Comparison of reservoir effect of topical corticosteroids in an experimental animal model by histamine-induced wheal suppression test.

INTRODUCTION: Topical corticosteroids used in various dermatological diseases are available in different potencies and different formulations. The reservoir effect of different potency corticosteroids in the stratum corneum will help the clinicians to choose an appropriate topical steroid to maximize their efficacy and safety as therapeutic agents. AIMS: This study was designed to compare the duration of reservoir of different potency topical corticosteroids experimentally in rabbits using histamine-induced wheal suppression test.
MATERIALS AND METHODS: The study was carried out in albino rabbits (as their skin is similar to humans) using four different concentrations of topical steroids, namely mometasone furoate ointment (0.1%), fluticasone propionate ointment (0.005%), betamethasone valerate cream (0.1%), and hydrocortisone butyrate cream (0.1%). These were applied on the back of rabbit on one side and the vehicle was applied on the other. One hour later, histamine-induced wheal suppression test was performed on both sides and wheal area was measured at 10 min for 7 days. Statistical analysis was done by ANOVA followed by post hoc test.
RESULTS: Maximum wheal suppression was seen on day 1 (P<0.001) in all four groups, both at test and at control sites. Interday comparison of mean wheal size by topical steroids showed that the reservoir of mometasone furoate ointment (0.1%) persisted till day 4 in the stratum corneum of the skin. In case of fluticasone propionate ointment (0.005%) and betamethasone valerate cream (0.1%), the reservoir persisted till day 2 and for hydrocortisone butyrate cream (0.1%), the reservoir was present only on day 1.
CONCLUSIONS: It is concluded that the duration of reservoir depends on the potency of topical steroids. Higher the potency more is the duration of reservoir in stratum corneum and vice versa.

Introduction

Topical glucocorticoids clinically have anti-inflammatory, immunosuppressive, antiproliferative, and vasoconstrictive effects.[1] These effects can be “immediate” due to direct steroid incorporation into membranes or “delayed” effects which are mediated by DNA transcription via a second messenger system. Due to regular and frequent usage of high potency steroids in various dermatoses, local and systemic adverse effects are likely. The local adverse effects with topical preparations include skin atrophy, telangiectasia,[2] purpura, striae, skin pigmentation, acneiform eruptions, rosacea, and dermatitis. Another important phenomenon which can occur with constant use of topical steroids is tachyphylaxis,[3] and abrupt discontinuation can lead to rebound dermatological condition. A decision about the choice of an appropriate steroid, its frequency of application, and the total duration of treatment therefore becomes difficult. Topical steroids have been classified according to their clinical potency which is assessed by vasoconstrictor assay.[4] In addition, an American classification divides them in seven groups, group I being super potent and group VII, least potent.[5]

Existence of a reservoir of topically administered steroids in stratum corneum has been demonstrated.[678] A depot effect based upon the pharmaceutical formulation of topical steroid has also been demonstrated.[9-13]

All these studies have used different assay methods and show varying results. Moreover, none of them have compared the reservoir effect of topical steroids of different potencies. We hypothesized that the higher potency topical steroids may have a reservoir effect for the greater duration and vice versa. Hence, this study was undertaken with four different potency topical steroids (according to American classification) to evaluate their reservoir effect and its duration using the histamine-induced wheal suppression bioassay method.

Materials and Methods

We used albino rabbits of either sex weighing between 1 and 2 kg. Animals were obtained from an authorized dealer and housed individually in metabolic cages under standard conditions in our central animal house. They were provided food and water ad libitum without any limitation exposed externally. Approval of Institutional Animal Ethics Committee was obtained.

Rabbits were chosen as their skin is akin to human skin.[14] Histamine-induced wheal suppression test[15] was employed. A pilot study was carried out initially to standardize the wheal size. Six albino rabbits of either sex were taken and weighed. The ear was first cleaned with spirit and then with xylene (for venous dilatation). Then 1 ml of 10 mg/ml of Evans blue dye (Central Drug House (P) Ltd, Mumbai [CDH]) was injected into the opposite marginal ear vein to facilitate identification of the histamine-induced wheal circumferences. Two identical sites were selected on either side on its back, near the anterior shoulder region of each rabbit. These areas (5 × 5 cm2)[11] were shaved and cleaned. After 30 min of injecting the dye, 0.05 ml of histamine dihydrochloride 1 mg/ml (0.1% w/v) was injected intradermally into the shaved area. The site was wiped with filter paper to remove the excess histamine solution and the wheal size was marked. The wheal area was clearly demarcated with the help of Evans blue dye.[16] The traced wheals were transferred to a transparent paper and then the wheal area was measured in cm2 by calipers at 10, 20, 30 min, and 1 h and readings were taken in each rabbit. Maximum wheal size was observed at 10 min.

Six readings were obtained and then the wheal size was standardized. After this, the test readings were taken without using the Evans blue dye.

Methodology

Forty-eight rabbits were taken for this experimental study and divided into four groups of 12 each. These rabbits were weighed and two areas (5×5 cm2 each) on the back, near the anterior shoulder of each rabbit, were shaved off with the help of a razor on either side. Right side area was taken as test and left area as control.

The following topical steroids were used. (Potency according to American classification):[5]

Group 1: Mometasone furoate ointment (0.1%), Elocon® (Fulford (India) Ltd, Mumbai, India.) (Class II potent)

Group 2: Fluticasone propionate ointment (0.005%), Flutivate® (GlaxoSmithKline, Mumbai, India.) (Class III potent)

Group 3: Betamethasone valerate cream (0.1%), Betnovate® (GlaxoSmithKline, Mumbai, India.) (Class V midstrength)

Group 4: Hydrocortisone butyrate cream (0.1%), Locoid® (Elder Health Care Ltd, Mumbai, India.) (Class VII least potent)

Half finger-tip unit (FTU)[17] of each topical steroid was applied on right side and its corresponding vehicle was applied on the left side and both sides were occluded with a plastic wrap for 1 h. After this, the plastic wraps were removed, and the remaining topical steroids from right side and vehicle from left side were wiped off gently with a clean cotton piece.[18]

The histamine-induced wheal suppression test was performed on both the sites.[15] Histamine solution containing 1 mg/ml (0.1% w/v) of histamine dihydrochloride was made in distilled water and 0.05 ml was injected intradermally with the help of an insulin syringe so that a bleb or wheal was raised on either side. The wheals were traced on a transparent paper and the wheal area was measured on either side with the help of calipers in cm2 at 10 min on days 1-7. The wheal area of test site was compared with the control site on each day.

Statistical Analysis

The data were analyzed using Statistical Package for Social Sciences (SPSS) for Windows Version 10 and ANOVA followed by post hoc test were applied. P value <0.05 was considered to be significant.

Results

Interday comparison of mean wheal size among the control and drug values at 10 min was done. There was no wheal suppression on the control side on day 1. Statistical analysis by post hoc test shows that there was no significant difference in the wheal size among all four vehicles if days 2-7 were compared with day 1 among the control values (P>0.05). Thus, there was no reservoir of any of the vehicles used. With mometasone furoate ointment (0.1%), maximum wheal suppression was seen on day 1. Days 2, 3, and 4 showed no significant difference in the wheal size (P>0.05) as compared to day 1 but days 5-7 showed significant difference in the wheal size (P <0.05) as compared to day 1 [Table 1]. This shows that the wheal was suppressed till day 4 by mometasone furoate ointment (0.1%); thus, the reservoir persisted till day 4. In case of fluticasone propionate ointment (0.005%) and betamethasone valerate cream (0.1%) also, maximum wheal suppression was seen on day 1. Day 2 showed no significant difference in the wheal size (P>0.05) as compared to day 1 but days 3-7 showed significant difference in the wheal size (P<0.05) as compared to day 1 [Table 2 and 3]. This shows that the reservoir of these topical steroids persisted till day 2. In case of least potent hydrocortisone butyrate cream (0.1%), maximum wheal suppression was seen on day 1. Days 2-7 showed significant difference in the wheal size (P<0.05) as compared to day 1. This shows that the wheal was suppressed only on day 1 and thus the reservoir was present only on day 1 of application [Table 4].

Table 1

Interday comparison of wheal size using mometasone furoate at 10 min (n = 12)

Table 2

Interday comparison of wheal size using fluticasone propionate at 10 min (n = 12)

Table 3

Interday comparison of wheal size using betamethasone valerate at 10 min (n = 12)

Table 4

Interday comparison of wheal size using hydrocortisone butyrate at 10 min (n = 12)

Discussion

With the availability of several corticosteroid formulations, it is important to know the potency of a particular preparation in relation to others, since the efficacy, response, patient compliance, and local and systemic adverse effects all depend on potency. An ideal topical corticosteroid should possess such characteristics which offer high concentrations or a greater reservoir effect on the skin at the site of application so that the topical efficacy is higher resulting in complete cure and lower absorption rate into the systemic circulation. Local side effects cannot be avoided totally but can be reduced by decreasing the frequency and the duration of application. Chemical structure and type of vehicle are important factors to decide about the potency of topical steroid preparation.[1920] Percutaneous absorption of topical corticosteroids (and hence their potency) is increased by occlusion as it increases hydration of skin. Thus, the use of occlusive vehicles, such as ointments or plastic bio-occlusive dressings increases the permeability of topical steroids.[21]

The effectiveness of the corticosteroids also depends on the rate of penetration into the stratum corneum. This rate of penetration and receptor binding in viable epidermis are greater with more lipophilic steroids. Hence, higher the lipid solubility, the more is likely to be the clinical potency.

Once the steroid penetrates the epidermis, it undergoes biotransformation by various enzymes into inactive metabolites. For increased effectiveness, the steroid must stay in the skin for sufficient period of time and must resist this biotransformation. The efficacy of a topical steroid is also affected by the condition of skin. Drug delivery is inversely proportional to the thickness of stratum corneum. Thus, maximal absorption occurs from mucous membranes, scrotum, eyelids, and hair follicles and least from palms, soles, and nails.[22]

There are various bioassay methods employed to study the potency, duration of action, reservoir effect of topical corticosteroids, and effect of various pharmaceutical formulations on the efficacy of topical drugs.

We preferred the histamine-induced wheal suppression test as it has many advantages over other known assay techniques. In this test, wheals are produced on the skin by a standard histamine solution and the ability of topical corticosteroids to reduce the wheal size induced by histamine is measured.[15] It is a simple, reliable, noninvasive, and reproducible method and therefore considered close to clinical practice. More than two compounds may be tested at a time on the same subject and hence less number of animals/patients may be required. The end point of this assay is easy to observe and skin color does not interfere with the interpretation of results as is the case with vasoconstriction assay where there may be difficulty in appreciating the pallor on a dark skin. Although it is a nonclinical assay, it has close similarity to processes involved in the development of clinical dermatoses. Hence, this method can be reproduced in human studies after conducting successful animal studies. The stratum corneum has both barrier and reservoir function for topically applied substances. We used the common market preparations and expected that the reservoir effect duration may be different for different potency topical steroids. The results of our study clearly demonstrated that the reservoir of mometasone furoate ointment (0.1%) persisted till day 4. It was 2 days for fluticasone propionate and betamethasone valerate and only a day for hydrocortisone butyrate. Thus, it is concluded that the duration of reservoir is more for high potency steroids and less for low potency steroids.

The potency of a steroid determines its efficacy and the reservoir effect will determine its duration of action. A longer reservoir function can lead to prolonged action of topical steroids, especially in case of high potency steroids. Therefore, the high potency steroids may be prescribed less frequently or intermittently. This will decrease the amount of drug used, curtail the cost as well as local and systemic adverse effects. A limitation of this study is that the actual amount of steroid reservoir was not determined.