0022-3565/02/3033-1283–1290$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 303:1283–1290, 2002
Vol. 303, No. 3
40105/1026548
Printed in U.S.A.
Inhibition of Allergic Dermal Inflammation by the Novel
Imidazopyridazine Derivative TAK-427 in a Guinea Pig
Experimental Model of Eczema
SHIGERU FUKUDA, KATSUO MIDORO, TAKAYUKI KAMEI, MICHIYO GYOTEN, YASUHIKO KAWANO, YASUKO ASHIDA,
and HIDEAKI NAGAYA
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan
Received June 21, 2002; accepted August 12, 2002
Atopic dermatitis is a chronically relapsing inflammatory
skin disease characterized by episodes of intense pruritus,
multiple lesions with erythema, excoriation, erosions, lichenification, papules, dry skin, and susceptibility to cutaneous
infection. Histopathological studies shown that the skin lesions of atopic dermatitis are characterized by acanthosis
and spongiosis in the epidermis, predominant infiltration by
CD1⫹ cells and activated CD4⫹ T cells in the dermis, and
extensive deposition of eosinophil granule proteins, such as
eosinophil major basic protein and eosinophil cationic protein
(Leiferman et al., 1985; Bruijnzeel et al., 1993). In addition,
serum levels of eosinophil cationic protein have been reported to correlate with the severity of disease (Tsuda et al.,
1992).
The study of atopic dermatitis has been hampered by the
lack of appropriate experimental models; however, spontaneously occurring dermatitis in mice has recently been reported
as a model (Matsuda et al., 1997). In humans, epicutaneous
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.040105.
dermatitis manifestations and epidermal damage. By contrast,
none of the antihistamines tested (azelastine, ketotifen, terfenadine, and cetirizine) suppressed the eosinophil infiltration or
dermatitis manifestations. To elucidate the mechanism by
which TAK-427 inhibited the development of eczema, we investigated cytokine expression in the affected skin. Both TAK427 and dexamethasone suppressed the increased mRNA expression of interleukin (IL)-13, granulocyte-macrophage
colony-stimulating factor, IL-1␣, tumor necrosis factor-␣, interferon-␥, and IL-8, but not IL-10, suggesting that TAK-427 inhibits allergic inflammation of the skin leading to the development of eczema by inhibiting the expression of proinflammatory
cytokines after antigen challenge.
application of aeroallergens, commonly referred to as the
atopy patch test, can provoke eczematous skin reactions in
atopic dermatitis patients (Clark and Adinoff, 1989). The
atopy patch test has been proposed as an in vivo model for the
study of allergic inflammation in atopic dermatitis (Langeveld-Wildschut et al., 1996) because the reactions to the
atopy patch test resemble atopic dermatitis lesions in terms
of both macroscopic appearance and histological characteristics (Bruijnzeel-Koomen et al., 1988). On the other hand,
intracutaneous administration of allergens induces a socalled late-phase reaction (Kaliner, 1984), which is limited to
the dermis and do not induce eczema-like changes in the
epidermis (Dolovich et al., 1973; Charlesworth et al., 1989a;
Bos et al., 1994).
The differences between the atopy patch test reaction and
late-phase skin reaction led to the hypothesis that epicutaneous exposure to protein allergens plays a role in the development of eczema in atopic dermatitis. According to the hypothesis, we developed a new animal model in which eczemalike lesions develop when an antigen is applied topically by
patch to epicutaneously sensitized guinea pigs.
ABBREVIATIONS: TAK-427, 2-[6-[[3-[4-(diphenylmethoxy)piperidino]propyl]amino]imidazo[1,2-b]pyridazin-2-yl]-2-methylpropionic acid dihydrate; OVA, ovalbumin; PCR, polymerase chain reaction; IL, interleukin; TNF-␣, tumor necrosis factor-␣; GM-CSF, granulocyte-macrophage
colony-stimulating factor; IFN-␥, interferon-␥; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; F, forward primer; R, reverse primer.
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ABSTRACT
Antigen challenge by patch ovalbumin emulsion induced an
eczema-like skin lesion in epicutaneously sensitized guinea
pigs. Diseased skin sites were macroscopically characterized
by manifestations of dermatitis, such as erythema, edema, and
papules, and microscopically characterized by acanthosis,
spongiosis, and dermal infiltration by eosinophils. Using such
lesions as a model of eczema, we evaluated the potential value
of TAK-427 [2-[6-[[3-[4-(diphenylmethoxy)piperidino]propyl]amino]
imidazo[1,2-b]pyridazin-2-yl]-2-methylpropionic acid dihydrate] as a therapeutic agent for atopic dermatitis by comparing
it with dexamethasone and antihistamines. TAK-427 (0.3–30
mg/kg, p.o.) and dexamethasone (3 and 10 mg/kg, p.o.) inhibited eosinophil infiltration into the skin and ameliorated the
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Fukuda et al.
We tested a number of compounds for an inhibitory effect
on allergic inflammation in the guinea pig model of eczema
and discovered that some imidazopyridazine derivatives had
an inhibitory effect. Among them, we selected TAK-427 (Fig.
1) as a candidate compound.
In this article, we describe histopathologic and pharmacological characteristics of the damaged skin in the eczema
model and the potential value of TAK-427 as a therapeutic
agent for atopic dermatitis. TAK-427 has antihistaminic activity as well as an anti-inflammatory effect, but its inhibitory effect on eczema formation was found to be unrelated to
its antihistaminic effect. To elucidate the anti-inflammatory
effect of TAK-427, we investigated expression of the mRNA of
several proinflammatory cytokines and chemokines in the
lesioned skin.
Materials and Methods
Fig. 1. Chemical structure of TAK-427.
Score
Erythema
No erythema
Very mild erythema
Well defined erythema with no to mild eschar formation
Moderate to severe erythema with mild to moderate eschar
formation
Severe erythema with severe eschar formation
Edema
No edema
Slight edema
Moderate to severe edema
Scratch formation
No scratches observed
Scratches observed
0
1
2
3
4
0
1
2
0
1
Histopathological Examinations. The OVA-challenged skin of
each animal was punched out (15-mm diameter) 48 h after antigen
application, and the specimens were fixed in 10% neutral buffered
formalin. After embedding in paraffin, 5-␮m sections were stained
with hematoxylin-eosin and Biebrich scarlet-iron hematoxylin (Luna
stain) to detect infiltrated eosinophils. The skin lesions were scored
(1 to 4) for crust formation, epidermal vacuolation, and eosinophil
infiltration of the epidermis and the crust. Eosinophils that had
infiltrated the dermis were counted in 10 to 12 consecutive highpower fields (200⫻) of each skin section, and the number per millimeter of length of the epidermal layer was calculated.
Passive Cutaneous Anaphylaxis Reaction. To determine the
IgE titer, serum from epicutaneously sensitized guinea pigs was
diluted with saline and intradermally injected into the shaved backs
of male guinea pigs at a volume of 0.05 ml. Seven days later, the
animals were intravenously challenged with 1 ml of 2.5% pontamine
sky blue dye containing 2.5 mg of OVA in saline and sacrificed by
bleeding 30 min after the challenge. The last dilution to give a
threshold reaction (5-mm diameter of blue spot) in two of three
guinea pigs was recorded as the OVA-specific IgE titer.
Quantitative PCR. At 24 h after the OVA-challenge, the skin of
the challenged sites was collected, and total RNA was extracted with
an RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol. cDNA was synthesized by incubating 4 ␮g of
total RNA with 33 ␮l of reaction mixture containing 0.2 ␮g of pd(N)6
primer (Amersham Biosciences UK, Ltd., Buckinghamshire, UK) at
37°C for 60 min using Ready-To-Go You-Prime First-Strand Beads
(Amersham Biosciences UK, Ltd.). The numbers of mRNA copies for
IL-8, IL-5, eotaxin, TNF-␣, GM-CSF, IL-1␣, IFN-␥, IL-10, IL-13, and
G3PDH were determined based on quantitative PCR standard
curves plotted by using data obtained with a sequence detector ABI
PRISM 7700 application (Applied Biosystems, Foster City, CA). All
primers and probes used in this study were designed with ABI
PRISM Primer Express 1.0 software (Applied Biosystems) and were
synthesized at Amersham Biosciences UK, Ltd. and PerkinElmer
Japan (Yokohama, Japan), respectively. The specificity of the PCR
primers was tested under normal PCR conditions in a thermal cycler
before quantitative PCR. The probes were labeled with a reporter
fluorescent dye, 6-carboxyfluorescein, at the 5⬘-end and a fluorescent
dye quencher, tetramethylrhodamine, at the 3⬘-end. The absolute
copy numbers of cytokine and chemokine mRNA in each sample were
calculated based on the standard curve for cDNA (from normal PCR
products), and the absolute copies of cytokine mRNA were then
normalized against those of G3PDH mRNA. The primers used were
G3PDH forward primer (F) 5⬘-CAAGGTCATCCCAGAGCTGAA-3⬘,
reverse primer (R) 5⬘-CCACAACCGACACATTAGGTG-3⬘, probe 5⬘AAGCTCACAGGTATGGCCTTCCGTGTAC-3⬘; IL-8 (F) 5⬘-GCTGCGATGCCAGTGTATTAAG-3⬘, (R) 5⬘-GGTCCACTCTCAATCACTTTCAGT-3⬘, probe 5⬘-CACACCACACCTTTCCACCCCAAATT-3⬘; IL-5
(F) 5⬘-AGCTGCACCTTTTGTAGCCA-3⬘, (R) 5⬘-CAGAGTTCGAT-
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Animals. Std:Hartley guinea pigs (4 –5 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan). The animals were
housed under controlled temperature (24 ⫾ 1°C) and humidity (55 ⫾
5%) conditions and given access to food and water ad libitum. The
care and use of the animals and the experimental protocol used in
this study were approved by the Experimental Animal Care and Use
Committee of Takeda Chemical Industries, Ltd. (Osaka, Japan).
Drugs and Materials. Sodium dodecylsulfate and Vaseline
(white, high-pure) were purchased from Wako Pure Chemical (Osaka, Japan), and ketotifen fumarate, terfenadine, ovalbumin (grade
III; OVA), and dexamethasone were from Sigma-Aldrich (St. Louis,
MO). Pontamine sky blue 6B was from Tokyo Kasei Kogyo (Tokyo,
Japan). Azelastine hydrochloride was extracted from AZEPTIN tablets (Eisai, Tokyo, Japan). TAK-427 (Fig. 1) and cetirizine were
synthesized at Takeda Chemical Industries, Ltd. All drugs were
suspended in a 5% gum arabic solution.
Epicutaneous Sensitization and Challenge. Female guinea
pigs were anesthetized by intramuscular injection of a combination
of ketamine (Sankyo, Tokyo, Japan) and xylazine (Bayer AG, Leverkusen, Germany), and 10 ␮g of OVA mixed with 0.5 mg of Al(OH)3
was injected intradermally at four sites on a shaved shoulder. One
week later, a lint patch (3 ⫻ 4 cm) spread with an emulsion mixture
containing 10% OVA, 10% sodium dodecylsulfate, 30% Vaseline, and
50% water was placed on the shaved shoulder and maintained in
position for 48 h by wrapping with cloth adhesive tape. Additional
sensitization 3 weeks after the OVA injection was performed in
essentially the same manner at same site with emulsion consisting of
10% OVA, 5% SDS, 37% Vaseline, and 48% water. One week after
the final sensitization, antigen challenge was carried out by bilateral
topical application to a shaved flank of a lint patch (1 ⫻ 1 cm) spread
with Vaseline or a 10% OVA emulsion containing 20% water and
70% Vaseline. The patches were maintained in position for 24 h by
wrapping with cloth adhesive tape and then removed. At 48 h after
the challenge, the sites were scored in terms of erythema, edema,
and scratch formation, as described in Table 1, and the sum of the
three scores was used as the dermatitis score. The drugs were administered to the guinea pigs orally by tube gavage in a volume of 0.2
ml/100 g b.wt. twice daily for 3 days beginning on the day before the
OVA challenge.
TABLE 1
Evaluation of eczema-like skin lesions (scale for scoring)
Effect of TAK-427 on Eczema-Like Skin Lesion in Guinea Pigs
Fig. 2. Gross appearance of OVA-induced eczema-like dermatitis in epicutaneously sensitized guinea pigs. OVA was patched for 24 h to shaved
flanks of epicutaneously sensitized guinea pigs. Eczema-like dermatitis
was induced on both flanks and evaluated at 48 h after OVA application.
The animals in the above photograph were three representatives in the
control group. Inset: magnification of squared area.
Results
Characteristics of OVA-Induced Eczema-Like Skin
Reactions in Epicutaneously Sensitized Guinea Pigs.
The OVA-specific serum IgE levels of the epicutaneously
sensitized guinea pigs measured by the passive cutaneous
anaphylaxis reaction titers were 307 ⫾ 48. Topical application of OVA to the epicutaneously sensitized guinea pigs
produced eczema-like skin reactions characterized by erythema and edema with scratches in half of the challenge sites
(Fig. 2). Histopathological examination 48 h after OVA application revealed that the skin at the OVA-challenged sites
was characterized by thickening of the epidermis and inflammatory cell infiltration of both dermis and epidermis (Fig. 3).
The epidermal cells in the OVA-patch sites had proliferated to approximately four to six cell layers thick compared
with one to two cell layers thick in the nonpatched sites (Fig.
3, C and D). The stratum granulosum, acanthosis, spongiosis,
edema, cyst formation, and crusting were prominent in the
epidermis (Fig. 3D).
Infiltration by inflammatory cells, predominantly eosinophils but neutrophils and lymphocytes as well, was detected
in the epidermis and the dermis at OVA-patched skin sites
(Fig. 3, C and D). Most of the inflammatory cells were located
in the dermal papillary layers, but a few were present in the
deeper dermal layers (Fig. 3, A and B). The time course study
revealed eosinophil infiltration as early as 6 h after OVA
challenge and a peak response at 48 to 72 h. In contrast to the
OVA-patched skin sites, infiltration by only a few eosinophils
was observed at all time points in the Vaseline-patched skin
sites (Fig. 4).
Effect of TAK-427, Dexamethasone, and Antihistamines on OVA-Induced Eczema-Like Skin Reactions in
Epicutaneously Sensitized Guinea Pigs. In animals
Fig. 3. Histopathological features of OVA-induced eczema-like dermatitis
in epicutaneously sensitized guinea pigs. OVA was patched for 24 h to
shaved flanks of epicutaneously-sensitized guinea pigs. Skin specimens
were collected 48 h after antigen challenge, and histological evaluations
were performed as described under Materials and Methods. A and C show
normal skin preparations. Significant cellular infiltration seen in the
upper dermis of OVA-challenged sites (B). Eczema-like lesions, such as
hypertrophy, spongiosis, and acanthosis, were seen in the epidermis of
the OVA-challenged sites (D). Staining with hematoxylin-eosin. Bar ⫽
100 ␮m (A and B) and bar ⫽ 50 ␮m (C and D).
treated with vehicle, according to the criteria described in
Table 1, the scores for erythema, edema, and scratches were
2.41 ⫾ 0.15, 1.91 ⫾ 0.06, and 0.41 ⫾ 0.13, respectively, and
the dermatitis score was 4.73 ⫾ 0.25. TAK-427 at doses of
0.3, 3, and 30 mg/kg, p.o. reduced the dermatitis score dose
dependently, and statistical significance was observed at 3
mg/kg and above (Table 2). In the animals treated with
dexamethasone, the edema and scratch formation in the
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GAGTAGAAAGCAGAG-3⬘, probe 5⬘-TGTCTGTGTCTGTGCCATCCCCAA-3⬘; Eotaxin (F) 5⬘-CAGACAGCCATTGTCTTTGAGA-3⬘, (R)
5⬘-GCATCCTGAACCCACTTCTTCT-3⬘, probe 5⬘-AAACCTGACAAAATGATATGTGCGGACCCC-3⬘; TNF-␣ (F) 5⬘-CTCATCTACTCCCAGGTCCTCTT-3⬘, (R) 5⬘-TGATGGCAGAGAGAAGGTTGAC-3⬘, probe 5⬘TCCTACCTGCTTCTCACCCATACCGTCA-3⬘; GM-CSF (F) 5⬘-CAGTCCTGGAAACACGTGGAT-3⬘, (R) 5⬘-TCATTCATCACAGCAGCCG-3⬘,
probe 5⬘-CATCAATGAAGCCCTGAGCCTCCTGA-3⬘; IL-1-␣ (F) 5⬘-ATGATCCGCTCCACGAGAA-3⬘, (R) 5⬘-GGATTCCTCTGAGTTTTCGTAGG-3⬘, probe 5⬘-TGTGGATGAGCCAGTGTCTCCGAA-3⬘; IFN-␥ (F)
5⬘-CCATCAAGGAACAAATCATTACTAAGTT-3⬘, (R) 5⬘-TTTGAATCAGGTTTTTGAAAGCC-3⬘, probe 5⬘-TTCAAAGACAACAGCAGCAACAAGGTGC-3⬘; IL-10 (F) 5⬘-CCCACATGCTTCGAGAGC-3⬘, (R)
5⬘-ATCCTGTGTTTGGAAGAAAGTCTTC-3⬘, probe 5⬘-CCGAGCTGCCTTTGGCAGGG-3⬘; and IL-13 (F) 5⬘-TCCAACTGCAGCGCCC-3⬘, (R)
5⬘-GGCCTTGTGCTGGCAAAG-3⬘ probe 5⬘-CCAGAGGACCCAGAAGATACTGAGCGG-3⬘. Quantitative PCR was performed with reverse
transcription products, TaqMan Universal PCR Mater Mix (Applied
Biosystems), forward primer (final, 0.3 ␮M), reverse primer (final, 0.3
␮M), probe (final, 0.2 ␮M), and distilled water in a total volume of 50 ␮l.
PCR was performed at 50°C for 2 min, at 95°C for 10 min and then for
40 cycles at 95°C for 15 s, and at 60°C for 1 min on the ABI PRISM 7700
detection system.
Statistical Analysis. Statistical analysis of the numbers of infiltrating eosinophils and mRNA levels of cytokines in the skin lesion
was performed by a Dunnett’s test, and dermatitis manifestations
were analyzed by the nonparametric Dunnett’s test. Values of P ⬍
0.05 were considered statistically significant. All statistical calculations were performed with the SAS statistical package in our laboratory.
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Fukuda et al.
patched-skin improved, but there was little effect on the
erythema. The drug decreased the dermatitis scores, and its
effect was significant at doses of 3 and 10 mg/kg (Table 2). On
the other hand, the antihistamines azelastine, ketotifen, terfenadine, and cetirizine did not significantly reduce the dermatitis scores (Table 4).
Histopathological Examination of OVA-Induced Eczema-Like Skin Lesions in Epicutaneously Sensitized
Guinea Pigs Treated with TAK-427, Dexamethasone,
and Antihistamines. In control groups treated with vehicle,
the number of eosinophils that had infiltrated the dermis was
138.6 ⫾ 18.7 cells/mm. The eosinophil infiltration of the
dermis was significantly inhibited by 40, 54, and 56% with
TAK-427 at doses of 0.3, 3, and 30 mg/kg, p.o., respectively
(Figs. 5, A and B, and 6A). TAK-427 also dose dependently
inhibited eosinophil infiltration into the crust and the epidermal layer, and the effects were significant at the 30 mg/kg
dose (Table 4). Vacuolation of epidermis tended to suppress
in response to TAK-427, although the effect was not statistically significant (Fig. 5, C and D; Table 4).
Dexamethasone dose dependently decreased the number of
eosinophils that infiltrated the dermis, and statistical significance was observed at doses of 3 mg/kg and above (Fig. 6B).
Fig. 5. Effects of TAK-427 on eosinophil infiltration and dermal injury at
the OVA challenged sites in epicutaneously sensitized guinea pigs. OVA
was patched to shaved flanks of epicutaneously sensitized guinea pigs for
24 h. Drugs were administered p.o. twice daily for 3 days beginning on the
day before antigen challenge. Skin specimens were collected 48 h after
patch challenge with OVA, and histological evaluations were performed
as described under Materials and Methods. Representative histologic
sections stained with Luna (A and B; bar ⫽ 50 ␮m) or hematoxylin-eosin
(C and D; bar ⫽ 100 ␮m; insets, bar ⫽ 50 ␮m) are shown. TAK-427
reduced eosinophil infiltration of the dermis (B; 3 mg/kg, p.o.) and tended
to suppress vacuolation (D; 30 mg/kg) at the OVA-challenged sites in
epicutaneously sensitized guinea pigs.
Dexamethasone also significantly inhibited epidermal vacuolation at a dose of 10 mg/kg, but it did not significantly
inhibit eosinophil infiltration of the epidermis or crust (Table
4). By contrast, azelastine (0.1 and 1 mg/kg), ketotifen (0.1
and 1 mg/kg), terfenadine (1 and 10 mg/kg), and cetirizine
(0.3 and 3 mg/kg) did not significantly suppress eosinophil
infiltration of the dermis in OVA-patched sites (Table 3), and
the opposite effect, a significant increase in number of infiltrated eosinophils, was observed in the group treated with
cetirizine at a dose of 0.3 mg/kg (Table 3).
Messenger RNA Expression of Cytokines in the OVAPatched Skin of Epicutaneously Sensitized Guinea
Pigs Treated with TAK-427 and Dexamethasone. To
elucidate the mechanism by which TAK-427 inhibits the
development of eczema, we investigated the effect of TAK427 on expression of the mRNA of cytokines such as IL-13,
TABLE 2
Effect of TAK-427 and dexamethasone on dermatitis manifestations in the OVA-induced eczema-like lesions of epicutaneously sensitized guinea
pigs
OVA was applied with a patch to the shaved flank of epicutaneously sensitized guinea pigs. Drugs were administered p.o. twice daily for 3 days beginning the day before
antigen challenge. Dermatitis manifestations 48 h after antigen challenge were scored according to the criteria described in Table 1. Results are expressed as means ⫾ S.E.M.
Dermatitis scores are the sums of the scores for erythema, edema, and scratches.
Drugs
Dose
n
Erythema
(0–4)
Edema
(0–2)
Scratch
(0–1)
Dermatitis Score
(0–7)
11
12
12
12
10
10
10
10
2.41 ⫾ 0.15
2.29 ⫾ 0.13
1.96 ⫾ 0.17
1.79 ⫾ 0.14
1.90 ⫾ 0.18
1.75 ⫾ 0.13
1.80 ⫾ 0.11
1.85 ⫾ 0.11
1.91 ⫾ 0.06
1.75 ⫾ 0.08
1.67 ⫾ 0.11
1.38 ⫾ 0.19
1.75 ⫾ 0.13
1.70 ⫾ 0.11
1.25 ⫾ 0.08
1.25 ⫾ 0.13
0.41 ⫾ 0.13
0.21 ⫾ 0.10
0.13 ⫾ 0.09
0.00 ⫾ 0.00
0.70 ⫾ 0.13
0.55 ⫾ 0.16
0.20 ⫾ 0.08
0.05 ⫾ 0.05
4.73 ⫾ 0.25
4.25 ⫾ 0.21
3.75 ⫾ 0.30*
3.17 ⫾ 0.30**
4.35 ⫾ 0.31
4.00 ⫾ 0.32
3.25 ⫾ 0.13*
3.15 ⫾ 0.20*
mg/kg, p.o.
Control
TAK-427
Control
Dexamethasone
0.3
3
30
1
3
10
Inhibition
%
* P ⬍ 0.05, **P ⬍ 0.01 vs. control (Dunnett’s test).
10
21
33
8
25
28
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Fig. 4. The time course of eosinophil infiltration of the dermis of OVAinduced eczema-like skin lesions in epicutaneously sensitized guinea
pigs. OVA or Vaseline was patched for 2 to 24 h to shaved flanks of
epicutaneously sensitized guinea pigs. Skin specimens were collected at
indicated time after patched challenge. Histological evaluations were
performed as described under Materials and Methods. The results are
expressed as means ⫾ S.E.M. for five animals.
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Effect of TAK-427 on Eczema-Like Skin Lesion in Guinea Pigs
Discussion
Fig. 6. Effect of TAK-427 and dexamethasone on eosinophil infiltration of
the dermis at OVA-challenged sites in epicutaneously sensitized guinea
pigs. OVA was patched for 24 h to shaved flanks of epicutaneously
sensitized guinea pigs. Drugs were administered p.o. twice daily for 3
days beginning the day before antigen challenge. Skin specimens were
collected 48 h after patch-challenge with OVA, and histological evaluations were performed as described under Materials and Methods. Results
are expressed as means ⫾ S.E.M. for 10 to 12 animals. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.01 versus control (Dunnett’s test).
The results of this study showed that topical antigen challenge by patch induced eczema-like lesions at the challenged
sites in epicutaneously sensitized guinea pigs and that TAK427 mitigated the development of eczema and suppressed
expression of proinflammatory cytokine/chemokine mRNA in
this experimental model. Histopathologically, the antigenchallenged sites showed erythema with papules, epidermal
hypertrophy, vacuolation of epidermal cells, and cellular infiltration. These results indicated that the eczema model in
the guinea pig shares several features with the skin lesions
in the acute phase of atopic dermatitis, suggesting that epicutaneous sensitization and challenge play an important role
in the development of eczema at the local sites. This notion is
supported by two recent studies in which application of the
antigen (OVA) to mouse skin resulted in the induction of
TABLE 4
Effect of several antihistamines on the dermatitis score and eosinophil infiltration of the dermis in the OVA-induced eczema-like lesions of
epicutaneously sensitized guinea pigs
OVA was applied to the shaved flank of epicutaneously sensitized guinea pigs with a patch. Drugs were administered p.o. twice daily for 3 days beginning the day before
antigen challenge. Dermatitis was scored 48 h after antigen challenge according to the criteria described in Table 1. Skin specimens were collected and histological
evaluations were performed as described under Materials and Methods. Results are expressed as means ⫾ S.E.M.
Drug
Dose
n
Dermatitis Score
(0–7)
10
10
10
10
9
10
10
10
10
10
4.65 ⫾ 0.30
4.35 ⫾ 0.34
4.30 ⫾ 0.24
4.35 ⫾ 0.25
4.56 ⫾ 0.27
4.70 ⫾ 0.27
4.00 ⫾ 0.22
4.40 ⫾ 0.24
4.55 ⫾ 0.29
4.10 ⫾ 0.24
mg/kg, p.o.
Control
Azelastine
Ketotifen
Control
Terfenadine
Cetirizine
0.1
1
0.1
1
1
10
0.3
3
* P ⬍ 0.05 vs. control (Dunnett’s test).
Inhibition
%
6
8
6
2
15
6
3
13
Infiltrated Eosinophils
Inhibition
cells/mm
%
108.0 ⫾ 20.4
120.6 ⫾ 21.6
104.8 ⫾ 14.8
145.3 ⫾ 24.1
121.3 ⫾ 14.3
169.0 ⫾ 14.3
178.0 ⫾ 14.8
165.7 ⫾ 17.0
246.0 ⫾ 29.0*
137.3 ⫾ 13.5
⫺12
3
⫺35
⫺12
⫺5
2
⫺46
19
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GM-CSF, IL-1␣, TNF-␣, IFN-␥, IL-8, and IL-10 in the skin at
OVA-patched sites. Since in the preliminary experiments
mRNA expression of these cytokines was found to be increased by OVA challenge and peak at 6 to 24 h after antigen
application, the effect of TAK-427 and dexamethasone was
evaluated at 24 h after OVA application. All cytokine mRNA
expressions were normalized as the number of copies of cytokine cDNA per copies of G3PDH cDNA (⫻ 10⫺5). Expression of IL-13, GM-CSF, IL-1␣, TNF-␣, IFN-␥, IL-8, and IL-10
mRNA in OVA-patched sites increased by 2.5-, 2.7-, 3.1-, 5.1-,
2.5-, 18-, and 10-fold, respectively, compared with the expression in the nonchallenged sites. TAK-427 completely suppressed the mRNA expression of IL-13 and significantly inhibited the mRNA expressions of GM-CSF, IL-1␣, and IL-8
by 72, 70, and 63%, respectively. TAK-427 clearly inhibited
the mRNA expression of IFN-␥ and TNF-␣, although the
differences were not statistically significant, but it did not
inhibit IL-10 mRNA expression (Fig. 7). Treatment with
dexamethasone significantly inhibited mRNA expression of
IL-13, GM-CSF, IL-1␣, TNF-␣, IFN-␥, and IL-8 in the OVAchallenged skin site but not that of IL-10 (Fig. 7). These
results indicated that both TAK-427 and dexamethasone inhibited the expression of proinflammatory cytokines after
antigen challenge but did not affect the anti-inflammatory
cytokine IL-10.
1288
Fukuda et al.
TABLE 4
Effect of TAK-427 and dexamethasone on epidermal injury and eosinophil infiltration of the epidermis or crust in the OVA-induced eczema-like
lesions of epicutaneously sensitized guinea pigs
OVA was applied with a patch to the shaved flank of epicutaneously sensitized guinea pigs. Drugs were administered p.o. twice daily for 3 days beginning the day before
antigen challenge. Skin specimens were collected 48 h after antigen challenge. The histological preparations were scored on a scale of 1 to 4. Results are expressed as means ⫾
S.E.M.
Epidermal Injury
Drug
Dose
n
Eosinophil Infiltration
Crust
formation
Vacuolation
Epidermis
Crust
2.7 ⫾ 0.2
2.5 ⫾ 0.2
2.3 ⫾ 0.2
2.3 ⫾ 0.1
3.0 ⫾ 0.3
2.6 ⫾ 0.2
2.9 ⫾ 0.2
2.4 ⫾ 0.2
2.7 ⫾ 0.2
2.3 ⫾ 0.1
2.4 ⫾ 0.3
2.1 ⫾ 0.1
3.0 ⫾ 0.2
2.5 ⫾ 0.2
2.5 ⫾ 0.2
2.1 ⫾ 0.3*
2.2 ⫾ 0.1
1.8 ⫾ 0.1
1.8 ⫾ 0.2
1.6 ⫾ 0.1*
2.2 ⫾ 0.1
2.1 ⫾ 0.1
2.1 ⫾ 0.1
2.0 ⫾ 0.0
2.9 ⫾ 0.3
2.9 ⫾ 0.3
1.9 ⫾ 0.3
1.7 ⫾ 0.3*
2.4 ⫾ 0.2
2.3 ⫾ 0.2
2.4 ⫾ 0.2
2.3 ⫾ 0.2
mg/kg, p.o.
Control
TAK-427
Control
Dexamethasone
0.3
3
30
1
3
10
11
12
12
12
10
10
10
10
* P ⬍ 0.05 vs. control (Dunnett’s test).
related to their amelioration of the manifestations of dermatitis in the guinea pig model.
The effect of cetirizine on eosinophil infiltration has been a
matter of controversy. Some groups have reported finding
that cetirizine is effective in inhibiting eosinophil infiltration/
migration into human skin in allergic conditions (Fadel et al.,
1987; Charlesworth et al., 1989b), whereas other groups have
reported that late-phase reactions, including eosinophil infiltration, are unaffected by cetirizine (Varney et al., 1992;
Atkins et al., 1997; Zweiman et al., 1997). In our eczema
model, cetirizine aggravated the eosinophil infiltration at
OVA-patched skin sites rather than suppressing it. The reason for the difference in the findings in these studies is
unclear.
The skin at the site of the lesions in atopic dermatitis
hyperexpressed several proinflammatory cytokines and chemokines, including IL-4, IL-5, IFN-␥, IL-13, GM-CSF,
TNF-␣, IL-1, IL-8, and eotaxin, and the anti-inflammatory
cytokine IL-10 (Van Joost et al., 1992; Hamid et al., 1994;
Ohmen et al., 1995; Pastore et al., 1997; Van der Ploeg et al.,
1997; Yawalkar et al., 1999). IFN-␥ has been reported to be
important for the development of skin hypertrophy in a murine model of dermatitis (Carroll et al., 1997; Spergel et al.,
1999). GM-CSF, IL-13, TNF-␣, and IL-8 works as a potent
stimulant of the recruitment of inflammatory cells (Nakajima et al., 1994; Erger and Casale, 1995; Pastore et al., 1997;
Ying et al., 1997). The present study has demonstrated that
the OVA-patched skin sites also hyperexpressed IFN-␥, IL13, GM-CSF, TNF-␣, IL-1, IL-8, and IL-10. We think that the
cytokines act together to produce the dermal inflammation
and eczema-like lesions in the guinea pig model. It is well
known that eotaxin and IL-5 are one of the most related
cytokine/chemokine to eosinophil infiltration, but their
mRNA expression did not increase at the sites of skin lesions
in this guinea pig eczema model (data not shown), suggesting
that local expression of eotaxin and IL-5 dose not play a
predominant role in the eosinophil infiltration in this model.
In this study, TAK-427 was found to significantly suppress expression of mRNA of IL-8, GM-CSF, IL-13, and
IL-1␣ and tends to suppress expression of the mRNA of
IFN-␥ (p ⫽ 0.0657) and TNF-␣ (p ⫽ 0.0639). Dexamethasone also inhibited mRNA expression of these proinflammatory cytokines. Neither TAK-427 nor dexamethasone,
however, suppressed mRNA expression of the anti-inflam-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
antigen-specific IgE antibodies and an eczema-like skin response (Wang et al., 1996; Spergel et al., 1998, 1999).
In addition, the eczema model in the guinea pig showed
similarities to atopic dermatitis pharmacologically because
dexamethasone was effective in suppressing dermal inflammation and eczema formation, but none of the four antihistamines tested (azelastine, ketotifen, terfenadine, and cetirizine) were effective. The highest dosage levels of
antihistamines used in this study were about 10 times the
ID50 values for their antihistaminic effects, and since they
were ineffective against the dermal lesions, histamine does
not seem to play an important role in terms of the development of eczema in this model. These observations suggest
that the guinea pig model may be a useful tool for evaluating
the potency of drugs for the treatment of atopic dermatitis.
TAK-427, a novel imidazopyridazine derivative, was discovered as a result of testing a number of compounds for
anti-inflammatory activity in the guinea pig model of eczema
and its antihistaminic activity in vivo and in vitro, and it is
currently under development as a new therapeutic agent for
atopic dermatitis. In this eczema model, TAK-427 significantly reduced the manifestations of dermatitis and eosinophil infiltration of the dermis and epidermis at the OVApatched sites. Although TAK-427 displays antihistaminic
activity (ID50, approximately 1 mg/kg), its inhibitory effect on
the development of eczema is not attributable to antihistaminic effect because none of the antihistamines exerted an
inhibitory effect in this experimental model.
Evidence of eosinophil infiltration and presence of eosinophil-derived major basic protein in the dermal layer have
been well documented in atopic dermatitis, although the
actual role of eosinophils in the pathogenesis of atopic dermatitis is poorly understood. Eosinophil major basic protein
and eosinophil cationic protein are known to have a cytotoxic
effect (Rothenberg, 1998), and a recent study indicated that
the absence of eosinophils in OVA-sensitized skin sites of
IL-5-deficient mice was associated with a lack of increase in
the thickness of the epidermis and dermis, a common feature
of the skin lesions in atopic dermatitis. These results suggest
that eosinophils play an important role in the cutaneous
hypertrophy in atopic dermatitis (Spergel et al., 1999), and
inhibition of eosinophil infiltration may improve dermal
symptoms in atopic dermatitis. The inhibitory effect of TAK427 and dexamethasone on eosinophil infiltration may be
Effect of TAK-427 on Eczema-Like Skin Lesion in Guinea Pigs
1289
matory cytokine IL-10. Similar results were observed in
atopy patch test lesions treated with topical glucocorticoids (Langeveld-Wildschut et al., 2000). Since we have
observed that TAK-427 has no effect on T or B cell proliferation in vitro in mice, it is not a general immunosuppressant (unpublished data). The inhibition of mRNA expression of proinflammatory cytokines may have
contributed to the anti-inflammatory effects of TAK-427 in
this experimental model. Although the precise mechanism
remains to be elucidated, since TAK-427 has the inhibitory
effect of TNF-␣ release from the mast cell by IgE-dependent mechanism (in preparation), this mechanism may
have contributed to the inhibition of mRNA expression of
proinflammatory cytokines. As T cells are one of the most
important source of cytokines, the effect of TAK-427 on T
cells migration into the challenged sites are now investigated.
In summary, the eczema-like skin lesions in this experimental model exhibit several features similar to those of the
acute phase of atopic dermatitis in terms of manifestations of
dermatitis, dermal inflammation, and epidermal injury, suggesting that this guinea pig model may be a useful tool for
evaluating the potency of drugs for the treatment for atopic
dermatitis. TAK-427 suppressed the allergic dermal inflammation that leads to eczema formation by inhibiting the
expression of proinflammatory cytokines at the lesion sites.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 7. Effects of TAK-427 and dexamethasone on cytokine mRNA expression at the OVA-challenged sites in epicutaneously sensitized guinea pigs.
OVA were patched for 24 h to shaved flanks of epicutaneously sensitized guinea pigs. TAK-427 (30 mg/kg) and dexamethasone (10 mg/kg) were
administered p.o. twice daily for 2 days beginning the day before antigen challenge. Skin specimens were collected 24 h after patched challenge with
OVA, and cytokine mRNA expression was evaluated as described under Materials and Methods. Results are expressed as relative quantity of mRNA
based on G3PDH (⫻ 10⫺5) expression, and the data are means ⫾ S.E.M. for 10 animals. Dex: dexamethasone. †, p ⬍ 0.05; ††, p ⬍ 0.01 versus no
patched control (Student’s t test); ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 versus control (Dunnett’s test).
1290
Fukuda et al.
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