Indian Journal of Dermatology
: 2010  |  Volume : 55  |  Issue : 4  |  Page : 329--333

Study of reservoir effect of clobetasol propionate cream in an experimental animal model using histamine-induced wheal suppression test

Afroz Abidi1, Farida Ahmad2, Satyendra K Singh3, Anil Kumar2,  
1 Department of Pharmacology, Subharti Medical College, Meerut, India
2 Department of Pharmacology, J.N. Medical College, A.M.U., Aligarh, India
3 Department of Dermatology and Venerology, Institute of Medical Sciences, BHU, Varanasi, India

Correspondence Address:
Afroz Abidi
C/o. S.I.S Abidi, Mass Products India Ltd., Mill Road, Aishbagh, Lucknow 226 004


Background: Topical corticosteroids used in various dermatological diseases several times a day led to an increase risk of side effects. By demonstrating a significant reservoir of corticosteroids in the stratum corneum, one can maximize their efficacy and safety as therapeutic agents. Aim: The study was designed to demonstrate a reservoir of topically applied corticosteroid clobetasol propionate cream experimentally in rabbits using histamine-induced wheal suppression test. Materials and Methods: The work was carried out on albino rabbits, as rabbit skin is akin to human skin, using a topical steroid. The topical steroid clobetasol propionate 0.05% cream was applied on the back of rabbit, and after 1-h occlusion histamine-induced wheal suppression test was performed and wheal area measured at 10 min till day 7. Statistical analysis was done by ANOVA followed by DQPost HocDQ test. Results: Maximum wheal suppression was seen on day 1 (P < 0.001). Interday comparison of mean wheal size showed no significant difference (P > 0.05) on day 2, 3, and 4 as compared to day 1. Day 5-7 show highly significant difference (P < 0.001) as compared to day 1, thereby suggesting that the reservoir effect of topical clobetasol propionate 0.05% cream persisted till day 4. Conclusions: This work demonstrated that histamine-induced wheal by the topical steroid clobetasol propionate 0.05% cream was suppressed till day 4, indicating that the reservoir of topical corticosteroid persisted till day 4.

How to cite this article:
Abidi A, Ahmad F, Singh SK, Kumar A. Study of reservoir effect of clobetasol propionate cream in an experimental animal model using histamine-induced wheal suppression test.Indian J Dermatol 2010;55:329-333

How to cite this URL:
Abidi A, Ahmad F, Singh SK, Kumar A. Study of reservoir effect of clobetasol propionate cream in an experimental animal model using histamine-induced wheal suppression test. Indian J Dermatol [serial online] 2010 [cited 2020 Jul 13 ];55:329-333
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Corticosteroids have been in use for over 50 years. Over time, they have become indispensable in controlling a variety of disease states. Topical glucocorticoids have both immediate effects which cause membrane stabilization and delayed effects which are due to glucocorticoid alteration of DNA transcription. Clinical effectiveness of glucocorticoids is related to its four basic properties antiproliferative/antimitotic effects, immunosuppressive, vasoconstrictive, and anti-inflammatory effects.

Antiproliferative effects of topical glucocorticoids is mediated by inhibition of DNA synthesis and mitosis on several cell types, but this antimitotic effect is non-cell cycle specific. [1]

Immunosuppresion may occur due to mast cell depletion in skin. It also causes local inhibition of chemotaxis of neutrophils in vitro and decreases the number of Langerhans cells in vivo. They induce monocytopenia, eosinopenia, lymphocytopenia, and increases circulating neutrophils. [2]

Topical steroids cause capillaries in the superficial dermis to constrict, thus reducing erythema. Ability to cause vasoconstriction correlates with anti-inflammatory potency and thus vasoconstriction assays are used to predict the clinical potency of the agent. [3]

Anti-inflammatory activity of steroids is non-specific. Glucocorticoids inhibit phospholipases-A 2 by inducing the production of lipocortins and also by directly inducing the phosphorylation of enzyme. [4] It may also be due to inhibition of phagocytosis and stabilization of lysosomal membranes. The topical corticosteroids also blocks the production and release of cytokines including interleukins IL-1,2,3,6 and tumor necrosis factor-alpha (TNF-α).[5] Hence, the vasoconstriction produced at the local site is caused by the topical steroid itself.

Stratum corneum, the outermost layer of epidermis is an impervious biological layer, which has barrier functions. Apart from this barrier function, the stratum corneum also has a reciprocal function of serving as a "reservoir" where the drugs are stored until changes in temperature or humidity affect their release into dermal vessels.

There is a wide range of clinical potency of topical corticosteroid formulations. Potency is the amount of drug required to produce a desired therapeutic effect. Various human and animal assays are available to grade the potency of topical steroids. The commonly used method is vasoconstriction assay described by Mc Kenzie and Stoughton in 1962. [6] They correlated the efficacy of steroid with its vasoconstriction effect which can be visualized as skin blanching. Another important test for measuring the potency of steroid is histamine-induced wheal suppression test. In this test, the ability of topical corticosteroids to reduce the size of wheal induced by histamine is used to assess their relative potencies. [7]

Topical corticosteroids have been classified on the basis of vasoconstriction assay and the clinical efficacy. There are two systems of classification: American [8] and British National Formulary Classification.

Adverse effects from the use of topical steroids have become more prevalent since the introduction of higher potency steroids. Local side effects such as epidermal thinning, dermal striae, atrophy, telangiectasia, purpura, and tachyphylaxis and systemic side effects such as suppression of HPA axis, stunting of growth in children and Cushing's syndrome have been reported.

The reservoir function of stratum corneum was first reported by Vickers in 1963. He demonstrated that topically applied corticosteroid forced into stratum corneum by occlusion for a few hours and remained there for as long as 7-14 days which was observed by the development of a physiological marker, vasoconstriction. [9]

There are other studies also which establish the reservoir function of topical corticosteroid. Among these studies, the ones which are worth mentioning are the studies by Rimbau and Lleonart, [10] Schaefer and coworkers, [11] Brickl et al., [12] Kellner et al., [13] Clarys et al., [14] and Teichman et al. [15]

By reviewing all these studies, this study was designed to observe the reservoir effect of topical corticosteroid clobetasol propionate 0.05% cream, the superpotent (Class I) steroid according to American classification. This study was carried out experimentally on rabbits as rabbit skin is akin to human skin, [16] by the method of histamine-induced wheal suppression test. It will be further correlated with human studies to help us maximize the efficacy and safety of topical steroid as therapeutic agents.

 Materials and Methods

The study was carried out taking albino rabbits of either sex weighing between 1 and 2 kg. Animals were housed individually in metabolic cages under standard conditions. They were provided food and water ad libitium. Approval of institutional animal ethics committee was obtained.

A pilot study was carried out to standardize the wheal size. For this, six albino rabbits were taken and weighed. The ear was cleaned with spirit and xylene (for venous dilatation) and 1 mL of 10 mg/mL of Evans blue dye (CDH) was injected into the marginal ear vein. Two identical sites were selected on its back, near the anterior shoulder region of each rabbit. These areas (5 Χ 5 cm 2 ) were shaved and cleaned. After 30 min of injecting the dye, 0.05 mL of histamine dihydrochloride 1 mg/mL (0.1%) was injected intradermally into the shaved area and the wheal size was marked. [17] The wheal area was clearly demarcated with the help of Evans blue dye. Then, the wheal size was measured in cm 2 by callipers at 10, 20, 30 min and 1 h and six readings were taken. Maximum wheal size was observed at 10 min.

After the wheal size had been standardized, the test readings were taken without using the Evans blue dye.

Clobetasol propionate 0.05% cream, Tenovate® (GlaxoSmithKline) was used as a Topical steroid.


For this study, 12 rabbits were taken. These rabbits were weighed and on anterior shoulder of each rabbit, two areas of 5Χ5 cm 2 were shaved with the help of a razor on either side. Right-sided areas were taken as test sites, and left-sided areas as control sites.

On the right side, the topical steroid, clobetasol propionate 0.05% cream, was used. One fingertip unit (FTU) of topical steroid was applied, and it was occluded with a plastic wrap for 1 h. On the control side, we applied non-greasy cream base, propylene glycol which was similarly occluded.

After 1 h, the plastic wraps were removed, and the remaining topical steroid and cream base were wiped off gently with a clean cotton piece. [18] Hence, the excess amount of the steroid remaining on the skin is removed.

Then, histamine-induced wheal suppression test was performed on both the sites. [6] Histamine solution containing 1 mg/mL (0.1%) 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.

Then, the wheal area was measured on either side with the help of callipers in cm 2 at 10 min on day 1, 2, 3, 4, 5, 6, and 7. The wheal area of test site was compared with the control site on each day [Figure 1].{Figure 1}

Statistical analysis

ANOVA followed by "Post Hoc" test. A P < 0.05 indicates the difference to be significant.


Interday comparison of mean wheal size among the control and drug values at 10 min was done. Mean difference of these control and drug values were calculated on each day. Statistical analysis by "Post hoc" test shows that there was no significant difference in the wheal size if day 2-7 was compared with day 1 among the control values (In each case P > 0.05). On using clobetasol propionate 0.05% cream [Table 1], maximum wheal suppression was seen on day 1. Day 2, 3, and 4 show no significant difference in the wheal size (P > 0.05) as compared to day 1. Day 5-7 show highly significant difference in the wheal size (P < 0.001) as compared to day 1. This shows that the wheal was suppressed till day 4, thereby suggesting that the reservoir of topical steroid clobetasol propionate 0.05% cream persisted till day 4.{Table 1}


Frequent use of topical corticosteroids leads to several local and systemic side effects. These adverse effects are more common with potent steroids and especially if the area of application is large. Besides these, other factors such as use of occlusion, site of application, status of stratum corneum, and application frequency also affect the occurrence of side effects. Various studies were undertaken to reduce the side effects of topical corticosteroids without affecting the efficacy of the molecule.

Reservoir effect of stratum corneum is now a well-known fact. Since we know that topical steroid is retained in stratum corneum for some time, we can reduce the frequency and the duration of application. Topical corticosteroids are absorbed through the skin into dermal blood vessels, with some of the compound being metabolized and excreted as occurs with systemic administration. Within the skin, concentration of drug is highest near the skin surface and lowest in dermis. Viable epidermis below stratum corneum is metabolically active and contains cytochrome P450 enzyme, which causes hydrolysis or sulfate conjugation of topically applied steroids within the epidermis. However, clobetasol is a fluorinated steroid and also contains a substituted 17-hydroxyl group hence it is not metabolized in the skin. Various factors affect absorption like anatomical site, age, skin hydration, occlusion, drug concentration, damage of stratum corneum, etc. Percutaneous absorption of topical corticosteroids is increased by occlusion as it increases hydration of skin. [19]

There are many methods to measure penetration of a topical steroid such as using a radiolabeled topical preparation, microdialysis, by taking serial strippings of skin, vasoconstrictor assay and by histamine-induced wheal suppression test.

Radiolabeling was not used as it is hazardous and was not ethically permitted by the institutional ethical committee.

Microdialysis is an in vivo sampling technique that can be used to measure the test substance in the dermis. An artificial probe is placed into the dermis, which consists of a semi-permeable structure allowing molecules to pass into the perfusate inside the probe by passive diffusion. The perfusate, now called the dialysate, containing the test substance is collected in small vials for analysis. This technique has been used in human volunteers as well as in animals. By this technique, one can analyze the presence of any substance in the dermis only and not in the upper epidermal layers. [20] However, the reservoir effect of topical steroids is seen in the superficial layer of epidermis, i.e., in the stratum corneum as has been demonstrated by various studies. [9],[11],[13],[14],[15] Moreover, it is an expensive, complicated, and invasive method. Other considerations are that the test substance may interact or stick to the probe, the membrane's permeability may not be suitable for penetration of the substance and whether the test substance is lipophilic or not-since very lipophilic substances prefer to stay outside the probe. It is also necessary to apply a high topical drug concentration to obtain measurable levels in the dialysate. Therefore, this method is not suitable for measuring the reservoir effect of topical steroids in the stratum corneum.

Many studies have used vasoconstrictor assay as a marker for reservoir of administered topical steroids. [3],[6] However, in our study, we have used histamine-induced wheal suppression test for determining the reservoir effect of a topical steroid. This histamine bioassay of topical steroids has many advantages over other known assay techniques. It is a simple, reliable, noninvasive, and reproducible procedure. It involves the suppression of the wheal produced by histamine; therefore, it is more likely to be nearer the clinical situation than vasoconstrictor assay which demonstrates the effect on normal blood vessels, as histamine will mimic the inflammatory condition in which steroids have to be used. More than two or three compounds can be tested at a time on the same subject, and hence the study can be conducted with a smaller number of patients. Moreover, on a dark skin, there is considerable difficulty in appreciating the pallor produced by local vasoconstriction but in this test the end point of the assay is easy to observe both in fair skin as well as in dark skin individuals. [7]

The existence of the reservoir effect for topical steroids in the stratum corneum (and not dermis) is well documented. The reservoir function of stratum corneum was first reported by Vickers (1963). He demonstrated a reservoir for 7-14 days for potent fluorinated steroids which was observed by the development of a physiological marker, vasoconstriction. This reservoir was in stratum corneum as no reservoir was demonstrated on skin stripped of stratum corneum. [9],[21] Vickers in 1980 claimed that the reservoir was in the stratum corneum, because a reservoir effect can still be demonstrated after the skin has been scrubbed with various detergents. [22]

Rimbau and Lleonart in 1975 compared the absorption kinetics of flumepasone and triamcinolone acetonide both labeled with tritium. The results showed a reservoir of corticosteroids at 400-700 mm depth and that flumepasone remains longer in skin than does triamcinolone acetonide. [10]

Schaefer and coworkers in 1978 observed the kinetics of absorption using radiolabeled drugs in vitro and in vivo. The main hindrance against penetration of drugs is by the horny layer, but the reciprocal function of stratum corneum, i.e., the reservoir function is also important for the efficiency of topical treatment. [11]

The presence of a reservoir in stratum corneum was also confirmed by Brickl et al. in 1980, who showed in the minipig that considerable proportions of radioactive corticosteroids may be recovered from stripped skin layers on the second and fifth day after application. [12]

Kellner et al. demonstrated in 1986 that topical application of corticosteroid prednicarbate (prednisolone-17-ethyl carbonate-21-propionate) which had been labeled with 14C in position four was retained in uppermost layer of stratum corneum and concentration fell with increasing depth of skin. This was demonstrated with the help of histo-autoradiographic studies. Thus, the horny layer serves both as a reservoir and as a barrier for topical prednicarbate. [13]

Clarys et al. in 1999 demonstrated that stratum corneum retention time is important for elaboration of optimal topical treatment by skin color measurements by chromametry for retention time of halcinonide. Significant reservoir for halcinonide was found up to 5 days of initial application. [14]

Teichman et al. developed an in vivo method to determine the stratum corneum reservoir by tape stripping technique 1 and 6 h after application. The results indicate that the reservoir of drugs is present in the stratum corneum, but the saturation level depends upon the individual volunteer, the topically applied substance, and the formulation used. [15]

In humans, the normal transit time for a basal cell, from the time it detaches from the basal layer to the time it enters the stratum corneum is at least 14 days. Transit through the stratum corneum and desquamation requires another 14 days. It means maximum depot affect of any topically applied medicine cannot be more than 14 days in non-proliferating human skin.

Thus, reservoir effect of topical corticosteroids was confirmed by these studies but different studies showed a wide variation in reservoir effect duration. Keeping all these facts in mind, we planned this study to find out the duration of depot effect. We chose rabbit as rabbit skin is similar to human skin, but will further correlate our study with human assays.

Our study confirmed the reservoir effect of topical corticosteroids. Since occlusive dressing has been applied for 1 h, and the remaining drug has been wiped off with a clean cotton, so bathing did not affect the outcome. [18] Moreover, drug does not remain on the skin, it is absorbed percutaneously.

Rabbits do not sweat through skin, and the area is occluded with a plastic wrap, hence there is no loss of drug due to sweating. Since the drug is absorbed into the skin and got stored in the stratum corneum, [9],[11],[13],[21],[22] so it may not be lost due to sweating. In humans also, the penetration of a topical drug is increased by occlusion so there can be no loss of the topical drug by sweating even in humans.

The FTU is the standard amount of topical steroid, which is advocated to be used in most of the dermatological diseases. Dermatology Working Groups in the UK [23] have recommended the guidance of FTU's for use of topical corticosteroids. This use of the FTU is greatly promoted worldwide to reduce the variation in usage of topical steroids and to encourage adherence to therapy. The FTU has been used to standardize the amount of cream being applied in clinical research studies in India also. [24] Hence we have used this standard amount in our study.

Rai et al. in 2004 [24] used 1 FTU of clobetasol propionate 0.05% cream on an area of 3 Χ 3 cm 2 in their study. On the basis of this study, we took a slightly larger area of 5 Χ 5 cm 2 for applying the topical steroid. The excess steroid left on the skin was wiped off after occlusion of 1 h; hence, the reservoir effect will only be seen by the amount of drug which is absorbed into the skin and not the total amount of drug which has been applied.

This study showed that the reservoir of topical steroid clobetasol propionate 0.05% cream persisted till day 4. Thus, the reservoir function is an important determinant of the duration of action of topical corticosteroids in skin, and will lead to prolonged action. Due to this cumulative depot effect, we can advocate less frequent application of topical corticosteroids, i.e., either alternate day or once every fourth day from the present advocated therapy of once a day. By doing so, the benefits of the therapy can be maximized, the cost can be reduced and local and systemic adverse effects of corticosteroids can be decreased. Even the phenomenon of tachyphylaxis [25] which occurs due to continuous use of topical steroids will be reduced. All these factors will improve the patient compliance.

Further, we can test the reservoir effect of different potency steroids and even with different formulations. We can also correlate it with clinical bioassays in humans. These areas need further evaluation.


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