JP2016503418A - 代謝障害を処置するアッカーマンシアの使用 - Google Patents
代謝障害を処置するアッカーマンシアの使用 Download PDFInfo
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- JP2016503418A JP2016503418A JP2015542276A JP2015542276A JP2016503418A JP 2016503418 A JP2016503418 A JP 2016503418A JP 2015542276 A JP2015542276 A JP 2015542276A JP 2015542276 A JP2015542276 A JP 2015542276A JP 2016503418 A JP2016503418 A JP 2016503418A
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- muciniphila
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- ackermansia muciniphila
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Abstract
Description
本発明では、以下の用語は以下の意味を有する。
「処置」は、ある疾患、障害または病態の少なくとも1つの有害作用を予防(すなわち発症しないようにする)、低減または軽減させることを意味する。したがってこの用語は、治療上の処置および予防または防止措置を指し、ここでの目的は、標的となる病態の状態または障害を予防または鈍化する(低減する)ことである。本発明の1つの実施形態では、処置を必要とする対象は、すでにこの障害を伴う対象、およびこの障害が生じやすい対象またはこの障害を予防すべき対象を含む。
本発明は、以下の実施例によりさらに例示される。
材料および方法
マウス
ob/ob実験:ob/ob対除脂肪の試験:6週齢のob/ob(n=5/グループ)マウス(C57BL/6バックグラウンド、ジャクソン研究所、アメリカ合衆国メイン州バー・ハーバー)を、2または3匹のマウス/ケージで、食物および水に自由にアクセスできる状況の中、管理環境下(12時間の明暗周期、午後6時に明かりを消す)で収容した。マウスに対照の食事(A04、Villemoisson−sur−Orge、フランス)を16週間与えた。盲腸の内容物を収集し、液体窒素に浸し、さらなるアッカーマンシア・ムシニフィラ解析のため−80℃で保存した。
全採血および組織のサンプリングの前にイソフルラン(Forene(登録商標)、Abbott、イギリスケント州クイーンズバラ)で動物に麻酔をかけ、次いで頚椎脱臼によりマウスを屠殺した。貯蔵脂肪(精巣上体、皮下および腸間膜)、肝臓を正確に解剖して重量を測定した。3つの脂肪組織の加算は、肥満指数に対応する。腸の部分(回腸、盲腸および結腸)、盲腸の内容物、ならびに脂肪組織を液体窒素に浸し、さらなる解析のため−80℃で保存した。
近位の結腸部分をすぐに除去し、カーノイズ溶液(エタノール−酢酸−クロロホルム、6/3/1 v/v/v)中で、4℃で2時間固定した。次いで試料を、パラフィン包埋の処理の前に、100%のエタノールに24時間浸した。5μmのパラフィン切片をアルシアンブルーで染色した。最低20の異なる測定を、実験グループについて知らされていない調査者により、区画ごとに内部の粘液層と直交して行った。ランダムに選択した5〜19の区画を、画像解析器(Motic−image Plus 2.0ML、Motic、中国)を使用することにより、合計2146の測定を各結腸について解析した。
総RNAを、トリピュア試薬(ロシュ)を使用して組織から調製した。総RNAの定量および完全性の解析を、1μlの各試料をAgilent 2100バイオアナライザー(Agilent RNA 6000ナノキット、アジレント社)にかけることにより実施した。
インスリン抵抗指数を、4週間の処置(A.muciniphilaの試験)の後に実施した経口グルコース負荷(kg体重あたり2gのグルコース)に続いて取得した血中グルコースおよび血漿インシュリンの両方の曲線下面積(0分および15分)を乗じることにより決定した。明暗周期の開始後から2時間後に食物を除去し、以前に報告されているようにマウスを6時間の飢餓期間の後に処置した(Everard et al. Diabetes 60, 2775−2786 (2011))。
門脈の血液のLPSの濃度を、試料中のエンドトキシン濃度に直接関連する色強度を測定するリムルス血球抽出液(Limulus amaebocyte Lysate、LAL)速度論的発色法(kinetic chromogenic methodology)に基づくEndosafe−MCS(Charles River Laboratories、フランス リヨン)を使用することにより決定した。反応中の干渉(阻害および亢進)を最小限にするためにエンドトキシンフリーバッファーで1/10に血清を希釈し、70℃で15分加熱した。各試料を、エンドトキシンフリーLAL試薬水(Charles River Laboratories)で1/70または1/100に希釈し、二重で処置し、各試料につき2つのスパイクを決定に含めた。全ての試料を、回収および変動係数について確認した。検出の下限は0.005EU/mlであった。血漿インシュリンの濃度を、製造者の説明にしたがってELISAキット(Mercodia、スウェーデン ウプサラ)を使用して25μlの血漿において決定した。
死後に回収したマウスの盲腸の内容物を−80℃で保存した。メタゲノムDNAを、製造者の指示にしたがってQIAamp−DNAツールミニキット(Qiagen、ドイツ ヒルデン)を使用して、盲腸の内容物から抽出した。
回腸の組織をCHCl3(10ml)中でホモジナイズし、重水素化標準物質を添加した。以前に報告されたように抽出曲線および較正曲線を作成し(Muccioli et al,Mol Syst Biol,2010,6,392)、データを組織試料の重量により正規化した。
アッカーマンシア・ムシニフィラを検出するために使用したプライマーおよびプローブは、16S rRNA遺伝子配列であるF−アッカーマンシア・ムシニフィラ CCTTGCGGTTGGCTTCAGAT(配列番号:29)、R−アッカーマンシア・ムシニフィラ CAGCACGTGAAGGTGGGGAC(配列番号:30)に基づくものであった。検出は、製造者の指示に従ったMesa Fast qPCR(商標)(Eurogentec、ベルギー セラン)を使用したStepOnePlus(商標)リアルタイムPCRシステムおよびソフトウェア(アプライドバイオシステムズ、オランダ デン・エイセル)を用いて行った。各アッセイは、同じ実験を2連で行った。次いで、各試料の周期の閾値を、ゲノムDNAを希釈すること(5倍の連続希釈)により作成した標準曲線(3連で実施)と比較した(DSMZ、ドイツ ブラウンシュヴァイク)。このデータを、log(細菌/盲腸の内容物(g))として表した。
マウス腸管チップ(MITチップ)、16S rRNA遺伝子の2つの超可変領域(V1領域およびV6領域)を標的とする3580の異なるオリゴヌクレオチドプローブからなる系統学的マイクロアレイを使用して、盲腸の内容物から抽出したDNAを解析した。以前に報告されたようにMITチップの解析を実施した(Everard et al. Diabetes 60, 2775−2786 (2011);Geurts et al. Front Microbiol. 2, 149 (2011))。簡単に述べると、微生物叢の解析を、遺伝子様レベルに対応するレベル2で実施した。多変量解析を、99の細菌のグループ(レベル2)の平均シグナル強度に関して、CANOCO 4.5シグナルソフトウェアパッケージ(Biometris、オランダ ヴァーヘニンゲン)で実施されるように、representational difference 解析(RDA)により実施した。すべての環境変数を、log(1+X)として変形した。999ランダム置換に基づくモンテカルロ法の並べ替え検定を使用して有意性を試験した。P値<0.05を有意とみなした。
商業的なキット(DiaSys、フランス コンドン)を使用して、エステルコレステロールの酵素的加水分解の後に存在するコレステロールを測定することにより、血漿試料をコレステロールについて解析した。
データを平均値±標準誤差(sem)として示す。2つのグループ間の差異を、対応のない両側スチューデントt検定を使用して評価した。2超のグループを含むデータセットをANOVAにより評価し、その後ニューマン・コイル ポストホック検定により評価した。ピアソン相関を使用して相関を解析した。異なる上付き文字を有するデータは、post−hoc ANOVA統計解析により有意に異なっている(p<0.05)。データはウィンドウズ(登録商標)用のGraphPad Prismバージョン5.00(GraphPad Software、アメリカカリフォルニア州サンディエゴ)を使用して解析した。結果は、p<0.05の場合に統計的有意であるとみなした。
本出願人は、A.muciniphilaの存在量がレプチン欠損肥満マウスにおいて除脂肪の同腹のマウスに対し3300倍少なかったことを見出した(図1a)。一致して、本出願人は、高脂肪(HF)摂食マウスにおいてA.muciniphilaの存在量が100倍低下したことを見出した(図1c)。両方のモデルで、プレバイオティクスは完全にA.muciniphila数を回復させた(図1b、c)。HF摂食マウスでは、プレバイオティクスは代謝性内毒血症を無効にし(図1d)、脂肪組織におけるマクロファージの亜集合CD11cを正常化し(図1e)かつ体脂肪量を低下させた(図1gおよび図3b)。これらの結果は、A.muciniphilaと有意にかつ逆比例して相関した(図1fおよび図3a、c)。しかしながら、これらの障害の発症の根底にある分子機構がA.muciniphilaの欠損に依存するかどうか、および対照的に、プレバイオティクス処置後の改善がより高い濃度のA.muciniphilaからもたらされるものかどうかは、まだ示されなかった。
Claims (17)
- それを必要とする対象での代謝性障害を処置する際に使用するためのアッカーマンシア・ムシニフィラまたはその断片。
- 前記代謝性障害が肥満である、請求項1に記載の代謝性障害を処置する際に使用するためのアッカーマンシア・ムシニフィラまたはその断片。
- 前記代謝性障害がメタボリックシンドローム、インスリン欠損性またはインスリン抵抗性関連障害、真性糖尿病(たとえば2型糖尿病など)、耐糖能障害、脂質代謝異常、アテローム性動脈硬化、高血圧、心臓性病態、脳卒中、非アルコール性脂肪肝疾患、高血糖症、脂肪肝、脂質異常症、体重過剰および肥満に関連する免疫系の機能不全、循環器疾患、高コレステロール、トリグリセリドの増加、喘息、睡眠時無呼吸、変形性関節症、神経変性、胆嚢疾患、シンドロームX、炎症性障害および免疫性障害、アテローム性脂質異常症ならびに癌を含む群から選択される、請求項1に記載の代謝性障害を処置する際に使用するためのアッカーマンシア・ムシニフィラまたはその断片。
- 対象のエネルギー消費を増大させるためのアッカーマンシア・ムシニフィラまたはその断片であって、好ましくは前記対象の食物摂取量に影響を与えることのないアッカーマンシア・ムシニフィラまたはその断片。
- 対象での満腹度を増大させるためのアッカーマンシア・ムシニフィラまたはその断片。
- アッカーマンシア・ムシニフィラの生細胞を、それを必要とする前記対象に投与する、請求項1〜5のいずれか1項に記載の使用のためのアッカーマンシア・ムシニフィラまたはその断片。
- アッカーマンシア・ムシニフィラを経口投与する、請求項1〜6のいずれか1項に記載の使用のためのアッカーマンシア・ムシニフィラまたはその断片。
- 1×104〜約1×1012cfu、より好ましくは約1×105〜約1×1011cfu、およびさらにより好ましくは約1×106〜約1×1010cfuの範囲のアッカーマンシア・ムシニフィラの量を前記対象に投与する、請求項1〜7のいずれか1項に記載の使用のためのアッカーマンシア・ムシニフィラまたはその断片。
- アッカーマンシア・ムシニフィラを少なくとも1週間に3回投与する、請求項1〜8のいずれか1項に記載の使用のためのアッカーマンシア・ムシニフィラまたはその断片。
- アッカーマンシア・ムシニフィラを、別のプロバイオティクス株および/または1つ以上のプレバイオティクスと同時に投与する、請求項1〜9のいずれか1項に記載の使用のためのアッカーマンシア・ムシニフィラまたはその断片。
- 代謝性障害を処置し、または対象のエネルギー消費を増大させ、または対象の満腹度を増大させるために使用するための組成物であって、請求項1〜10のいずれか1項に記載のアッカーマンシア・ムシニフィラまたはその断片を、賦形剤を伴って含む、組成物。
- 前記組成物が栄養性組成物である、請求項11に記載の使用のための組成物。
- 前記組成物を経口投与する、請求項11または12に記載の使用のための組成物。
- 対象の代謝性障害を処置し、対象のエネルギー消費を増大させ、または対象での満腹度を増大させる医薬組成物であって、請求項1〜10のいずれか1項に記載のアッカーマンシア・ムシニフィラまたはその断片を薬学的に許容可能なビヒクルを伴って含む、組成物。
- 対象の代謝性障害を処置し、対象のエネルギー消費を増大させ、または対象の満腹度を増大させる薬物であって、請求項1〜10のいずれか1項に記載のアッカーマンシア・ムシニフィラまたはその断片を含む、薬物。
- それを必要とする対象の体重の減少を促進する、アッカーマンシア・ムシニフィラまたはその断片の使用。
- それを必要とする対象での体重の減少を促進する、アッカーマンシア・ムシニフィラまたはその断片を含む美容組成物。
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