ARTICLE
Auteur(s) : Tomohiro Sakuta,
Toshiji Kanayama
Shiseido Pharmaceutical Research Center 2-12-1 Kanazawa-ku
Fukuura, Yokohama, Japan 236-8643
accepté le 23 Août 2005
Acne vulgaris is a common, multifactorial disease affecting the
pilosebaceous unit of the skin. It is now widely accepted that acne
is a result of the following four processes: hyperkeratinization of
the upper region of the follicle; increased sebum production by the
sebaceous gland; bacterial (Propionibacterium acnes) colonization
of the follicle; and induction of inflammatory responses by
bacterial antigens and cell signals [1]. Treatment of acne is
usually with topical therapeutic agents, though in severe cases, a
combination of topical and systemic therapy is required [2]. These
therapies are directed against major pathological processes of acne
and representative topical agents include retinoids, azelaic acid,
benzoyl peroxide, topical antibiotics, salicylic acid, and zinc, as
well as combinations of these agents [3].Topical retinoids, both
naturally occurring and synthetic compounds, which exert biological
activities resembling that of vitamin A, are the mainstay for
treating common varieties of acne vulgaris. They also prevent
development of comedos, halting progression to inflammatory lesions
[3, 4]. Indeed, all-trans-retinoic acid (RA), a classical RAR-pan
agonist, was first used in the topical treatment of acne more than
30 years ago [5]. Retinoids exert their effects through two
distinct families of nuclear receptors, retinoic acid receptors
(RARs) and retinoid X receptors (RXRs), that act as
ligand-dependent transcription factors [6]. Each of them can be
divided into three subtypes (α, β and γ) that show distinct
spatio-temporal tissue distributions [7-12]. Recently, ligands that
are highly specific for individual RAR subtypes have been
developed, and some of them are used for the treatment of skin
diseases.A new synthetic retinoid, adapalene, which has higher
affinity for RARβ and RARγ than for RARα, is effective in the
treatment of mild to moderate acne vulgaris [13]. In the treatment
of acne vulgaris, 0.1% adapalene gel has similar efficacy to 0.025%
RA gel [14]. Another RARβ/γ-selective retinoid, tazarotene, is also
efficacious in the treatment of patients with psoriasis, acne
vulgaris, and photoaging [15, 16]. The skin tolerability of these
receptor-selective retinoids is superior to that of RA, while the
pharmacological effects are equivalent to those of RA. Several
studies have shown that RARγ selectivity contributes to the
therapeutic effect in the rhino mouse model, which is a mutant
strain of hairless mouse with horn-filled utricles reminiscent of
human microcomedos [17, 18].In this report, we describe the
comedolytic effects of ER36009 in the rhino mouse model, to obtain
further evidence for the in vivo efficacy of this retinoid.
Materials and methods
Chemicals
All-trans-retinoic acid (RA) was purchased from BASF (Wyandotte,
MI). The synthetic retinoid ER36009 ( (figure 1) ) was kindly
provided by Eisai Tsukuba Research Laboratories.
Differine® (0.1% adapalene gel) and Tazorac®
(0.1% tazarotene gel) were purchased from Galderma Laboratories LP
(Fort Worth, Texas) and Allergan Inc. (Irvine, California)
respectively.
Animals
Fifty-five female rhino mice were obtained from CLEA Japan, Inc.
(Tokyo, Japan). The mice were 9 weeks old at the beginning of the
study and were divided into eleven groups of five animals each.
Groups I, II, III and IX received topical application of ER36009 at
concentrations of 0.00025%, 0.0001%, 0.000025% and 0.00001%,
respectively. Groups V, VI, VII and VIII received topical
application of RA at concentrations of 0.05%, 0.01%, 0.005% and
0.001%, respectively. Group IX received acetone only, group X
received 0.1% adapalene gel and group XI received 0.1% tazarotene
gel.
Treatment
Non-formulated test compounds were dissolved in acetone. Each stock
solution was prepared to a concentration of 0.1% in subdued light,
and diluted as required just before application. Between
applications, stock solutions were stored under argon at 4 °C.
Formulated materials were used as supplied. Aliquots of 100 μl of
test materials were applied with a pipette or a gloved finger, on
the dorsal skin of the animal, once daily for 5 consecutive days
per week (excluding Saturday and Sunday) during 2 weeks. A control
group was treated with acetone alone.
Preparation of epidermal sheets
Approximately 72 h following the final topical treatment, the
mice were sacrificed by cervical dislocation. The dorsal trunk skin
was removed and soaked in 0.5% acetic acid for approximately
18 h at 4 °C for whole mount processing. An area of skin,
approximately 1 × 1 cm, was cut with a blade and the epidermis
was carefully peeled using a flat-ended spatula and fine forceps.
The removed epidermis was placed on a glass slide with the dermal
side facing up, and air-dried. Then, specimens were dehydrated by
immersion in increasing concentrations of ethanol (75%, 95%, 100%),
followed by xylene, and mounted dermal side up on glass slides
using Pro-Texx.
For each whole mount, the diameters of 10 utricles in 5 random
fields were measured with an image analysis system (ATTO, Tokyo).
The mean utricle diameter was calculated for each treatment
group.
Histology
Skin samples approximately 6 mm in diameter were obtained
postmortem from the skin of each animal, from the mid-line of the
anterior portion of the dorsum skin. All specimens were fixed in
10% formalin, embedded in paraffin and sectioned at 5 microns.
Hematoxylin/eosin staining was used for routine examination of the
tissue.
Detection of apoptosis
Apoptotic staining (terminal deoxynucleotidyl transferase-mediated
nick-end labeling) was performed on paraffin sections using
DermaTACSTM (Trevigen, Inc., Gaithersburg, MD), which is
based on the labeling of fragmented DNA ends, according to the
manufacturer’s protocol [19].
Statistical analysis
The significance of intergroup differences of utricle diameter was
assessed by using the Tukey’s multiple comparison tests.
Results
Histology
In the control mice ( (figure 2A) ), most of the
utricles opened to the surface and often contained keratin-like
sheets. Sebaceous glands were associated with the utricles and
subcutaneous cysts. The epidermis was thin and convoluted. The
anucleate stratum corneum was relatively thick, with a loose
‘basket-weave’ appearance.
After 0.000025% ER36009 treatment, the epidermis was thicker and
less undulating, and utricles were reduced in both size and number
( (figure 2B) ).
The ER36009-treated epidermis was hyperplastic with an increase in
the number of cell layers of both the follicular epithelium and the
epidermis. Changes were observed mainly at the granular layer and
the stratum corneum, resulting in restored desquamation. Compared
with controls, the sebaceous glands seemed more obvious after
retinoid treatment, probably due to the reduced utricle size.
With increasing doses of ER36009, the utricle walls became
thicker, the number of utricle cell layers increased, and the
utricles became more elongated and started to resemble normal pilar
units ( (figure
2C) ). In addition, an apparent decrease in the number of
utricles was observed in the vertical sections in dose-dependent
manner. At the dose of 0.00025% ER36009 ( (figure 2D) ), no
utricle-like structures were seen, these having been replaced by
essentially normal-looking pilar units. The dose-dependent changes
observed with ER36009 were comparable to those seen with RA.
However, at the lowest dose of ER36009 (0.00001%), the utricle
histology was indistinguishable from that of the controls (data not
shown).
Progressive changes in utricle structure were induced by all the
retinoids tested. As shown in ( figure 2 ), 0.5% RA ( (figure 2E) ), 0.1%
adapalene gel ( (figure
2F) ) and 0.1% tazarotene gel ( (figure 2G) ), transformed
the horn-filled utricles of the rhino mouse into normal follicles.
On the other hand, no change in the histology of the subcutaneous
cysts was seen in any of the groups.
Effects of retinoids on utricle size
Measuring diameters from vertical sections is limited by the fact
that the plane of the section may not intersect the maximum
diameter or the opening of the utricle. We used Mezick’s method
[20] to observe the effects of retinoids on utricle size and
distribution.
In the control mice, numerous circular-shaped utricles were
observed in the epidermal sheets ( (figure 3A) ). ER36009
decreased the size of utricles in a dose-dependent manner ( (figure 3B, C, D) ).
0.00025% ER36009 showed superior effect compared with 0.05% RA (
(figure 3E) ).
As shown in table 1( Table 1 ), all
tested retinoids, exclusive of the lowest dose of ER36009,
significantly reduced the diameter of utricles compared with the
control. The 0.00025% ER36009-treated group showed the greatest
reduction to 29.6% relative to the controls. This effect is
equivalent to that of 0.05% RA, 0.1% adapalene gel or 0.1%
tazarotene gel.
Dose-response regression lines of both ER36009 and RA are
plotted in ( figure
4 ). The ED50 value is defined as that dose which reduced
the utricle diameter by one-half relative to the mean utricle
diameter of control animals. Table 2( Table
2 ) summarizes the topical ED50 values and relative potency
of ER36009 and RA for utricle size reduction. As can be seen from
table 2, ER36009 is 96 times more potent than RA in this rhino
mouse model.
Table 1 Utricle diameter after topical application of
retinoids
|
Utricle diameter (μm)
|
% of reduction of diameter
|
|
Control
|
110.7 ± 8.2
|
100.0
|
|
0.000010% ER36009
|
113.9 ± 9.1
|
102.9
|
|
0.000025% ER36009
|
53.1 ± 3.9**
|
48.0
|
|
0.000100% ER36009
|
43.5 ± 3.2**
|
39.3
|
|
0.000250% ER36009
|
32.8 ± 2.5**
|
29.6
|
|
0.001% RA
|
61.8 ± 4.2**
|
55.8
|
|
0.005% RA
|
48.7 ± 0.8**
|
44.0
|
|
0.010% RA
|
40.8 ± 2.4**
|
36.9
|
|
0.050% RA
|
34.9 ± 3.2**
|
31.6
|
|
Differine®
|
36.5 ± 1.8**
|
33.0
|
|
Tazorac®
|
33.9 ± 1.5**
|
30.3
|
Table 2 Topical potency of ER36009 and RA for utricle
reduction of rhino mouse. The ED50 value is defined as that dose
which reduced the utricle diameter by one-half relative to the mean
utricle diameter of control animals
|
Retinoid
|
ED50
|
Relative potencies
|
|
RA
|
2.00E-03
|
1
|
|
ER36009
|
2.10E-05
|
96
|
ER36009 Treatment results in the elimination of apoptosis in
the rhino follicular epithelium
At the completion of the first hair cycle, the rhino mouse
follicular papillae fail to follow the regressing follicle [21].
The lack of epithelial-mesenchymal interaction prevents the
reinitiation of a new hair cycle and causes the follicular
epithelium to undergo extensive apoptosis. This is in contrast to
epidermal apoptosis at the granular layer, which is minimal,
implying abnormal epidermal maturation. DermaTACSTM,
which labels the ends of DNA fragments, revealed an unusually high
level of apoptotic bodies in the follicular epithelium in the
control mice ( (figures
5A and 5B) ). On the other hand, ER36009 treatment resulted
in the elimination of all the apoptotic bodies within the
follicular epithelium in a dose-dependent manner (( figures 5C and 5D) ). This
effect is specific to the utricles. Apoptosis within the epidermis
was restored by ER36009 as epidermal differentiation was restored.
Discussion
In this study, we have demonstrated that ER36009, an RARγ selective
retinoid, has potent comedolytic activity in the rhino mouse model.
Topical treatment with ER36009 also eliminated extensive apoptosis
in the follicular epidermis and restored normal apoptosis at the
granular layer. These results suggest that ER36009 influences
differentiation of the follicular epithelium and exerts comedolytic
activity.
Several studies have shown that RARγ selectivity contributes to
the therapeutic effect of retinoids in the rhino mouse model [17,
18]. Chen et al. revealed that utricle reduction activity
correlated with RARγ transactivation activity.
In the previous report, the selectivity of ER36009 for each
receptor was indicated as relative 50% inhibitory dose
(IC50), obtained by dividing the IC50 value
of ER36009 for a given receptor by that of RA. Kikuchi et al.
showed that ER36009 exhibits a greater affinity for RARγ (relative
IC50 3.6) than for RARα (relative IC50 83) or
RARβ (relative IC50 21) [22]. They also reported that
the EC30 value (that dose which provides 30% of the
maximal transactivation activity in the cotransfection assay) of
ER36009 for RARγ was 0.34 relative to that of RA. (The relative
EC30 values for RARα and RARβ were given as 5.2 and
0.39, respectively.)
Here we demonstrated that ER36009 was 96 times more potent than
RA in the utricle diameter reduction assay. Thus, our experiment
confirms that RARγ plays a crucial role in the comedolytic effect
of retinoids. Even RARβ/γ-selective retinoids, adapalene and
tazarotene, provide a therapeutic advantage over RA [23, 24]. In
the present study, we found that the comedolytic effect of 0.00025%
ER36009 was approximately equivalent to that of the above
commercially available formulations. Although it is not appropriate
to make a simple comparison between the acetone solution of ER36009
and the above formulations, ER36009 showed an equivalent effect at
a much lower concentration as compared with adapalene or
tazarotene. On the basis of published data, ER36009 has a higher
affinity for RARγ than adapalene or tazarotene [22, 25, 26]. Thus,
the pharmacological advantage of ER36009 may be associated with its
specificity for RARγ.
The pathogenesis of acne is complex and depends on the interplay
of multiple factors. In acne treatment, retinoids influence cell
proliferation and differentiation, modify immune reactions, exert
anti-inflammatory effects, regulate DNA synthesis with consequent
differential expression of specific proteins, and suppress the
production of sebum from the sebaceous glands [27]. In particular,
Bernerd et al. suggested that the effect of retinoids on the
terminal differentiation of keratinocytes plays an important role
in the comedolytic activity [28]. Indeed, failure of follicular
keratinocytes to differentiate results in hypergranulosis that
resembles an impermeable outer skin layer, resulting in the
formation of microcomedos. Topical retinoids and synthetic
retinoids such as adapalene and tazarotene help to normalize cell
turnover and cell cohesion [29]. Here we have shown that ER36009
treatment restores the balance between cell death and proliferation
in the follicular epithelium and the epidermis. This result
suggests that activity of ER36009 is associated with changes in the
differentiation of the follicular epithelial cells. In addition, we
have further demonstrated that ER36009 decreases the expression of
bcl-2, an anti-apoptotic factor, and affects the apoptosis pathway
in the visceral adipose tissue (manuscript in preparation).
On the other hand, inflammatory responses lead to the
development of increasingly severe inflammatory forms of acne. Both
non-specific and specific immune responses are operative in the
inflammatory and possibly earlier stages of acne vulgaris [30-32].
Topical RA and retinoid analogues have exhibited significant
anti-inflammatory effects in experimental trials. Inhibition of
various immune factors, including the activity of leukocytes [13,
33, 34], the release of proinflammatory cytokines and other
mediators [13, 34-37], and the expression of transcription factors
[38] and toll-like receptors [39] involved in immunomodulation, has
emerged in both in vitro studies and in vivo animal experiments
[25, 34, 40]. However, it is not yet clear whether ER36009 has
anti-inflammatory effects.
In conclusion, we have shown that the novel synthetic retinoid
ER36009 has potent comedolytic effects on the rhino mouse skin
compared with RA. Topically applied ER36009 induces modifications
similar to those obtained with both 0.1% adapalene gel and 0.1%
tazarotene gel. Thus, we consider that ER36009 has a potent
anti-acne activity. Further work is required to determine the
molecular mechanisms of ER36009 action.
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