JP7389151B2 - 乾癬治療用薬品の製造におけるCaMK4阻害剤の使用 - Google Patents
乾癬治療用薬品の製造におけるCaMK4阻害剤の使用 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/18—Sulfonamides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/11—Protein-serine/threonine kinases (2.7.11)
- C12Y207/11017—Ca2+/Calmodulin-dependent protein kinase (2.7.11.17)
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Description
本発明は乾癬治療用薬品の製造におけるCaMK4阻害剤の使用を提供する。
本発明は、免疫組織化学(immunohistochemistry)、リアルタイム定量のPCR、Western blot等の手段により、CaMK4を乾癬患者の皮膚とイミキモド(IMQ)に誘発されたネズミの乾癬病の皮膚に用いるときの発現状況を分析する。その結果によるとCaMK4は乾癬病の発展に係っている。野生(Camk4+/+)ネズミと比較してみると、IMQでCaMK4欠陥型(Camk4-/-)ネズミの乾癬モデルを構成することを誘発することにより、皮膚の厚さ、スポット及び表皮の厚さを有効に低減することができる。また、ネズミの体内の実験において、骨髄由来(bone marrow-derived)の大食細胞(BMDM)の伝送、IL-10とCaMK4の阻害剤KN-93の外因性供給により、免疫細胞または表皮角化細胞中のCamk4の発現を抑制し、皮膚の厚さ、スケール及び表皮の厚さを減少させ、かつ炎症誘発性サイトカインを低減することができる。IMQの誘発により大食細胞の特異性CaMK4をノックアウト(knock out)した(Camk4fl/fl Lyz2-Cre)ネズミの乾癬病フェノタイプは対比型(Camk4fl/fl)ネズミより低下する。そのため、本発明は乾癬治療用薬品を製造するときドラッグターゲットとして使用するCaMK4を提供する。
実験の材料と方法
(1)乾癬患者の皮膚のサンプル
中国安徽医科大学第一付属医院の皮膚科の患者、病症が典型的な乾癬である患者(33名)を選択する。常用の方法により病症部位の皮膚と病症周囲の健康皮膚を9例を取って生理的食塩水に浸入させ、サンプルを当日に処理することによりスキンバイオプシー(skin biopsy)を獲得する。常用の方法により血液を採り、血液とTrizolを1:3に混合した混合液体を-80℃温度で冷凍し、RNAを抽出する。
国際において常用する乾癬病ネズミのモデルを採用する。満6~8歳であるC57BL/6ネズミを用意し、毛を剃ったネズミの背中の皮膚に5%のIMQを塗る。一日に5%のIMQを62.5mgを塗り、それを五日間続ける。満6~8歳であるC57BL/6ネズミを対比用ネズミにする。
Tritzol方法により組織または細胞の総RNAを抽出する。具体的に、1mLのTrizolを組織または細胞に送入した後、ホモジェナイザーで組織を均質状態にし、細胞の複数回の刺激により組織を充分にソリューションする。つぎに、200μLのクロロホルム(chloroform)を注入し、15sのボルテックスオシレーション(Vortex oscillation)を実施した後、室温下において10min静止させる。つぎに、それを12000gを取り、4℃の温度下において15minの遠心処理を実施した後、500μLのsupernateを収集してEPチューブに注入する。つぎに、4℃の温度で予め冷却させた500μLのイソプロパノール(isopropanol)をEPチューブに注入し、それらを均一に混合させた後、室温下において10min静止させる。つぎに、それを12000gを取り、4℃の温度下において15minの遠心処理を実施し、1mLの75%のアルコールを注入することにより2回の洗浄を実施する。最後に、それらを乾燥させた後、20μL DEPCで処理した水を注入し、Nanodrop2000で濃度と純度を測定する。
1mlの抗凝血(Anticoagulant)を取って15mlの遠心管(centrifugal tube)を注入し、5mlの赤血球ソリューションソルーション(erythrocyte lysis solution)に注入し、それらを均一に混合させた後、氷の上において5minのソリューションを実施する(その間にそれらを複数回混合させることができる)。PBSをいっぱいに注入することにより反応を停止させ、500gの原料を取って4℃の温度下において10minの遠心処理を実施し、細胞の沈殿を収集してPBSで一回洗浄した後、洗浄される細胞を収集する。細胞の数量を統計し、1×106 の細胞を取り、Fcレセプタを密閉させ、抗体V500-CD45、PerCP/Cy5.5-CD3、FITC-CD4、PE/Cy7-CD8、PE-CD14を入れることにより表面ランドマークの染色を実施する。つぎに、固定と破裂を実施した細胞にウサギ抗ヒト抗体(Rabbit anti human antibody)と標記AF647付きヤギアラビット(goatantirabbit)を順に加入する。染色が終わると、PBSで一回洗浄し、200μLPBS再浮遊細胞(Resuspended cells)を加入し、フローサイトメトリーでそれを測定する。
パラフィンセクション(paraffin section)に対して脱蝋(dewaxing)を実施した後、0.01M(pH=6.0)のクエン酸ナトリウムにより抗原リトリーバル(antigen retrieval)を実施する。3%のH2O2で内因性ペルオキシダーゼ(Endogenous peroxidase)であるブロッキングバッファ(blocking buffer)により非特異蛋白(nonspecific protein)をバッファする。一次抗体(CaMK4抗体)に対して4℃の一夜のインキュベーションを実施し、二次抗体に対して37℃のインキュベーションを30min実施し、DABで顕色をし、Largetrophozoitesstainedwithironhematoxylinを実施した後、乾燥と包装を実施する。
構成される前記ネズミのモデルを採用し、五日目に皮膚のサンプルを採集することにより実験をする。
IMQはTLR7により乾癬病炎症を誘発し、TLR7はいろいろな骨髄由来(bone marrow-derived)の自然免疫細胞上において発現をする。また、IMQはネズミの皮膚のF4/80+とCD11c+細胞中のCaMK4の上昇を誘発する(実施例1を参照)。本発明は所定の骨髄性細胞(myeloid cell)亜集団の変化も検出する。まず、免疫細胞を分離させ、具体的なステップはつぎのとおりである。
大食細胞はIL-10を形成することにより調節をし、IL-10は、中枢抗炎症メディエーター(Central anti inflammatory mediators)であり、過度な炎症と制御不可能な炎症に刺激されることによる組織の損傷を減少させ、かつホストが過度な炎症による影響を受けることを防止することができる。大食細胞がIL-10を形成することとCaMK4が足りないことによる乾癬病症を確定するため、皮膚と大食細胞中のIL-10の発現を検出する必要がある。
大食細胞中のサイトカインを測定する。
外因性供給(Exogenous supply)によりネズミの体内にIL-10とCamk4-/-BMDMを送入することにより養子移入(adoptive transfer)実験をする。
Camk4fl/fl Lyz2-Creネズミの乾癬病フェノタイプと皮膚中の骨髄性細胞を検出する。
TNF-αとIL-17AでHaCaT細胞を刺激することにより乾癬細胞モデルの検出を模擬する。
ネズミ体内においてCaMK4の阻害剤としてKN-93を用いるときの治療の効果を評価する。
Claims (1)
- 乾癬治療用薬品の製造におけるCaMK4阻害剤の使用であって、
前記CaMK4阻害剤はKN-93であり、
前記CaMK4阻害剤の外因性供給により、CaMK4をドラッグターゲットとして、CaMK4のタンパク質発現またはmRNA発現を阻害し、且つCaMK4の遺伝子レベル及び/又は蛋白質レベルを下げることにより、IL-10の回復を招き、乾癬を治療するためのものである、使用。
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