, Nonclinical Research CenterEﬀect of STAT inhibitor on the murine chronic graft-versus-hostdisease (GVHD) scleroderma model using an X-ray irradiation device○ Shiruku Hosoi, Yumi Wako, Hajime Wada, Kousuke Morizumi, Seiichi Katayama, Naoyuki Hironaka,Katsuhide Nishi

Summary in Japanese

Objective
Chronic graft-versus-host disease (cGVHD) is the fatal complication of allogeneic transplantation
occurred by immunological response. Scleroderma-like symptoms are major in cGVHD such as
progressive sclerosis of skin, peripheral circulatory failure, and multiple organ failure. The
current treatment methods for sclerodermatous-GVHD (Scl-GVHD) are only conservative andmore eﬀective drugs are waited. The murine GVHD model induced by irradiation and allogeneic
cell transplantation is known as one of the models which develops Scl-GVHD-like cutaneous
symptoms. The aim of this study is to conﬁrm the pathologic state of the Scl-GVHD model andevaluate the eﬀectiveness of agents on this model.
We evaluated Scl-GVHD score, hydroxyproline (HYP) content of the skin and histopathologic
examinations. The results indicate that the allogeneic transplantation groups (cell amount A-C)
develop cutaneous symptoms compared to the syngeneic group. Eﬀects of Niclosamide wereevaluated on this model. Niclosamide is reported for the suppressive eﬀect on Scl-GVHD by
inhibiting STAT3 pathway which involved in ﬁbrogenesis.

慢性移植片対宿主病（chronic graft versus host disease，cGVHD）は同種異系間移植の合併症の一つであり，生体内の免疫応答により発症する症状の総称である．強皮症様症状は cGVHD の主要な症状であり，皮膚の硬化，末梢循環障害，多臓器不全を呈する．
その治療法は対症療法のみであり，治療薬の開発が待ち望まれる疾患の一つである．

本研究では放射線照射と同種異系細胞移植による GVHD 強皮症モデルの作製を検討し，さらに本モデルを用いて薬効評価検討を行った．評価は病態スコア，皮膚中 HYP 量測定および病理組織学的検査にて行った．
モデル作製は BALB/c マウスを X 線照射後，B10.D2 マウス細胞を移植した．細胞移植後
Day 14 前後で皮膚及び全身症状の発症が始まり，Day 56 までは病態の維持が確認された．
このモデルに対してニクロサミドを投与し，薬剤の皮膚線維化に対する効果を評価した．ニク
ロサミドは STAT3 の阻害により線維形成を抑制することが報告されている．

本試験条件下より，ニクロサミド群では Control 群との有意差は認められず，抗線維化効果を捉えることはできなかった．今後，モデルの病態発症の程度や薬効評価方法について引き続き検討を行っていく．

Materials and Methods
Experiment 1【Animals】

Experiment 2【Animals】

10 week-old B10.D2, female
10 week-old BALB/c, female

Donner:Recipient:【Method】

the same as Experiment 1【Method】

the same as Experiment 1 except for concentration of administrated cell suspension which was
Sp: 1×106 cells/body and BM: 5×105 cells/body.

X-ray irradiation for recipients:
7 Gy, whole body irradiation using MX-160 Labo (mediXtec Japan Corp.).
Donner cell transplantation:
Donner spleen and bone marrow cell suspension was administrated intravenously via tailvein after 6 hours from irradiation.

【Administration】

Niclosamide:
1 mg/mL, 10 mL/kg, administrated intraperitoneally 3 times a week
(every Monday, Wednesday and Friday) from Day 14 (total 12 times).

【Evaluation】● Scl-GVHD Score:
determined by evaluating dorsal skin alopecia and other cutaneous symptoms twice a weekfrom Day 12 using following criteria.
- Criteria (ranging from 0 to 9)
Dorsal skin:
0: no sign, 0.5: thin hair region<1 cm2, 1: thin hair region>1 cm2, 1.5: alopecia<1 cm2,2: alopecia>1 cm2
Other cutaneous symptoms:
add 0.5 for each item with cutaneous symptoms: face, auricle, tail, and/or limbs.
General symptoms:
add 1 for each following item: piloerection, hunched posture or lethargy, vasculitis (one ormore purpural lesions on ears or tail), eyelid sclerosis (blepharophimosis), and/or diarrhea.
● Dorsal connective tissue thickness:dermal connective tissue thickness was measured on histopathological specimen.
● Histopathological analysis:
Day 56
Test Groups

X-rayirradiation

Donner (B10.D2) cellamount (cells/body)

n--

3

7 Gy

Sp*: 4x106, BM*: 2x106
Sp: 1x106, BM: 5x105
Sp: 2x106, BM: 1x106
Sp: 4x106, BM: 2x106

5888

1 Normal
2345Syngeneic
Cell amount A
Cell amount B
Cell amount C

Experiment 1 Schedule-the same as Experiment 1 except for following:● Scl-GVHD Score:
determined by evaluating dorsal skin alopecia and other cutaneous symptoms twice a weekfrom Day 14 using following criteria.
- Criteria (ranging from 0 to 4.5)
Dorsal skin:
0: no sign, 0.5: thin hair region<1 cm2, 1: thin hair region>1 cm2, 1.5: alopecia<1 cm2,2: alopecia 1-2 cm2, 2.5: alopecia 2-3 cm2, 3: alopecia>3 cm2
Other cutaneous symptoms:
add 0.3 for each item with cutaneous symptoms: snout, periocular, auricle, tail, and/or limbs.
● Histopathological analysis:
Day 42Test
groupsAdministration

1 Syngeneic
2 Control
3 Niclsamide

X-ray
Donner (B10.D2) cellirradiation amount (cells/body)i.p.
10 mL/kg

7 GynExperiment 2 Schedule

Day 0Day 7
1W

Sp: 1x106, BM: 5x105

88Day 00W

Day 7
1W

Day 14 Day 21 Day 28 Day 32 Day 42 Day 49 Day 56
2W

3W

4W

5W

6W

7W

X-ray irradiation
Cell transplantation
Scl-GVHD score (twice a week)
Dissection

Sp*: 1x106, BM*: 5x105 5

Sp: Spleen, BM: bone marrow*: recipient (BALB/c) cells were transplanted to Syngeneic group0W

Sp: Spleen, BM: bone marrow*: recipient (BALB/c) cells were transplanted to Syngeneic group

X-ray irradiation
Cell transplantation
Scl-GVHD score (twice a week)
Dissection【Evaluation】

Day 142W

Day 21
3W

Day 284W

Day 325W

Day 42
6W

Niclosamide administration: i.p., 3 times/ week

8W

Results
Experiment 1

Experiment 2
Figure 2 HYP Content of Skin

9168

146
5

Cell amount C4Cell amount B

3210Syngeneic
1219263340

Cell amount A
4754

**

**

412108642Control

3.5
32.5

Niclosamide

21.5
10Day

Figure 4 Scl-GVHD score

4.5

**

Scl-GVHD score

7HYP content (mg/g Skin)

Scl-GVHD score

Figure 1 Scl-GVHD score

Normal Syngeneic

AB
Cell amountC

0.5

Mean±SEM, n = 3〜８**: P<0.01, signiﬁcant diﬀerence from syngeneic group.
(Dunnett's multiple test)

Cell amount A0Syngeneic
14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
DayDay 20

Syngeneic:
Score 0Day 21

Cell amount A:
Score 1Day 56

Syngeneic

Day 42Control:
Score 0.5

Syngeneic

Niclosamide:
Score 1.5

Figure 5 HYP Content of Skin

Syngeneic

Cell amount A:
Score 2.5

Figure 3 Body weight ratio

120

NormalRatio to Day0 (%)

110

Cell amount A

Syngeneic

100
Cell amount C

90

Cell amount B:hair follicle

11010864100
90

Control

80

20Syngeneic

Niclosamide
Syngeneic

Control70

Niclosamide0

Mean±SEM, n = 5
no signiﬁcant diﬀerence from syngeneic group. (Studentʼs -test)
no signiﬁcant diﬀerence from control group. (Studentʼs -test)

4812 16 20 24 28 32 36 40 44
Day

Cell amount A

80
70120.054

Niclosamide:
Score 2.4

Figure 6 Body weight ratio

120Ratio to Day0 (%)

Syngeneic:
Score 0

HYP content(mg/g Skin)

14Control:
Score 3.104

8 12 16 20 24 28 32 36 40 44 48 52 56
Day・ There was signiﬁcant increase in Scl-GVHD score and HYP content of skin of Scl-GVHD group compared to theNormal group (Fig. 1, 2).
・ Histopathological analysis in dorsal skin showed the remarks seen in human patient of Scl-GVHD such asswelling of collagen ﬁbers and decrease of hair follicles in all model groups.
・ Transplanted cell amount aﬀect the development speed of pathological condition and the systemic conditionof animals (Fig. 1, 3).
・ Scl-GVHD score reached plateau on Day 30 for cell amount B and C groups, and Day 37 for cell amount A groupwhile the appearance (alopecia and/or other cutaneous symptoms) of animals change afterwards.
・ From the results of Experiment 1, the cell transplantation was conducted with cell suspension prepared withconcentration of cell amount A (Sp: 1x106, BM: 5x105) in Experiment 2. For Scl-GVHD score, we changed thecriteria to enable the score to evaluate more than 37 days.

Conclusion
・ The Scl-GVHD model which develops dermal ﬁbrosis and histopathologicalchanges seen in human was produced.
The transplanted cell amount aﬀect the development speed of pathologicalcondition.
・ Results of Experiment 2 revealed that Niclosamide (10 mg/kg) showedno signiﬁcant improving eﬀect on this model (Fig. 4-6).
・ We continue to revise the method of induction to achieve stable
pathological conditions of dermal ﬁbrosis for a better model, which could be
used for the evaluation and development of new anti-ﬁbrosis agents.