Development and validation of a minimally invasive and image-guided tape stripping method to sample atopic skin in children.

BACKGROUND: Molecular skin profiling techniques, typically performed on skin samples taken by punch biopsy, have enhanced the understanding of the pathophysiology of atopic dermatitis (AD), thereby enabling the development of novel targeted therapeutics. However, punch biopsies are not always feasible or desirable, and novel minimally invasive methods such as skin tape stripping have been developed. AIM: To develop, optimize and validate a novel tape stripping method guided by noninvasive in vivo skin imaging to sample atopic skin in children. METHODS: Skin tape stripping-based procedures were compared and optimized using data from 30 healthy controls (HCs: 5 adults, 25 children) and 39 atopic children. Evaluations were guided by high-resolution photography, reflectance confocal microscopy, optical coherence tomography and transepidermal water loss measurements. We assessed and compared adverse events (AEs), the time needed to perform the sampling and the cDNA levels obtained from the tapes. RESULTS: Tape stripping methods based on previously described protocols resulted in erosions in all participants and required a median time of 65 min to perform (range 60-70 min), but provided good cDNA yield. Shorter durations appeared less invasive but provided lower cDNA yield. The final optimized tape stripping protocol, using 11 tapes of 22 mm in diameter, each applied twice for 5 s with 90° rotation, did not produce significant AEs, was completed within a median time of 7 min (range 5-15 min) and provided good cDNA yield both in HCs and atopic children. CONCLUSION: Our minimally invasive method is safe and reliable, and provides reproducible acquisition of cDNA in atopic children. In addition, it enables rapid sample collection, a crucial factor in clinical practice.

Stratum Corneum Sampling to Assess Bioequivalence between Topical Acyclovir Products.

PURPOSE: To examine the potential of stratum corneum (SC) sampling via tape-stripping in humans to assess bioequivalence of topical acyclovir drug products, and to explore the potential value of alternative metrics of local skin bioavailability calculable from SC sampling experiments. METHODS: Three acyclovir creams were considered in two separate studies in which drug amounts in the SC after uptake and clearance periods were measured and used to assess bioequivalence. In each study, a "reference" formulation (evaluated twice) was compared to the "test" in 10 subjects. Each application site was replicated to achieve greater statistical power with fewer volunteers. RESULTS: SC sampling revealed similarities and differences between products consistent with results from other surrogate bioequivalence measures, including dermal open-flow microperfusion experiments. Further analysis of the tape-stripping data permitted acyclovir flux into the viable skin to be deduced and drug concentration in that 'compartment' to be estimated. CONCLUSIONS: Acyclovir quantities determined in the SC, following a single-time point uptake and clearance protocol, can be judiciously used both to objectively compare product performance in vivo and to assess delivery of the active into skin tissue below the barrier, thereby permitting local concentrations at or near to the site of action to be determined.

Comparison of patient (POEM), observer (EASI, SASSAD, TIS) and corneometry measures of emollient effectiveness in children with eczema: findings from the COMET feasibility trial.

BACKGROUND: Eczema affects around 20% of children, but multiple different outcome measures have hampered research into the effectiveness of different treatments. OBJECTIVES: To compare the change in scores and correlations within and between five measures of eczema severity: Patient-Orientated Eczema Measure (POEM), Eczema Area and Severity Index (EASI), Six Area, Six Sign Atopic Dermatitis (SASSAD), Three Item Severity (TIS) and skin hydration (corneometry). METHODS: Data from a feasibility trial that randomized young children with eczema to one of four emollients were used. Participants were followed for 3 months (84 days). Descriptive statistics (by emollient over time) and Spearman's correlation coefficients comparing scores at each time point and absolute change (between adjacent time points) for each outcome measure were calculated. RESULTS: In total, 197 children, mean ± SD age 21·7 ± 12·8 months, were randomized. POEM and TIS appeared to capture a range of eczema severity at baseline, but only POEM had close approximation to normal distribution. Mean POEM, EASI, SASSAD and TIS scores improved month by month, with POEM showing the greatest sensitivity (effect size 0·42). Correlations within POEM, EASI, SASSAD and TIS were moderate to good, decreasing over time. Correlations between measures were strongest for EASI, SASSAD and TIS. By contrast, corneometry scores were more variable, correlated less well over time and were poorly correlated with the other measures. CONCLUSIONS: Except for corneometry, all measures appear to change in relation to emollient use over time and correlate well with themselves. POEM demonstrated the greatest range of scores at baseline and change in eczema severity over the first 28 days.

Skin--'that unfakeable young surface'.

The 20- to 25-year period from the mid-1960s to the early 1990s represents, in many ways, a 'golden era' in the understanding of skin barrier function, percutaneous penetration and (trans)dermal drug delivery. From the ground-breaking mechanistic work of Scheuplein and Blank, and the 'gold standard' in vivo skin absorption experiments of Feldmann and Maibach, through the unravelling of the stratum corneum's structural and physicochemical characteristics that make it such an incredible feat of bioengineering, to the elegant biophysical and modelling studies of Potts, Francoeur and many others, the idea of administering drugs rationally to treat both local and systemic diseases was transformed from something close to the ravings of a lunatic to the reality of significant therapeutic and commercial success. This short article attempts to pick out some of the highlights along the way from the viewpoint of an interested observer and occasional participant in the 'action'. © 2013 S. Karger AG, Basel.

Optimisation of cosolvent concentration for topical drug delivery III--influence of lipophilic vehicles on ibuprofen permeation.

Previously, we have reported the effects of water, ethanol, propylene glycol and various binary and ternary mixtures of these solvents on the permeation of ibuprofen in model membranes and in skin. The present study investigates the influence of lipophilic vehicles on the transport of ibuprofen in silicone membrane and in human skin. The permeation of ibuprofen was measured from mineral oil (MO), Miglyol® 812 (MG) and binary mixtures of MO and MG. The solubility of ibuprofen was 5-fold higher in MG than in MO, however, the permeation of ibuprofen from the pure vehicles and combinations of both was comparable in silicone membrane. Additionally, there were no significant differences in skin permeation for MO and MG vehicles. When the permeation of various hydrophilic and lipophilic vehicles is considered, a trend between flux values for the model membrane and skin is evident (r(2) = 0.71). The findings suggest that silicone membrane may provide information on qualitative trends in skin permeation for vehicles of diverse solubility and partition characteristics.

Assessment of Drug Delivery Kinetics to Epidermal Targets In Vivo.

It has proven challenging to quantify 'drug input' from a formulation to the viable skin because the epidermal and dermal targets of topically applied drugs are difficult, if not impossible, to access in vivo. Defining the drug input function to the viable skin with a straightforward and practical experimental approach would enable a key component of dermal pharmacokinetics to be characterised. It has been hypothesised that measuring drug uptake into and clearance from the stratum corneum (SC) by tape-stripping allows estimation of a topical drug's input function into the viable tissue. This study aimed to test this idea by determining the input of nicotine and lidocaine into the viable skin, following the application of commercialised transdermal patches to healthy human volunteers. The known input rates of these delivery systems were used to validate and assess the results from the tape-stripping protocol. The drug input rates from in vivo tape-stripping agreed well with the claimed delivery rates of the patches. The experimental approach was then used to determine the input of lidocaine from a marketed cream, a typical topical product for which the amount of drug absorbed has not been well-characterised. A significantly higher delivery of lidocaine from the cream than from the patch was found. The different input rates between drugs and formulations in vivo were confirmed qualitatively and quantitatively in vitro in conventional diffusion cells using dermatomed abdominal pig skin.

Effect of Aqueous Cream BP on human stratum corneum in vivo.

BACKGROUND: Aqueous Cream BP is widely prescribed to patients with eczema to relieve skin dryness. The formulation contains sodium lauryl sulphate (SLS), a chemical that is a known skin irritant and a commonly used excipient in personal care and household products. The chronic effects of Aqueous Cream BP application on skin barrier function have not been determined. OBJECTIVES: To characterize and assess skin barrier function of healthy skin after application of Aqueous Cream BP and to study the physical effects of the formulation on the stratum corneum (SC). METHODS: The left and right volar forearms of six human volunteers were each separated into treated and control sides. The treated sides of each forearm were subjected to twice daily applications of Aqueous Cream BP for 4 weeks at the end of which concomitant tape stripping and transepidermal water loss (TEWL) measurements were made. The untreated sides of the forearms were not exposed to any products containing SLS during the study period. RESULTS: Changes in SC thickness, baseline TEWL and rate of increase in TEWL during tape stripping were observed in skin treated with Aqueous Cream BP. The mean decrease in SC thickness was 1·1 µm (12%) (P = 0·0015) and the mean increase in baseline TEWL was 2·5 g m⁻² h⁻¹ (20%) (P < 0·0001). Reduced SC thickness and an increase in baseline TEWL, as well as a faster rate of increase in TEWL during tape stripping, were observed in 16 out of 27 treated skin sites. CONCLUSIONS: The application of Aqueous Cream BP, containing ∼1% SLS, reduced the SC thickness of healthy skin and increased its permeability to water loss. These observations call into question the continued use of this emollient on the already compromised barrier of eczematous skin.

Extraction and quantification of amino acids in human stratum corneum in vivo.

BACKGROUND: Amino acid (AA) levels in stratum corneum (SC) are potential biomarkers of skin health while their systemic levels may be used to diagnose inherited metabolic diseases. OBJECTIVES: To examine reverse iontophoresis, in human volunteers, as a minimally invasive tool to analyse AAs within the skin and subdermally. METHODS: In four volunteers, the amounts of iontophoretically extracted AAs were compared with those determined in the SC following repetitive tape stripping and with the plasma concentrations. Glucose levels, evaluated in the different compartments, were used as a control. RESULTS: SC concentrations of 13 essentially zwitterionic AAs were ∼100-fold higher than the respective plasma levels. Passive and reverse iontophoretic extraction for 4 h did not deplete the SC depot of AAs, a fact reinforced by postextraction tape stripping, which revealed that AAs remained in the SC at this time. In contrast, glucose was much less abundant in the SC and was fully and relatively quickly extracted by reverse iontophoresis. CONCLUSIONS: It follows that reverse iontophoresis is useful for quantifying AAs in the SC and these data are highly correlated with levels obtained by tape stripping. However, reverse iontophoresis is impractical for the routine monitoring of AA plasma concentrations (unlike the situation for glucose, the skin reservoir of which is much smaller).

Disposition of charged nanoparticles after their topical application to the skin.

The objective of this preliminary investigation was to examine the disposition of charged nanoparticles on and within the skin following their topical application and to explore whether the formulations have potential utility for the local delivery of an associated 'active' substance. Three nanoparticles (approx. 100 nm in diameter) were investigated: cationic amino-functionalized polystyrene, an anionic carboxyl-functionalized polystyrene and anionic poly-(L-lactide), into each of which the fluorophore N-(2,6-diisopropylphenyl)perylene-3,4-dicarboximine (PMI) was incorporated. Formulations were applied to excised porcine skin in vitro for 6 h. After cleaning the skin surface following treatment, the skin was either examined by laser scanning confocal microscopy or subjected to repeated tape-stripping and subsequent analysis of the removed stratum corneum (SC) for the presence of PMI. The cationic nanoparticles showed clear affinity for the negatively charged skin surface (in contrast to the anionic carriers) and delivered a significantly greater amount of the model 'active' agent (PMI) into the SC.

Pharmacodynamics and dermatopharmacokinetics of betamethasone 17-valerate: assessment of topical bioavailability.

BACKGROUND: The bioavailability of most topically delivered drugs is difficult to quantify, but is generally believed to be very low. With the exception of the vasoconstrictor assay for corticosteroids, no methodology to quantify the rate and extent of drug delivery to the skin has been validated. Recent research has examined the dermatopharmacokinetic (DPK) technique, which is based on stratum corneum (SC) tape-stripping. OBJECTIVE: To compare the in vivo bioavailability of different topical formulations of betamethasone 17-valerate (BMV) using the vasoconstrictor assay and the DPK method. METHODS: BMV was formulated in different vehicles and the drug concentration was adjusted to either (i) equal thermodynamic activity, or (ii) a range of values up to that corresponding to 80% of maximum thermodynamic activity. Vasoconstriction, an accepted and widely used method to determine bioavailability and bioequivalence of topical steroids, was quantified with a chromameter over 24 h post-removal of the formulation. Drug uptake into the SC was assessed by tape-stripping. RESULTS: BMV at the same thermodynamic activity in different vehicles provoked similar skin blanching responses, while DPK profiles distinguished between the formulations. Further, skin blanching responses and drug uptake into the SC clearly depended upon the absolute BMV concentration applied. However, while the saturable nature of the pharmacodynamic response was clear, the tape-stripping method distinguished unequivocally between the different formulations and different concentrations. CONCLUSIONS: The DPK approach offers a reliable metric with which to quantify transfer of drug from the vehicle to the SC, and may be useful for topical bioavailability and bioequivalence determinations.

Prediction of chemical absorption into and through the skin from cosmetic and dermatological formulations.

BACKGROUND: To date, risk assessment following topical exposure to cosmetic/dermatological formulations cannot be precisely evaluated. OBJECTIVES: To provide a tool for optimization of active permeation into/through skin and for risk assessment. METHODS: A predictive model was developed for estimating the cumulative mass of a chemical absorbed into and across the skin from a cosmetic/dermatological formulation. Account was taken of (i) the ionization state of the chemical, to correct the skin/vehicle partition coefficient; and (ii) the nature of the cosmetic/dermatological formulation. Three specific assumptions were made: firstly, steady-state transport across the skin was achieved despite application of a finite dose of chemical; secondly, vehicle effects were small relative to the precision of the prediction; and, thirdly, each formulation could be treated as an oil-in-water emulsion, in which only that fraction of the chemical in the aqueous phase was available to partition into the stratum corneum. A database of 101 ex vivo human skin experiments involving 36 chemicals was analysed. RESULTS: For 91% of the data, the difference between predicted and experimental values was less than a factor 5; when the aforementioned corrections were not used, on the other hand, only 26% of the data was well predicted. The model was successfully applied to predict skin absorption of two compounds not included in the database, for which in vitro percutaneous penetration from cosmetic vehicles have been measured. CONCLUSION: A model has been developed to predict the mass of a chemical absorbed into and through the skin from a cosmetic or dermatological formulation.

Effects of various vehicles on skin hydration in vivo.

The stratum corneum, the outermost layer of the skin, regulates the passive loss of water to the environment. Furthermore, it is well accepted that drug penetration is influenced by skin hydration, which may be manipulated by the application of moisturizing or oleaginous vehicles. Measurements of transepidermal water loss (TEWL), and of skin hydration using a corneometer, were used to assess the effect of different vehicles on stratum corneum barrier function in vivo in human volunteers. A microemulsion significantly increased skin hydration relative to a reference vehicle based on medium chain triglycerides; in contrast, Transcutol(R) lowered skin hydration. TEWL measurements confirmed these observations.

Optimisation of cosolvent concentration for topical drug delivery - II: influence of propylene glycol on ibuprofen permeation.

The influence of formulation components on drug thermodynamic activity and on membrane properties has not been extensively studied to date. Propylene glycol (PG) has a long history of use as a component of topical pharmaceutical and cosmetic preparations. In the present study we investigated the influence of PG on the solubility and membrane permeability characteristics of ibuprofen in silicone and skin using binary (PG:water) and ternary (ethanol:PG:water) solvent systems. Fluxes were maximum for 70:30 PG:water systems in silicone membrane; however, for experiments conducted with skin, the flux of ibuprofen systematically increased with increasing amounts of PG. For silicone membrane, the flux values of ibuprofen from ternary ethanol:PG:water (25:25:50 and 50:25:25) systems were 1.2- and 1.5-fold higher than the highest values observed from the PG:water systems. A comparison of the data from occluded versus non-occluded studies suggested that the ethanol content was the critical determinant of flux enhancement in these systems. For human skin, a PG:water (50:50) mixture enhanced ibuprofen flux to the same extent as the corresponding ethanol:PG:water (25:25:50) system. However, the ternary system, ethanol:PG:water (50:25:25), demonstrated the greatest enhancement. An analysis of the partition and diffusion parameters for experiments conducted with skin confirmed that the influence of PG in the PG:water systems is primarily on the solubility and partitioning behaviour of ibuprofen. The ternary systems also appear to influence skin partition behaviour rather than diffusivity when compared with the permeation parameters of ibuprofen from saturated aqueous solutions.

Influence of ethanol on the solubility, ionization and permeation characteristics of ibuprofen in silicone and human skin.

In the context of topical and transdermal delivery, cosolvents may be expected to enhance solubility, modify drug partitioning behaviour and alter skin barrier properties. In the present study, we have investigated systematically the influence of increasing amounts of the cosolvent ethanol on the solubility, ionization, and permeability characteristics of ibuprofen in silicone and in skin. The possibility of ibuprofen self-association was also examined. As the amount of ethanol was raised from 0 to 100%, ibuprofen solubility was enhanced 5,500-fold relative to its aqueous solubility. Up to 50%, the pK(a) of ibuprofen shifted from 4.44 to 5.68. Infrared spectroscopy confirmed the presence of ibuprofen in both monomer and dimer forms in vehicles containing 75 and 100% ethanol. In silicone membranes, the flux of ibuprofen increased 8-fold over the range of ethanol concentrations studied (0-100%) relative to aqueous solutions, with the highest flux observed for 100% ethanol. However, in skin, the flux of ibuprofen was optimal for 50:50 and 75:25 ethanol-water vehicles (>10-fold flux enhancement). The lower ibuprofen flux from pure ethanol may reflect the alcohol's ability to dehydrate human skin. In general, the flux data confirm that increasing ethanol content enhances the solubility of ibuprofen in silicone and human skin as well as in the vehicle itself.

In vivo infrared spectroscopy studies of alkanol effects on human skin.

Many studies investigate the permeation of actives through the skin and ignore the role of excipients. The solvents used in formulations will undoubtedly penetrate the skin where they can have a number of effects. They can extract skin lipids, they can alter the fluidity of the lipids and they can alter the polarity of the skin. The degree to which they do this and the depth into the skin where this occurs will depend on the uptake kinetics. The problem is to distinguish the different effects. Using ATR-FTIR and deuterated materials this can be achieved in vivo. The aim of the present study was to study the higher alkanols (hexanol, octanol, decanol) in vivo using a combination of ATR-FTIR spectroscopy and tape stripping. Studies conducted in vivo using deuterated vehicles confirmed the lipid extraction effects of d-hexanol and d-octanol, whereas d-decanol did not change skin lipid content. The uptake of d-decanol was higher than for the other vehicles consistent with previous observations on mouse skin for alkanols of increasing chain length. In general, solvent uptake was proportional to the induced shift in the C-H stretching frequency. Lipid disorder was induced by all vehicles studied in vivo and was proportional to the amount of vehicle present in the skin.

Transdermal delivery of peptides by iontophoresis.

Transdermal administration by iontophoresis (enhanced transport via the skin using the driving force of an applied electric field) has been successfully demonstrated but no formal relationship between peptide sequence/structure and efficiency of delivery has been established. There are notable examples, such as the lipophilic leutinizing hormone releasing hormone (LHRH) analogs, Nafarelin and Leuprolide, that exhibit down-regulation of their own transport across the skin under the influence of an iontophoretic current. The hypothesis that this phenomenon is due to neutralization of the skin's net negative charge by these cationic peptides was examined with LHRH oligopeptides. The impact of these compounds on the electroosmotic flow of solvent into the skin, which is induced by iontophoresis and which contributes significantly to the electrotransport of large, positively charged ions, was examined and quantified. Close juxtaposition of cationic and lipophilic residues profoundly inhibited electroosmosis and, presumably, peptide flux. The results indicate that the lipophilicity of the oligopeptides facilitates van der Waals interactions with hydrophobic patches along the transport route, thereby permitting the positively charged oligopeptide to interact with carboxylate side chains that give the skin its net negative charge at neutral pH. The lipophilic, cationic oligopeptide, therefore, becomes anchored in the transport path, neutralizing the original charge of the membrane, and completely altering the permselective properties of the skin.

Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

The threshold of toxicological concern (TTC) has been used for the safety assessment of packaging migrants and flavouring agents that occur in food. The approach compares the estimated oral intake with a TTC value derived from chronic oral toxicity data for structurally-related compounds. Application of the TTC approach to cosmetic ingredients and impurities requires consideration of whether route-dependent differences in first-pass metabolism could affect the applicability of TTC values derived from oral data to the topical route. The physicochemical characteristics of the chemical and the pattern of cosmetic use would affect the long-term average internal dose that is compared with the relevant TTC value. Analysis has shown that the oral TTC values are valid for topical exposures and that the relationship between the external topical dose and the internal dose can be taken into account by conservative default adjustment factors. The TTC approach relates to systemic effects, and use of the proposed procedure would not provide an assessment of any local effects at the site of application. Overall the TTC approach provides a useful additional tool for the safety evaluation of cosmetic ingredients and impurities of known chemical structure in the absence of chemical-specific toxicology data.

Topical bioavailability of diclofenac from locally-acting, dermatological formulations.

Assessment of the bioavailability of topically applied drugs designed to act within or beneath the skin is a challenging objective. A number of different, but potentially complementary, techniques are under evaluation. The objective of this work was to evaluate in vitro skin penetration and stratum corneum tape-stripping in vivo as tools with which to measure topical diclofenac bioavailability from three approved and commercialized products (two gels and one solution). Drug uptake into, and its subsequent clearance from, the stratum corneum of human volunteers was used to estimate the input rate of diclofenac into the viable skin layers. This flux was compared to that measured across excised porcine skin in conventional diffusion cells. Both techniques clearly demonstrated (a) the superiority in terms of drug delivery from the solution, and (b) that the two gels performed similarly. There was qualitative and, importantly, quantitative agreement between the in vitro and in vivo measurements of drug flux into and beyond the viable skin. Evidence is therefore presented to support an in vivo - in vitro correlation between methods to assess topical drug bioavailability. The potential value of the stratum corneum tape-stripping technique to quantify drug delivery into (epi)dermal and subcutaneous tissue beneath the barrier is demonstrated.

In situ detection of salicylate in Ocimum basilicum plant leaves via reverse iontophoresis.

The quantitative analysis of salicylate provides useful information for the evaluation of metabolic processes in plants. We report a simple, noninvasive method to measure salicylate in situ in Ocimum basilicum leaves using reverse iontophoresis in combination with cyclic voltammetry at disposable screen-printed electrodes and the concentration of salicylate in basil leaves was found to be 3 mM.