Evaluation of the Effects of Corticosteroids on Histamine Release by <i>ex Vivo</i> Cutaneous Microdialysis

doi:10.4236/jcdsa.2013.33035

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Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 228-233

http://dx.doi.org/10.4236/jcdsa.2013.33035 Published Online September 2013 (http://www.scirp.org/journal/jcdsa)

Evaluat io n o f th e E ff ec ts of Corticosteroids on Histamine

Release by ex Vivo Cutaneous Microdialysis

Carol Courderot-Masuyer1, Sophie Robin1, Hélène Tauzin1, Sylvain Harbon2, Sophie Mac-Mary3,

Alexandre Guichard4, Patrice Muret5, Philippe Humbert6*

1Bioexigence SARL, Espace Lafayette, 8 rue Alfred de Vigny, Besançon, France; 2Plastic Surgery, 9 rue Klein, Saint Vincent Clinic,

Besançon, France; 3Skinexigence SAS, immeuble Bioparc, 2 rue du Dr Paul Milleret, Besançon, France; 4Research and Clinicl Cen-

ter on the Tegument (CERT); Clinical Investigation Center (CIC BT506), Department of Dermatology, Besançon University Hospi-

tal, Besançon, France; 5Laboratory of Clinical and Toxicology Pharmacology, Jean Minjoz University Hospital, Besancon, France;

6University of Franche-Comté, Inserm U1098, SFR FED 4234 IBCT, Besançon, France.

Email: *[email protected]

Received August 2nd, 2013; revised August 27th, 2013; accepted September 2nd, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The purpose of the study was to evaluate the effects of corticosteroids on histamine release and to compare their po-

tency with the MacKenzie classification based on their vasoconstrictor effects. Thank s to ex vivo cutaneous microdialy-

sis, we studied histamine-induced release over a period of time on excised abdominal skin from women. Eight corticos-

teroids were topically applied with occlusiv e dressing onto the skin, above prob es, before anti-IgE injection. Histamine

levels were assessed by an EIA method. In order to compare the different corticosteroids, AUC was calculated allowing

an estimation of the amount of released histamine for 60 min of ex vivo cutaneous microdialysis. Diflucortolone 0.1%

and micronized betamethasone dipropionate 0.05% are considered as corticosteroids with high potency in MacKenzie

classification. Betamethasone dipropionate associated with propylene glycol 0.05%, belongs to a stronger class in

Mackenzie classification. Our results showed that the decrease in histamine release was more important with difluocor-

tolone than with both of these corticosteroids. Therefore there was no correlation between the vasoconstrictor potency

of topical corticosteroids and their ability to inhib it histamine release.

Keywords: MacKenzie Test; Corticosteroids; ex Vivo Cutaneous Microdialysis; Histamine

1. Introduction

The McKenzie’s classification of topical corticosteroids

is based on the ability to produce the blanching of the

skin when they are in contact with them [1,2]. The inten-

sity of skin blanching has been correlated with drug po-

tency and the degree of drug deliv ery th rough th e stratum

corneum. The vasoconstrictor assay has also been used to

measure the bioavibility and bioequivalence of topical

corticosteroids formulations and was adopted in 1995 for

bioequivalence determination by the US Food and Drug

Administration (FDA) [3,4]. The technique consisted of

the application of corticosteroid solutions on the skin

covered with plastic occlusive bandages. The results

were evaluated 16 hours after application. This technique

described a quantitative comparison of the vasoconstric-

tor effect of topically applied corticosteroids in healthy

human skin. However, there is no direct proof that the

vasoconstrictor and anti-inflammatory activities are

correlated. Amongst potentially suitable surro gates for in

vivo clinical tests, there are in vitro dermatopharmaco-

kinetic methods such as the Franz cells system [5,6] and

ex vivo cutaneous microdialysis, in which the rate and

extent of permeation through ex vivo skin is measured. In

Franz cells, the subcutaneous fat was removed, not in ex

vivo cutaneous microdialysis allowing a model closer to

the in vivo co nditions. Even thoug h the blood flow is not

present, we can compare the cutaneous permeation of

different formulations of corticosteroids. The microdialy-

sis (MD) principle can be compared to an artificial blood

vessel. MD sampling is performed by placing a tubular

MD membrane into the dermis, parallel to the skin’s sur-

face. The probe, which is permeable to water and small

molecules, is continuously perfused with a physiological

buffer solution at a low flow rate. Unbound substances

*Corresponding author.

Evaluation of the Effects of Corticosteroids on Histamine Release by ex Vivo Cutaneous Microdialysis 229

present in the skin can cross the membrane and enter the

lumen probe in proportion to a concentration gradient [7].

Leveque et al. compared Franz cells and microdialysis

for the skin absorption of salicylic acid and showed that

the permeation profiles of salicylic acid determined by

the both methods were similar [8]. MD can follow the

evolution of dermis concentrations of endogenous com-

ponents as well as exogenous components over a period

of time. Topical corticosteroids are commonly used in

the treatment of immuno-allergic conditions. We decided

to carry out an experiment in order to measure directly

the ability of topical corticosteroids to inhibit histamine

release in an original ex-vitro model using cutaneous

microdialysis because the integrity of skin barrier func-

tion is maintained. We compared the McKenzie classi-

fication of corticosteroids to this new classification ac-

cording their effect on histamine release. Indeed mast

cells are well known to play a central role in the forma-

tion of allergic inflammation and con tribute to the patho-

genesis of allergic diseases, including atopic dermatitis

and psoriasis. So histamine plays a pivotal role in the

pathogenesis of allergic inflammation. It is known that

the dermis of psoriatic plaques contains an increased

number of mast cells [9]. Krogstad et al. reported that

histamine concentration and release are increased in le-

sional skin of psoriatic patients [10]. Indeed mast cells

reside in all vascularized tissues and particularly high

mast cell numbers can be found at the interfaces of host

and environment, such as the skin. Release of histamine

and “slow reacting substance of anaphylaxis” from mast

cells following crosslinking of cell-bound IgE by aller-

gen was demonstrated in 1985 by Ishizaka et al. [11].

Our study is based on experiments performed by Peter-

sen et al. who induced histamine release from resident

dermal mast cells of excised female abdominal skin with

an intradermal injection of a solution of anti-IgE with a

maximum peak around 15 minutes of microdialysis [12].

Thus it is possible to calculate the area under the curve

for the release of histamine and to use this data to clas-

sify the corticosteroids in accordance with their effects

on histamine release.

2. Materials and Methods

Products

Eight corticosteroids creams were obtained from phar-

macy:

 micronized betamethasone dipropionate 0.05% (Dip-

rosone®) (0.0 5 % mBD)

 betamethasone dipropionate 0.05% with propylene

glycol (Diprolene®) (BdD + PG 0.05%)

 desonide 0.05% (Tridesonit®) (DES 0.05%)

 desoni de 0.1% (Locap r ed ®) (DES 0.1%)

 betam e thasone valerate 0.1% ( B e t n eva l®) (BV 0.1%)

 diflucortolone val e rate 0.1% (N eri sone®) (DF 0.1%)

 hydrocortisone butyrate 0.1% (Locoid®) (HB 0.1%)

 clobetasol propionat e 0.05% (Dermoval®). (CP 0.05%)

Anti-IgE solution was purchased from Dako (France).

Histamine EIA kits were obtained from Beckman Coulter

(Paris, France).

Skin speci m en s

For these experiments, 16 excised abdominal skin

(operative wastes) were obtained from surgery procedure

and were used as soon as possible after their excision

(mean age of women: 41.0 ± 2.3 years). The allergic

status of the donors was not known. Informed consent

was obtained from each subject before surgery. Skin

fragments were cut and placed immediately in phosphate

buffer (PBS, PAA laboratories, France) and the tem-

perature of 37˚C of phosphate buffer was maintained

during the experiments. The cutting of skin fragments

makes it possible to repeat the measurements of hista-

mine release twelve times for each corticosteroid after

the application of the cream.

Microdia lysis Experi m ent

The microdialysis system consisted of a CMA/100®

syringe pump (Phymed, Paris, France) and CMA/140

microfraction collector, which collected samples. The

microdialysis probes (CMA/20®) had a 20-kD cutoff

with a polycarbonate membrane (length 10 mm. For each

corticosteroid, a total of 12 microdialysis probes were

used to determine their potency on the inhibition of his-

tamine release after anti-IgE antibody injection. So each

skin fragment was cut into several parts. Histamine re-

lease was induced after 1 hour of stabilization following

the cutting of the skin and the insertion of microdialysis

probes.

Microdialysis samples were collected 5 minutes before

the induction of histamine liberation (T0) and the micro-

dialysis lasted 1 hour with sampling of extracellular der-

mal fluid every 5 minutes. Probes were used in order to

determine basal histamine release after anti-IgE inject-

tion.

The corticosteroids tested were topically applied with

occlusive dressing (TegadermTM, 3M, France) on the skin

above each probe, four hours before anti-IgE injection.

Microdialysis started 5 minutes before the histamine re-

leased and lasted 1 hour with sampling every 5 minutes

(Figure 1).

Determination of histamine in dialysates

The concentration of histamine in the dialysate was

determined by EIA using a commercially available ana-

lytic kit for histamine. In order to compare the different

corticosteroids, AUC (area under the curve) was calcu-

lated allowing an estimation of the amount of released

histamine during the period of 60 min of microdialysis

on excised skins. For each skin fragment, the kinetic

Evaluation of the Effects of Corticosteroids on Histamine Release by ex Vivo Cutaneous Microdialysis

230

Figure 1. Schematic representation of experimental protocol of histamine-induced model.

curve of histamine release without the presence of cor-

ticosteroids (control) was plotted. After 20 minutes, the

release of histamine reached a maximum and was con-

sidered as 100%. The percentage of histamine was cal-

culated for each time in each curve with an d without cor-

ticosteroids.

release in the presence of corticosteroids such as clobe-

tasol propionate (CP 0.05%) (Figure 2). During the 60

minutes of the experiment, histamine dermal levels de-

creased in the presence of each corticosteroid and made it

possible to calculate Area Under the Curve (AUC) for

each corticosteroid. The area under the plot of dermal

concentration of histamine against time after corticoste-

roid administration is used in the estimation of the bioa-

vailability of histamine in th e dermis showing the effects

of corticosteroids in the treatment of immuno-allergic d i-

seases. The classification of corticosteroids according to

the value of AUC showed that micronized betamethasone

dipropionate (mBdP 0.05%), betamethasone dipropionate

with propylene glycol (BdP + PG 0.05%) or desonide,

(DES 0.05%) were less powerful than in McKenzie clas-

sification (Figure 3 and Table 1). Difluocortolone (DF

0.1%) showed to have the more potent effect after clobe-

tasol propionate (CP 0.5%) on histamine release.

For each curve, area under the curve was calculated

using GraphPAD Prism software. The results were ex-

pressed as the percentage in comparison to AUC of con-

trol curve

3. Statistics

Results are expressed as means ± SEM. Statistical analy-

ses were performed by using one way or two ways vari-

ance analysis followed if necessary by Fischer test. Val-

ues were considered significantly different when p <

0.05.

4. Results 5. Discussion

Twelve skin fragments were used in order to test each

corticosteroid cream on the histamine release. The hista-

mine release induced by anti-IgE antibody was recorded

after a stabilization period following the cutting of ex-

cised female abdominal skin and the insertion of the pro-

be. This stabilization period makes it possible to deter-

mine the basal quantity of histamine present in skin fra-

gments. Histamine concentrations increased and a pla-

teau was obtained 20 minutes after anti-IgE induction.

Thus it is possible to calculate the area under the curve

for the release of histamine. The second part of the ex-

periment consisted of applying the different corticoster-

oids creams 4 hours before the induction of histamine

release. The results demonstrated that the plateau was

lower than the control after the induction of histamine

An allergy is an overreaction of the immune system fol-

lowing contact with a substance usually foreign to the

body. Mast cells play a key role in the immediate phase

of allergic reaction s and also in th e inflammatory pro cess.

When activated by direct contact, by cross-linking of IgE

receptors, or by activated complement proteins, a mast

cell rapidly releases numerous chemical mediators such

as serotonin, histamine, tryptase or heparin by a mecha-

nism of exocytosis. Mast cells express a high-affinity re-

ceptor for the Fc region of IgE. This receptor is of such

high affinity that binding of IgE molecules is essentially

irreversible. As a result, mast cells are coated with IgE.

IgE molecules are produced by plasma cells and are

specific to one particular antigen. Petersen et al. deve-

Evaluation of the Effects of Corticosteroids on Histamine Release by ex Vivo Cutaneous Microdialysis 231

Time (minutes)

0 102030405060

Histamine (%)

100

Control

CP 0.05%

*** ***

*** *** ***

*p<0.05 et ***p<0.001 versus Control

***

Figure 2. Skin release of histamine after anti-IgE injection in the presence or not of PC 0.05%). Propionate clobetasol 0.05%

(Sampling intervals were 5 min. Histamine concentrations in dialysates are shown over 60 min. *p < 0.05 and ***p < 0.001

versus control.

Corticosteroids

**p<0.01 and ***p<0.001 versus Control

Control

CP 0.05%

DF 0.1%

BdP + PG 0.05%

BV 0.1%

DES 0.1%

HB 0.1%

mBdD 0.05%

DES 0.05%

AUC (%/Control)

100

120 <50%50-70 %70-80%>80%

***

*** *** ***

*** **

Figure 3. Classification of corticosteroids using the estimation of the amount of released histamine (AUC) during the period

of 60 min of microdialysis on excised skins in the presence of corticosteroids. **p < 0.01 and ***p < 0.001 versus control.

loped a microdialysis technique allowing the measure-

ment of histamine release from intact human skin mast

cells ex vivo [12] using intradermal injection of anti-IgE.

The kinetics of histamine release in the skin ex vivo by

anti-IgE showed a maximum peak around 15 minutes of

microdialysis comparable to that obtained in the present

study. So this microdialysis technique made it possible to

test the potency of several topical corticosteroids on ex

Evaluation of the Effects of Corticosteroids on Histamine Release by ex Vivo Cutaneous Microdialysis

232

Table 1. Potency of topical corticosteroids. Comparison between MacKenzie classification and the new classification using

histamine-induced model

Potency Mackenzie

classification Topical corticosteroid Formulation French trade name Potency Inhibition of

histamine release

Ultra high Clobetasol propionate (C P 0.05%) Cream, 0.05%Dermoval® Ultra high

High Micronized betamethasone dipropionate (mBdD 0.05%)Cream, 0.05%Diprosone® Low

Ultra High Betamethasone d ipropionate

with propylene glycol (BdD + PG 0.05%) Cream, 0.05%Diprolene® Moderate

High Betamethasone v alerate (BV 0.1%) Cream, 0.1% Betneval® Moderate

High Diflucortolone (D F 0.1%) Cream, 0.1% Neris one® High

Moderate Desonide (DES 0.1%) Cream, 0.1% Locapred® Moderate

Moderate Desonide (DES 0.05%) Cream, 0.05%Tridesonit® Low

Moderate Hydrocortisone butyrate (HB 0.1%) Cream, 0.1% Locoid® Moderate

vivo model of histamine release. Indeed topical cortico-

ster o id ap p li cat io n to the sk in i s a s ta nd ar d th er ap y in im-

muno-allergic diseases. Up till the present moment, it

was thought that the effectiveness of topical corticoste-

roids results especially in a vasoconstriction of superfi-

cial dermis and a decrease in capillary permeability,

which results in the rapid reduction of edema and erythe-

ma. The degree of vasoconstriction seems to be related to

the potency and the absorption of the topically applied

steroid and has been used in the Mckenzie vasocon-

strictor assay [1].

This assay, introduced by McKenzie and Stoughton

[1], involves the evaluation of the degree of skin blan-

ching over a period following the application of topical

corticosteroid prod ucts to the skin of healthy human sub-

jects. It can be carried out by visual assessment of the

degree of blanching at the application site or by an in-

strumental method of assessment using a chromameter.

The intensity of the skin blanching response is related to

the amount of corticosteroid that has penetrated into the

skin [13]. Various publications have shown that the pre-

cision, sensitivity and repeatability of this assay are ade-

quate for the assessment of the bioequivalence of topical

corticosteroid formulations [14-19]. The degree of hydra-

tion of the horny layer of epidermis affects its permea-

bility for corticosteroids and their transport through the

skin. Occlusion increases the permeability of th e skin for

corticosteroids, resulting in an increased degree of skin

hydration [20]. There is also a relationship between the

vehicule composition of corticosteroids and the skin

blanching. Indeed the release of corticosteroids from

creams depends on the composition of the vehicule and

can contribute to the blanching. Propylene glycol associ-

ated with betamethasone dipropionate (Diprolene®) in-

creases the solubilization of its molecule and improves

the blanching compared to the formulation without pro-

pylene glycol (Diprosone®). So the skin blanching was

used a s a measur e of the per cutaneous abso rption of cor-

ticosteroids from topical formulations. Up to now, human

skin blanching is used to evaluate the efficacy of topical

corticoids prior to their final testing during clinical trials.

However there is no direct proof that the vasocon-

strictor and anti-inflammatory activities are correlated.

Since topical corticosteroids are commonly used in the

treatment of immuno-allergic conditions, we firstly car-

ried out in the present study, an experiment in order to

measure directly the ability of topical corticosteroids to

inhibit histamine release in an original ex-vitro model

which allowed studies of the bioequivalence of topical

corticoids formulations without the presence of blood

flow. Secondly we compared the McKenzie classification

of corticosteroids to this new classification according

their effect on histamine release from resident dermal

mast cells. This original model consists in an ex vivo skin

treated by injection of anti-IgE antibody because mast

cells also reside in the skin. Th e results showed that topi-

cal corticosteroids inhibited directly the release of hista-

mine in addition to vasoconstriction properties. A pre-

treatment of the skin by corticosteroids for 4 hours with

occlusive dressing made it possible to decrease the der-

mal histamine levels. So McKenzie classification of cor-

ticosteroids is not correlated to their potency of inhibition

on histamine release. Micronized betamethasone dipro-

pionate (mBdP 0.05%), betamethasone dipropionate with

propylene glycol (BdP + PG 0.05%) or desonide, (DES

0.05%) were less powerful than in McKenzie classifica-

tion. Our model also showed that the presence of propy-

lene glycol induced an increase in the activity of beta-

methasone on histamine release. It is noteworthy that

difluocortolone (DF 0.1%) showed to have a more potent

effect after clobetasol propionate (CP 0.5%) on histamine

release.

6. Conclusion

In conclusion, we did not find the same results concern-

Evaluation of the Effects of Corticosteroids on Histamine Release by ex Vivo Cutaneous Microdialysis 233

ing the potency of corticosteroids according to MacKen-

zie classification and the inhibition of histamine release.

These results show us that a new classification of the po-

tency of corticosteroids should be established to treat

correctly the patients according to the origin of the ob-

served disturbances. These results invite us to consider

the power of topical corticosteroids differently depending

on their target action (anti-allergic, anti-inflammatory...).

New ex vivo models should be developed in order to ob-

serve the direct effects of corticosteroids on the release of

inflammatory mediators. The method, which we have de-

veloped, should lead to the study of clinical disease mo-

dels for immuno-allergic clinical validation.

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