Importance of a suitable working protocol for tape stripping experiments on porcine ear skin: Influence of lipophilic formulations and strip adhesion impairment.

The tape stripping method is a very important tool for dermopharmacokinetic experiments in vitro and the accurate measurement of the removed corneocytes is key for a reliable calculation of a drug's skin penetration behavior. Therefore, various methods to quantify the amount of corneocytes removed with each tape strip have been employed, ranging from gravimetric approaches to protein assays and recently near infrared densitometry (NIR) has become very widely used. As this method is based on a reduction of light intensity, interference of formulation components seems conceivable, as they could scatter light and change the results. In this study, NIR measurements were compared to a protein assay and in addition, the influence of highly lipophilic formulations on the results of tape stripping experiments was investigated as impairment of the adherence of strips has been reported. To this end, different tape stripping protocols were employed. The obtained results ensure suitability of the NIR method and moreover suggest a more pronounced influence on adherence with increasing lipophilicity in applied formulations. The results show that adaptation of the tape stripping protocol to the specifications of envisioned experiments is important for reliable results. Two protocols were found favorable and are presented in this work.

Size analysis of nanoparticles extracted from W/O emulsions.

Nanosized particles are frequently used in many different applications, especially TiO2 nanoparticles as physical filters in sunscreens to protect the skin from UV radiation. However, concerns have arisen about possible health issues caused by nanoparticles and therefore, the assessment of the occurrence of nanoparticles is important in pharmaceutical and cosmetic formulations. In a previous work of our group, a method was presented to extract nanoparticles from O/W emulsions. But to respond to the needs of dry and sensitive skin, sunscreens of the water-in-oil emulsion type are available. In these, assessment of present nanoparticles is also an important issue, so the present study offers a method for extracting nanoparticles from W/O emulsions. Both methods emanate from the same starting point, which minimizes both effort and cost before the beginning of the assessment. By addition of NaOH pellets and centrifugation, particles were extracted from W/O emulsions and measured for their size and surface area by laser diffraction. With the simple equation Q=A/S a distinction between nanoparticles and microparticles was achieved in W/O emulsions, even in commercially available samples. The present method is quick and easy to implement, which makes it cost-effective.

Influence of a multiple emulsion, liposomes and a microemulsion gel on sebum, skin hydration and TEWL.

OBJECTIVE: In this study, the influence of three cosmetically relevant, priorly characterized vehicles on skin hydration, sebum content and transepidermal water loss was investigated. The chosen vehicles included a liposomal pre-formulation, a multiple W/O/W emulsion and a microemulsion gel. The in vivo effects of these vehicles were demonstrated and compared among them. METHODS: The stability of the prepared vehicles was determined visually, microscopically, rheologically by pH measurements and particle size. Interactions with skin were assessed by non-invasive biophysical techniques using the Corneometer(®), Aqua Flux(®) and Sebumeter, measuring skin hydration, TEWL and skin sebum content, respectively. RESULTS: All vehicles remained stable over an observation period of 6 weeks. The multiple emulsion increased sebum content and skin hydration. In case of the liposomes, each monitored parameter remained almost constant. In contrast, the microemulsion gel lowered skin hydration and increased TEWL values, but even 1 week after termination of the treatment TEWL decreased almost close to control levels. CONCLUSION: All produced vehicles were proven to remain physically stable over the duration of this study. The used multiple emulsion showed very skin-friendly properties by increasing sebum and skin hydration. Likewise, the liposomal pre-formulation exhibited no negative effects. On the contrary, the investigated microemulsion gel seemed to have skin dehydrating and TEWL increasing features. However, the multiple emulsion as well as liposomes was identified to be well-tolerated vehicles for skin which might qualify them for the use in cosmetic formulations.

Dilution of semi-solid creams: influence of various production parameters on rheological properties and skin penetration.

UNLABELLED: In order to customise treatment for patients, topical formulations are often diluted with drug-free cream bases to adjust the drug dose and thereby the formulations' activity to the patients' needs. However, the process of dilution influences properties of the formulations. Stability can be reduced as well as the microbial stability and most importantly, efficacy and skin penetration behaviour can be severely and unpredictably changed. The present study investigates the effects of production parameters on creams, namely incorporation of an API (active pharmaceutical ingredients) into an OW cream with prior mixing with propylene glycol or without and subsequent automated or manual dilution of the resulting creams with three different cream bases. Effects were measured by influence on microscopic appearance, measurement of chemical stability, skin penetration and rheological behaviour. RESULT: suggest strong influence of the cream bases used for dilution of the formulations. Mixture of equal amounts of the employed OW and WO cream proved unfavourable due to inferior penetration behaviour and less appealing microscopic and macroscopic appearance. Prior mixing with PG was of negligible importance for the characteristics of the dilutions, however, the type of API and manner of dilution had an influence on the viscosity of the formulations.

Size analysis of nanoparticles in commercial O/W sunscreens.

Nanoparticles are employed in a variety of applications and especially in cosmetics the issue is discussed whether or not they can be regarded as safe. Analysis of nanosized structures and morphology studies prove to be difficult in many aspects. Nevertheless, there is the demand for new, cost-effective and simple yet reliable methods of analysis to assess the occurrence of nanoparticles in cosmetics in order to evaluate the possible risks conditioned by nanosized structures. In the present study, a simple method was developed to extract particles from commercial sunscreens that are O/W emulsions to measure the particle size of suspended material by laser diffraction. A following, simple calculation based on the specific surface area and particle size distribution allows distinguishing agglomerated nanoparticles from larger particles and thereby contributes well to the tools in analysis of cosmetic products. It was possible to create a simple, fast and cost-effective method to obtain an overview whether nanoparticles are included in a cosmetic product or not.

Influence of drug content, type of semi-solid vehicle and rheological properties on the skin penetration of the model drug fludrocortisone acetate.

Throughout Europe, topical creams containing corticosteroids are diluted with various neutral cream bases to meet the specific needs of patients. Even though this practice has been common for years, its effect has not been thoroughly investigated and so the effectiveness of the diluted topical steroidal creams is difficult to predict. In the present study, the model drug fludrocortisone acetate was incorporated into three cream bases of different hydrophilicity that are commonly used in Austria. Different final drug concentrations were chosen for comparative studies. Additionally, a semi-solid preparation developed by our group was investigated for comparison. These formulations were tested in diffusion and tape stripping experiments. Diffusion cell studies showed that changes in drug concentration do not necessarily change the skin permeation behaviour in vitro. The tape stripping protocol was successfully optimised for investigation of semi-solid preparations to provide reproducible and accurate results despite the challenges of investigating semi-solid formulations. The results showed that tape stripping experiments are more suitable to elucidate subtle differences between formulations. The composition of the cream bases exhibited stronger effects on the skin penetration of the steroidal drug irrespective of its concentration than the rheological properties. No correlation between formulation viscosity and skin penetration was found.

Corneocyte quantification by NIR densitometry and UV/Vis spectroscopy for human and porcine skin and the role of skin cleaning procedures.

Optical methods of corneocyte quantification during tape stripping experiments on the skin are useful tools for the rapid evaluation of the skin penetration potential of dermally applied substances. However, a comparative investigation of the different methods proposed for this task, namely NIR densitometry and UV/Vis spectroscopy, is still missing. Thus, the aim of the present work was to employ these two techniques in comparative tape stripping experiments both in vivo on human forearm skin and in vitro on porcine ear skin. Standard tape stripping experiments were performed in the absence and presence of a marketed formulation containing flufenamic acid as a model drug. In the context of these methodological investigations, different methods of skin cleaning prior to the tape stripping procedure were evaluated to identify the most appropriate working protocol among the approaches proposed in the respective literature. The results showed that the investigated methods of NIR densitometry and UV/Vis spectroscopy deliver highly comparable results. Both optical methods are suitable to determine the skin penetration profiles of active substances during in vivo and in vitro tape stripping, especially if a simple working protocol without any cleaning procedures is maintained.

Facilitating in vitro tape stripping: application of infrared densitometry for quantification of porcine stratum corneum proteins.

Infrared (IR) densitometry is a highly practical method recently proposed for protein analysis during in vivo tape stripping. However, this method has not yet been validated for the quantification of porcine stratum corneum (SC) proteins. Therefore, the aim of this study was to establish calibration curves for the analysis of adhesive tapes removed from porcine ear skin. To this end, the protein absorption (as determined via IR densitometry) was correlated with the protein content determined with the Micro BCA™ protein assay after extraction of the tapes. The obtained linear regressions confirm that IR densitometry is suitable for the quantification of not only human, but also porcine, SC proteins. The pattern of protein removal observed with porcine skin differs from that of human skin due to more pronounced corneocyte clustering and deep 'canyons', which necessitates specific evaluation of porcine skin samples and a working protocol that takes this into account. The presented data will facilitate future analysis of porcine SC proteins during in vitro tape stripping.

[Topical application of morphine and buprenorphine gel has no effect in the sunburn model]. / Keine Wirkung bei topischer Applikation von Morphin- und Buprenorphingel im Sonnenbrandmodell.

BACKGROUND: The aim of this placebo-controlled double-blinded cross-over study was to investigate the antihyperalgesic effect of topical morphine and buprenorphine in the sunburn pain model. MATERIAL AND METHODS: The study was designed as a double-blind, placebo-controlled cross-over trial, separated into 2 parts each with 16 volunteers. In part A morphine dissolved in Ultrabas-ultrasic ointment at 3 concentrations (0.1, 0.2, 0.4%) and placebo ointment were applied to 4 UVB-induced erythemas on the thighs. In part B buprenorphine at 3 concentrations (0.01, 0.02 and 0.1%) and placebo dissolved in a gel for transcutaneous application, was applied to 4 erythemas on the thighs. Thermal and mechanical hyperalgesia were assessed in the respective erythema by standardized quantitative sensory testing and opioids were compared to the placebo. RESULTS: Neither morphine nor buprenorphine showed any significant reduction of hyperalgesia in comparison to the placebo. CONCLUSION: The topical application of opioids in this form has no effect on inflamed skin.

Skin permeation and stability studies of 5-aminolevulinic acid in a new gel and patch preparation.

A new cubic gel as well as a patch system containing additional carrageenan as matrix were used as vehicles for 5-aminolevulinic acid (5-ALA). Standard diffusion experiments of 5-ALA using Franz-type diffusion cells and porcine skin were performed. Drug stability was monitored by analysing the 5-ALA content in the formulations. The analysis of 5-ALA as non-fluorescent probe was performed after chemical reaction leading to a fluorescent derivative. The 5-ALA permeation through porcine skin after 48 h was the highest from the patch formulation with 80.3% and the second highest from the cubic gel with 66.4%, however only about 40% of 5-ALA were chemical stable after 14 days storage in the patch formulation, whereas no degradation of 5-ALA was measured in the cubic gel over 90 days of observation.

Phloretin and 6-ketocholestanol: membrane interactions studied by a phospholipid/polydiacetylene colorimetric assay and differential scanning calorimetry.

The aim of this study was to investigate membrane interactions of phloretin and 6-ketocholestanol using different methods. A previously reported colorimetric assay with phospholipid/polydiacetylene (PDA) vesicles was used to examine a possible interaction of phloretin and 6-ketocholestanol with this target. During this interaction the used aggregates of lipids and conjugated PDA undergo a visible and quantifiable blue to red color transition. A positive result is indicative for a reaction response with membrane lipids of a simplified bilayer structure instead of the complex bilayer system of the stratum corneum. Results of this test confirm previous proposed membrane interactions by skin diffusion studies. Additional differential scanning calorimetry studies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes confirm a membrane interaction and indicates that phloretin and 6-ketocholestanol interact with the lipid layer and change structural parameters. They strongly decrease the lipid phase transition temperature of DMPC and DPPC liposomes by at least about 6.6 degrees C and maximally about 13.9 degrees C which refers to a higher fluidity of the membrane.

Influence of lipophilic counter-ions in combination with phloretin and 6-ketocholestanol on the skin permeation of 5-aminolevulinic acid.

In this study, the effect of lipophilic counter-ions on the permeation of 5-aminolevulinic acid (ALA) in combination with skin impregnation by phloretin and 6-ketocholestanol was evaluated. Standard in vitro permeation experiments with porcine skin were performed analysing the ALA content by HPLC and fluorescent detection after ALA derivatisation. The shake flask method in combination with a trinitrobenzensulfonic acid test for ALA analysis was performed to calculate the apparent partition coefficient (logP(Oct)). The permeation of ALA was enhanced by cetylpyridinium chloride and benzalkonium chloride at pH 7.0 and by sodium-1-octanesulfonic acid, sodium-1-heptanesulfonic acid and sodium-1-pentanesulfonic acid monohydrate at pH 4.0. Corresponding effects of these additives were observed on the partitioning of ALA. Pre-impregnation of porcine skin with phloretin and 6-ketocholestanol increased the ALA diffusion about 1.7-fold at pH 7.0. Moreover, this transport enhancement by 6-ketocholestanol was 3.5-fold higher when a combination of ALA and cetylpyridinium chloride was used as donor.

Development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone.

The purpose of the present study was to design a novel carrier system based on a mucoadhesive polymer exhibiting improved properties concerning drug delivery to the vaginal mucosa. This was reached by the covalent attachment of L-cysteine to commercially available polyacrylic acid (Carbopol 974P). Mediated by a carbodiimide, increasing amounts of L-cysteine were covalently linked to the polymer. The resulting thiolated polyacrylic acid conjugates (NaC974P-Cys) displayed between 24.8 and 45.8 micromol thiol groups per gram of polymer. Because of the formation of intra- and/or intermolecular disulfide bonds, the viscosity of an aqueous thiolated polymer gel (3%) increased about 50% at pH 7.0 within 1 h. In oscillatory rheological measurements, it was shown that this increase in viscosity is mainly due to the increase in elasticity. Tensile studies carried out on freshly excised cow vagina demonstrated a significant (P<0.05) increase in the total work of adhesion (TWA) compared to the unmodified polymer. An amount of 24.8 micromol thiol groups per gram of polymer resulted in a 1.45-fold increase in the TWA, whereas an amount of 45.8 micromol showed an even 2.28-fold increase. These improved mucoadhesive properties can be explained by the formation of disulfide bonds between the thiolated polymer and cysteine rich subdomaines of the mucus layer. The release rate of the model drug progesterone from tablets based on microcrystalline cellulose serving as the reference was approximately 1% per hour, whereas it was 0.58% per hour for the unmodified polymer (NaC974P) and 0.12% per hour for the thiolated polymer (NaC974P-Cys). Therefore, this thiolated polymer is a promising carrier for progesterone providing a prolonged residence time and a controlled drug release.

Thiolated polymers: development and evaluation of transdermal delivery systems for progesterone.

PURPOSE: To evaluate the possible use of polycarbophil-cysteine (PCP-Cys) as polymeric matrix for transdermal progesterone application. METHODS: Thiolated polycarbophil was synthesised by the covalent attachment of cysteine to the basis polymer. The adhesive properties of PCP-Cys in comparison to polyvinylpyrrolidone/hydroxypropylmethylcellulose (PVP/HPMC) and polyvinylpyrrolidone/polyvinylalcohol (PVP/PVA) were investigated by testing the total work of adhesion (TWA) on porcine skin. Release studies in Franz diffusion cells and standard in vitro permeation experiments with porcine skin were performed analysing the progesterone content by high-performance liquid chromatography. RESULTS: Films based on PCP-Cys displayed very high cohesive properties due to the formation of interchain disulfide bonds. The TWA of the thiolated polymer on porcine skin was significantly (P <0.05) the highest. In addition progesterone permeation was also the highest from PCP-Cys compared with PVP/HPMC and PVP/PVA within 24 hours. CONCLUSION: PCP-Cys--a partly thiolated polymer--might be a novel polymer matrix for transdermal progesterone delivery with excellent adhesiveness on porcine skin.

Influence of phloretin and 6-ketocholestanol on the permeation of progesterone through porcine skin.

In this study the effect of phloretin (PH) and 6-ketocholestanol (KC) on the permeation of progesterone through porcine skin has been examined. Both PH and KC were incorporated into unilamellar L-alpha-phosphatidylcholine (PC) liposomes at different concentrations (7.5, 15, 30 and 60 mol%). In diffusion experiments with porcine skin, both substances, to a different degree, enhanced the steady state flux of progesterone. It was increased up to 2.4-fold using 15 mol% KC, and 1.4-fold using 30 mol% PH. The results indicate an interaction of these two compounds with the lipid components of the stratum corneum. In order to visualise the interaction, differential scanning calorimetry (DSC) measurements were performed on porcine skin, which had been impregnated with KC and PH. Both showed a lowering ( approximately 5-6 degrees C) in the lipid phase transition temperature that occurs around 75 degrees C in porcine skin.

Effect of urea and pantothenol on the permeation of progesterone through excised rat skin from polymer matrix systems.

Standard in vitro permeation experiments, using excised rat skin, were carried out to establish the release profile and permeation behavior of progesterone from polymethacrylate (PMA), polyvinylpyrrolidon (PVP), and polyvinylalcohol (PVA) transdermal systems. Data obtained show significant differences in release characteristics from each polymer systems. The greatest amount of progesterone was released from the PVA system. The influence of urea and pantothenol on progesterone release was also investigated. Release data were compared with the permeation rates of progesterone across excised rat skin. The highest permeation rates were measured from PVA matrices containing 5% urea (860 +/- 138 micrograms/cm2; cumulative amount permeated in 24 hr) and from PVP matrices containing 6% pantothenol (660 +/- 73 micrograms/cm2; cumulative amount permeated in 24 hr). A good correlation between release and permeation data was found with the polymer matrices; however, this was not the case when additives were included.

Effect of phloretin on the percutaneous absorption of lignocaine across human skin.

The potential use of phloretin, a polyphenolic compound, as a penetration enhancer in the transdermal delivery of lignocaine hydrochloride (L-HCl) has been investigated. Standard in vitro skin permeation methods, using excised human skin, were used to characterize the percutaneous absorption of L-HCl. Initially, phloretin was applied to the skin surface as a methanolic solution. The skin samples were treated 12 h prior to application of the lignocaine donor solution, which was buffered at pH 4.0 and 7.0. The data obtained from the methanolic solutions at pH 4.0 show a 3.2-fold increase of the cumulative amount permeated after 24 h compared with the control. A second series of experiments were conducted using unilamellar phosphatidylcholine liposomes instead of methanol as a vehicle for the phloretin. The L-HCl amount permeated from liposomal-pretreated skin was 5.4-fold (p < 0.05) higher than the control within 24 h. In addition to the diffusion experiments, pressure area isotherms were recorded on a Langmuir-Blodgett trough using the model skin lipid ceramide-2. They showed a slight increase in the area occupied per lipid molecule of 1.04 nm(2) at constant surface pressure. This result indicates an interaction between the model lipid and phloretin. The results suggest the potential use of phloretin as penetration enhancer in the delivery of L-HCl through skin.

pH, pK(a) and dermal delivery.

The effect of pH on the permeation of ibuprofen and lignocaine through human skin has been modelled using a modification to the equation derived by Potts and Guy, which is normally applied to unionized entities. The results show that permeation is related to the distribution coefficient. The physicochemical properties have been predicted ab initio using commercially available software and compared to literature values. The approach is successful and shows that there is significant permeation of the ionized drugs through a lipophilic pathway, possibly as a result of ion pairing. Since the aqueous solubility of the ionized material is significantly higher than the unionized, the maximum flux through the skin may occur at a pH where ionization is high. Optimum topical or transdermal formulations may not therefore be for the free acid or free base.

The dermal delivery of lignocaine: influence of ion pairing.

The purpose of the present study was to determine the significance of ion pairing on the permeation of lignocaine. Results of diffusion studies through polydimethylsiloxane (PDMS) at different pH values 4. 0, 6.0, 7.0, 8.0 indicated that lignocaine hydrochloride (L-HCl) flux significantly increased with the amount of unionized base. In order to see if similar results could be obtained using human skin, permeation runs were performed with human skin at pH of 4.0, 5.5 and 7.0. These values were chosen to simulate an appropriate range of physiological conditions. Results of the experiments with human epidermis showed increasing L-HCl flux with increasing pH, confirming the trends seen with PDMS membranes. A linear relationship was found between the apparent partition coefficient and the steady state flux. Further experiments were conducted at donor pH 4.0 to minimise the contribution of the unionized species. Although an excess of different ions such as nitrate, mesylate and bromide increased the apparent partition coefficient, the steady state flux was not significantly increased. The steady state lignocaine flux was increased up to 2.45-fold using different counter ions. The highest flux was measured from lignocaine morpholinopropane sulfonate (L-mps). It is possible to enhance the flux of salts across lipophilic membranes by using an ion pair approach. The degree to which this is possible depends on the lipophilicity of the counter ion, the medium in which the ion pair forms, and the ionic strength.

Peroral administration of enzymes: strategies to improve the galenic of dosage forms for trypsin and bromelain.

In this study, we investigated the presystemic metabolism of trypsin and bromelain and the influence of these proteolytic enzymes on the mucus layer covering the gastrointestinal (GI) epithelia. In vitro studies demonstrated that 77.3% +/- 4.0% (mean +/- SD, n = 3) of trypsin is autodegraded within 2 hr, whereas autodegradation of bromelain was negligible. In contrast to the metabolization of bromelain by all pancreatic serine proteases, trypsin is only degraded to some extent by elastase. Both therapeutically used enzymes remained stable after incubation with an excised porcine mucosa, demonstrating that proteolysis caused by brush border membrane-bound enzymes is negligible. Trypsin and bromelain were highly mucolytic active, thereby reducing the diffusion barrier based on the mucus gel layer. Strategies to improve the galenic of dosage forms for trypsin and bromelain include the use of bioadhesive polymers such as hydroxyethylcellulose or slightly modified chitosan-EDTA, providing strongly improved stability of these enzymes toward proteolytic degradation in vitro. The given information represents a good starting point to improve the galenic of dosage forms for orally administered proteolytic enzymes.