Skin Blanching Research Articles

Evaluation of Bioequivalence and Pharmacokinetic Profiles for Topical Desonide Cream Using Chinese Skins

Introduction: Skin-blanching assay has been established as a surrogate method for assessing bioequivalence of topical corticosteroids. This study aimed to apply the skin-blanching assay to evaluate the bioequivalence of a test desonide cream (T) compared with the reference Desonide® (R) using Chinese skins. Additionally, the pharmacokinetics and safety profiles were also assessed. Methods: By detecting the degree of skin blanching under different dose durations in a pilot dose-duration-response study, the area under the observed effect-time curve (AUEC) and half of the maximum effect (ED50) was calculated. Based on this, the skin color of different time points after a dose duration of ED50, D1 (0.5 × ED50), and D2 (2 × ED50) were detected as a pharmacodynamic indicator to compare between test and reference creams. Single-center, single-dose, randomized, open-label, two-cycle crossover pharmacokinetic studies were designed to make sure the exposure of tested formulationswas not higher than that of the reference formulations. Subjects experiencing adverse events (AEs) were monitored and utilized for safety analysis. Results: These studies involved 12 subjects for the dose-duration-response study, 100 subjects for the bioequivalence study, and 12 subjects for the pharmacokinetic study. The results showed that the population ED50 was 0.88 ± 0.45 h; the mean ratio of area under effective curve from 0 to 24 h (AUEC0–24h) of test and reference preparations was 0.95, with a 90% confidence interval as 88.09–101.72%, indicating the bioequivalence of the test formulation and Desonide®. The maximum plasma concentration (Cmax) and area under the concentration time curve from time 0 to the last time point (AUC0–t) of T and R were 20.8 ± 11.5 pg/mL versus 19.7 ± 10.1 pg/mL, respectively, and 451.04 ± 363.65 pgh/mL versus 541.47 ± 581.41 pgh/mL, respectively. The systemic exposure of a single dose of the test cream was not higher than that of the reference preparation. All of the volunteers experienced grade 1 AEs, suggesting that single administration of the test desonide cream is well tolerated in the Chinese healthy population. Conclusions: This study demonstrated the applicability of skin-blanching assay in Chinese skins and established the bioequivalence of test and reference desonide creams.

Fitting Pharmacodynamic Data to the Emax Model to Assess the Inherent Potency of Topical Corticosteroids.

The widespread use of topical corticosteroids (TCs) in dermatotherapy requires a consideration of their potency and benefit/risk ratios. Although there are a variety of topical corticosteroid products (TCPs) available on the market and their potencies are ranked using different classification systems, to our knowledge, no classification system to rank the inherent potencies of TC active pharmaceutical ingredients (APIs) currently exists. Most of the published classification systems for TCPs are based on randomized clinical comparative studies and/or vasoconstrictor assay (VCA) data. The objective was to apply the US FDA's VCA to classify the inherent potencies of several TCs using standardized doses to make appropriate comparisons of the relevant APIs in solutions of the same molar concentrations. Six TC APIs were assessed for their relative potencies using healthy human participants. The Emax model was used to fit skin blanching data following application of the respective TCs, and the parameters, Emax and ED50, were derived. Emax values were used as the metric to assess potency. Statistical analyses of the data revealed that the inherent potencies of fluticasone propionate, mometasone furoate, and hydrocortisone butyrate were similar. However, there was no significant difference between hydrocortisone butyrate and clobetasol propionate, while there was a significant difference between clobetasol propionate, fluticasone propionate, and mometasone furoate. Hence, the potency of hydrocortisone butyrate appears to overlap two potency classes. Furthermore, the potencies of betamethasone valerate and methylprednisolone aceponate were similar but lower than those of all of the other APIs. The application of the VCA to classify inherent potency provides a reliable method to establish a classification system for TCs. Inherent potency assessment of TCs provides information that will be useful when choosing an appropriate TC for the development of a TCP for a specific clinical indication.

Application of Emax model to assess the potency of topical corticosteroid products.

The objective of this study was to compare the potencies of two topical corticosteroid products (TCPs) using the Emax model to fit the skin blanching responses obtained from the US FDA's vasoconstrictor assay (VCA) and to illustrate the influence of formulation on potency. The potencies of two marketed TCPs, Dermovate® cream containing clobetasol propionate (CP) and Elocon® cream containing mometasone furoate (MF), were assessed using healthy human subjects. In order to investigate the influence of formulation and associated vehicle properties, the creams were compared with their respective topical corticosteroids (TCs) from a previously published study wherein the inherent potencies of those TCs were assessed using a validated VCA method. Whereas the inherent potency of MF (Emax = -94.45 ± 0.21) was found to be greater than CP (Emax = -58.80 ± 15.65), when formulated as creams, the TCP containing CP had a higher potency (Emax = -86.15 ± 0.17) than that containing MF (Emax = -42.61 ± 26.04). This reversal of potency may be attributed to the effect of formulation factors. The comparison of the potencies of TCPs with inherent potencies of their corresponding TCs confirmed the influence of formulation parameters on the potency of those products.

Assessment of dermal bioavailability: predicting the input function for topical glucocorticoids using stratum corneum sampling

Predicting the dermal bioavailability of topically delivered drugs is challenging. In this work, minimally invasive stratum corneum (SC) sampling was used to quantify the delivery of betamethasone valerate (BMV) into the viable skin. Betnovate® cream (0.1% w/w BMV) was applied at three doses (2, 5, and 10 mg cm−2) to the ventral forearms of 12 healthy volunteers. The mass of drug in the SC was measured using a validated tape-stripping method (a) after a 4-h “uptake” period, and (b) following a 6-h “clearance” period subsequent to cream removal. Concomitantly, the skin blanching responses to the same doses were assessed with a chromameter over 22 h post-application. BMV uptake into the SC was significantly higher for the 5 mg cm−2 dose compared to those of 2 and 10 mg cm−2. In all cases, ~30% of the drug in the SC at the end of the uptake period was cleared in the subsequent 6 h. From the SC sampling data, the average drug flux into the viable epidermis and its first-order elimination rate constant from the SC were estimated as 4 ng cm−2 h−1 and 0.07 h−1, respectively. In contrast, skin blanching results were highly variable and insensitive to the dose of cream applied. The SC sampling method was able to detect a 50% difference between two applied doses with 80% power; detection of a 20% difference would require a larger sample size. SC sampling enabled quantitative metrics describing corticosteroid delivery to the viable epidermis to be determined.Graphical abstract

A Novel Approach to Assess the Potency of Topical Corticosteroids.

The potencies of topical corticosteroid products have mainly been classified using clinical data but in some instances, the US Food and Drug Administration’s (FDA’s) vasoconstrictor assay (VCA) to assess the skin blanching response has also been used. However, the reported skin blanching response data were often based on a single visual reading and lack information on the dose (amount/quantity) or dose duration. Although several lists classifying potencies of various topical corticosteroid products have been published, the inherent potencies of topical corticosteroid raw materials used as active pharmaceutical ingredients (APIs) have not been investigated. The objective was to rank the inherent potencies of topical corticosteroid APIs and to standardize dosing such that the relevant compounds could be compared on a normalized molar basis. The potencies of clobetasol propionate, halcinonide, mometasone furoate, and fluocinolone acetonide were compared using the resulting Emax data following the fitting of the relevant response data to the Emax model where mometasone furoate > fluocinolone acetonide = clobetasol propionate > halcinonide. This ranking lists the respective inherent potencies of the APIs, which will facilitate the choice of a suitable candidate for incorporation into an appropriate topical corticosteroid product for a specific clinical indication.

Conversion of fisheries processing by-product into salted-egg fish skin chips

Fisheries processing produces fish skin as a by-product. By using simple technology, the skin can be processed to produce high-protein snack foods, that the taste can be adjusted to the public tastes, such as chips. The economic value of these skin chips can be improved if they are packaged attractively and processed to a contemporary taste. The purpose of this study was to innovate technology in the utilization of fishery by-products to produce the salted egg fish skin chips and analyze its characteristics before and after storage. Fish skin used was tilapia fish (Oreochromis niloticus). The methods include the fish skin blanching using water at a temperature of 90°C for approximately 2 minutes, draining, and soaking in a 0.5% sodium bicarbonate solution at 21-23°C for 15 minutes, then salt 2% and garlic 2.5% (w/w) adding then deep frying with an oil temperature of 170-180°C for 75 seconds. Salted egg fish skin chips were packed in aluminum foil pouches and stored for 84 days. The parameters observed were yield, water content, ash content, protein content, fat content, and rancidity (TBA), physical tests (crispness, i.e., hardness and breaking force also aw), and microbiological analysis (total mold). The results showed that the yield of dried tilapia skin was 37.30%; water content increased during the storage and the same condition for ash content, protein content, fat content, and rancidity, but they did not change significantly. Likewise, crispness values were decreased, but aw values were increased during storage. While the total mold increased, but the value was still within safe limits. The salted egg chips from tilapia fish skin could still be developed to be accepted and safe for consumption by the consumers.

Clinical study -The Facial Skin Beauty and Oral Mucosa Changes Due to Effect of Local Anesthesia Per Oral Surgery

Facial beauty and attractiveness have been linked to social perception, where people with more attractive faces are perceived to have higher athletic, social, and leadership skills. Smile and eyes were reported to be the most important factors regarding the hierarchy of characteristics for determining facial beauty, the purpose of this study was to determine the changes in the facial skin, facial aesthetic, oral mucosa. And record the changes in the facial expressions as a result of the effect of local anesthetic prior to the oral surgery and assess if the smile would be influenced by the local anesthetics, This Clinical study is performed in Oral and maxillofacial surgery department and dental clinics of Saudi Arabia. After the administration of the local anesthetic (4.0 ml of 2% lidocaine with 1: 80000 epinephrine) 9.8% of the patient showed an immediate area of facial blanching; perioral (4.9%), cheeks (3.3%), and periorbital (1.6%). whereas 90.2% of the examined patients showed no facial skin blanching, upon examination Signs indicating blanching were observed and area of skin blanching was noted in the forehead, periorbital, nose, chin, cheeks, perioral and periauricular areas of skin.

Vasoconstrictor potency of fixed-dose combination calcipotriol (50 μg/g) and betamethasone dipropionate (0.5 mg/g) cutaneous foam versus other topical corticosteroids used to treat psoriasis vulgaris

Background: It is important to determine the vasoconstrictor potencies of topical corticosteroids used to treat psoriasis to ensure appropriate clinical use.Objective: To compare the vasoconstrictive potencies of fixed-dose combination calcipotriol (50 μg/g) and betamethasone dipropionate (0.5 mg/g) (Cal/BD) cutaneous foam with other topical corticosteroids.Methods: In this Phase I, single-center, healthy volunteer study, Cal/BD foam, clobetasol propionate 0.05% cream (CP; very potent), BD 0.05% ointment (potent), mometasone furoate 0.1% cream (MF; potent), hydrocortisone-17-butyrate 0.1% ointment (HB; moderately potent), and foam vehicle were applied, then removed after 16 h. Skin blanching was visually assessed 2 h later (scale of 0–4).Results: Thirty-six volunteers were randomized. Skin blanching with Cal/BD foam (median [range], 2.00 [0.75–3.00]) was significantly lower than CP cream (3.00 [1.75–4.00]; p < .001), was not significantly different from BD ointment (1.75 [0.75–3.00]; p = .30) and MF cream (2.00 [1.00–3.75]; p = .22), and was significantly greater than HB ointment (1.25 [0.50–3.00]; p < .001) and vehicle (0 [0–0.50]; p < .001). There were no local tolerability reactions or adverse events.Conclusions: The corticosteroid potency of Cal/BD foam was not significantly different from BD ointment and MF cream, significantly stronger than HB ointment, but weaker than CP cream in healthy volunteers.

Efficacy and Safety of EMLA Cream for Pain Control Due to Venipuncture in Infants: A Meta-analysis.

To determine the efficacy and safety of EMLA in infants <3 months of age requiring venipuncture in comparison with nonpharmacological interventions in terms of pain reduction, change in physiologic variables, and methemoglobinemia. Medline, Embase, Cochrane Central Register of Controlled Trials, Cumulative Index to Nursing and Allied Health Literature, Web of Science, and gray literature were searched from inception to August 2017, without language restrictions. We selected randomized controlled trials in which researchers compared EMLA with nonpharmacological interventions. Two reviewers independently performed abstract screening and full-text review, and extracted the data and assessed the risk of bias. Ten randomized controlled trials (907 infants) were included. EMLA revealed little or no effect in reduction of pain (standardized mean difference: 0.14; 95% confidence interval [CI]: -0.17 to 0.45; 6 trials, n = 742; moderate-quality evidence) when EMLA was compared with sucrose, breastfeeding, or placebo. In comparison with placebo, EMLA revealed a small-to-moderate effect on increasing methemoglobin levels (mean difference: 0.35; 95% CI: 0.04 to 0.66; 2 trials, n = 134; low-quality evidence). There was an increased risk of blanching of the skin in the EMLA group (relative risk: 2.63; 95% CI: 1.58 to 4.38; 2 trials, n = 123; I2 = 84%, very low-quality evidence). Our results may not be applicable to older infants. EMLA reveals minimal benefits in terms of reduction of pain due to venipuncture procedure in comparison with placebo and no benefit in comparison with sucrose and/or breastfeeding. Moreover, it produced an elevation in methemoglobin levels and skin blanching.

Revisiting FDA's 1995 Guidance on Bioequivalence Establishment of Topical Dermatologic Corticosteroids: New Research Based Recommendations.

As per the US FDA guidance issued on June 2, 1995, the establishment of bioequivalence for topical dermatologic corticosteroids is based on comparing the pharmacodynamic (PD) effects of Test and Reference products at the dose duration corresponding to the population ED50, determined either by naïve pooled data or nonlinear mixed effect modeling (NLME). The guidance was introduced using a study case example where the expectation maximization (EM) NLME algorithm, as implemented in P-PHARM®, was used. Although EM methods are relatively common, other methods such as the First-Order Conditional Estimation (FOCE) as implemented in the NONMEM® software are even more common. The objective of this study was to investigate the impact of using different parametric population modeling/analysis methods and distribution assumptions on population analysis results. The dose duration-response data from 11 distinct skin blanching blinded pilot studies were fitted using FOCE (NONMEM®) and an EM algorithm (ADAPT5® (MLEM)). Three different Emax models were tested for each method. Population PD estimates and associated CV%, and the agreement between model predicted values and observed data were compared between the two methods. The impact of assuming different distributions of PD parameters was also investigated. The simple Emax model, as proposed in the FDA guidance, appeared to best characterize the data compared to more complex alternatives. The MLEM method in general appeared to provide better results than FOCE; lower population PD estimates with less inter-individual variability, and no variance shrinkage issues. The results also favored ln-normal versus normal distribution assumptions. The population ED50 estimates were influenced by both the type of population modeling methods and the distribution assumptions. We recommend updating the FDA guidance with more specific instructions related to the population approach to be used (EM-like versus FOCE-like methods) and to the normality assumptions that need to be set (ln-normal versus normal distribution).

Hypoperfusion following the injection of epinephrine in human forearm skin can be measured by RGB analysis but not with laser speckle contrast imaging

BackgroundThe time taken for epinephrine to achieve its optimal effect during local anesthesia has recently become the subject of debate. The time from injection to commencement of surgery is traditionally quoted to be 7 to 10 min, while recent reports claim that it may take 30 min to achieve maximum hypoperfusion, which would prolong the time required for surgical procedures. The discrepancy may be related to difficulties associated with the techniques used to measure blood perfusion. The aim of this study was to test two methods of determining the time to maximum hypoperfusion. MethodsLaser speckle contrast imaging (LSCI) and red, green, blue (RGB) analysis of images obtained with a commercial digital camera, were used to monitor the effect of infiltration with commonly used local anesthetic preparations: lidocaine (20 mg/ml) + epinephrine (12.5 μg/ml), lidocaine (10 mg/ml) + epinephrine (5 μg/ml), and lidocaine (20 mg/ml) alone, in healthy subjects. ResultsLSCI showed a paradoxical increase in signal after the injection of local anesthetics containing epinephrine, probably due to a change in the laser penetration depth resulting from blanching of the skin. However, RGB analysis of digital photographs gave more reliable results, showing skin blanching that corresponded to the expected effect of epinephrine in local anesthetics. The time to maximum effect was found to be 7 (range 5–10) minutes for 12.5 μg/ml epinephrine, and 9 (range 7–13) minutes for 5 μg/ml epinephrine in lidocaine. ConclusionsRGB analysis of digital images proved to be a valid technique for monitoring the effect of local anesthetics with epinephrine in human skin. The technique requires only a commercial digital camera and constitutes a cheap, simple method. The optimal delay between epinephrine injection and incision, to minimize bleeding, was found to be 7 to 9 min, which is in good agreement with common surgical practice.

The Face – A Vascular Perspective. A literature review

Vascular supply is key for maintenance of healthy tissue conditions but also with regard to healing following trauma or therapeutic interventions. The face is probably the most exposed part of the body and any changes of vascularity are readily visible (skin blanching, ecchymosis, hematoma, edema). With regard to the arterial supply, all vessels reaching the facial skin originate from the bilateral common carotid arteries. The ophthal­mic artery is considered the major arterial shunt between the internal and external carotid artery systems. Main arterial contributors to the face include the facial, transverse facial, and infraorbital arteries. In general, homonymous veins accompany the arteries, but there are some exceptions (inferior ophthalmic vein, retromandibular vein). Furthermore, the facial vein demonstrates a consistently more posterior course compared to the facial artery. Lymphatic vessels including lymph nodes play an important role for facial drainage.

The Face – A Vascular Perspective. A literature review

Vascular supply is key for maintenance of healthy tissue conditions but also with regard to healing following trauma or therapeutic interventions. The face is probably the most exposed part of the body and any changes of vascularity are readily visible (skin blanching, ecchymosis, hematoma, edema). With regard to the arterial supply, all vessels reaching the facial skin originate from the bilateral common carotid arteries. The ophthal­mic artery is considered the major arterial shunt between the internal and external carotid artery systems. Main arterial contributors to the face include the facial, transverse facial, and infraorbital arteries. In general, homonymous veins accompany the arteries, but there are some exceptions (inferior ophthalmic vein, retromandibular vein). Furthermore, the facial vein demonstrates a consistently more posterior course compared to the facial artery. Lymphatic vessels including lymph nodes play an important role for facial drainage.

Use of remote photoplethysmography in assessment of topical corticosteroid-induced skin blanching

Use of remote photoplethysmography in assessment of topical corticosteroid-induced skin blanching

A Randomized, Double-Blind Trial to Determine if Vapocoolant in the Adult Population Improves Patient Perception of Pain With Peripheral Intravascular Access.

The purpose of this study was to investigate whether the use of a topical anesthetic spray (vapocoolant) at the site of intravenous access reduces pain and anxiety associated with peripheral intravenous (PIV) catheter insertion in an adult emergency department population. A randomized, double-blind, placebo-controlled, single-center trial, conducted from July to August, in an adult emergency department where 72 patients with orders for PIV insertion receiving either topical vapocoolant spray (n = 38) or placebo spray (n = 34). Vapocoolant or placebo was applied to the intravenous site and allowed to evaporate before cleansing and insertion. The primary outcome was patient perception of pain and anxiety with PIV needle insertion using a 0-10 Likert scale. Secondary outcomes included patient/staff preference or desire for vapocoolant/placebo use in future procedures and staff perception of patient pain and anxiety. Patient groups did not vary significantly in the history of intravenous access, nor anxiety preprocedure, and the sprays did not affect ability to obtain intravenous access. Patient perception of pain did not vary significantly between vapocoolant (2) and placebo populations (2.5), nor did the scores vary significantly for patient-forecasted anxiety in future procedures using the same methods (0.5 for vapocoolant, 0 for placebo; p > 0.05). Eighty-nine percent of nurses and patients would use vapocoolant in future procedures, whereas 74% would use placebo; the difference was not significant (p > 0.05). No skin blanching or lesions were noted. Among adult patients in the Parkland Emergency Department receiving PIV access, no significant differences in pain relief or alleviation of anxiety were found between treatment using a topical anesthetic spray or placebo.

Pharmaceutical development of a generic corticoid semisolid formulation

The development of bioequivalent topical products is still a challenge, partly due to a lack of general methods available to perform this assessment. Several tests are proposed to fulfil this objective, such as dermatopharmacokinetic and pharmacodynamic evaluation. The aim of this study is the evaluation of a topical corticoid emulsion as a candidate to undergo further skin blanching tests. 0.1% mometasone furoate pickering emulsion was produced and evaluated regarding appearance, pH, viscosity assay and impurity D values, under three experimental conditions, 25°C/60% HR, 30°C/65% HR and 40°C/75% HR., in vitro drug release and ex vivo skin absorption profiles have also been studied. All the parameters evaluated remained within specifications in time. Based on impurity D levels, expected shelf life is over five years for formulations stored at 25 °C and three years for formulations stored at 30 °C. A moderate range of viscosity (16785–94611 mPa s) was found between batches, but this variability did not lead to statistical differences in Higuchi's release constants. Mometasone skin absorption parameters were found to be statistically equivalent to the reference formulation (elocom® emulsion), therefore, the present formulation is a good candidate for skin blanching tests.

Enhanced vasoconstrictor potency of the fixed combination calcipotriol plus betamethasone dipropionate in an innovative aerosol foam formulation vs. other corticosteroid psoriasis treatments.

BackgroundAn aerosol foam formulation of fixed combination calcipotriol 50 μg/g (Cal) and betamethasone 0.5 mg/g (as dipropionate; BD) has been developed for psoriasis vulgaris treatment.ObjectiveTo compare Cal/BD aerosol foam pharmacodynamic activity with Cal/BD ointment and with other topical corticosteroids of different potencies by assessing vasoconstrictor potential.MethodsA Phase I, single‐centre, investigator‐blinded, vehicle‐controlled, intra‐individual comparison vasoconstriction study. Healthy volunteers received a single application on selected sites of: Cal/BD aerosol foam, clobetasol propionate 0.5 mg/g cream (CP; very potent), Cal/BD ointment (potent), fluocinolone acetonide 0.25 mg/g ointment (FA; moderately potent), BD aerosol foam and aerosol foam vehicle. A seventh untreated site acted as a negative control. Skin blanching was assessed by visual (primary response criterion) and colorimetric a* and L* measurements (secondary criteria), and was analysed over time (6–32 h post‐application).ResultsThirty‐five healthy volunteers were included. All active treatments led to significantly greater skin blanching than control. By visual assessment, skin blanching with Cal/BD aerosol foam was significantly less compared with CP cream [mean AUC0–32 2560 vs. 3831; mean difference = −1272; 95% confidence interval (CI): −1598, −945; P < 0.001], similar to BD aerosol foam (mean AUC0–32 2560 vs. 2595; mean difference = −35; 95% CI: −362, 292; P = 0.83) and significantly greater than Cal/BD ointment (mean AUC0–32 2560 vs. 2008; mean difference = 552; 95% CI: 225, 878; P = 0.001) and FA ointment (mean AUC0–32 2560 vs. 1981; mean difference = 578; 95% CI: 251, 905; P < 0.001). Colorimetric assessments a* and L* also indicated significantly reduced skin blanching with Cal/BD aerosol foam compared with CP cream. No adverse events (AEs) were reported.ConclusionCal/BD aerosol foam can be considered a more potent formulation than Cal/BD ointment and the moderately potent FA ointment, but less potent than the very potent corticosteroid, CP cream.

Enhanced Activity of Topical Hydrocortisone by Competitive Binding of Corticosteroid-Binding Globulin

Atopic dermatitis of sensitive areas such as the face, particularly in children, is a difficult disease to treat as the standard therapeutic, topical steroids, is contraindicated for this application in children. Hydrocortisone (HC) can be used in these instances because it has been shown to be safe, but is often ineffective as it is a relatively weak steroid, especially at over-the-counter concentrations. To enhance the local topical activity of HC, the terminal inactive metabolite of prednisolone, Δ1-cortienic acid (Δ1-CA), is added to HC, as Δ1-CA preferentially binds transcortin, liberating more HC to elicit its therapeutic effect. Skin blanching studies, which are used to evaluate the potency of topical steroids, were employed to assess the ability of Δ1-CA to enhance the activity of HC. The results demonstrate that Δ1-CA, when applied in combination with HC, does indeed potentiate the vasoconstriction effect of topically applied HC, while having no effect alone. Thus, addition of the inert prednisolone metabolite Δ1-CA can increase the therapeutic effect of over-the-counter concentrations of HC when applied topically.

Bioequivalence of topical corticosteroids: Design and data analysis challenges with the vasoconstrictor assay

T vasoconstrictor assay (VCA) method published in FDA’s 1995 Guidance on bioequivalence of topical dermatologic corticosteroids is currently the only pharmacodynamic method approved by FDA for demonstrating bioequivalence of topical corticosteroid products (gels, creams, lotions, ointments, foams, tapes and sprays). The Guidance recommends a pilot dose durationresponse study to determine the appropriate dose duration for use in the pivotal study. In general, evaluation of bioequivalence by the VCA method has worked well over the last 20 years, but there remain several challenges in design and data analysis. For example, for the dose-response study the fit of the simple Emax model is not always optimal with low potency products, with products that show delayed vasoconstrictor (skin blanching) response, or with subjects that have low skin blanching (low Emax); for the pivotal study a too short or very long ED50 duration products can pose operational challenges. Furthermore, the sample size recommendation in the Guidance of 40-60 qualifiers for pivotal studies may be too low for those products that show high intra-subject variability in vasoconstrictor response. Successful bioequivalence studies depend on the clinic’s ability to recruit a fair-skinned population of subjects that show high and consistent skin blanching to even low potency topical steroids so that within-subject variability in vasoconstrictor response is minimized. This presentation will discuss approaches for minimizing operational issues, estimating sample size, and evaluating reliability of blanching profiles for pivotal studies and will provide examples of design and data analysis challenges for dose-response and pivotal studies.

Effects of topical corticosteroids on cell proliferation, cell cycle progression and apoptosis: In vitro comparison on HaCaT

Topical-corticosteroids are mainly used for the treatment of inflammatory or hyperproliferative skin diseases. The in vivo assay to rank topical-corticosteroids potency, based on the skin blanching, is not adapted to compare their anti-proliferative efficacy. We have compared the antiproliferative effect of six topical-corticosteroids on a model of hyperproliferant keratinocytes (HaCaT). Betamethasone-dipropionate; clobetasol-propionate; betamethasone-valerate; desonide; hydrocortisone-butyrate and hydrocortisone-base, at different concentrations (10−8–10−4M) have been compared. HaCaT proliferation has been evaluated by MTT-assay and the mechanism of the death was evaluated by annexin V/propidium iodide staining and cell cycle phases analysis. Topical corticosteroids reduced cell growth in a dose-dependent manner. At 10−4M, betamethasone dipropionate was the most antiproliferative compound while hydrocortisone-butyrate was the less. Hydrocortisone-base which is usually considered as the less potent topical-corticosteroids showed a clear cytotoxic effect. Betamethasone-dipropionate and betamethasone-valerate induced more apoptosis than necrosis whereas the reverse has been observed for other topical-corticosteroids. All topical-corticosteroids, except clobetasol-propionate, arrested cell cycle mainly in G2-phase. Clobetasol-propionate arrested cell cycle in S-phase population. At 10−8M, topical-corticosteroids induced HaCaT proliferation. In terms of antiproliferative effect at 10−4M, we propose to rank topical corticosteroids as follow: betamethasone-dipropionate>desonide≥betamethasone-valerate=hydrocortisone-base=clobetasol-propionate>hydrocortisone-butyrate. This classification differs from the current ranking, based on the vasoconstrictive effect, but is more adapted for hyperproliferative disease treatment.