Blanching Assay Research Articles

Enhanced dermal delivery of diflucortolone valerate using lecithin/chitosan nanoparticles: in-vitro and in-vivo evaluations

The objective of this study was to prepare a suitable formulation for dermal delivery of diflucortolone valerate (DFV) that would maintain the localization in skin layers without any penetration and to optimize efficiency of DFV. Drug-loaded lecithin/chitosan nanoparticles with high entrapment efficiency (86.8%), were successfully prepared by ionic interaction technique. Sustained release of DFV was achieved without any initial burst release. Nanoparticles were also incorporated into chitosan gel at different ratios for preparing a more suitable formulation for topical drug delivery with adequate viscosity. In ex-vivo permeation studies, nanoparticles increased the accumulation of DFV especially in the stratum corneum + epidermis of rat skin without any significant permeation. Retention of DFV from nanoparticle in chitosan gel formulation (0.01%) was twofold higher than commercial cream, although it contained ten times less DFV. Nanoparticles in gel formulations produced significantly higher edema inhibition in rats compared with commercial cream in in-vivo studies. Skin blanching assay using a chromameter showed vasoconstriction similar to that of the commercial product. There were no barrier function changes upon application of nanoparticles. In-vitro and in-vivo results demonstrated that lecithin/chitosan nanoparticles in chitosan gel may be a promising carrier for dermal delivery of DFV in various skin disorders.

Effect of small changes in natural origin-based emulsion systems on hydrocortisone skin absorption and performance: a comparison of two in vivo methods

Context: Alkyl polyglucoside surfactants (APG) remain prominent natural origin stabilizers offering a prospect of combining satisfactory stability with mild dermatological properties and complete biodegradability.Objective: With the purpose of adjusting the dose to a patient’s needs, dilution of commercial corticosteroid formulations is a practice which may modify efficacy uncontrolledly. The rational of the study was to investigate whether a simple change in ready-to-use bases (co-solvent addition) could address these needs in a more predictive manner.Methods: Hydrocortisone (HC) delivery from such emulsion systems was comparatively assessed employing two in vivo methods: the established human skin blanching assay versus skin stripping technique.Results: HC permeation data obtained after three dose durations showed better overall performance of the APG-stabilized bases relative to reference ones. Although the solubility study showed that all the assessed active samples retained equal thermodynamic activity, diverse HC permeation/penetration implies the importance of the applied base’s colloidal structure and/or changes endured. Isopropyl alcohol (IPA) addition offered faster drug penetration enhancement, while glycerol as a moisturizing agent influenced HC penetration through the increase in skin hydration.Conclusion: Although the performed in vivo methods cannot be considered alternative, skin stripping technique proved to be a cost-efficient mode of percutaneous penetration assessment, providing additional information on vehicle–skin interactions.

Effects of phosphatidylcholine on the topical bioavailability of corticosteroids assessed by the human skin blanching assay.

A non-occluded multiple application skin blanching assay has been used to determine the effect of applied phosphatidylcholine (PC) on the bioavailability of corticosteroids. One forearm of each of ten volunteers was treated twice daily for one week with PC presented as a liposomal suspension in Sørensen's (pH 5.0) phosphate buffer (2.5 mg PC/0.5 mL) while the other arm was treated with 0.5 mL of phosphate buffer. For the following two weeks this treatment regimen was continued but in addition, each of four corticosteroid formulations (containing (i) hydrocortisone 0.1%, (ii) clobetasone butyrate 0.05%, (iii) betamethasone 0.1% and (iv) clobetasol propionate, 0.05%) was applied to sites on both arms. 5 +/- 1 mg of each cream was applied twice daily to the sites on day 1, then once daily for a further four days; after two days of no application, the protocol was repeated. Estimation of pallor was usually made four times daily. At the end of the second week of corticosteroid application the blanching response to all four formulations on the PC treated arms was significantly higher than on the buffer treated arm. Tachyphylaxis to the applied corticosteroids was markedly less apparent on the lipid-treated arms. It is proposed that the applied phospholipid either supplements the lipid content of the skin or provides a thin film in intimate epidermal contact. Such a film may promote hydration of the stratum corneum and also provide an environment into which corticosteroids initially partition before a subsequent, more controlled, release to the underlying tissue.

Quantitative skin blanching assay of corticosteroid creams using tristimulus colour analysis.

The potencies of four proprietary corticosteroid creams were ranked using blanching measured by a tristimulus colour analyser as an index. Intra-subject variation was measured in a trial using a single subject while in a second study inter-subject variation was quantified using six volunteers. Discriminative parameters were derived from the extended multiple-point chromaticity coordinates L* and a* recorded after application of the steroid creams under occlusion. Analysis of variance of the data using the linear regression model was followed by Tukey's multiple comparison tests. Ranking of the creams using parameters of the pharmacodynamic response corresponded with the generally accepted rank order of the potencies of the corticosteroid creams. It is proposed that this multiple-point skin blanching assay of topical corticosteroids using an internationally accepted clear measurement standard and the subsequent data analysis be adopted as a standard protocol.

Comparison of tape stripping with the human skin blanching assay for the bioequivalence assessment of topical clobetasol propionate formulations.

A draft guidance on tape stripping for assessing the bioavailability/bioequivalence of topical formulations was issued by the United States Food and Drug Administration in 1998 but has since been withdrawn. This was due to problems associated with the method and also inconsistencies and variability in the resulting data. The purpose of this study was to re-visit the tape stripping technique, incorporate refinements to reduce variability and validate the method using bioequivalence data obtained from the assessment of a topical corticosteroid cream containing 0.05% clobetasol propionate using the human skin-blanching assay. A pilot tape stripping study was conducted to establish the variability of the formulations.The bioequivalence of two different commercially available clobetasol propionate cream formulations and a clobetasol propionate ointment formulation were subsequently investigated using the tape stripping method. The data from the pilot tape stripping study correlated well with data from the human skin-blanching assay. A subsequent pivotal tape stripping study confirmed bioequivalence between the two cream formulations whereas bio-inequivalence was demonstrated between the cream and ointment formulations. These studies show that the results from tape stripping concur with data from the human skin blanching assay and demonstrate the potential of a well-controlled tape stripping study as an option for the assessment of bioequivalence of topical corticosteroid formulations.

Strategies for the Bioequivalence Assessment of Topical Dermatological Dosage Forms

The assessment of the bioequivalence of topical products not intended for absorption into the systemic circulation has presented a formidable challenge over the years. In particular, dermatological dosage forms such as creams, ointments, lotions and gels, apart from those containing topical corticosteroids, cannot readily be assessed for bioequivalence using “conventional” methodology and the only recourse to-date has been to undertake tedious, time consuming and expensive clinical end-point trials for such products. Although the human skin blanching assay (HSBA), also known as the vasoconstriction assay (VCA) has been successfully used for dermatological products containing topical corticosteroids and the methodology has found formal acceptance by a number of regulatory agencies, e.g. the US FDA amongst others, no surrogate methodology for the bioequivalence assessment of other topical dermatological products such as those containing non- steroidal anti-in fl ammatory drugs, anti-fungals, antibiotics and antivirals has yet found favour with regulatory agencies. Application of the HSBA, Tape Stripping (TS) and Dermal Microdialysis (DMD) for the assessment of bioequivalence is described and the theoretical basis and prognosis for each technique is presente

Measurement and prediction of the rate and extent of drug delivery into and through the skin

Skin diseases are prevalent and can significantly affect quality of life. Empirical mathematical models retrospectively analyse data to predict skin permeation from the physico-chemical properties of drugs. Quantitative structure permeability relationships are discussed, along with alternatives to linear modelling. Mechanistic mathematical models derived from first principles are also considered. Further, in vitro experiments allow predictions to be made using suitable membranes (cultured cell lines or excised skins). In vivo methods to assess (trans)dermal drug delivery aim to minimise clinical studies, especially to determine whether formulations are bioequivalent. Microdialysis is discussed, together with the FDA-approved skin blanching (pharmacodynamic) assay for corticosteroids. The progress made with the tape stripping methodology is reviewed. Two distinct strategies have emerged, the first where the total amount of drug in the stratum corneum (SC) at one uptake and one clearance time are compared; and the second which generates drug permeation profiles across the SC, and allows dermatopharmacokinetic parameters to be derived.

An alkylpolyglucoside surfactant as a prospective pharmaceutical excipient for topical formulations: The influence of oil polarity on the colloidal structure and hydrocortisone in vitro/in vivo permeation

There is a growing need for research into new skin- and environment-friendly surfactants. This paper focuses on a natural surfactant of an alkylpolyglucoside type, which can form both thermotropic and lyotropic liquid-crystalline phases. The aim of this study was to relate some physicochemical properties (characterised by polarisation and transmission electron microscopy, thermal analysis and rheology) of the three formulations based on cetearyl glucoside and cetearyl alcohol, to the results of in vitro and in vivo bioavailability of hydrocortisone (HC). The three formulations contained oils of different polarity (medium chain triglycerides: MG, isopropyl myristate: IPM and light liquid paraffin: LP), respectively. In vitro permeation was followed through the artificial skin constructs (ASC), while the parameters measured in vivo were erythema index: EI (using instrumental human skin blanching assay), transepidermal water loss (TEWL) and stratum corneum hydration (SCH). The vehicles based on cetearyl glucoside and cetearyl alcohol showed a complex colloidal structure of lamellar liquid-crystalline and lamellar gel-crystalline type, depending on oil polarity. Rheological profile of the vehicle was directly related to the in vitro profile of the HC permeation. In vivo results suggested that the vehicle with MG retarded the HC permeation, whereas less polar IPM and non-polar LP enhanced it. It is suggested that the enhancement is achieved either by a direct interaction with lipid lamellae of the SC or indirectly by improving skin hydration. There were no adverse effects during in vivo study, which indicates a good safety profile of this alkylpolyglucoside surfactant.

Vehicles based on a sugar surfactant: Colloidal structure and its impact on in vitro/in vivo hydrocortisone permeation

An emerging class of natural surfactants, named alkylpolyglucosides, which can form both, the thermotropic and the lyotropic liquid crystalline phases, were focused. The aim of the study was to integrate some physicochemical properties (characterised through the polarization and transmission electron microscopy, wide-angle X-ray diffraction, thermal analysis and rheology) of the three formulations based on cetearyl glucoside and cetearyl alcohol, with the in vitro (the artificial skin constructs) and in vivo bioavailability of hydrocortisone (HC), in comparison with a standard pharmacopoeial vehicle. The parameters measured in vivo were erythema index (an instrumental human skin blanching assay), transepidermal water loss (TEWL) and stratum corneum hydration. A complex colloidal structure of lamellar liquid crystalline and lamellar gel crystalline type was deduced for sugar surfactant-based vehicles. In dependence on surfactant/water/oil ratio, several thermodinamically variable fractions of water were predicted. Rheological profile of the vehicle appeared to influence the in vitro profile of permeation. A surplus of total amount of drug permeated in vitro from the alkylpolyglucoside-based vehicles coincided with the more pronounced increase of TEWL and less marked blanching action of HC from the selected alkylpolyglucoside-based vehicle tested in vivo, related to the pharmacopoeial one. These findings imply an enhanced delivery of HC from this vehicle and its putative penetration enhancing effect, probably dependent on specific distribution of the vehicle's inherent water.

Preparation and Evaluation of Liposomal Flucinolone Acetonide Gel for Intradermal Delivery

Hydroxypropyl methylcellulose (HPMC) K4M gels containing free flucinolone acetonide, liposomal-encapsulated flucinolone acetonide and physical mixture of the drug and lipids were prepared and evaluated in in-vitro drug release studies using rat skin to determine diffusion parameters. Data were analysed to calculate the quantity of the drug remaining in the skin (Qm) and in the blood (Csf) and the ratio of Qm to Csf. The in-vivo skin blanching assay performed with these formulations in human volunteers showed low blanching scores for liposomal gel formulations indicating low absorption of the liposomal-encapsulated drug into the blood stream and leading to the accumulation of the drug in the skin. This result is in agreement with the high Qm/Csf ratios obtained in the in-vitro experiments. Of all the formulations, gels containing liposomal-encapsulated flucinolone acetonide, prepared with drug, phosphatidyl choline and cholesterol in the ratio of 4:8:1, had the highest Qm/Csf ratio and minimum blanching score. The results indicate the potential of topical preparations containing liposomal-encapsulated drugs for selective accumulation of the drug in the skin.

Postural Skin Colour Changes during the Corticosteroid Blanching Assay

Objective: To assess the effect of the position of a limb (dependency) on the cutaneous vasoconstrictor (blanching) assay (VCA) using topical corticosteroid preparations. Method: Two studies were performed on the forearms of healthy volunteers using tristimulus reflectance colorimetric assessments. The first one conducted in 60 normal adults aimed at quantifying the range of variation in the skin chromaticity a* when the arm was successively positioned horizontally and vertically, either in the upward or downward direction. In the second study, 16 volunteers were selected according to a weak spontaneous postural dependency of the limb in a* (<2 units). The blanching effect of 0.1% mometasone furoate, 0.1% betamethasone valerate and petrolatum were compared to the colour of an untreated site. Chromaticity a* and the colorimetric variable (ΔtL*² + Δta*²)^0.5 were measured in time following a 2-hour application. Results: The postural variations in skin colour varied among subjects. The majority of them (80%) presented a difference in a* higher than 2 units between the upright and downward arm positions. The upright position appeared to be the most sensitive to show significant differences in the VCA. Mometasone furoate exhibited the most prominent effect, significantly higher than betamethasone valerate and controls. Conclusion: The corticosteroid VCA is influenced by the position of the limb. The upright position increases the sensitivity of the test. Mometasone furoate is more potent than betamethasone valerate.

Analysis of chromameter results obtained from corticosteroid-induced skin blanching assay: comparison of visual and chromameter data

In a Guidance document, the American FDA recommends the use of a Minolta chromameter rather than the human eye for the quantitative assessment of the pharmacodynamic blanching response produced by topical application of corticosteroids. The purpose of this study was to compare the appropriateness of the human eye and two models of chromameter for the estimation of skin blanching, in terms of the quality of the data generated by each method. The corticosteroid-induced skin blanching from four different betamethasone 17-valerate cream formulations was compared in a typical human skin blanching trial. The optimized assay methodology routinely practised in our laboratories was utilized. The blanching responses were assessed visually by three trained, independent observers and recorded by two chromameters (Minolta model CR-200 and model CR-300). The topical availability of the four creams was determined using visual scoring and chromameter measurements. All data were manipulated in such a manner as to produce a blanching response versus time profile from which AUBC analysis could be performed. Good correlation was observed between the visual assessments made by three independent observers. In contrast, moderate correlation was determined between visual, CR-200 and CR-300 measurements. Surprisingly, no direct linear relationship between the AUBCs produced by the two chromameters was observed indicating that the quality of the data obtained from the two instruments may not be equal. This investigation also indicated that the use of the chromameter is not completely objective. Visual scoring and chromameter measurement produce data sets that differ in quality. Each procedure needs to be validated and investigators have to be trained for both visual assessment and the operation of the chromameter, particularly with regard to the manipulation of the measuring head of the instrument.

Effect of concentration and degree of saturation of topical fluocinonide formulations on in vitro membrane transport and in vivo availability on human skin.

The thermodynamic activity of drugs in topical vehicles is considered to significantly influence topical delivery. In vitro diffusion across a synthetic membrane was shown to be correlated to the degree of saturation of the drug in the applied vehicle and therefore offers a potential for increased topical drug delivery. Fluocinonide a topical corticosteroid, was chosen as a model compound to investigate in vitro and in vivo availability from formulations with different degrees of saturation. Sub-, as well as, supersaturated drug solutions were prepared using PVP as an antinucleant agent. In vitro membrane diffusion experiments across silicone membrane and in vivo pharmacodynamic activity assessments, using the human skin blanching assay, were carried out. Over the concentration range studied, the in vitro membrane transport of fluocinonide was proportional to the degree of saturation of the respective formulations. The in vivo pharmacodynamic response in the human skin blanching assay was related to the concentration of the drug in the vehicle irrespective of the degree of saturation. From the membrane permeation experiment it can be concluded, that the drug flux might be increased supra-proportionally with increasing donor concentration, drug (super-)saturation (proportional), beyond what would be anticipated based on ideal donor concentration and partition coefficient considerations only. These findings could not be confirmed in the in vivo investigation, probably due to additional vehicle effects (e.g., enhancement, irritation, drug binding) which have to be expected and could have altered the integrity of the stratum corneum and therewith topical bioavailability of the drug.

Evaluation of Topical Application of Clobetasol 17-Propionate from Various Cream Bases

The effect of clobetasol 17-propionate (CP), a potent corticosteroid, in various cream bases on the permeation through artificial membrane was sought. Four formulations were then chosen for a further in vivo skin blanching assay. After calculation of the relationship between in vivo flux0–8hr determined from a surface recovery technique and in vitro release rate0–8hr of CP from various formulations, a high correlation coefficient of 0.9996 was achieved. Therefore, the in vitro release study could be used as an index to predict and evaluate the in vivo penetration capacity of CP cream to screen the effective formulation preclinically. After a series of in vivo investigations in this study, it was concluded that myristic acid-added formulations may show a bioequivalence with commercial Dermovate®. Furthermore, the flux calculated from the surface recovery technique and ΔE* detected from the skin blanching assay may be useful as parameters evaluating the quality and effectiveness of CP cream.

Analysis of chromameter results obtained from corticosteroid-induced skin blanching. I: Manipulation of data.

One of the unresolved issues in the FDA Guidance document for topical corticosteroid bioequivalence testing is the method of manipulation suggested for the chromameter data. The purpose of this study was to manipulate the instrumental data from a typical blanching study in a number of ways to investigate the appropriateness of these procedures for comparison with the subjective visually-assessed results. The human skin blanching assay methodology routinely practiced in our laboratories was utilised and the vasoconstriction produced by two corticosteroid formulations of different potency was assessed visually and instrumentally by use of a Minolta chromameter. The instrumental data were corrected for zero-time and unmedicated site readings. In addition, Euclidean distances were calculated using all data generated by the instrument. Individually the a-, b- and L-scale chromameter values are imprecise and there is negligible vasoconstriction response recorded for the moderately potent formulation. Arithmetical manipulation of the data as suggested by the FDA does not appear to improve the quality of the data in any way. Euclidean distance analysis more closely resembles the visual data and appears to have better precision. It is clear that mathematical correction of chromameter data is unnecessary, especially since the instrumental data are extremely imprecise. Furthermore, the assessment of each individual chromameter index does not adequately characterise the blanching response profile. It is therefore suggested that Euclidean distance may be a better measure on which to base an analysis of bioequivalence than the truncated data set methodology currently suggested by the FDA.

The human skin blanching assay for in vivo topical corticosteroid assessment: I. Reproducibility of the assay

The human skin blanching (vasoconstriction) assay for the assessment of topical corticosteroids has been in use for over 30 years, the intensity of the drug-induced blanching being assessed subjectively by eye. Both arms of several male and female volunteers are used for product application and more than one observer is used to estimate the degree of induced blanching. There are, therefore, numerous variables which are inherent in the assay procedure. This investigation consisted of three identical trials performed at 8-week intervals, utilising the same 18 volunteers and the same three observers in an attempt to address the question of reproducibility of the assay. From the results obtained it is clear that the assay methodology is capable of consistently distinguishing, on a rank order basis, between preparations which show similar blanching (chemically-equivalent formulations). The similarity of the results for the three individual trials gives considerable confidence to results produced using this methodology. An experiment designed to test the reproducibility of the blanching scores showed that the observers are capable of producing identical results even though visual observation is highly subjective.

The human skin blanching assay for in vivo topical corticosteroid assessment: II. Subject- and observer-dependent variation in blanching responses

The human skin blanching (vasoconstriction) assay for the assessment of topical corticosteroids uses the skin pallor induced at the site of application as an indicator of the potency of the drug or efficacy of the delivery vehicle. Usually several volunteers and several visual observers are used in the bioassay to counteract the subjectiveness of the methodology. Given the inherent biological variability of the population, the minimum number of volunteers and observers required to give valid results in this assay has not been firmly established. This investigation consisted of three identical trials performed at 8 week intervals, utilising the same 18 volunteers and the same three observers in an attempt to address the question of intra- and inter-volunteer and observer reproducibility of the results from three replicate experiments. The results indicate that for any assay utilising this methodology, the number of application sites for a topical corticosteroid formulation should be at least 48, the number of volunteers (of either sex) should be not less than nine and the number of observers not less than two.

The skin blanching assay with halcinonide, influence of halcinonide concentration and application time ⋆

AbstractObjective Instrumental colour readings (Minolta Chromameter CR200) were performed in order to quantify corticosteroid induced skin blanching.Methods Different Halcinonide concentrations in the same vehicle ranging from 0.005% to 0.200% were tested on the volar part of the forearms. Besides dose‐response relationships we studied the influence of application time by applying the formulations under occlusion during 2, 12 and 18 h.Results All evaluated formulations provoked a discernible blanching as indicated by the significant increase of the L* parameter and the decrease of the a* parameter. Discrimination between the different Halcinonide concentrations was possible only in the 2 h application experiment. With longer application times a similar blanching was recorded for all Halcinonide concentrations.Conclusion Our results indicate that a shorter application time may lead to a more sensitive skin blanching assay.

The skin blanching assay with halcinonide, influence of halcinonide concentration and application time

Objective Instrumental colour readings (Minolta Chromameter CR200) were performed in order to quantify corticosteroid induced skin blanching.Methods Different Halcinonide concentrations in the same vehicle ranging from 0.005% to 0.200% were tested on the volar part of the forearms. Besides dose-response relationships we studied the influence of application time by applying the formulations under occlusion during 2, 12 and 18 h.Results All evaluated formulations provoked a discernible blanching as indicated by the significant increase of the L∗ parameter and the decrease of the a∗ parameter. Discrimination between the different Halcinonide concentrations was possible only in the 2 h application experiment. With longer application times a similar blanching was recorded for all Halcinonide concentrations.Conclusion Our results indicate that a shorter application time may lead to a more sensitive skin blanching assay.

Topical 0.050% betamethasone dipropionate. Pharmacokinetic and pharmacodynamic dose-response studies in humans.

Effective topical drug therapy requires drug delivery into the skin to produce the desired pharmacodynamic response. For topical corticosteroids, the visual skin-blanching assay has been used to rank the potency of the corticosteroids and their overall efficacy. While vehicles have been shown to influence the resulting blanching response, the dose of drug applied has not always produced proportional differences in the blanching assay. The mechanism of the nonproportional pharmacodynamic response to the corticosteroid dose is unclear. We describe four methods for assessing the dose-response relationship of topical betamethasone dipropionate on the ventral forearm of six human subjects: duration, concentration, film thickness, and surface area. Drug uptake analysis in human stratum corneum and the resulting pharmacodynamic response, measured visually and with a chromameter, were performed with each method to quantify the dose-response relationship. Only the concentration and duration methods demonstrated an increase in mean drug uptake with increasing dose. The maximal mean pharmacodynamic response reflected the mean drug uptake with all four methods. Application conditions for maximal pharmacodynamic activity of topical betamethasone dipropionate in the skin include short duration of treatment (< or = 2 hours), a lower concentration than commercially marketed, and thin film thicknesses (1 to 5 microns). A dose response can be produced by increasing the drug concentration or the duration of application time. Achievement of steady-state betamethasone dipropionate uptake into the stratum corneum was not commensurate with the maximal pharmacodynamic response. Very small amounts of this potent corticosteroid within the skin appear to maximize the receptor response to drug.