripping
Raman Spectroscopy
DMD
OFM
In vitro In vivo Full-thickness skin Dermatomed skin Limited time consumption Minimally invasive Continuous sampling Multiple application sites Simple design Low cost Good reproducibility
+
+
+ + +
+ + +
+ + +
+ + +
+ + +
+ + + + ++
+ + + + ++
+ + + + + ++
+ +++ + + +
+ + +
+ + +
?
+
+
Furthermore, due to the increasing number of chemicals to which human beings are exposed, data from clinical and experimental studies will not be able to satisfy the need for quantitative information, and mathematical models for predicting penetration will therefore be necessary. The validity of these in silico models will, however, still depend on the continued input of relevant data from experimental studies on percutaneous penetration. For all the above reasons, studies on percutaneous penetration are highly relevant. In these cases, the OECD guidance is to combine knowledge from different experimental models (multimodel approach) to mitigate the drawbacks from relying on a single model. Skin penetration research can be considered very versatile due to the different areas of interests mentioned previously. Over time, different models have been used to gain knowledge of kinetics related to pharmaceuticals, pesticides and other industrial chemicals and products. All models have their advantages and limitations. The model of choice will depend on the research question to be answered, because different models may provide the researcher with different types of information. When planning experimental studies, awareness of the advantages, limitations and model-specific challenges makes it possible to design an experimental study in agreement with the scientific question to be answered and the penetrant of choice. Skin Structure The skin is the largest single organ of the body, accounting for more than 5.5% of the body mass; in average about 4 kg covering 1.7 m² depending on the height and weight of the individual [1]. The human skin surface is continually exposed to chemicals, mechanical injury, microorganisms, UV-light, temperature variations and water, and the most important function of the skin is to act as a barrier against these exposures. Besides barrier properties against exogenous exposures, the skin helps maintaining homoeostasis. The skin can be divided into the upper epidermis, underneath is the more vascular dermis and below the subcutaneous layers (fig. 1). Topically applied substances have to penetrate the avascular and
+ + + ++
Fig. 1. Schematic illustration of the skin structure.
lipophilic SC and continue through the more aqueous lower epidermis and dermis to reach the systemic circulation. Lipophilic substances will easily cross the SC, but the penetration rate will decrease as it reaches the hydrophilic epidermis and dermis – leading to increased deposition and giving the appearance of a reservoir – the reservoir effect. Determinants of Percutaneous Penetration Percutaneous penetration will – for the vast majority of exogenous chemicals – occur via passive diffusion. The exception to this generalization concerns larger or protein-bound molecules, which may use carrier-mediated transportation besides passive diffusion. The passive diffusion process follows Fick’s law, which defines flux as the multiplication of the concentration differences across the membrane and a concentrationindependent constant, the chemical-specific permeability coefficient Kp. Thus, the penetration rate (flux) will be proportional to the experimentally chosen concentration gradient across the membrane and if the concentration gradient diminishes during the experimental period, the flux will also be reduced. This is evident in experiments with finite doses applied on the skin and/or if the concentration of the chemical
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is allowed to become significant on the lower side of the skin. The latter can be practically avoided by having a sufficiently large volume of receptor fluid or by continuously replacing the receptor fluid with fresh receptor fluid. The permeability coefficient (Kp) has been described to depend mainly on the molecular weight (MW), the octanol/water partition coefficient (Kow) and molecular size (stereochemistry), whereas other chemical characteristics such as melting point, hydrogen bonding acceptor capability [H(a)] only marginally affect penetration rates [2,3]. Besides these determinants, vapour pressure, ionization (which is pH-dependent) and susceptibility to protein binding will affect the concentration of unbound and uncharged chemical available for percutaneous penetration at the surface of the skin at any time during the experimental period. Thus, if a chemical has a sufficient vapour pressure, a substantial part of the applied chemical may evaporate and thereby significantly reduce the concentration gradient and the observed flux. Besides the chemical-specific characteristics, the passive diffusion will also depend on experimental conditions related to the physicochemical environment at the experimental setting. In the in vivo situation, the absorbed chemical will reach the lymph or blood circulation after penetrating the upper layers of the skin. To mimic this situation, the sampling fluid applied experimentally for sampling the penetrant should have relevant physico-chemical characteristics related to solubility and pH; otherwise, experiments may under- or overestimate the true in vivo flux. Likewise, if a chemical is not applied as neat chemical, it will be dissolved in a solvent, which may in turn affect the penetration characteristics. This is why it needs to be well argued if the chemical is not applied under user-relevant conditions. The above-mentioned determinants are general to all in vivo and in vitro experimental models, but model-specific characteristics posing challenges to the transferal of experimentally observed penetration rates or time lag also exist. To facilitate a harmonized interpretation of in vitro data from different experimental models, a number of guidance documents have been published [4–7]. In the following sections, some of these models will be discussed under each type of experimental model. Skin Penetration Models for in vitro Studies The first in vitro model to study skin penetration was developed in the 1940s in response to the threat of World War II chemical warfare agents intended for inhalational as well as dermal exposure [8]. The original model resembles the presently used static and flow-through diffusion cells. The static diffusion cell, also known as the Franz diffusion cell (fig. 2), has been one of the most used in vitro models in skin penetration research since 1975 [9]. In the mid-eighties, the flow-through system was developed (fig. 3), and it is used for the same purposes as the static model. Both static and flow-through diffusion cells will work with full thickness as well as epidermal skin barriers [10,11]. Various guidelines indicate that the skin samples that may be used during in vitro studies are split thickness [6] or when justified, full thickness
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Fig. 2. Illustration of the static diffusion cell. The penetrating substance is diffusing from the donor chamber through the skin to the receptor chamber from where it is collected through the collection pipe. The ‘○’ symbolizes the penetrating substance.
Fig. 3. Illustration of the flow-through system. The penetrating substance symbolised by ‘○’ is sampled in the passing receptor fluid and collected in vials from the outlet tube.
up to 1 mm. Full thickness means that the upper approximately 1 mm of the skin including SC, epidermis and part of dermis is mounted in the diffusion cell, whereas epidermal barriers mean that the upper 200–400 lm skin has been separated (e.g. through techniques such as dermatome or heat separation) before being mounted in the diffusion cell. In both models, a skin sample is mounted between a donor chamber and a receptor chamber with the SC side towards the donor chamber. Skin from both human donors and experimental animals may be used, but human skin samples are preferred as it will avoid the use of experimental animals and consequently also avoid extrapolation between species when data are being used. Irrespective of the donor source, the barrier integrity should be checked before application of test substances. The basic procedure is that the penetrant of choice is applied on the donor side, and percutaneous penetration can be measured after sampling from the receptor side [6] – figs 2 and 3. Specific OECD guidelines have been developed [6] for both models, and for regulatory purposes both models are acceptable, though each of them have their advantages and limitations [12,13].
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In vitro techniques are ideal for screening percutaneous penetration of large numbers of topically applied substances, as experimental cost and time consumption are low. Ethical considerations are fewer than for in vivo studies and relates to the use of experimental animals or the consent to use of human skin samples obtained from operative procedures, that is, typically surgical waste. Many studies would be hazardous if conducted in vivo, for example, studies regarding chemical warfare agents [14]. When comparing data between studies, the in vitro methods have the advantage of a simple design, great predictability and little variability. Consequently, these methods have been easy to standardize. Another advantage can be the possibility of working with radiolabelled substances, which is associated with easy quantification of penetration of the substance. However, the researcher needs to take notice that the radiolabel may be separated from the substance itself due to enzymatic degradation/metabolism during the penetration pathway, and quantification of penetration by counting the radiolabel will – in that case – be erroneous. Due to a lack of biochemical, physiological and immunological systems (missing skin metabolism, lack of blood flow, etc.) in vitro studies do not reproduce real physiological conditions. However, in vitro studies have – when thoughtfully designed – been shown to be able to predict skin penetration in vivo [15–17]. As most substances penetrate the skin by passive diffusion, an in vitro method may be an excellent method for initial studies or, as mentioned previously, for high-throughput screening of topically applied drugs and other substances. The two in vitro methods mentioned previously – the static diffusion cell and the flow-through system – both have advantages and challenges [12] and the choice of cell needs to be carefully considered in relation to the research question posed. The flow-through system mimics the microcirculation in the skin as the continuous flow in the receptor chamber removes the substance once it has permeated the skin. This is an advantage when dealing with substances with a low solubility in the receptor fluid, and sink conditions are also easier maintained in studies running for longer time periods compared with the method based on static diffusion cells, where the concentration of the penetrant in the receptor chamber slowly increases during the experimental period. In the flow-through system, the length of the outlet tubing will affect the time lag, especially at low flow rates, and the complexity of the method – compared with the simpler and less expensive version based on the static cells – is technically more challenging. Both in vitro models will work with finite and infinite dosing. Use of infinite doses will allow estimation of time lag, and subsequently, the maximal flux from which the apparent permeability coefficient, Kp, can be calculated. Infinite dosing will, however, also very often keep the topical side of the skin covered with donor fluid throughout the experimental period, which is oftentimes different form the in vivo exposure situation. However, in transdermal therapeutic delivery systems (TTS), the occlusive environment underlying the patch will make the underlying skin moist, and in this case, the resemblance to a live situation may be closer. This is also the case in occupational toxicology where risk assessment in occlusive/
occluded environments is relevant, for example, when employees use gloves when handling toxic substances, which may – due to incorrect choice or use of gloves or damage to the glove membrane – become trapped between glove and skin. Use of finite dosing will more closely resemble most in vivo situations in relation to infrequent short-term occupational exposures (e.g. splashes) or the use of cosmetic or medicinal products. Use of finite doses will allow an estimate of the fractional permeation of the applied dose. The amount of a substance permeating the skin depends on the area of skin available for penetration, and calculation of different measures for permeation is therefore adjusted to that area. If the entire area of the donor cell is not covered, the adjustment is flawed, and the calculations will overestimate the actual permeation rate. The present OECD guideline [6] suggests adding 25 lL/cm2 to the donor chambers in flowthrough as well as static cells, which corresponds to a thickness of the donor fluid of 250 lm. If the natural surface tension in the donor solution is not reduced by adding detergents or solvents, this volume is (at least in our hands) too small to assure full coverage of the entire area, especially as the skin surface, besides having a natural uneven surface, has a tendency to be slightly thicker in the middle of the donor chamber. Our suggestion is to increase the volume applied to the cells to 50 lL/cm2. Both methods are also ideal for studies exploring the reservoir effect of the skin [18]. Furthermore, the effect of different penetration enhancers, which are often applied to a solution in order to change the skin permeability, can easily and inexpensively be studied in different applications. Enhancers or detergents present in the receptor solution may interact with the SC by causing either disruption of the SC lipids, structural protein changes or improved partitioning of the drug [19]. Ethanol, as an example, makes reversible changes to the skin barrier described in different ways as intercellular lipid removal [20], lipid fluidization [21] lipid disordering or lipid extraction and modulation of the lipid barrier [22]. The effect of any enhancer or enhancer-added solution can be studied in vitro without ethical considerations. Skin Penetration Models for in vivo Studies In the development of drugs for human topical use or in risk assessment in occupational settings, the most directly transferrable kinetic research is conducted by in vivo human studies, because there is no need for extrapolation to human conditions as is the case after animal or in vitro studies. Some methods can be used both in vitro and in vivo. These methods can be used in, for example, the development of new drugs from the laboratory concept to the final product tested in clinical settings. By avoiding a change in methodology during the path of development, an intermethodological variability can be avoided. Among the presently developed in vivo methods, a few also have the advantage of chronological real-time continuous sampling – dermal microdialysis and open-flow microperfusion are mentioned below – which is a relevant feature in pharmaco- and toxicokinetic research.
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Tape Stripping Tape stripping (TS) has become one of the traditional investigative methods in in vivo skin penetration research and is known as a non-invasive method, which can be used to determine the kinetics of penetrants. By repeated application and removal of adhesive tape to the same site on the skin, it is possible to remove and sample successively layers of the SC and determine the absorption profile and potential temporary deposit within the upper SC of the penetrant at the time of sampling (fig. 4). After stripping a specified skin area, the penetrant is extracted from the tape and subsequently analysed with traditional analytical methods. The sampling method has some manual steps, which requires trained personnel to avoid significant interpersonal and interlaboratory variability [23]. Spectroscopy In the early 1990s, spectroscopy was developed for skin penetration studies as a method for SC research. Spectroscopic methods are optical methods based on light scattering and generally have until recently had only but a limited depth range confined to the SC or just below the SC. More recently, imaging techniques based on confocal fluorescence microscopy have been developed that provide a pictorial description of skin structures including visualization of the different skin strata, which enables optical sectioning of the specimen to be performed in a non-invasive manner. Two major confocal fluorescence microscopy techniques have been used in skinrelated experiments: laser scanning confocal fluorescence microscopy (LSCM) and two-photon excitation fluorescence microscopy (TPEFM). Both techniques are suitable to explore the epidermis; however, TPEFM has particular advantages over LSCM. Two-photon excitation is a non-linear process in which a fluorophore absorbs two photons simultaneously. These photons are generally of low energy compared with the one-photon absorption process in conventional fluorescence microscopy techniques, resulting in an overall low extent of photobleaching and photodamage to the specimens [24]. In addition, as infrared light is used as the excitation source in TPEFM, the penetration depth in thick specimens can be up to 800 lm, allowing in-depth, three-dimensional examination of biological specimens. This penetration depth is far superior to
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that obtained using LCSM, where a maximum depth of approximately 150–200 lm is generally obtained [25]. The first publication showing concordance between drug penetration investigated by Confocal Raman spectroscopy (Confocal microscopy in combination with Raman spectroscopy) and the TS methodology was recently published [26]. Other spectroscopic methods such as Near IR and Terahertz spectroscopy are currently being developed [27,28]. A challenge for all these methods is that they are not quantitative by nature, though analytical ways to semi-quantitative measurements are being developed. The methods are fully non-invasive and can provide detailed information about the layers studied. Given that a test molecule can act as a fluorophore, it is expected to become possible to study specific depositional patterns during the absorption process for such molecules. As the methods are applicable in vivo as well as in in vitro settings, it should be possible to use these imaging techniques to study experimentally induced structural artefacts in experimental settings. Microdialysis Dermal MD (DMD) was first described by Anderson et al. in 1991 in a human study concerning percutaneous absorption of solvents, using ethanol as penetrating substance and MD sampling in the dermis [29]. Since then, the DMD method (fig. 5) has been used for sampling of a large number of topically applied drugs and other skin penetrants [30] in both healthy and damaged/diseased skin in human beings as well as in animals. DMD can sample endogenous and exogenous substances in all types of tissue by use of a thin catheter with a semi-permeable membrane imitating a small blood vessel. This vessel (the microdialysis probe) is connected to an inlet and an outlet tube perfused with a tissue compatible fluid (perfusate). The exchange of molecules across the membrane occurs by passive diffusion driven by the concentration gradient. The microdialysis probes have specific pore sizes, which set upper limits (cut-off value) for the molecules that can be sampled, but also excludes larger molecules and proteins from entering the sampling fluid. Thus, unless protein has been added to the perfusate, which is only done if it enhances recovery of the substance of interest, then DMD sampling delivers protein and enzyme-free samples, which makes the pre-analytical steps relatively uncomplicated. The method requires prior considerations of the suitability of the substance of interest for microdialysis sampling, as the typical combination seen in many topical medical treatments, that is, a high or very high lipophilicity of the drug and a low drug concentration in the topical product, both make DMD sampling challenging as the resulting samples may be of very low (below LOQ) concentration [31]. Open-flow Microperfusion:
Fig. 4. Tape stripping. Illustration of the sampling of the upper layers of the skin – the stratum corneum (SC) - by repeated tape application and removal of successive layers of SC cells.
In 1997, a similar technique – OFM – was introduced by Trajanoski et al. [32].. OFM is also designed as a continuous tissue-specific sampling method. Where the DMD probe has a
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enzymes and some cells will be included in the sample fluid collected by the catheter. Thus, the resulting sample requires technically more demanding pre-analytical steps before analysis of the sample fluid. The use of in vivo methods has significant ethical implications. In the case of TS, the skin trauma invoked when removing the SC layers is most often negligible leaving only a small temporarily sore area. For DMD and OFM, the considerations concern the necessary skin trauma inflicted when inserting the probe/catheter horizontally in the dermis (figs 5 and 6). Lasting tissue damage is not seen, but the insertion trauma is followed by a histamine release, causing a reversible wheal and flare reaction [35]. Fig. 5. Illustration of the microdialysis probe placed in the dermis, which is sampling increasing dermal drug concentrations after topical drug penetration (modified from Benfeldt and Serup 1999). The ‘○’ symbolizes the penetrating substance.
semi-permeable membrane, the OFM sampling catheter has a membrane-free macroscopically perforated area with unrestricted access to and exchange of solutes in the peri-catheter tissue. Therefore, the OFM technique does not have the same limitations regarding sampling efficacy towards large and/or protein-bound penetrants as the MD technique. Since 2006, the OFM method has been utilized for dermal sampling [33,34] (fig. 6). The method is, due to the open exchange area, relevant for sampling of large and/or lipophilic penetrants, which is where the DMD sampling methodology is often challenged. However, OFM is a more demanding method both technically and labourwise, as the method needs a push- as well as a pull-pump function connected to the sampling catheters to counteract the tendency to induce oedema in the tissue surrounding the probe due to the open exchange area. As a consequence of the open exchange area, proteins,
Fig. 6. Illustration of the open-flow microperfusion catheter placed in the dermis. The topically added substance symbolized by ‘○’ is penetrating through the skin and sampled in the dermis. The push/pull system is connected to the probe and the samples are collected in an exchangeable glass capillary. Markings on the probe make a correct positioning in the skin easy.
From Experimental Observations to the Real World When conducting experimental studies, in vitro as well as in vivo, researchers often take pride in following specified guidelines and standard procedures in the laboratory. This approach increases repeatability and reduces variability in results. However, the human population is most often better characterized by heterogeneity than homogeneity, and a significant fraction of the population suffers from a diseased or otherwise damaged skin barrier for exogenous or endogenous (including genetic) reasons. When we study skin penetration, it is therefore important not only to create reliable results in relation to a normal skin profile but also to adjust the experimental studies to real-life conditions, which are often not as straightforward as in the ‘standard operation procedures’ applied in the laboratory. The use of an experimental model that can be adapted to a real-life situation would therefore be most relevant for the study of situations that may deviate from the average. Known exceptions are damaged or diseased skin [36]. Irrespective of whether the affected skin barrier is caused by blunt trauma, wet work, delipidization of the SC or the presence of dermatitis, a decrease in skin barrier integrity has proven to increase percutaneous penetration [37–40]. A mutation in the filaggrin gene, which is carried by approximately 9% of the European population, is associated with a decrease in natural moisturizing factor of the skin, a key parameter for skin barrier function and a risk factor for the development of dermatitis [41–43]. Patients suffering from dermatitis have previously been shown to have impaired skin barrier function in lesional [44] as well as non-lesional skin [45]. The impact of an impaired skin barrier function may be seen in both the occupational setting but also in penetration and/or reaction to transdermally administered medical products, topical formulations as well as cosmetic products. In some situations, the experimental focus is on percutaneous penetration of the active ingredient and not on the commercial product that consumers are exposed to, which is often a mixture of different substances. The tested individual ingredients are then used to formulate non-tested mixtures. These mixtures have often been demonstrated to have a higher penetration rate than the tested active ingredient [46,47]. The research results from testing pure chemicals are in these
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situations potentially misleading and may affect the risk assessment in occupational settings where an added detergent/ enhancer affects the skin integrity and thereby increase skin permeability [48,49]. This is an area that still lacks relevant experimental data to support mathematical modelling, which will allow valid predictions of the percutaneous penetration of formulated products including if the formulations of already approved products are changed. Often individuals are exposed to several products at the same time. Interactions between two or more unintended dermal exposures in occupational settings have been described, causing increased absorption just as well as an absence of any interaction of practical importance [50,51]. In the occupational setting, prevention of dermal absorption to single chemicals has been handled through the implementation of hazard indicators raising awareness for individuals handling/using chemicals with a potential for dermal absorption. Following description of clear inconsistencies [52], much work has been done internationally to develop better and more consistent hazard indicators [53], but still only taking into account the single substances and not addressing the potential for interaction. Models to handle known occupational co-exposures to several chemicals need to be developed and handled preventively through regulations. However, one difficulty in obtaining this goal is if one of the exposures is not perceived as a dermal exposure to a chemical. Such an exposure could be through the use of sunscreens including nanomaterials or other cosmetic products. On the topic on interaction between unintentional occupational exposures and intentional use of skin care products, much more experimental evidence is needed as most people at work places are not aware of the potential interactions, and because the hazard indicators used at work sites (i.e. skin notations) do not take this potential enhancement into account. Use of topical creams has been demonstrated to change the lipid composition in the skin, and thereby potentially also the percutaneous penetration of other substances [54]. How much this may quantitatively affect penetration rate and time lag is presently not known. Likewise, it is not known for how long after exposure to the cosmetic product that the lipid composition will be affected. Given the very frequent and often long-term use of topical cosmetics, this is an issue that as previously mentioned deserves more attention. The examples described previously demonstrate the continuing need for more experimental data. The challenge will not be to generate in vivo or in vitro experimental data on all imaginable combinations, but to generate sufficient data to allow valid predictions and modelling. The use of mathematical models is, however, not without caveats. Several models have been developed and refined over the last decades to predict steady-state flux or the permeability coefficient (Kp). These models are most often based on physicochemical properties related to partition coefficients between octanol and water, MW, and different measures of molecular volume and steric structures [55–57]. The original algorithm from 1992 based on the Flynn database has been revisited several times, but the overall r2 values for the quantitative structure–activity relationship (QSAR) models related to
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percutaneous penetration seldom exceed 0.7 except when excluding specific outliers, in which case the r2 value may approach 0.9. However, from a preventive perspective, it seems equally important to be able to identify the outliers than to create sophisticated models. Basically, regulatory guidelines based solely on QSAR models may miss the outliers and potentially produce a significant underestimation of the true penetration potential of a chemical. However, for specific groups of chemicals, predictive models may be used to predict Kp or maximal flux for other chemicals belonging to the same group. Prediction of Kp or maximal flux is relevant, but from a preventive perspective, the time lag is almost equally important, as a high Kp not always correlates with a short lag time. Thus, a short time lag and a medium flux may produce higher total absorption during a short exposure period than a higher maximal flux but a long time lag. Further, lack of knowledge on lag time may jeopardize biological monitoring based on blood sampling shortly after dermal exposures to a chemical with long time lag by producing significant underestimation of the actual exposure. Moreover, most modelling is presently performed based on well-known physiochemical determinants predicting the percutaneous penetration of single chemicals [3,55], but efforts are needed to allow also for valid predictions on absorption into and through compromised skin after exposure to combinations of chemicals. It can be concluded that we continue to be in need of data on percutaneous penetration from well-validated experimental models to help refine in silico models and to find and describe those chemicals and/or exposure situations, where reality deviates from the predictive mathematical models. A range of experimental in vitro and in vivo approaches exist, and each model has strengths and limitations. Before planning and conducting the experimental work, some initial and important considerations and questions therefore need to be addressed. What are the reasons for conducting this particular study? What question is the study expected to answer, and more importantly, what can we use it for? Is it possible to relate the experimental set-up to a real-life scenario? Does the study method chosen meet the required conditions for extrapolations and predictions - and will our results reflect the real world scenarios or can they potentially mislead us, thereby allowing unacceptable exposure to individuals who are not aware of an existing hazard? Acknowledgement Supported by COST Action BM0903 (SKINBAD). References 1 Goldsmith LA. My organ is bigger than your organ. Arch Dermatol 1990;126:301–2. 2 Magnusson BM, Anissimov YG, Cross SE, Roberts MS. Molecular size as the main determinant of solute maximum flux across the skin. J Invest Dermatol 2004;122:993–9. 3 Nielsen JB, Sorensen JA, Nielsen F. The usual suspects - influence of physicochemical properties on lag time, skin deposition, and percutaneous penetration of nine model compounds. J Toxicol Environ Health A 2009;72:315–23. 4 EFSA. EFSA guidance on dermal absorption. EFSA J 2012;10:10.
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