JP2021519763A - 炎症性腸疾患を治療するための組成物及び方法 - Google Patents
炎症性腸疾患を治療するための組成物及び方法 Download PDFInfo
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Abstract
Description
本出願と共に提出された、ASCIIテキストファイルで電子的に送信された配列表の内容(名称:4268.014PC01_SequenceListing_ST25.txt;サイズ:8,737バイト;作成日:2019年3月29日)は、その全体が参照により本明細書に組み込まれる。
本開示は、精製された細菌集団を含む組成物を対象とし、ここで、精製された細菌集団は、Flavonifractor_SC49、Clostridium leptum、またはそれらの組み合わせを含み、組成物は、対象に投与されたときに二次胆汁酸のレベルを調節することができる。特定の実施形態では、精製された細菌集団は、Flavonifractor_SC49を含む。他の実施形態では、精製された細菌集団は、Clostridium leptumを含む。さらなる実施形態では、精製された細菌集団は、Flavonifractor_SC49とClostridium leptumの両方を含む。
本明細書に記載されているのは、対象における1つまたは複数の胆汁酸のレベルを調節する能力を有するヒト胃腸管微生物叢の細菌及び細菌の組み合わせである。いくつかの実施形態では、胆汁酸レベルの調節は、胃腸障害、例えば、炎症性腸疾患(例えば、潰瘍性大腸炎またはクローン病)に関連する1つまたは複数の徴候または症状を治療、予防、遅延、または改善することができる。特定のメカニズムに限定されることなく、特定の胆汁酸の増加及び/または減少は、活性化された細胞(例えば、単球、PBMC、または腸上皮細胞)によって産生される炎症誘発性メディエーター(例えば、TNF−αまたはIL−8)の量を減らすことができると考えられている。いくつかの実施形態では、特定の胆汁酸の増加及び/または減少は、活性化された細胞によって産生される抗炎症メディエーター(例えば、IL−10)の量を増加させることができる。
本明細書でさらに提供されるのは、製剤を必要とするヒト及び他の対象(例えば、IBD患者、例えば、潰瘍性大腸炎患者)に投与するための製剤である。一般に、本明細書に記載の細菌組成物は、追加の活性物質及び/または最終製品を製造するための不活性材料と組み合わされ、これは、単回投与単位または複数回投与形式にすることができる。
本明細書に開示される製剤は、例えば、疾患の1つまたは複数の徴候または症状を改善すること(例えば、寛解を誘導すること)によって、及び/または活動性疾患の再発を減らすため(例えば、寛解を維持するため)に、炎症性腸疾患(IBD)(例えば、潰瘍性大腸炎またはクローン病)の治療に使用することができる。
炎症性腸疾患(IBD)における胆汁酸の役割を理解し始めるために、7α−デヒドロキシラーゼ経路に関連する二次胆汁酸のレベル(すなわち、DCA、LCA、3−オキソLCA、3α 12−オキソ−デオキシコール酸、及び3β 12α−デオキシコール酸)は、様々な治療レジメンを受けた潰瘍性大腸炎患者から得られた糞便試料で定量化された。治療レジメンは、以下のうちの1つを含んだ:(A)プラセボのみ;(B)プラセボに続いて、健康なヒトの糞便に由来する胞子集団(「HHSP」)の毎週の投与;(C)バンコマイシンとそれに続くHHSPの毎週の投与;(D)バンコマイシンとそれに続くHHSPの毎日の投与。胆汁酸濃度は、4つの異なる時点で測定された:(1)ベースライン(すなわち、バンコマイシンまたはHHSPの投与前)(「来院1」);(2)バンコマイシン治療直後(必要な場合)(「来院4」);(3)HHSPの投与開始から2週間後(必要な場合)(「来院6」);(4)胞子形成画分の投与を開始してから8週間後(必要な場合)(「来院12」)。
ヒトの糞便試料を等分し、秤量し、10倍(w/v)の抽出緩衝液中に均質化した(水中50%メタノール)。試料を氷上で1時間抽出した後、等量の冷アセトニトリルでさらに抽出した。次いで、抽出物を遠心分離し、液体クロマトグラフィー−タンデム質量分析(LC−MS)により分析する前に、上清を0.22μmフィルターで濾過した。ラベル付けされた胆汁酸標準は、代謝物の抽出と分析の品質管理を提供するために、抽出の前後にスパイクされた。各試料のアリコートも秤量し、次いで乾燥させて試料の含水量と乾燥重量を決定した。
胆汁酸は、0.2μmプレカラムフィルターが先行するMicrosolv二座C18カラムを備えたAgilent 1260HPLCを使用して分離した。分離は、0.1%ギ酸を含む水とアセトニトリルの勾配を流速0.4ml/分で使用して行った。試料は5μLの量で注入された。HPLCシステムは、Agilentの低質量チューニングミックスを使用して50〜1700m/zの質量範囲に校正されたBrukerCompass(商標)qTOF質量分析計に結合された。各分析は、各分析の開始時に注入された参照質量溶液に対してさらに較正された。胆汁酸はネガティブモードで検出され、既知の純粋な標準と比較した固有のm/z及び保持時間によって同定された。ピーク下面積は、Brukerデータ分析ソフトウェアを使用して決定された。胆汁酸は、0.001μM〜100μMの範囲の濃度の純粋な標準から生成された検量線を使用して定量化された。LC−MSで検出された胆汁酸を以下の表1に列挙する。
胆汁酸レベルと疾患の寛解との間の潜在的な関係を評価するために、寛解している潰瘍性大腸炎患者(「寛解者」)と活動性疾患を患っている患者(「非寛解者」)の間で異なる胆汁酸のレベルを比較した。潰瘍性大腸炎患者は、実施例1に記載された治療レジメンの1つを受けていた。胆汁酸は、糞便試料から抽出され、上述の実施例1に記載されたように定量化された。
特定の二次胆汁酸のレベルの上昇と潰瘍性大腸炎の寛解との関係の可能性をよりよく理解するために、単球とPBMCを、抱合型と非抱合型の様々な濃度の異なる一次胆汁酸と二次胆汁酸の存在下でLPSにより活性化した。活性化細胞が産生するTNF−α及びIL−10の量を以下のように測定した。
ヒトのバフィーコートはBioreclamationから入手し、氷上で一晩輸送した。バフィーコートをPBSで1:1に希釈し、50mLファルコンチューブ内のFicoll−Paque(GE Healthcareカタログ番号17−1440−03)の上部に重ねた。試料は、ブレーキなしで室温で20分間500×gで回転させた。PBMCをパーコール勾配層で吸引し、PBSで3回洗浄した。血球計算盤で細胞を計数し、細胞の生存率と濃度を決定した。細胞を96ウェルプレートに播種し、37℃、5%CO2で1時間インキュベートした。これに続いて、LPS(1ng/mL)の添加前に、胆汁酸を最終濃度12.5μM、25μM、及び50μMで1時間添加した。16〜20時間のインキュベーション後、サイトカイン分析のために細胞培養培地を収集した。Milliplex Human Cytokineキット(Luminex;Millipore)を使用してサイトカインをアッセイし、IL−10及びTNF−αをアッセイした。Miltenyi Biotec Pan単球分離キット(カタログ番号:130−096−537)を製造元の指示に従って使用して、PBMCから単球を分離し、上述のようにアッセイした。
IBDは主に腸組織に影響を与えるため、単球及びPBMCで観察された抗炎症効果が腸上皮細胞(HT29)でも当てはまるかどうかを以下のように評価した。
10%FBS、GlutaMAX、及びPen/Strepを補充したマッコイズ培地で培養したHT29細胞を、96ウェルフォーマット中に5万細胞/ウェルの密度で播種し、完全にコンフルエントになるまで5日間増殖させた。培地は2日ごとに交換した。5日目に、細胞を250μM、125μM、または62.5μMの胆汁酸化合物で1時間前処理した後、1.25ng/mLのTNF−αに曝露した。細胞を一晩(16時間)インキュベートし、培養上清をELISAによるIL−8タンパク質の定量化のために収集した。試験試料のIL−8レベルは、1.25ng/mLのTNF−αに曝露された炎症性対照、DMSO前処理試料(すなわち、胆汁酸なし)に対して正規化された。
上述の実施例で説明した抗炎症効果に関連する胆汁酸の増加の原因となる可能性のある細菌を特定するために、様々な潰瘍性大腸炎患者(すなわち、実施例1に記載される治療レジメンのうちの1つを受けた寛解者と非寛解者の両方)からの糞便試料を、Flavonifractor_SC49及びClostridium leptumの存在に基づいて分割した。次いで、実施例1で前述したように、7α−デヒドロキシラーゼ経路に関連する二次胆汁酸(すなわち、DCA、3α 12−オキソ−デオキシコール酸、3β 12α−デオキシコール酸(3−イソデオキシコール酸)、LCA、及び3−オキソ−LCA)の量を測定した。
Claims (22)
- 精製された細菌集団を含む組成物であって、前記精製された細菌集団が、Flavonifractor_SC49、Clostridium leptum、またはそれらの組み合わせを含み、対象に投与されたときに二次胆汁酸のレベルを調節することができる、前記組成物。
- 前記精製された細菌集団が、Flavonifractor_SC49を含む、請求項1に記載の組成物。
- 前記精製された細菌集団が、Clostridium leptumを含む、請求項1に記載の組成物。
- 前記精製された細菌集団が、Flavonifractor_SC49とClostridium leptumの両方を含む、請求項1に記載の組成物。
- 前記Flavonifractor_SC49が、参照Flavonifractor_SC49 OTUの16S rDNA配列(配列番号1、3、または4)に対し、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、または100%同一である、16S rDNA配列を含む、請求項1、2、及び4のいずれか1項に記載の組成物。
- 前記Clostridium leptumが、参照Clostridium leptum OTUの16S rDNA配列(配列番号2)に対し、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、または100%同一である、16S rDNA配列を含む、請求項1、及び3〜5のいずれか1項に記載の組成物。
- 前記二次胆汁酸が、デオキシコール酸(DCA)、3α 12−オキソ−デオキシコール酸、3β 12α−デオキシコール酸(3−イソデオキシコール酸)、7α 3−オキソ−ケノデオキシコール酸、リトコール酸(LCA)、3−オキソLCA、またはそれらの組み合わせを含む、請求項1〜6のいずれか1項に記載の組成物。
- 前記二次胆汁酸が、ウルソデオキシコール酸(UDCA)を含む、請求項1〜6のいずれか1項に記載の組成物。
- 二次胆汁酸のレベルを調節することを必要とする対象における二次胆汁酸のレベルを調節する方法であって、有効量の請求項1〜8のいずれか1項に記載の組成物を前記対象に投与することを含む、前記方法。
- 炎症性腸疾患(IBD)の1つもしくは複数の徴候もしくは症状を改善するか、またはそれを必要とする対象においてIBDの寛解を維持する方法であって、有効量の請求項1〜8のいずれか1項に記載の組成物を前記対象に投与することを含む、前記方法。
- 前記二次胆汁酸が、デオキシコール酸(DCA)、3α 12−オキソ−デオキシコール酸、3β 12α−デオキシコール酸(3−イソデオキシコール酸)、7α 3−オキソ−ケノデオキシコール酸、リトコール酸(LCA)、3−オキソLCA、またはそれらの組み合わせを含む、請求項9または10に記載の方法。
- 前記投与が前記対象における前記二次胆汁酸のレベルを増加させる、請求項11に記載の方法。
- 前記二次胆汁酸のレベルが、参照(例えば、前記組成物を投与されなかった対象の対応するレベル)と比較して、前記対象において、少なくとも10%、少なくとも20%、少なくとも30%、少なくとも40%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または、少なくとも90%増加する、請求項12に記載の方法。
- 前記二次胆汁酸のレベルの増加が、前記IBDの寛解と相関している、請求項12または13に記載の方法。
- 前記二次胆汁酸が、in vitroでリポ多糖(LPS)により刺激された単球における、TNF−αの産生を減少させることができ、及び/またはIL−10の産生を増加させることができる、請求項11〜14のいずれか1項に記載の方法。
- 前記二次胆汁酸が、in vitroでLPSにより刺激された末梢血単核球(PBMC)における、TNF−αの産生を減少させることができ、及び/またはIL−10の産生を増加させることができる、請求項11〜15のいずれか1項に記載の方法。
- 前記二次胆汁酸が、in vitroでTNFαにより刺激された腸上皮細胞におけるIL−8の産生を減少させることができる、請求項11〜16のいずれか1項に記載の方法。
- 前記二次胆汁酸が、ウルソデオキシコール酸(UDCA)を含む、請求項9または10に記載の方法。
- 前記投与が前記対象におけるUDCAのレベルを低下させる、請求項18に記載の方法。
- 前記UDCAのレベルが、参照(例えば、前記組成物を投与されなかった対象の対応するレベル)と比較して、前記対象において、少なくとも10%、少なくとも20%、少なくとも30%、少なくとも40%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または、少なくとも90%低下する、請求項19に記載の方法。
- 前記UDCAのレベルの低下が、前記IBDの寛解と相関している、請求項19または20に記載の方法。
- 前記IBDが、潰瘍性大腸炎またはクローン病である、請求項10〜21のいずれか1項に記載の方法。
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JP2013147469A (ja) * | 2012-01-20 | 2013-08-01 | Calpis Co Ltd | 腸内酪酸産生菌増加剤 |
JP2016509002A (ja) * | 2013-02-04 | 2016-03-24 | セレス セラピューティクス インコーポレイテッド | 病原性細菌生育の抑制のための組成物および方法 |
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CA3095402A1 (en) | 2019-10-03 |
EP3773643A1 (en) | 2021-02-17 |
RU2020135433A (ru) | 2022-04-29 |
MX2020010177A (es) | 2021-01-15 |
CN112118852A (zh) | 2020-12-22 |
KR20200138333A (ko) | 2020-12-09 |
AU2019245430A1 (en) | 2020-10-22 |
WO2019191694A1 (en) | 2019-10-03 |
BR112020019979A2 (pt) | 2021-03-09 |
US20210121505A1 (en) | 2021-04-29 |
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