JP2018534235A - 細菌株を含む組成物 - Google Patents
細菌株を含む組成物 Download PDFInfo
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- JP2018534235A JP2018534235A JP2018504969A JP2018504969A JP2018534235A JP 2018534235 A JP2018534235 A JP 2018534235A JP 2018504969 A JP2018504969 A JP 2018504969A JP 2018504969 A JP2018504969 A JP 2018504969A JP 2018534235 A JP2018534235 A JP 2018534235A
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Abstract
【選択図】なし
Description
本発明の組成物は、Blautia属の細菌株を含む。実施例は、この属の細菌が、内臓過敏を処置または防止するのに有用であることを実証している。好ましい細菌株は、Blautia hydrogenotrophica、Blautia stercoris及びBlautia wexlerae種のものである。本発明で使用される他の好ましい細菌株は、Blautia producta、Blautia coccoides及びBlautia hanseniiである。
好ましい実施形態において、本発明の組成物は、内臓過敏を処置する際の使用のためのものである。内臓過敏は、胸部、骨盤内、または腹部臓器(器官)の侵害受容器の活性化から結果として生じる、腹部領域に位置する、主観的に疼痛を伴う知覚によって特徴付けられる特定のタイプの疼痛である。内臓過敏は、概して拡散し、局所化が困難であるため、概してより鋭くかつより局所化している体性痛とは対照をなしている。また、内臓過敏は、体性痛とは違って、具体的な構造的病変とは概して関連しない。内臓侵害受容器は、皮膚及び大部分の他の非内臓侵害受容器とは本質的に異なる[22]。
好ましくは、本発明の組成物は、本発明の細菌株による腸への送達及び/または腸への部分もしくは全体定着を可能にするために胃腸管に投与されるべきである。概して、本発明の組成物は、経口で投与されるが、これらは、経直腸的に、鼻腔内に、または頬側もしくは舌下経路を介して投与されてよい。
概して、本発明の組成物は、細菌を含む。本発明の好ましい実施形態において、組成物は、凍結乾燥形態で調合される。例えば、本発明の組成物は、本発明の細菌株を含む顆粒またはゼラチンカプセル、例えば、硬質ゼラチンカプセルを含んでいてよい。
本発明における使用のための細菌株は、例えば、参照文献[32、33、34]に詳述されている標準の微生物学的技術を使用して培養され得る。
本発明の実施は、別途示さない限り、当業者の範囲内の、化学、生化学、分子生物、免疫学及び薬理学の従来の方法を用いる。かかる技術は、文献において完全に説明されている。例えば、参照文献[35]及び[36、37、38、39、40、41、42]などを参照されたい。
実施例1−内臓過敏のラットモデルにおける細菌接種の効能
概要
ラットに、内臓過敏を示すヒトIBS対象からの糞便微生物叢を接種した。次いで、ラットに、本発明による細菌株を含む組成物を投与し、次いで、膨張アッセイを使用して試験して、内臓過敏を測定した。本発明の組成物は、膨張に対するラットの応答を低減することが分かり、これは内臓過敏の低減を示唆した。
Blautia hydrogenotrophica(BH)株DSM10507/14294。
BH培養物(16H)または凍結乾燥物−経口胃管栄養法によって投与
対照溶液を経口胃管栄養法によって投与
IBS対象からのヒト腸微生物叢を接種した。
14日目−ラットにIBS対象からのヒト腸微生物叢を接種した
0〜28日目−BH培養物もしくは凍結乾燥物、または対照溶液を連日投薬
0、14及び28日目−糞便サンプル中のBH集団のqPCR
14日目と28日目との間−腹部内に電極を埋め込む操作(膨張アッセイ用)
28日目−膨張アッセイ、盲腸サンプルを硫化物及び短鎖脂肪酸(SCFA)分析用に収集し、選択培地における糞便サンプル中の微生物叢を計数した
図1は、対照溶液(IBS)またはBH凍結乾燥物(IBS+BH)を投与したラットからの糞便サンプル中のBH集団のqPCR分析の結果を表す。BH集団の増加は、ラットにBH凍結乾燥物が与えられた14及び28日目に見られ、成功裏の定着が確認された。
Blautia hydrogenotrophicaを含む組成物の投与は、膨張アッセイを使用して測定される、成功裏の定着及び内臓過敏の顕著な低減をもたらした。この効果は、Blautia hydrogenotrophicaを培養物として及び凍結乾燥物として投与したときに観察された。Blautia hydrogenotrophicaの投与もまた、SRB及び硫化物産生の低減が観察されつつも、微生物叢の構成及び発酵に顕著な効果があった。これらのデータは、Blautia hydrogenotrophicaが、内臓過敏、特にIBSに関連する内臓過敏を低減するのに有用であり得ることを示唆している。内臓過敏の低減は、観察されるSRB及び硫化物産生の低減と関連し得る。
実施例1の観察を、Blautia hydrogenotrophica(BH)株DSM10507/14294の凍結乾燥物及びIBSのラットモデルを使用してさらなる実験において確認した。図7及び8に示すように、BH凍結乾燥物の投与は、膨張に応答した腹部収縮の数の統計的に有意な低減を付与し、内臓過敏の低減を示唆した。図7において、1つのIBS対象からの糞便サンプルを使用してラットに接種した。図8において、3つのIBS対象からの糞便サンプルを使用してラットに接種し、これらのIBS対象の1つは、図7において使用したIBS対象と同じであった。さらに、図9及び10に示すように、BH凍結乾燥物の投与は、硫化物の統計的に有意な低減を付与した。図9において、1つのIBS対象からの糞便サンプルを使用してラットに接種した。図10において、3つのIBS対象からの糞便サンプルを使用してラットに接種し、これらのIBS対象の1つは、図9において使用したIBS対象と同じであった。
健常なHIMラットに対するBlautia hydrogenotrophica(BH)株DSM10507/14294の凍結乾燥物の投与の効果を研究した。結果を図11〜14に報告する。実験に関するさらなる詳細は図の説明において上記に付与している。図11は、ラットにおけるBHの適切な用量が109細胞/日以上であることを示している。図12は、これらの実験においてBHがラット消化管に永久には定着しなかったことを示している。図13は、BHが盲腸において主に見られることを示している。図14は、BHの投与が酢酸及び酪酸産生の増加を誘発することを示している。
IBSのラットモデルに対するBlautia hydrogenotrophica(BH)株DSM10507/14294の凍結乾燥物の投与の効果をさらに調査した。無菌ラットにC−IBS(便秘あり)またはU−IBS(非亜型)患者からの糞便サンプルを接種した。実験の大部分を、内臓過敏(圧調節器によって測定したVH)を示しているIBS患者からの糞便サンプルを用いて実施した。結果は図15及び16に報告されており、実験に関するさらなる詳細は図の説明において上記に付与している。図15において、BH凍結乾燥物の投与が硫酸塩還元細菌の統計的に有意な低減を引き起こすことが確認されている。予期されるように、BHの増加もまた観察される。図16は、BH投与が、IBS HIMラットによって産生されるH2Sの量の統計的に有意な減少を誘発したことを示している。腸内微生物叢による盲腸でのH2Sの過剰産生は、内臓過敏に関連する。
Blautia hydrogenotrophica(受託番号DSM10507及び受託番号DSM14294でも寄託されている株「Blautix」)を、過敏性腸症候群(IBS)を有するヒト患者に投与する第一相臨床試験を行った。患者にBlautixを投与期間(1〜16日目)に投与し、ウオッシュアウト期間が19〜23日目であった。Blautixは、安全かつ耐容性良好の両方であることが分かった。4つの症状をモニタリングし、そのうちの1つが腹部疼痛であった。研究において、患者がこれらの症状の改善を経験したか、変化が無かったか、または悪化したかを記録した。Blautixが投与された患者からの結果を、プラセボが投与された患者を使用して得たものと比較した。症状を3つの時点:研究の1日目、15/16日目、及び最後;でモニタニングした。結果を図17及び18に示す。
概要
16匹の無菌ラット群(対照群における8匹のラットと処置群における8匹のラットとを含む)にヒトIBS対象からの糞便微生物叢を接種した(IBS−HMAラット)。3つの連続実験を3の異なるIBS患者からの糞便サンプルを使用して実施した。2つの他のラット群(n=10)に、健常な対象(n=2対象;2つの健常なHMAラット群)の糞便サンプルを内臓感受性対照として接種した。こうして、24のIBS−微生物叢関連ラット(対照)、Blautixで処置した24のIBS微生物叢関連ラット、及び20の健常な微生物叢関連ラットとした。IBS−HMAラットの半分に、本発明によるB.hydrogenotrophicaの細菌株を含む組成物を28日間投与しながら、他の半分の動物には対照溶液を与えた。投与の28日後、全てのHMAラットを、結腸膨張アッセイを使用して試験し、内臓感受性を測定した。本発明の組成物は、膨張に対するIBS−HMAラットの応答を低減することが見出され、健常な−HMAラットにおいて観察されたように正常な感度に達した内臓過敏の低減を示唆した。
Blautia hydrogenotrophica(BH)株DSM10507T/14294。
BH凍結乾燥物を1010細菌/mlの濃度で無菌鉱液に懸濁した。2mlのこの懸濁液を経口胃管栄養法によって28日間、IBS−HMAラットごとに連日投与した。
無菌雌Fisherラット(10週齢)にIBS対象(IBS−HMAラット)からのヒト糞便微生物叢を接種した。16匹のラットに同じヒト糞便接種材料を接種した。3つの連続実験を3つの異なるIBS対象からの糞便サンプルによって実施した。10匹のラットのうちの2つの他の群に2の健常な対象(正常な感度の対照群)からの糞便サンプルを接種した。
14日目−ヒト糞便微生物叢を無菌ラットに接種。
0〜28日目−経口胃管栄養法によりBH凍結乾燥物(アッセイ群)または対照溶液(対照群)を連日投薬
14日目と22日目との間−腹部内に電極を埋め込む操作(膨張アッセイ用)
22〜28日目−ラットを適応させて膨張試験に関連するストレスを回避する。
28日目−膨張アッセイ、ならびに動物の安楽死により硫化物及び短鎖脂肪酸(SCFA)分析用の盲腸サンプルを収集する。
0、14及び28日目−微生物の分析用の糞便サンプルの収集:BH集団及び他の片利共生微生物群の評価のためのqPCR、ならびに選択培地及び偏性嫌気性方法を使用した微生物の機能群の計数。
図19は、対照溶液またはBH凍結乾燥物が与えられたIBS−HMAラットからの糞便サンプルのB.hydrogenotrophica集団のqPCR分析の結果を表す。BH凍結乾燥物が与えられたラットにおいて投与期間の最後(28日目)にBH集団の有意な増加が観察され、結腸におけるBHの成功裏の送達を確認した。
Blautia hydrogenotrophicaを含む組成物の投与は、膨張アッセイを使用して測定される内臓過敏の有意な低減をもたらした。処置後、IBS−HMAラットの内臓感受性は、健常な−HMAラットにおいて測定したものと同様であることが見出された。B.hydrogenotrophicaを含む組成物の投与は、正常なものへのIBS−HMA動物の内臓感受性を復元することができる。Blautia hydrogenotrophicaの投与もまた、微生物叢の構成及び発酵に対する有意な効果があり、SRB及び硫化物産生の重要な低減を特に誘発した。これらのデータは、Blautia hydrogenotrophicaが、内臓過敏、特にIBSに関連する内臓過敏を低減するのに有用であり得ることを示唆している。内臓過敏の低減は、観察されるSRB及び硫化物産生の低減と関連し得る。
本明細書に記載されている少なくとも1つの細菌株を含有する本明細書に記載されている組成物を、25℃または4℃の密閉容器に貯蔵し、該容器を30%、40%、50%、60%、70%、75%、80%、90%または95%の相対湿度を有する雰囲気に置く。1ヶ月、2ヶ月、3ヶ月、6ヶ月、1年、1.5年、2年、2.5年または3年後、標準のプロトコルによって決定されるコロニー形成単位で測定したとき、細菌株の少なくとも50%、60%、70%、80%または90%が残存する。
配列番号1(Blautia stercoris株GAM6−1 16SリボソームRNA遺伝子、部分配列−HM626177)
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Claims (31)
- 内臓過敏を処置または防止する方法における使用のための、Blautia属の細菌株を含む組成物。
- 前記内臓過敏が、IBS、クローン病、潰瘍性結腸炎、機能性消化不良または乳児疝痛に関連する、請求項1に記載の組成物。
- 前記内臓過敏が、IBS、クローン病、潰瘍性結腸炎または機能性消化不良に関連する、請求項2に記載の組成物。
- IBS、クローン病、潰瘍性結腸炎、機能性消化不良または乳児疝痛と診断された対象において内臓過敏を処置または防止する際に使用するためのものである、請求項1に記載の組成物。
- IBS、クローン病、潰瘍性結腸炎または機能性消化不良と診断された対象において内臓過敏を処置または防止する際に使用するためのものである、請求項4に記載の組成物。
- 内臓過敏の処置または防止において、硫酸塩還元細菌(SRB)による胃腸管への定着を低減する際の使用のためのものである、請求項1〜5のいずれかに記載の組成物。
- 内臓過敏の処置または防止において、胃腸管におけるH2Sレベルを低下させる、またはH2Sレベルの上昇を防止する際の使用のためのものである、請求項1〜6のいずれかに記載の組成物。
- 胃腸管、特に結腸または直腸の疼痛を伴う膨張に苦しむ患者において内臓過敏を処置または防止する際の使用のためのものである、請求項1〜7のいずれかに記載の組成物。
- 前記細菌株が、Blautia hydrogenotrophicaの細菌株である、請求項1〜8のいずれかに記載の組成物。
- 前記細菌株が、Blautia stercorisの細菌株である、請求項1〜8のいずれかに記載の組成物。
- 前記細菌株が、Blautia wexleraeの細菌株である、請求項1〜8のいずれかに記載の組成物。
- 前記細菌株が、Blautia hydrogenotrophicaの細菌株の16s rRNA配列と少なくとも95%、96%、97%、98%、99%、99.5%または99.9%同一である16s rRNA配列を有する、請求項1〜8のいずれかに記載の組成物。
- 前記細菌株が、配列番号5と少なくとも97%、98%、99%、99.5%もしくは99.9%同一である16s rRNA配列を有する、または配列番号5の16s rRNA配列を有する、請求項12に記載の組成物。
- 前記細菌株が、Blautia stercorisの細菌株の16s rRNA配列と少なくとも95%、96%、97%、98%、99%、99.5%または99.9%同一である16s rRNA配列を有する、請求項1〜8のいずれかに記載の組成物。
- 前記細菌株が、Blautia wexlerae の細菌株の16s rRNA配列と少なくとも95%、96%、97%、98%、99%、99.5%または99.9%同一である16s rRNA配列を有する、請求項1〜8のいずれかに記載の組成物。
- IBSと診断された対象において内臓過敏を処置または防止する方法における使用のための、Blautia hydrogenotrophica種の細菌株を含む、請求項1に記載の組成物。
- 内臓過敏の処置または防止において、硫酸塩還元細菌(SRB)による胃腸管への定着を低減する、胃腸管におけるH2Sレベルを低下させる、またはH2Sレベルの上昇を防止する際の使用のための、Blautia hydrogenotrophica種の細菌株を含む、請求項1に記載の組成物。
- IBSと診断された対象において内臓過敏を処置または防止する方法における使用のための、Blautia stercoris種の細菌株を含む、請求項1に記載の組成物。
- 内臓過敏の処置または防止において、硫酸塩還元細菌(SRB)による胃腸管への定着を低減する、胃腸管におけるH2Sレベルを低下させる、またはH2Sレベルの上昇を防止する際の使用のための、Blautia stercoris種の細菌株を含む、請求項1に記載の組成物。
- IBSと診断された対象において内臓過敏を処置または防止する方法における使用のための、Blautia wexlerae種の細菌株を含む、請求項1に記載の組成物。
- 内臓過敏の処置または防止において、硫酸塩還元細菌(SRB)による胃腸管への定着を低減する、胃腸管におけるH2Sレベルを低下させる、またはH2Sレベルの上昇を防止する際の使用のための、Blautia wexlerae種の細菌株を含む、請求項1に記載の組成物。
- 経口投与用である、請求項1〜21のいずれかに記載の組成物。
- 1以上の薬学的に許容可能な賦形剤または担体を含む、請求項1〜22のいずれかに記載の組成物。
- 前記細菌株が、凍結乾燥されている、請求項1〜23のいずれかに記載の組成物。
- 前記細菌株が、生存可能である、請求項1〜24のいずれかに記載の組成物。
- Blautia属の単一株を含む、請求項1〜25のいずれかに記載の組成物。
- Blautia属の細菌株を微生物共同体の部分として含む、請求項1〜26のいずれかに記載の組成物。
- 請求項1〜27のいずれかに記載の使用のための、請求項1〜27のいずれかに記載の組成物を含む食品。
- 請求項1〜27のいずれかに記載の使用のための、請求項1〜27のいずれかに記載の組成物を含むワクチン組成物。
- 内臓過敏を処置または防止する方法であって、Blautia属の細菌株を含む組成物を、それを必要とする患者に投与することを含む、前記方法。
- Blautiaが、Blautia hydrogenotrophicaである、請求項30に記載の方法。
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