JP7466253B2 - フィーカリバクテリウム・プラウスニッツイを含むアトピー性疾患の予防又は治療用の薬学的組成物 - Google Patents
フィーカリバクテリウム・プラウスニッツイを含むアトピー性疾患の予防又は治療用の薬学的組成物 Download PDFInfo
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Description
実施例1:フィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株の分離及び同定
1.1.菌株の分離及び同定
健康な韓国人(女性、9歳、BMI 15.5)の糞便からフィーカリバクテリウム・プラウスニッツイを分離するために、Martin方法によって、嫌気性チャンバーを用いて、厳格な無酸素条件(5%H2、15%CO2及び80%N2)の下で、0.5%酵母エキス、0.1%D-セロビオース、0.1%D-マルトースを添加したYBHI培地(brain heart infusion medium supplemented with 0.5% yeast extract)(Difco社製、米国デトロイト)を用いて培養した後、EOS(Extremely Oxygen Sensitivity)菌種を選別した後、分離した。
分離された菌株がフィーカリバクテリウム・プラウスニッツイ菌株であるかを確認するために、分離された菌株を顕微鏡で観察した。図1に示すように、標準菌株であるフィーカリバクテリウム・プラウスニッツイA2-165標準菌株(A)及びフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株(B)を1000倍の倍率に拡大して観察した結果、菌株の形状がいずれもまっすぐな又は曲がっている棒状を有し、互いに類似した形状を有することを確認した。
フィーカリバクテリウム・プラウスニッツイ菌株であるかを確認するために、分離された菌株を表1のFP-特異性プライマー(配列番号2及び配列番号3)を使用してPCR分析を実施した。その結果、図2に示すように、陽性対照群の菌株であるフィーカリバクテリウム・プラウスニッツイA2-165菌株と類似したバンドパターンを現したことを確認することができた。
前記ように分離されたフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株が既に報告された同種のフィーカリバクテリウム・プラウスニッツイA2-165標準菌株と同じ菌株であるかを検証するために、分子タイピングの一種であるRAPDを実施した。このために、菌体から抽出したゲノムDNAを対象として、下記の表2の汎用プライマーを用いてDNAを増幅した後、1%寒天ロースゲルで1時間30分間電気泳動し、UVトランスイルミネーターでDNA分節パターンを比較し、その結果を図3に示した。
分離された菌株がフィーカリバクテリウム・プラウスニッツイであるかを確認するために、16S RNAの塩基配列を分析した後、BLAST(Basic local alignment search tool)で確認した結果、フィーカリバクテリウム・プラウスニッツイ種と99%以上一致することを確認した。
前記ように分離されたフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株の全長(full-length)16S rRNA遺伝子の塩基配列分析のために、下記の表3の27F及び1492Rプライマーを用いて16S rRNA遺伝子を増幅した後、3730xl DNA分析器を用いて塩基配列を決定した。このように得られた本発明のEB-FPDK11菌株及び既に公表された同種の他の菌株の16S rRNA遺伝子の塩基配列を用いて系統樹(phylogenetic tree)を作成して図4に示した。
2.1.機能性代謝体(短鎖脂肪酸)の分析
分離されたフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株の機能性代謝体を確認するために、培養液に含有された短鎖脂肪酸(SCFA、short chain fatty acids)の含有量をガスクロマトグラフィーで分析した。このために、YBHI培地[Brain-heart infusion medium supplemented with 0.5%w/v yeast extract(Difco)、0.1%w/v Dcellobiose、0.1%w/v D-maltose)に菌株を24時間培養した後、12,000Хgで5分間遠心分離して上澄み液を回収し、上澄み液は0.2μmシリンジフィルターで濾過した後、分析に使用した。FFAP column(30m×0.320mm、0.25μm phase)が装着されたガスクロマトグラフィー(Agilent 7890N)を用い、条件は表4のように設定した。
前記ように分離されたフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株の抗菌剤感受性を把握するために、Clinical & Laboratory Standard Institute(CLSI)ガイドラインの微量液体希釈法(broth microdilution method)によって、嫌気性細菌用抗菌剤をピペラシリン-タゾバクタム(PTZ)、セフチゾキシム(CTZ)、クロラムフェニコール(CHL)、クリンダマイシン(CLI)、メロペネム(MEM)、モキシフロキサシン(MXF)、メトロニダゾール(MTZ)、シプロフロキサシン(CIP))の最小阻止濃度(minimum inhibitory concentration、MIC)を決定し(CLSI、2017)、その結果を下記の表5に示した。
前記ように分離されたフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株の安全性検証のために、溶血活性を有するかを評価した。このために、トリプティックソイ寒天(17.0g/Lカゼインの膵臓消化物、3.0g/L大豆の膵臓消化物、2.5g/Lデキストロース、5.0g/L塩化ナトリウム、2.5g/Lリン酸カリウム、15g/L寒天)に5%w/vの脱線維素血液(defibrinated sheep blood)を添加して製造した血液寒天培地を用いて菌株を培養し、その結果は図6に示した。
3.1.菌株試料
本実験に使用したフィーカリバクテリウム・プラウスニッツイA2-165標準菌株及びフィーカリバクテリウム・プラウスニッツイEB-FPDK11生菌は、1×108CFU/150μl PBS(25%グリセロール、0.05%システイン/PBS)の濃度に製造した。
アトピー性皮膚炎病変を観察するために、6週齢の体重21~25g程度の雄性NC/Ngaマウス(SLC、Inc.、Japan)をデハンバイオリンク(韓国、忠清北道)から供給された。動物実験はInstitutional Animal Care and Use Committee (IACUC)のAnimal use and Care Protocolを遵守して遂行した。一週間の適応期間を経た後、9週間飼育し、飼育環境は、一定の温度(22℃)及び相対湿度(40~60%)を維持し、12時間の周期で明暗を調節しながら飼育した。
6週齢のNC/Ngaマウスの背中部位をきれいに除毛した後、皮膚の微細傷が治癒されるように、24時間放置した。1%の2,4-ジニトロクロロベンゼン(DNCB)溶液(Sigma-Aldrich Korea)をマウスの背中部位に1週間に2回ずつ、3週間塗布して免疫反応を誘発した後、0.5%DNCB溶液を一週に2回塗布して接触性皮膚炎を誘導した。本実施例で使用されたDNCBはアセトンとオリーブオイルとが3:1で混合された溶液に0.5%及び1%に希釈して使用した。
DNCBによってアトピー性皮膚炎を誘発させた後、デキサメタゾン(DEX)、ATCCBAA-835菌株及びEB-AMDK19菌株を含有する調製品で6週間の治療を行い、臨床症状を確認するために、皮膚の状態と、これを点数化した皮膚炎指数(dermatitis score)を確認した。
実験終了の後、マウスをCO2で麻酔して犠牲させた後、厚さ測定器(Digimatic thickness gauge、547-301、Mitutoyo、Japan)を用いて、速度変化法でマウスの両耳の浮腫の程度を測定し、その結果を図9及び図10に示した。
実験終了の前日にそれぞれのかゆみによる患部引っかき回数を測定した。引っかき回数(scratching frequency)の測定は、マウスに10分間の適応期間を与えた後、10分間の引っかき回数をカウンターで測定した。
脾臓は、1次及び2次リンパ器官の特性を全て示し、赤血球を濾過する赤脾髄(red pulp)と体液性免疫及び細胞性免疫活性を示す白髄(white pulp)とから構成されており、免疫反応で他の重要な器官である(Mebius、R E and Kraal、G 2005 Structure and function of the spleen Nat Rev Immunol 5、pp606-616)。
実験終了の際、CO2で麻酔した後、心穿刺によって血液を採取し、採取した血液は、10,000rpmで5分間遠心分離して血清(serum)を分離し、IgE濃度を測定し、その結果を図14に示した。IgE濃度の測定は、ELISAキット(IgEマウスuncoated ELISA kitcat#88-50460、Invitrogen、CA、USA)を用いた。
実験終了の際、マウスを犠牲させた後、皮膚を摘出し、10%ホルムアルデヒド溶液に固定し、パラフィンブロックを製作して薄片を製造した後、ヘマトキシリン及びエオシン(hematoxylin & eosin、H&E)染色を実施し、表皮層及び真皮層の厚さ変化を光学顕微鏡で200倍拡大して観察し、その結果を図15に示した。
アトピー性皮膚炎誘導の後、本発明のフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株の投与による免疫反応を確認するために、Th1及びTh2に関連したサイトカインの濃度を測定した。全ての実験の統計は、GraphPad Prism7.04を使用してOne-way ANOVAを遂行した。
本実験では、Th1サイトカインであるIFN-γ及びそのインデューサ(inducer)であるIL-12を測定した結果、図17に示すように、フィーカリバクテリウム・プラウスニッツイEB-FPDK11群でこれらのサイトカインの全て有意に増加することが分かった。IL-12の濃度を測定した結果、正常群に比べて、アトピー性皮膚炎誘導群(DNCB)は有意に減少し(P=0.03)、アトピー性皮膚炎誘導群(DNCB)に比べて、陽性対照群(P=0.008)、フィーカリバクテリウム・プラウスニッツイA2-165標準菌株群(P=0.03)、EB-FPDK11群(P=0.004)では有意に増加した。
免疫学的観点で、アレルギー性アトピー性皮膚炎は、Th1及びTh2の免疫反応が過度に発生してバランスが崩れて生じる疾患である。したがって、先に測定したサイトカインの濃度に基づいてTh2/Th1サイトカインの比で表現して図18及び図19に示した。
Claims (10)
- フィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株(Faecalibacterium prausnitzii)(KCCM12621P)を有効成分として含む、
アトピー性疾患の予防又は治療用の薬学的組成物。 - 前記フィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株は配列番号1で示される16s rDNA配列を有する、
請求項1に記載のアトピー性疾患の予防又は治療用の薬学的組成物。 - 前記フィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株は生菌株又は低温殺菌された菌株である、
請求項1に記載のアトピー性疾患の予防又は治療用の薬学的組成物。 - 前記薬学的組成物はビタミン又は免疫抑制剤を更に含む、
請求項1に記載のアトピー性疾患の予防又は治療用の薬学的組成物。 - 前記免疫抑制剤は、グルココルチコイド(Glucocorticoid)、シクロスポリン(Cyclosporine)、タクロリムス(Tacrolimus)、ピメクロリムス(Pimecrolimus)、及びISA(TX)247を含むカルシニューリン(calcineurin)阻害剤、ラパマイシン(Rapamycin)、IV型PDE阻害剤、ミコフェノール酸モフェチル(Mycophenolate Mofetil)、及びデキサメタゾンからなる群から選択される、
請求項4に記載のアトピー性疾患の予防又は治療用の薬学的組成物。 - 前記アトピー性疾患の予防又は治療用の薬学的組成物は、有効成分としてフィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株を108~1012CFUの含有量で含むか、又は同CFUの生きている菌又は低温殺菌された菌を有する培養物を含む、
請求項1に記載のアトピー性疾患の予防又は治療用の薬学的組成物。 - 前記アトピー性疾患は、喘息、アトピー性皮膚炎、じんましん、アレルギー性鼻炎、アナフィラキシー又は食物アレルギーである、
請求項1に記載のアトピー性疾患の予防又は治療用の薬学的組成物。 - フィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株(Faecalibacterium prausnitzii EB-FPDK11)(KCCM12621P)を有効成分として含む、
アトピー性疾患の予防又は改善用の健康機能性食品。 - フィーカリバクテリウム・プラウスニッツイEB-FPDK11菌株(Faecalibacterium prausnitzii EB-FPDK11)(KCCM12621P)を有効成分として含む、
アトピー性疾患の緩和又は改善用の化粧品組成物。 - 前記アトピー性疾患は、皮膚アレルギー、皮膚じんましん、アトピー性皮膚炎、乾癬又は湿疹である、
請求項9に記載の化粧品組成物。
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