JP7421504B2 - 胃腸のマイクロバイオームの検出および操作のためのデバイスおよびシステム - Google Patents
胃腸のマイクロバイオームの検出および操作のためのデバイスおよびシステム Download PDFInfo
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- JP7421504B2 JP7421504B2 JP2020566919A JP2020566919A JP7421504B2 JP 7421504 B2 JP7421504 B2 JP 7421504B2 JP 2020566919 A JP2020566919 A JP 2020566919A JP 2020566919 A JP2020566919 A JP 2020566919A JP 7421504 B2 JP7421504 B2 JP 7421504B2
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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- A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
- A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Description
本出願は、2018年6月1日に出願された米国仮出願第62/679,659号、2018年10月26日に出願された米国仮出願第62/751,209号、および2019年4月4日に出願された米国仮出願第62/829,225号の利益を主張し、それらの各々は、それらの全体が参照により本明細書に組み込まれる。
本出願は、参照により、以下の同時係属中の米国特許出願を組み込んでいる:「Ingestible Medical Device」と題され、かつ2014年8月15日に出願されたUSSN14/460,893、「Electromechanical Pill Device with Localization Capabilities」と題され、かつ2017年3月24日に出願されたUSSN15/514,413、「Systems and Methods for Obtaining Samples using Ingestible Devices」と題され、かつ2017年8月18日に出願されたUSSN15/680,400、「Sampling Systems and Related Materials and Methods」と題され、かつ2017年8月18日に出願されたUSSN15/680,430、「Electromechanical Ingestible Delivery of a Dispensable Substance」と題され、かつ2017年9月8日に出願されたUSSN15/699,848、「Gastrointestinal Tract Detection Methods,Device and Systems」と題され、かつ2017年12月7日に出願されたUSSN15/835,270、「Gastrointestinal Tract Detection Methods,Device and Systems」と題され、かつ2017年12月7日に出願されたUSSN15/835,237、「Gastrointestinal Tract Detection Methods,Device and Systems」と題され、かつ2017年12月7日に出願されたUSSN15/835,292、「Ingestible Device and Associated Methods」と題され、かつ2017年12月15日に出願されたUSSN15/844,349、「Ingestible Device and Associated Methods」と題され、かつ2017年12月15日に出願されたUSSN15/844,381、「Ingestible Device and Associated Methods」と題され、かつ2017年12月15日に出願されたUSSN15/844,427、「Systems and Methods for Extracting a Sample from an Ingestible Device」と題され、かつ2018年3月15日に出願された15/694,458、「Localization Systems and Methods for an Optoelectromechanical Pill Device」と題され、かつ2018年3月29日に出願されたUSSN15/940,407、および「Ingestible Device With Relatively Large Payload Volume」と題され、かつ2018年3月13日に出願されたUSSN62/642,544。
抗菌剤を含む有効量の医薬製剤を対象に経口投与し、それにより、対象のSIBO、SIBO関連状態、またはSIBOを示唆する症状を治療することを含み、
抗菌剤が、メロペネム、セフトリアキソン、エルタペネム、およびピペラシリン-タゾバクタムからなる群から選択される、方法を提供する。
本明細書で別段の定義がない限り、本開示で使用される科学的および技術的用語は、当業者によって一般的に理解される意味を有するものとする。一般に、本明細書に記載の、化学、細胞および組織培養、分子生物学、細胞および癌生物学、神経生物学、神経化学、ウイルス学、免疫学、微生物学、薬理学、遺伝学およびタンパク質および核酸化学に関連して使用される命名法およびそれらの技術は、当技術分野で周知であり、一般的に使用されているものである。
本明細書に記載のデバイスおよび方法は、対象のGI障害(GID)の診断および/または治療に使用され得る。いくつかの実施形態では、GI障害の症状を有するまたは提示する対象を診断することができ、および/または本明細書に記載の方法を使用して、対象の治療過程を決定することができる。いくつかの実施形態では、治療過程は、抗菌剤を含む有効量の医薬製剤を対象に経口投与して、それにより、対象のGI障害またはGI障害に関連する状態を治療することを含む。
いくつかの実施形態では、対象は、機能性腸疾患(FBD)の症状を有するか、または示している。FBDは、ローマIVの診断基準に従って、次の機能カテゴリに分類される:過敏性腸症候群(IBS)、機能性便秘(FC)、機能性下痢(FDr)、機能性腹部膨満/膨張、不特定の機能性便秘、およびオピオイド誘発性便秘(OIC)(例えば、その各々の内容全体が、参照により本明細書に組み込まれる、Lacy et al.(2016)Gastroenterology 150:1393-1407、およびDrossman et al.(2016)Gastroenterology 150:1262-79を参照)。機能性腸疾患の症状には、腹痛、腹部膨満、膨張、および/または腸機能障害(例えば、便秘、下痢、または便秘と下痢の混合)が含まれるが、これらに限定されない。いくつかの実施形態では、FBDを有する対象はまた、障害のあるライフスタイルまたは社会的混乱に苦しんでいる。
過敏性腸症候群(C1)の診断基準(症状の発症は、診断の少なくとも6か月前に発生し、症状は、診断前の3か月間に存在する必要がある):
●次のうちの2つ以上と関連付けられる、平均して、過去3か月間に週に少なくとも1日の反復性腹痛、
-排便に関連する
-便の頻度の変化と関連付けられる
-便の形(外観)の変化と関連付けられる。
●対象は、次のうちの2つ以上を有している必要がある、
-排便の25%を超える間の緊張
-排便の少なくとも25%でゴツゴツした、または硬い便
-排便の25%を超える不完全な排便の感覚
-排便の25%を超える肛門直腸閉塞/遮断の感覚
-排便の25%を超えるものを促進するための手動操作(例えば、デジタル排便、および骨盤底のサポート)
-週に3回未満の自然な便通
●下剤を使用しないと、緩い便が現れることはめったにない、
●IBSの基準が不十分。
●緩いまたは水っぽい便、主な腹痛または厄介な膨満感がなく、便の25%を超えるもので発生する(下痢型過敏性腸症候群の基準を満たす患者を除く)。
●対象は、次のうちの両方を有している必要がある、
-再発性の膨満および/または膨張が平均して週に少なくとも1日発生し、他の症状よりも腹部膨満および/または膨張が優勢である(軽度の膨満関連の痛みおよび軽度の排便異常が存在する場合がある)
-IBS、機能性便秘、機能性下痢、または食後苦痛症候群の診断のための不十分な基準。
●IBSまたは機能性便秘の基準を満たさない器質的病因に起因しない腸症状;下痢、または腹部膨満/膨張障害。
●オピオイド療法を開始、変更、または増加する際の便秘の新しい、または悪化する症状であって、これには、次のうちの2つ以上が含まれている必要がある、
-排便の25%を超える間の緊張
-排便の25%を超えるゴツゴツした、または硬い便
-排便の25%を超える不完全な排便の感覚
-排便の25%を超える肛門直腸閉塞/遮断の感覚
-排便の25%を超えるものを促進するための手動操作(例えば、デジタル排便、骨盤底のサポート)
-週に3回未満の自然な排便
●下剤を使用しないと、緩い便が現れることはめったにない。
主な便秘を伴うIBS(IBS-C):
●ブリストルスツールフォームタイプ1または2での(ブリストルスツールフォームスケール(BSFS)を使用して決定される)25%を超える便通、およびブリストルスツールフォームタイプ6または7での25%未満の便通
●あるいは、対象は、異常な便通は通常便秘であると報告している(ブリストルスツールフォームタイプ1または2)
主な下痢を伴うIBS(IBS-D):
●ブリストルスツールフォームタイプ6または7での25%を超える便通、およびブリストルスツールフォームタイプ1または2での25%未満の便通
●あるいは、対象は、異常な便通は通常下痢であると報告している(ブリストルスツールフォームタイプ6または7)
混合排便習慣のあるIBS(IBS-M):
●ブリストルスツールフォームタイプ1または2での25%を超える便通、およびブリストルスツールフォームタイプ6または7での25%を超える便通
●あるいは、対象は、異常な便通は通常便秘および下痢であると対象は報告している(25%を超える便通は便秘であり(ブリストルスツールフォームタイプ1または2)、および25%を超える便通は下痢であった(ブリストルスツールフォームタイプ6または7))
未分類のIBS:
●対象は、IBSの診断基準を満たしているが、排便習慣をIBS-C、IBS-D、またはIBS-Mのうちの1つに正確に分類できない
いくつかの実施形態では、対象は、GI管の慢性炎症性自己免疫状態である「炎症性腸疾患」または「IBD」の症状を有するか、または示している。IBDの原因は不明なままであるが、遺伝的、感染性、および免疫学的感受性などのいくつかの要因が関与している。IBDは、白人、特にユダヤ人の子孫でかなり一般的である。
特定の実施形態では、対象は、小腸細菌異常増殖(SIBO)の症状を有するか、または示している。小腸は、健康な状態で103未満のバクテリア/mLを収容する。腸内細菌叢の恒常性が破壊されたり異常になったりすると、腸内細菌叢の様々な機能が制御されなくなる。例えば、各々が参照により組み込まれる、Shreiner et al.(2016)Curr.Opin.Gastroenterol.31(1):69-75、Bures et al.(2010)World J.Gastroenterol.16(24):2978-2990、およびAdike and DiBaise(2018)Gastroenterol.Clin.North Am.47(1):193-208を参照。小腸内の細菌および/または古細菌の過剰なレベル(例えば、105細菌/mLを超える)、ならびに異常なタイプの細菌および/または古細菌は、SIBOの発症につながる可能性がある。SIBOは、慢性の下痢、腹部の不快感、膨満、吸収不良、鼓腸、および意図しない体重減少と関連付けられる。グラム陽性細菌は、典型的には、小腸に見られるが、SIBOに苦しむ対象は、グラム陰性細菌を含む様々な細菌を小腸に有しており、これらの細菌は通常、ごく少数しか存在しないか、または小腸内にはまったく存在しない。例えば、SIBOに存在する細菌は、粘膜に損傷を与える毒素を分泌したり、または胆汁酸塩を代謝したりする可能性があり、吸収不良および腹部膨満につながる可能性がある。24~50歳の対象および61歳以上の対象のSIBOの有病率を比較した研究では、SIBOは若い対象と比較して高齢の対象でより多く見られた(それぞれ15.6%および5.9%)(Parlesak et al.(2003)J.Am.Geriatr.Soc.51(6):768-773)。SIBOは、体重が減少した対象でもより頻繁に見られた。SIBOを発症するリスク要因には、代謝障害(例えば、糖尿病、低塩酸症)、栄養失調、過敏性腸症候群(IBS)、セリアック病、クローン病、肝硬変、腎不全、胃不全麻痺、小腸運動障害、GI管の構造異常(例えば、空腸憩室)、胃切除および免疫不全が含まれる。追加の危険因子には、特定の薬剤(例えば、抗生物質、胃酸分泌阻害剤)の使用が含まれる。例えば、Dukowicz et al.(2007)Gastroenterol.Hepatol.3(2):112-122を参照。いくつかの実施形態では、SIBOを有する対象は、腸通過時間を遅らせた(Cuoco et al.(2002)Hepatogastroenterology 49:1582-1586)。いくつかの実施形態では、SIBOを有する対象は、腸通過時間を加速させた(Van Citters and Lin(2006)Clin.Nutrition in Gastrointestinal Disease.Thorofare:Slack Inc;2006;271-280)。
本明細書に記載の方法のうちのいずれかのいくつかの実施形態では、対象は、片利共生および/または非片利共生(例えば、病原性)微生物の感染および/またはコロニー形成と関連付けられるGIDを有する。ほとんどのヒトは、通常は無害で病状を誘発しない細菌などの微生物によってコロニーを形成している。例えば、一部の細菌は、共生するヒトの腸管に存在し、免疫を促進し、病原性病原体による感染のリスクを低減する。微生物の病原性は、特定の宿主細胞、組織および/または器官への侵入およびアクセスの経路、微生物の固有の毒性、感染の可能性のある特定の部位に存在する細菌の量、ならびに宿主生物の健康などの様々な要因に依存する。したがって、一部の微生物は通常無害であり、宿主生物の内因性フローラの一部を形成するが、感染に有利な条件が与えられると病原性になる。
いくつかの実施形態では、本明細書に記載の方法は、1つ以上のアッセイを実行して、GID症状を有する対象が、GIDを発症するかまたは発症するリスクがあるかどうかを決定および/または特定することを含む。アッセイは、熟練した開業医(例えば、医師)が、対象のGI管機能、ならびに特定のGIDまたはGI病原体と関連付けられる1つ以上のバイオマーカーの存在または非存在を評価するために対象をスクリーニングし、対象を正確に診断し、および/または適切な治療を提供することを可能にする。例えば、本明細書に記載のアッセイ/試験は、熟練した開業医が、特定のバイオマーカーまたは機能的特徴の特定に基づいて、GID症状を示す対象を診断することを可能にするであろう。
いくつかの実施形態では、本明細書に記載の方法は、対象の胃腸バリア機能を決定するためのアッセイを実行することを含む。腸バリアは、栄養素、電解質、および水の吸収を可能にしながら、腸内細菌叢、抗原、および毒素を含む腸管腔の内容物から保護する複雑なシステムである。本明細書で使用される場合、「腸バリア」という用語は、腸管腔を対象の内部環境から分離する機能システムを指し、機械的構成要素(例えば、粘液および上皮層)、免疫学的構成要素(例えば、ディフェンシン、IgA、リンパ球、および自然免疫細胞)、筋肉成分、および神経学的成分を含む。欠陥のある腸バリアは、腸透過性の増加をもたらす可能性があり、それにより、管腔内容物の露出および腸の炎症を促進する免疫学的応答の誘発を可能にする。腸内細菌叢の変化、粘液層の変化、および上皮の損傷、ならびにアルコールおよびエネルギー密度の高い食品の摂取を含む多くの要因が、腸透過性を変化させる可能性がある(Bischoff et al.(2014)BMC Gastroenterol.14:189)。一般に、腸透過性をもたらす腸バリア機能の変化は、炎症と関連付けられている。例えば、過敏性腸症候群、脂肪性脂肪炎、急性膵炎、多臓器不全、大手術、および外傷のある患者では、腸透過性の変化が報告されている(Michielan and D’Inca(2015)Mediators Inflamm.2015:628157)。さらに、腸透過性の変化は、腸の炎症の病因、ならびにIBD、クローン病、および潰瘍性大腸炎を含むいくつかのGFIDの重症度に重要な役割を果たす(例えば、Antoni et al.(2014)World J.Gastroenterol.20(5):1165-79を参照)。腸バリア機能の変化はまた、腸の感染症、アレルギー性食品または毒性化合物の摂取、分泌型IgAの欠乏、外傷、内毒素血症、ならびに非ステロイド性抗炎症薬(NSAID)によって引き起こされる可能性がある。
いくつかの実施形態では、本明細書に記載の方法は、対象からの糞便サンプル中に存在するカルプロテクチンの濃度を決定するためのアッセイを実行することを含む。カルプロテクチン(S100A8/A9)は、ヒト好中球およびマクロファージの細胞質ゾルに一般的に見られるカルシウムおよび亜鉛結合タンパク質である。細胞のストレスおよび損傷の際、カルプロテクチンは、便中に検出され得る。したがって、カルプロテクチンレベルは、腸の炎症の敏感なマーカーと見なされる。
いくつかの実施形態では、本明細書に記載の方法は、対象からのサンプル(例えば、血液、血清または血漿)中に存在するC反応性タンパク質の濃度を決定するためのアッセイを実行することを含む。CRP(C反応性タンパク質)は、炎症または感染部位で劇的に増加する(最大1,000倍)急性炎症性タンパク質バイオマーカーである。したがって、いくつかの実施形態では、CRPレベルを使用して、対象が炎症を有するかどうかを決定することができる。いくつかの実施形態では、CRPレベルを使用して、対象が感染症を有するかどうかを決定することができる。このタンパク質は、ネイティブCRP(nCRP)と呼ばれるホモ五量体タンパク質として生成されるが、単量体CRP(mCRP)と呼ばれる5つの単量体に解離する可能性がある(Sproston and Ashworth(2018)Front.Immunol.9:754)。健康な対象の循環中のCRPレベルは低い(1mg/L未満)が、急性炎症の期間にはレベルが100倍に上昇する(300~400mg/Lに達することさえある)可能性がある(Chang et al.(2015)World J.Gastroenterol.21(40):11246-59)。
いくつかの実施形態では、本明細書に記載の方法は、対象からのサンプル(例えば、血液、血清または血漿)中に存在する7α-ヒドロキシ-4-コレステン-3-オン(7αC4)の濃度を決定するためのアッセイを実行することを含む。7αC4の測定は、胆汁酸合成の律速酵素である肝コレステロール7α-ヒドロキシラーゼの酵素活性のモニタリングを可能にし、胆汁酸吸収不良(BAM)を検出するための代理として使用され得る(例えば、参照によりその全体が本明細書に組み込まれる、Galman et al.(2003)J.Lipid.Res.44:859-66、およびCamilleri et al.(2009)Neurogastroeterol.Motil.21(7):734-43を参照)。
いくつかの実施形態では、本明細書に記載の方法は、血清学的試験(例えば、ELISA)を実行して、抗グリアジン抗体(AGA)、抗筋内膜抗体(EmA)、抗組織トランスグルタミナーゼ抗体(tTGA)、抗脱アミド化グリアジンペプチド抗体(DGPA)、および対象からのサンプル(例えば、血液、血清、血漿)中の総血清IgAのうちの1つ以上を検出および/または定量化することを含む。いくつかの実施形態では、AGA、EmA、tTGA、またはDGPAは、IgG抗体(例えば、IgG AGA、IgG EmA、IgG tTGA、およびIgG DGPA)である。いくつかの実施形態では、AGA、EmA、tTGA、またはDGPAは、IgA抗体(例えば、IgA AGA、IgA EmA、IgA tTGA、およびIgA DGPA)である。AGA、EmA、tTGA、およびDGPAは、セリアック病の血清学的マーカーである。したがって、これらの抗体の検出および/または定量化を使用して、対象がセリアック病を発症しているか、または発症するリスクがあるかどうかを決定することができる。
いくつかの実施形態では、本明細書に記載の方法は、対象からのサンプル(例えば、糞便または血液サンプル)中の微生物(例えば、GI病原体)の存在または特定を検出するためのアッセイを実行することを含む。GIDの症状を示す対象がGI病原体(例えば、細菌性病原体、原生動物、寄生虫、真菌、およびウイルス)に感染しているかどうかを評価するために、当技術分野で知られている任意の方法を、本明細書に記載されているように使用することができる。例示的なGI病原体には、以下の属のうちの1つの細菌が含まれるが、これらに限定されない:ブドウ球菌(例えば、黄色ブドウ球菌)、赤痢菌(例えば、志賀赤痢菌、フレキシネル赤痢菌、ボイディ赤痢菌、ソネ赤痢菌)、サルモネラ菌(例えば、サルモネラエンテリカおよびサルモネラボンゴリ)、エシェリキア(例えば、腸管毒素原性大腸菌(ETEC)を含む大腸菌)、腸管侵襲性大腸菌(EIEC)、腸管病原性大腸菌(EPEC)、または腸管出血性大腸菌(EHEC))、ビブリオ(例えば、コレラ菌、V.ブルニフィカスおよびV.パラヘモリチカス)、アエロモナス(例えば、アエロモナスハイドロフィラ)、プレジオモナス(例えば、プレジオモナスシゲロイデス)、カンピロバクター(例えば、カンピロバクタージェジュニ)、クロストリジウム(例えば、クロストリジウムディフィシル)、ヘリコバクター(例えば、ヘリコバクターピロリ)、バチルス(例えば、バチルスセレウス)、およびエルシニア(例えば、Y.エンテロコリチカおよびY.シュードツベルクロシス)、ロタウイルスA、ノロウイルスGI/GII、およびアデノウイルス40/41などのウイルス、ならびにクリプトスポリジウム属、赤痢アメーバ、およびランブル鞭毛虫などの寄生虫。例えば、いくつかの実施形態では、対象からのサンプル中のGI病原体の存在は、xTAG(登録商標)胃腸病原体パネル(GPP)アッセイ(Luminex Corporation,Austin,TX,USA、luminexcorp.com/clinical/infectious-disease/gastrointestinal-pathogen-panel/も参照)を使用して検出される。GPPアッセイは、細菌(サルモネラ属、赤痢菌属、ビブリオコレラエ、イェルシニアエンテロコリチカ、カンピロバクター属、クロストリジウムディフィシル、大腸菌O157、志賀毒素生成性大腸菌および腸管毒素原性大腸菌)、ウイルス(ロタウイルスA、アデノウイルス40/41、およびノロウイルスGI/GII)、ならびに寄生虫(クリプトスポリジウム属、赤痢アメーバ、およびランブル鞭毛虫)を含む、胃腸炎の原因となる最大15の異なる病原体を検出する多重核酸試験である。
いくつかの実施形態では、本明細書に記載の方法は、対象がSIBOを有するかどうかを決定するためのアッセイまたは試験を実行することを含む。いくつかの実施形態では、小腸または空腸吸引物は、対象から得られ(例えば、特別なゾンデを使用して、または小腸内視鏡検査を介して)、直接的に培養される。サンプリングは、挿管後、十二指腸、空腸、または回腸の内腔を含む腸管腔の内容物の掻き取り、生検、または吸引によって達成され得る。さらに、細胞、流体、糞便、または気体の性質の材料を含む腸管腔の内容物のいずれか、またはサンプリングが管腔壁自体のものであることが、サンプリングされ得る。細菌の異常増殖を検出するためのサンプルの分析は、顕微鏡検査、培養、および/または細胞数え上げ技術を含む従来の微生物学的技術によるものである。流体の約1×103CFU/mLよりも大きい、約1×104CFU/mLよりも大きい、または約1×105CFU/mLよりも大きい検出は、SIBOを示すことができる。
一態様では、本明細書で提供されるのは、対象のGIマイクロバイオームを特徴付ける方法である。上記で議論したように、GI管には、細菌、ウイルス、原生動物、および他の寄生虫を含む何百もの微生物種が含まれている。腸内細菌の大部分は、バクテロイデス門およびフィルミクテス門の2つの門に属しており、他の門は、プロテオバクテリア、放線菌、シネルギステス門、フソバクテリア門を含む。健康なマイクロバイオータは、有害な病原体によるコロニー形成への耐性、難消化性炭水化物の代謝、ビタミン生成、および宿主免疫応答の調節を含む、多くの利点を提供する(Browne et al.(2017)Nat.Rev.Microbiol.15(9):531-43)。腸内毒素症とも呼ばれる腸内細菌叢の破壊は、GI管障害、ならびに代謝障害、および脳機能障害を引き起こす可能性がある(参照により本明細書に組み込まれる、Lin and Zhang(2017)BMC Immunol.18:2を参照)。したがって、腸内細菌叢の監視および特徴付けは、GIDに関連する診断/治療ツールの武器庫における重要なツールである。
いくつかの実施形態では、本明細書に記載のデバイスおよび方法を使用して、(例えば、インビボまたはエクスビボで)対象のGI管内の硫黄代謝微生物、または硫酸化代謝物(例えば、胆汁酸、ポリフェノールおよび生体アミン)の存在量を特定し、特徴付けし、および/または定量化する。例えば、活動性疾患のIBD患者のマイクロバイオームは、エシェリキア、赤痢菌、およびフソバクテリウムなどの硫黄代謝に関与する微生物分類群に富んでいる場合があり、デスルフォビブリオおよびカンピロバクターなどの硫酸塩還元細菌の割合が高い場合がある。
いくつかの実施形態では、本明細書に記載のデバイスおよび方法を使用して、(例えば、インビボまたはエクスビボで)対象のGI管内のムチン分解微生物、またはムチンまたはムチン代謝物の存在量を特定し、特徴付けし、および/または定量化する。例えば、潰瘍性大腸炎およびクローン病患者のマイクロバイオームは、R.トルクおよびR.グナバスに富む可能性があり、一方で、A.ムシニフィラは、潰瘍性大腸炎およびクローン病患者において減少する可能性がある。
いくつかの実施形態では、本明細書に記載のデバイスおよび方法を使用して、(例えば、インビボまたはエクスビボで)対象のGI管内のメタン生成微生物を同定し、特徴付けし、および/または定量化することができる。古細菌は、確認された、天然に存在する唯一のメタンの生物学的供給源である。メタン生成古細菌は、水素を酸化してメタンを生成する。Methanobrevibacter smithiiは、人間の腸内の主要なメタン生成菌であるが、Methanosphaera stadtmanaeおよびMethanobacterium ruminatumなどの他のメタン生成古細菌も存在する可能性がある。IBS-Cと高呼吸メタンレベルとの間の関連が報告されており、実験的証拠は、メタンが腸の通過および収縮性を遅らせる可能性があることを示唆している(Triantafyllou et al.(2014)J.Neurogastroenterol.Motil.20:31-40、およびGoettlieb et al.(2016)Aliment.Pharmacol.Ther.43(2):197-212)。さらに、SIBOの抗生物質治療に反応した対象では、メタン生成の減少(ラクツロース呼気試験でメタンを使用して評価された)間の相関が観察された(Gatta and Scarpignato(2017)Aliment.Pharmacol.Ther.45(5):604-16)。したがって、メタン生成古細菌は、IBSおよびSIBOを含む多くの胃腸疾患で重要な役割を果たす可能性がある。
いくつかの実施形態では、上記の培養物から得られた個々の細菌分離株は、特定の抗菌剤に対するそれらの耐性および/または感受性を決定するために、抗菌薬感受性試験に供される。この情報は、例えば、特定の病原体によるGI感染症の治療、対象のGI管内の特定の細菌株/種の量の減少/増加、および/または対象のSIBOの治療に適切な抗生物質治療を決定するために使用され得る。したがって、目的の細菌分離株について抗菌プロファイルが得られたら、必要に応じて、適切な抗菌剤を対象に投与することができる。
いくつかの実施形態では、本明細書に記載の方法は、GID(例えば、SIBO)を発症する、または発症するリスクがあると特定された対象への、1つ以上の治療、例えば、抗生物質の投与を含む。方法はまた、分析物(例えば、特定の微生物)の存在もしくは不存在に基づいて、または分析物の量に基づいて、GIDを有するか、またはGIDを発症するリスクがあると決定される対象のための治療を選択することを含み得る。
一態様では、小腸細菌異常増殖(SIBO)、SIBO関連状態、またはSIBOを示唆する症状の治療を必要とする対象における小腸細菌異常増殖(SIBO)、SIBO関連状態、またはSIBOを示唆する症状を治療するための方法であって、方法は、抗菌剤を含む有効量の医薬製剤を対象に経口投与することと、それにより、対象のSIBO、SIBO関連状態、またはSIBOを示唆する症状を治療することと、を含み、抗菌剤が、メロペネム、セフトリアキソン、エルタペネム、およびピペラシリン-タゾバクタムからなる群から選択される、方法が本明細書で提供される。SIBO、SIBO関連状態、およびSIBOを示唆する症状(それらの診断を含む)が、本明細書に記載される。
本明細書に記載の組成物および方法は、非アルコール性脂肪性肝疾患(NAFLD)、非アルコール性脂肪性肝炎(NASH)、または肝硬変と関連付けられる胃腸管内の分析物を検出、分析、および/または定量化するために使用され得る。例えば、BMIおよび性別を一致させた健康な対照と比較して、NAFLD対象の大腸からのFaecalibacterium prausnitziiの存在量が減少する可能性がある。したがって、対象が非アルコール性脂肪性肝疾患(NAFLD)を発症するリスクがあるかどうかを決定するためのデバイスおよび方法であって、これには、NAFLDを有する疑いのある対象のGI管から(例えば、大腸から)サンプルを収集するために、本明細書に記載の摂取可能なデバイスを投与することと、GI管からのサンプルに存在する1つ以上の微生物を同定するための試験を実行することと、が含まれ、ここで、健康な対象と比較してFaecalibacterium prausnitziiの存在量が減少していることが、NAFLDの存在を示す、デバイスおよび方法が本明細書で提供される。別の態様では、肝硬変を有する対象が肝細胞癌(HCC)または肝性脳症などの合併症を発症するリスクがあるかどうかを決定するためのデバイスおよび方法が、本明細書で提供される。肝硬変は一般に、肝臓の構造を破壊する進行性のびまん性の線維化状態を指す(例えば、Heidelbaugh and Bruderly(2006)Am.Fam.Physician 74(5):756-62を参照)。いくつかの実施形態では、方法は、肝硬変を有する対象を特定することを含む。アスパラギン酸アミノトランスフェラーゼ対血小板比指数(APRI)、FIB-4指数、アスパラギン酸アミノトランスフェラーゼ(AST)対アラニンアミノトランスフェラーゼ(ALT)比、音響放射インパルス(ARFI)、トランジェントエラストグラフィ(TE)、および磁気共鳴エラストグラフィなどの血清マーカーを含む、肝硬変を有する対象を特定するためのいくつかの非侵襲的方法が開発されている(例えば、Nishikawa and Osaki(2015)Mediators Inflamm.2015:872152を参照)。
本明細書に記載の組成物および方法は、ヒト対象における様々な分析物を検出、分析、および/または定量化するために使用され得る。本出願で使用されるような「分析物」は、サンプル中で検出される化合物または組成物を指す。本出願での使用に適した例示的な分析物には、米国特許第6,251,581号に記載されているものが含まれ、参照によりその全体が本明細書に組み込まれる。大まかに言えば、分析物は、検出することができる任意の物質(例えば、1つ以上の抗原を有する物質)であり得る。分析物の例示的かつ非限定的なリストには、リガンド、タンパク質およびそれらのフラグメント、血液凝固因子、ホルモン、サイトカイン、多糖類、核酸、炭水化物、ムコ多糖類、脂質、脂肪酸、微生物(例えば、細菌)、微生物抗原、ならびに治療薬(そのフラグメントおよび代謝物を含む)が含まれる。
以下に記載される特定の検出方法は、サンプル中の分析物を検出するために、少なくとも1つの分析物結合剤を利用することができる。「分析物結合剤」は、特定の分析物に結合する分子である。いくつかの分析物結合剤は、以下に説明する方法を使用して検出される別の分子に結合する分析物の能力に従って、分析物(例えば、上記の分析物)を含み得る。例えば、いくつかの実施形態では、分析物結合剤は、抗体が特異的に結合する抗原を検出および/または定量化するための試薬として使用される場合に、抗体を含む。しかしながら、いくつかの実施形態では、抗体は、分析物(例えば、TNFα抗体などの薬物である抗体)であり、分析物結合剤は、抗体が特異的に結合する抗原を含み、それにより、抗体を検出および/または定量化するための試薬としての使用が可能になる。いくつかの実施形態では、分析物結合剤は、微生物(例えば、病原性細菌)の特定の属、種、または株に特異的な分析物に結合する。いくつかの実施形態では、分析物結合剤は、分析物に特異的に結合し、それによって分析物の特定の空間的および極性組織と相補的であると定義される表面または空洞内の領域を有する。いくつかの実施形態では、分析物結合剤および対応する分析物は、免疫学的対(抗原-抗体など)、ビオチン-アビジン対、ホルモン-ホルモン受容体対、核酸二本鎖、IgG-プロテインAペア、DNA-DNA、DNA-RNAなどのポリヌクレオチドペアなどであるがこれらに限定されない、結合対を形成する。いくつかの実施形態では、分析物結合剤は、抗体(例えば、モノクローナル抗体)、アフィマー、アプタマー、抗原、受容体、小分子、および核酸(例えば、DNA分子またはRNA分子)を含む。いくつかの実施形態では、結合対のいずれかのメンバー(例えば、分析物結合剤および/または分析物)は、本明細書に記載されるように検出可能に標識され得る。
摂取可能なデバイスおよびそれらの使用は、例えば、以下の米国特許出願に記載されており、これらの各々は、参照により本明細書に組み込まれる:「Ingestible Medical Device」と題され、かつ2014年8月15日に出願されたUSSN14/460,893、「Electromechanical Pill Device with Localization Capabilities」と題され、かつ2017年3月24日に出願されたUSSN15/514,413、「Systems and Methods for Obtaining Samples using Ingestible Devices」と題され、かつ2017年8月18日に出願されたUSSN15/680,400、「Sampling Systems and Related Materials and Methods」と題され、かつ2017年8月18日に出願されたUSSN15/680,430、「Electromechanical Ingestible Delivery of a Dispensable Substance」と題され、かつ2017年9月8日に出願されたUSSN15/699,848、「Gastrointestinal Tract Detection Methods,Device and Systems」と題され、かつ2017年12月7日に出願されたUSSN15/835,270、「Gastrointestinal Tract Detection Methods,Device and Systems」と題され、かつ2017年12月7日に出願されたUSSN15/835,237、「Gastrointestinal Tract Detection Methods,Device and Systems」と題され、かつ2017年12月7日に出願されたUSSN15/835,292、「Ingestible Device and Associated Methods」と題され、かつ2017年12月15日に出願されたUSSN15/844,349、「Ingestible Device and Associated Methods」と題され、かつ2017年12月15日に出願されたUSSN15/844,381、「Ingestible Device and Associated Methods」と題され、かつ2017年12月15日に出願されたUSSN15/844,427、「Systems and Methods for Extracting a Sample from an Ingestible Device」と題され、かつ2018年3月15日に出願された15/694,458、「Localization Systems and Methods for an Optoelectromechanical Pill Device」と題され、かつ2018年3月29日に出願されたUSSN15/940,407、および「Ingestible Device With Relatively Large Payload Volume」と題され、かつ2018年3月13日に出願されたUSSN62/642,544。
図73は、摂取可能なデバイスからサンプルを抽出するための方法の例示的な実施形態を示している。この方法は、摂取可能なデバイスのハウジングに開口部を作成させることを含む。開口部を作成する実施例を、図73150に示している。この方法は、摂取可能なデバイスのハウジングの開口部をチューブに接続する取り付けられたスリーブに少なくとも部分的に摂取可能なデバイスを挿入することを含む。このプロセスの実施例を、図73152および図73154に示している。方法はまた、摂取可能なデバイスを取り付けられたスリーブに少なくとも部分的に挿入しながら、摂取可能なデバイスを遠心分離して、サンプリングチャンバに含まれるサンプルの少なくとも一部分をチューブに移行することを含む。遠心分離プロセスの実施例を、図73156、図73158、および図73160に示している。
生細胞色素
本明細書に記載の特定のシステムは、自律的で摂取可能なデバイス、または他の同様のサイズのデバイスにおいて、蛍光を総細菌数(TBC)に正確かつ確実に相関させることが見出された方法、組成物および検出システムを使用する。本明細書で使用される場合、「総細菌数」という用語は、細菌および/または古細菌細胞の量を指すために使用される。いくつかの実施形態では、本明細書に記載の方法およびデバイスを使用して、対象の胃腸管からのサンプル中のTBCを検出して、対象がGI障害(例えば、SIBO)を発症するかまたは発症するリスクがあるかどうかを決定することができる。組成物は、非細菌性および/または非古細菌細胞、ならびにそうでなければ、生きた細菌および/または古細菌細胞の検出または定量化が困難になる他の成分を含む、サンプル中の生菌および/または古細菌細胞の選択的染色を可能にする色素、緩衝液および界面活性剤の新規の組み合わせを含む。いくつかの実施形態では、システムは、細菌がほぼリアルタイムで定量化され、結果がデバイスの外部で遠隔測定的に共有されることを可能にする。上記に、このセクションで説明する方法を使用して検出することができる、様々なタイプの細胞(例えば、細菌細胞および古細菌細胞)が開示されている。
いくつかの実施形態では、本開示は、対象の胃腸(GI)管内のインビボで細胞および/または分析物を取得、培養、および/または検出する方法を提供する。関連するデバイスがまた、開示されている。説明される方法およびデバイスは、対象から流体サンプルを取得および/または分析するための多くの利点を提供する。いくつかの実施形態では、流体サンプルを希釈することにより、分析物検出のダイナミックレンジが増加し、および/またはサンプル内のバックグラウンド信号もしくは干渉が減少する。例えば、干渉は、非標的分析物の存在、またはサンプル内の色素もしくは標識の非特異的結合によって引き起こされる可能性がある。いくつかの実施形態では、サンプルを培養することは、細胞(例えば、特定のタイプの細胞)および/または細胞によって生成される分析物(例えば、目的の特定の分析物)の濃度を増加させ、それによって、それらの検出および/または特徴付けを容易にする。上記に、本明細書に記載されるように検出および/または特徴付けられ得る様々なタイプの分析物が、開示されている。
いくつかの実施形態では、本出願は、サンプル中の分析物を検出するための摂取可能なデバイスを提供し、ここで、摂取可能なデバイスは、(1)複数のドナー粒子であって、複数のドナー粒子の各々が、光増感剤を含み、かつそれに結合して、分析物に結合する第1の分析物結合剤(例えば、抗原結合剤)を有し、ここで、光増感剤が、その励起状態で、一重項酸素を発生させることができる、複数のドナー粒子と、(2)複数のアクセプター粒子であって、複数のアクセプター粒子の各々が、化学発光化合物を含み、かつそれに結合して、分析物に結合する第2の分析物結合剤(例えば、抗原結合剤)を有し、ここで、その化学発光化合物が、一重項酸素と反応して、発光することができる、複数のアクセプター粒子と、を含む組成物を保持するように構成されたサンプリングチャンバを含む。いくつかの実施形態では、第1および第2の分析物結合剤は、抗原結合剤(例えば、抗体)である。いくつかの実施形態では、第1および第2の抗原結合剤は、分析物(例えば、タンパク質)の同じエピトープに結合する。いくつかの実施形態では、第1および第2の抗原結合剤は、空間的に重複する分析物(例えば、タンパク質)の別個のエピトープに結合する。いくつかの実施形態では、第1および第2の抗原結合剤は、空間的に重複しない分析物(例えば、タンパク質)の別個のエピトープに結合する。いくつかの実施形態では、第1および/または第2の分析物結合剤(複数可)は、抗体である。いくつかの実施形態では、第1および/または第2の分析物結合剤(複数可)は、アフィマーである。いくつかの実施形態では、第1および/または第2の分析物結合剤(複数可)は、抗原結合剤であり、アプタマーである。
I.要約
様々な抗生物質のインビトロ活性は、様々なSIBO生物について決定され得る。個々の抗生物質に特徴的な他の薬力学的パラメータとともに、インビトロ活性は、動物モデルで評価される用量の枠組みを提供する。エルタペネム、メロペネム、セフトリアキソン、およびピペラシリン-タゾバクタムは安全であり、経口投与した場合の全身吸収が低く、SIBO生物に対して活性がある。これらの抗菌剤およびSIBO生物に対する既知の活性の要約を、以下の表1に示す。
ラットおよびミニブタは、ラットがIBS/SIBOの動物モデルとして使用されており、ミニブタがヒトと同様のGIシステムを有しているため、前臨床/有効性の種になる可能性がある。
●ラットおよびミニブタの経口PK研究は、異なる用量のメロペネムで実施される。血漿および糞便を収集して、高用量であってもこれら2つのサンプル間の曝露を実証および比較する。
●異なるメロペネム製剤は、便の粘稠度の変化を監視することにより、ラットおよびミニブタで評価される。ラットでは、安楽死後に小腸液中の薬物および微生物レベルが定量化される。
●メロペネムのインビトロ活性は、SIBO生物に対して測定される。
○MIC研究
○タイムキル研究
○抵抗力発達研究
SIBOのPimentel(2008)Campylobacter jejuniラットモデルを使用して、抗菌製剤の選択を評価し、優先順位を付ける。Pimentel et al.2008。新しいラットモデルは、過敏性腸症候群の2つの現代的な理論を結び付けている。Dig Dis Sci.53:982-989。簡単に言えば、ラットはC.jejuniに感染し、3か月後、便の粘稠度の変化および体重の減少に基づいて、どの動物がSIBOの証拠を有しているかが決定される。その動物は、対照グループを含む異なるグループに分けられ、動物の体重および便の粘稠度が正常化するかどうかを監視するために、異なる抗菌製剤が投与される。これに続いて、Pimentel(2008)に記載されているように、PCRによる空腸内容物の細菌定量化が行われる。さらに、インビボで発達したメロペネムに対する耐性が、もしあれば監視される。
I.研究設計の概要
参加する対象は、抗菌薬(「治験薬」とも呼ばれる)またはプラセボのいずれかを受け取るために、1:1の比率でランダム化される。対象は、2~4週間の治療段階で治験薬を受け取り、その後、10~12週間の治療後段階が続く。治験薬の投与計画および治療期間は、薬物動態およびシンチグラフィーの研究データ、公表された文献、SIBOのソートリーダーとの協議、ならびに実施例Aに記載されているようなインビトロおよびインビボ研究の結果からの考慮に基づいて選択される。フォローアップ期間は、SIBOで治験薬を評価するためのFDAの推奨に基づいて延長または短縮され得る。
含めるために選択された患者または対象は、SIBOを示唆する症状を示している。FDAがSIBOに対して設定している現在の診断基準は、小腸流体サンプルで≧105CFU/mL以上であり、しかしながら、このカットオフ値は、変更される可能性がある。例えば、それは、小腸流体サンプル中で≧104または≧103CFU/mLに低下され得る。小腸の細菌レベルを確認するために、特別なゾンデを使用して、または小腸内視鏡検査を介して、対象から空腸流体をサンプリングし、直接的に培養することができる。あるいは、摂取可能なデバイスを使用して、空腸流体サンプルの希釈液中の細菌増殖の存在を検出することができる。≧105CFU/mLは、(例えば、表2に列挙された細菌種、または本明細書の他の箇所を参照)SIBOと関連付けられている細菌種のいずれかを含むことができる。治験薬によっては、対象集団は、空腸流体で検出された細菌種によってさらに制限され得る。例えば、治験薬がメロペネムである場合、インビトロおよびインビボの予備研究でメロペネムに反応することが知られている細菌種の異常増殖を有する対象のみが、試験に含まれる。
提案された研究の有効性エンドポイントは、SIBO症状(例えば、SIBOを示唆する症状)に関する毎週の問題に対する対象の反応、ならびに膨満、下痢、鼓腸、便の頻度の増減、腹痛、便秘、体重減少、発熱、腹部圧痛、吐き気、胃のうっ血、および脂肪便などのSIBO症状に対する症状固有の毎日の有効性測定に対する反応に基づいている。有効性の評価は、対象の観察の12~16週間全体にわたって収集される。
この研究では、リファキシミンと比較したメロペネムおよびセフトリアキソンのインビトロ活性が、SIBOに関与している一連の好気性および嫌気性細菌に対して決定された。最小発育阻止濃度(MIC)値は、臨床検査標準協会のガイドライン(CLSI、(1)“Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard;11th ed.”CLSI standard M07-A11,2018、(2)“Performance Standards for Antimicrobial Susceptibility Testing;Twenty-Eighth Informational Supplement,”CLSI document M100-S28,2018、および(3)“Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria;Approved Standard-Eighth Edition,”CLSI document M11-A8,2012)に従って、ブロス微量希釈または寒天希釈によって決定された。
試験化合物
試験剤は、Micromyx(Kalamazoo、MI)から提供され、使用前に製造元の指示に従って保管された。ストック溶液は、CLSI(1)が推奨する溶媒中の各試験剤から、アッセイで試験するのに望ましい最高濃度の100倍で作製した。すべてのストック溶液は、自動滅菌に使用する前に少なくとも1時間放置した。サプライヤー、カタログ番号、ロット番号、溶剤、希釈剤は、次のとおりである。
試験生物は、Micromyxリポジトリからの臨床分離株またはアメリカンタイプカルチャーコレクション(ATCC、バージニア州マナッサス)から取得した基準株で構成されていた。それらは最初にMicromyxで受け取られ、隔離のためにストリークされた。コロニーを滅菌綿棒で採取し、凍結防止剤を含む適切なブロスに懸濁した。懸濁液を極低温バイアルに分注し、-80℃に維持した。試験の前に、分離株を、冷凍バイアルから適切な寒天上にストリークした。好気性菌には、sheep bloodが5%含まれるトリプチケースソイ寒天培地(TSA)を使用した、嫌気性菌は、5%のsheep blood、ヘミン、ビタミンK1を含むブルセラ寒天培地で培養した。ストリークされた分離株は、成長に最適な条件下でインキュベートされた。Streptococcus spp.は、anaerobesが嫌気的にインキュベートしたのに対し、3%CO2でインキュベートされた。品質管理分離株は、CLSIガイドライン(1)~(3)に従って試験中に含まれていた。
●Escherichia coli、n=50
●Klebsiella spp.、n=50
●Pseudomonas aeruginosa、n=50
●Streptococcus spp.、n=50
●Staphylococcus aureus、n=50
●Enterococcus faecalis、n=50
●Enterobacter aerogenes、n=50
●Proteus mirabilis、n=50
寒天希釈により嫌気的に試験した:
●Prevotella spp.、n=30
●Veillonella spp.、n=30
●Clostridium sporogenes、n=10
●Bacteroides fragilis、n=30
●Bacteroides spp.、n=30(B.vulgatusを含む)
好気性細菌のブロス微量希釈試験は、陽イオン調整ミューラーヒントンブロス(MHBII)で実施された。連鎖球菌を試験するために、溶解したウマの血液(LHB)を、最終濃度3%でMHBIIブロスに添加した。LHBを使用した場合は、それを濾過後に追加した。
ブロス微量希釈MICアッセイ
96ウェルマイクロプレートでのブロス微量希釈感受性試験は、CLSI法(1)および(2)に従って実施された。Multidrop384(Labsystems、Helsinki、Finland)、Biomek2000、およびBiomek FX(Beckman Coulter、Fullerton、CA)を含む自動液体ハンドラーを使用して、段階希釈と液体移送を実施した。標準的な96ウェル微量希釈プレート(Costar3795)のカラム2から12のすべてのウェルに、150μLの適切な希釈液を充填した。次に、300μLの試験剤を、試験する最高最終濃度の100倍でプレートのカラム1のウェルに加えた。Biomek2000を使用して、11列目までの行全体で連続2倍希釈を行った。カラム12のウェルには薬物が含まれておらず、成長制御ウェルとして機能した。このプレートは、MICアッセイプレートまたは「ドータープレート」を作製するための「マザープレート」として機能した。
嫌気性生物のMIC値は、CLSI(3)が推奨する寒天希釈法を使用して決定された。試験剤は、滅菌チューブ内に5%のLaked Sheep Bloodを含む19.5mLの寒天に対する0.5mLの40X試験剤の比率で溶融添加ブルセラ寒天(50~55℃)と混合され、穏やかに混合し、次に滅菌ペトリ皿に手動で注いだ。プレートを室温で約1時間固化させた。薬物を含まないプレートを成長対照としてインキュベートした。0.5マクファーランド標準懸濁液を滅菌生理食塩水で調製し、ステンレス鋼レプリケーターブロックのウェルに移送し、適切な薬物濃度を含む寒天プレートにスタンプした。インキュベーション後、プレートを、BactronII嫌気性チャンバ(Sheldon Manufacturing Co.;Cornelius,OR)の嫌気性環境内で35℃で、42~48時間インキュベートした。MICは、CLSIガイドライン(1)に従って読み取られた。
メロペネム、セフトリアキソン、リファキシミン、およびSIBOに関与する生物に対するコンパレーターのインビトロ活性を以下の表3~34に示す。ここで、MEM=メロペネムであり、CTR=セフトリアキソンであり、RFX=リファキシミンであり、MXF=モキシフロキサシンであり、およびPTZ=ピペラシリン/タゾバクタムである。これらのデータは、図91~図106にグラフで示されている。
表3に示すように、本研究での50のEnterobacter aerogenesに対して、メロペネムは、0.015~2μg/mlのMIC範囲で、0.03μg/mlのMIC50および0.06μg/mLのMIC90を有していた。このパネルの分離株の98%は、メロペネムに感受性があり、2%は、中程度の耐性を示した。セフトリアキソンのMIC50/90は、0.12/32μg/mlであり、その範囲は、生物のこのセットに対して0.03~64μg/mlであり、ここで、リファキシミンについて、MIC50は16μg/mLであり、MIC90は64μg/mLであり、MIC範囲は、8~>64μg/mLであった。このパネルでは、分離株の26%が、セフトリアキソン耐性であった。モキシフロキサシンおよびピペラシリン-タゾバクタムがまた、このパネルに対して試験され、メロペネムが、評価された最も活性の高い薬剤であった。図91は、これらのデータをグラフで表示しており、表4は、各MICでの分離株の数および累積パーセンテージを示している。
表13は、28のメチシリン耐性(MRSA)菌株を含む50のStaphylococcus aureus分離株に対して試験薬を評価した結果を示している。メロペネムは、1μg/mlのMIC50、8μg/mLのMIC90、および0.06~>16μg/mlのMIC範囲を示した。セフトリアキソンについて、MIC50は、32μg/mlであり、MIC90は、>64μg/mlであり、その範囲は、2~>64μg/mLであった。28のMRSA分離株の存在は、メロペネム、セフトリアキソン、ピペラシリン-タゾバクタム、およびオキサシリンに対する感受性の低下に寄与した。これらの分離株に対するリファキシミンMIC50は、0.015μg/mlであり、MIC90は、0.008~32μg/mlのMIC範囲で0.06μg/mLであった。この研究では、モキシフロキサシン、ピペラシリン-タゾバクタム、バンコマイシン、およびオキサシリンがコンパレーターであり、リファキシミンが、試験された最も活性の高い薬剤であった。図96は、これらのデータをグラフで表示しており、表14は、各MIC値での分離株の数および累積パーセンテージを示している。
この研究では、10のClostridium spp.のパネルは、C.sporogenesの4つの分離株、C.ramosumの2つの分離株、およびC.innocuumの4つの分離株を含んでいた。表25に示すように、本研究での10のすべてのClostridium spp.に対して、メロペネムは、1μg/mlのMIC50および2μg/mLのMIC90、ならびに0.06~2μg/mlのMIC範囲を有していた。このパネルの分離株の100%は、メロペネムに感受性であった。セフトリアキソンのMIC50/90は、8/16μg/mlであり、その範囲は、生物のこのセットに対して0.5~16μg/mlであり、ここで、リファキシミンについて、MIC50は>64μg/mLであり、MIC90は>64μg/mLであり、MIC範囲は、2~>64μg/mLであった。このパネルでは、すべての分離株が、セフトリアキソンに感受性であった。この研究のコンパレーターは、メトロニダゾールであり、メロペネムおよびメトロニダゾールは、同様の活性を有していた。図102は、これらのデータをグラフで表示しており、表26は、各MICでの分離株の数および累積パーセンテージを示している。
要約すると、メロペネム、セフトリアキソン、およびリファキシミンが、SIBOに関連しているとされている広範囲のグラム陽性およびグラム陰性の好気性および嫌気性細菌に対するインビトロ活性の適切なコンパレーターとともに評価された。リファキシミンは、典型的に、局所抗生物質として臨床的に使用されているため、耐性ブレークポイントは利用できなかった。メロペネム、セフトリアキソン、およびコンパレーター剤の耐性ブレークポイントは、全身投与用に決定された(CLSI(1)および(2)を参照)。結果は、メロペネムがE.aerogenes、E.coli、P.aeruginosa、Streptococcus spp.、Clostridium spp.、Prevotella spp.、およびVeillonella spp.に対して最も活性な薬剤であり、セフトリアキソンがP.mirabilisに対して試験された最も活性な薬剤であったことを示していた。これらの薬剤のMIC90値は、Klebsiella spp.について試験した最高濃度よりも大きかったので、効力の順番を決定することができなかった。しかしながら、メロペネムのMIC50値が、最も低かった。
SIBOの病因に関与する細菌に対する抗菌剤メロペネムおよびセフトリアキソンのタイムキル動態を、SIBOの治療に使用されるリファキシミンと比較した。メロペネム、セフトリアキソン、およびリファキシミンは、最小発育阻止濃度(MIC)の倍数(MICの2倍、4倍、および8倍)で、Escherichia coli、Bacteroides spp、およびStreptococcus spp.の各々3つの分離株に対して評価された。MIC値は、臨床検査標準協会(CLSI;(1)“Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard;11th ed.,”CLSI standard M07,2018、(2)“Performance Standards for Antimicrobial Susceptibility Testing;28th ed.,”CLSI supplement M100,2018)からのガイドラインに従ってブロス微量希釈によって決定された。
試験化合物
メロペネム、リファキシミン、およびセフトリアキソンは、Micromyx(Kalamazoo、MI)から提供された。
Escherichia coli、Bacteroides spp、およびStreptococcus spp.の各々の3つの分離株を使用した。最初の受け取り時に、分離株を適切な寒天培地にストリークした。E.coli分離株を35℃で一晩インキュベートした。Streptococcus分離株は、CO2インキュベーター内で35℃で一晩インキュベートし、嫌気性菌は、35℃で48時間Bactron嫌気性チャンバ内でインキュベートした。これらのプレートからの増殖を使用して、20%滅菌グリセロールを含むトリプチケースソイブロス(嫌気性生物用のブルセラブロス)で凍結ストックを調製し、-80℃で保存した。
E.coliのMICおよびタイムキル試験中に使用された培地は、陽イオン調整ミューラーヒントンブロス(CAMHB;Becton-Dickinson[BD],Sparks,MD)であった。Streptococcusの場合、培地は、3%のウシ溶血血液(Hemostat Laboratories;Dixon,CA)を含むCAMHBであり、嫌気性菌の場合、培地は、5μg/mLのヘミン(Sigma,St.Louis,MO)、1μg/mLのビタミンK1(Sigma)、および5%のウシ溶血血液を含む補足ブルセラブロス(Becton Dickinson)であった。すべての培地は、CLSIガイドライン(2)に従って準備および保存された。
メロペネム、リファキシミン、およびセフトリアキソンは、最初に、CLSIガイドライン(1)および(2)に従って実施された適切なQC生物を用いたブロス微量希釈MIC試験にかけた。自動液体ハンドラー(Biomek2000およびBiomek FX、Beckman Coulter、Fullerton CA)を使用して、段階希釈および液体移送を実施した。
タイムキルは、試験剤の殺菌活性を評価するために、CLSI((3)”Methods for Determining Bactericidal Activity of Antimicrobial Agents;Approved Guideline,”CLSI document M26-A,1999)によって記述された方法を使用して決定された。すべての化合物は、以前の試験で決定されたMICの2倍、4倍、および8倍で試験された。
メロペネム、セフトリアキソン、およびリファキシミンは、小腸細菌異常増殖(SIBO)に関与していると考えられる生物に対するインビトロ殺菌活性について評価され、殺菌は、24時間の薬物曝露後のコロニー形成単位(CFU)の≧3logの減少として定義された。試験分離株に対するこれらの抗生物質およびコンパレーター薬のMIC結果を表35に示し、このデータを使用して、以下に説明するMICの倍数での薬物曝露を決定した。MIC値が示されているが、メトロニダゾールおよびバンコマイシンは、E.coliに対して臨床的に役立つ抗生物質ではないことに留意されたい。
メロペネム、セフトリアキソン、およびリファキシミンのブロス微量希釈MIC値は、E.coli ATCC25922に対してそれぞれ0.015、0.06、および8μg/mLであった。この生物に対するメロペネム、セフトリアキソン、およびリファキシミンのタイムキル動態を図107に示し、対応するCFU/mL、log10CFU/mL、およびベースラインに対するlog10CFU/mLの変化を表36に示している。2倍、4倍、および8倍のMICのメロペネムは、24時間で2つの高濃度で急速な死滅および殺菌活性を示した。セフトリアキソンは、24時間で8倍のMICで殺菌性であったが、リファキシミンは、2~6時間で静菌効果を示し、6~24時間で再増殖した。
メロペネム、セフトリアキソン、およびリファキシミンはすべて、S.pneumoniae ATCC49619に対して0.06μg/mLのMIC値を有していた。この生物に対するメロペネム、セフトリアキソン、およびリファキシミンのタイムキル動態を図110に示し、対応するデータを表39に示している。メロペネムおよびセフトリアキソンの場合、試験した3つの薬物濃度すべてが24時間で殺菌効果を示したが、リファキシミンの場合、24時間で薬物なしの対照レベルに回復した6時間の時点で3つの濃度すべてで、約2.3log10CFUの減少が観察された。
B.fragilis ATCC25285に対するメロペネム、セフトリアキソン、およびリファキシミンのMIC値は、それぞれ0.06、32、および0.05μg/mLであった。この生物について、タイムキル動態および対応するデータをそれぞれ図113および表42に示している。メロペネムは、8倍のMICで6、24、48時間、4倍のMICで24、48時間、および2倍のMICで24時間で、この生物に対して急速な殺菌活性を示した(2倍のMICでは、再成長が48時間で観察された)。リファキシミンは、この菌株に対する死滅の証拠を示さなかった。この生物に対するMICは32μg/mLであったため、セフトリアキソンのタイムキル動態は実施されなかった。
メロペネム、セフトリアキソン、およびリファキシミンを、SIBOに関与する生物であるE.coli、Streptococcus spp.、およびBacteroidesspp.の各々3つの分離株に対する殺菌活性について評価した。全体として、メロペネムおよびセフトリアキソンの両方が、評価された分離株に対して殺菌活性を示したのに対し、静菌活性および再成長は典型的に、リファキシミンで観察された。メロペネムおよびセフトリアキソンの殺菌活性は、特にGI状態の治療のための標準的な抗生物質と一般に考えられているリファキシムと比較して驚くべきものであった。リファキシミンの作用がいかに不十分であったことについて驚くべきことであっただけでなく、おそらくさらに驚くべきことは、メロペネムおよびセフトリアキソンがいかに細菌を死滅し、かつ持続的な殺菌活性を提供したことであり、これは規制当局、臨床医、および支払者にとって重要である可能性がある。
リファキシミンと比較したメロペネムおよびセフトリアキソンに対する耐性発現の能力は、自然突然変異頻度アッセイを使用して、Escherichia coli、Bacteroides spp.、およびStreptococcus spp.の各々の3つの分離株の最小発育阻止濃度(MIC)の倍数で決定された。MICの初期値は、臨床検査標準協会(CLSI;(1)“Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard;11th ed.,”CLSI standard M07,2018、(2)“Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria;Approved Standard-Eighth Edition,”CLSI document M11-A8,2012.)からのガイドラインに従って寒天希釈によって決定された。
試験化合物
試験剤は、Micromyx(Kalamazoo、MI)から提供された。リファキシミン、セフトリアキソン、およびメロペネムは、適切な溶媒中で、寒天希釈MICアッセイの100倍の最高試験濃度のストック濃度で調製された。自然突然変異アッセイでは、試験した100倍の最高最終濃度で薬物ストックを調製した。自然突然変異アッセイでのリファキシミンの試験では、最終DMSO濃度(v/v)は、1%であった。
試験生物は、以下の通りであった。
●E.coli ATCC25922
●E.coli ATCC35218
●E.coli MMX1312
●S.pneumoniae ATCC49619
●S.pyogenes ATCC49399
●S.agalactiae ATCC13813
●B.fragilis ATCC25285
●B.vulgatus MMX3483
●B.ovatus MMX3503
ミューラーヒントン寒天培地(MHA II;BD;Sparks,MD)、5%のヒツジ溶血血液を含むMHA II(Hemostat)、または補足されたブルセラ寒天培地(BD;Sparks,MD)を、寒天希釈MICおよび自然突然変異アッセイの試験培地として使用した。
自然突然変異アッセイを実施する前に、メロペネム、セフトリアキソン、およびリファキシミンのMICを、寒天希釈法を使用して各分離株について決定した。得られたMIC値を使用して、自然突然変異アッセイの選択濃度を決定した(4倍および8倍の寒天希釈MIC)。
メロペネム、セフトリアキソン、およびリファキシミンの段階希釈は、適切な希釈剤で行われた。寒天プレートを1.0mLの濃縮薬物と49.0mLの適切な溶融(47℃)寒天の比率で混合することによって調製し、薬剤を添加した寒天を混合し、15x150mmのペトリ皿に注ぎ、固化させて室温で乾燥させてから接種した。4倍および8倍の寒天希釈MICで、薬剤を含む複製プレートを調整した。各プレートを室温で少なくとも1時間冷却させた。
分離株を適切な寒天培地にストリークし、20時間(好気性)または48時間(嫌気性条件下のBacteroides)インキュベートした。滅菌綿棒を使用して、いくつかの十分に分離されたコロニーを除去し、重い細胞密度で滅菌生理食塩水に再懸濁した。各プレートに、0.5mLの細胞懸濁液をインキュベートした(各試験濃度を2回インキュベートした)。各自然突然変異プレートに対する目標接種材料は、109~10CFUであった。さらに、各分離株からの接種材料の一部分を、希釈し、寒天上に広げ、コロニーをカウントすることによって数えた。
薬剤選択プレート上で成長するコロニーが選択抗生物質に対してMIC値が上昇したかどうかを決定するために、ブロス微量希釈による感受性試験は、CLSI(1)、(2)、および(3)が推奨する方法論に従って(“Performance Standards for Antimicrobial Susceptibility Testing;Twenty-Eighth Informational Supplement.,”CLSI document M100-S28,2018)単離された変異体で実施された。
試験剤は、Micromyxから提供された。試験中に使用したすべての試験剤、製造元、ロット番号、保管場所、および溶媒は、次のとおりである。
5%のsheep bloodを含むトリプティックソイ寒天プレートを使用して、好気性菌(BD/BBL)をストリークし、陽イオン調整ミューラーヒントンブロス(CAMHB II;BD)をアッセイで好気性細菌分離株を試験するための培地として使用した。Bacteroides spp.を5%のヘミン(Sigma)および1mg/mLのビタミンK1(Sigma)を含むブルセラプレート(BD/BBL)にストリークし、5mg/mLのヘミン(Sigma)、1mg/mLのビタミンK(Sigma)、および5%のウシ溶血血液(Hemostat)を添加したブルセラブロス(BD/BBL)で試験した。
マイクロタイタープレートは、CLSI(1)~(3)に従って調製された。試験プレートを準備するために、Multidrop384(Labsystems、Helsinki、Finland)、Biomek2000、およびBiomek FX(Beckman Coulter、Fullerton、CA)を含む自動液体ハンドラーを使用して、段階希釈と液体移送を実施した。
評価された生物に対するメロペネム、セフトリアキソン、およびリファキシミンの寒天希釈MIC値を表45に示す。結果は、すべてのQC分離株について確立されたQC範囲内であり、リファキシミンMIC値は、QC生物を用いた以前の試験結果(上記の実施例CおよびDを参照)に基づいて予想されたとおりであった。自然突然変異率については、以下の各薬物について説明する。
メロペネムは、評価された試験分離株全体で強力な活性を示し、MIC値は、0.008~0.12μg/mLであった(上記の表45を参照)。メロペネムの自然突然変異の頻度は、表46に生物ごとにまとめられている。Streptococcus spp.またはBacteroides spp.コロニーは選択されておらず、一方で、以下に説明するように、E.coli MMX1312のみが増殖を示した。
上記の表45に示すように、セフトリアキソンは、Streptococcus spp.に対して強力な活性を示し、E.coli MMX1312を除いてE.coliのMIC値は、0.03~0.06μg/mLであり、ここで、4μg/mLのMIC値が観察された。Bacteroides spp.に対しては比較的少ない活性が観察され、MIC値は、4~128μg/mLであった。
リファキシミンは、0.03~0.5μg/mLのMIC値での評価されたStreptococcus spp.およびBacteroides spp.に対して最も活性があったが、≧4μg/mLのMIC値でのE.coliに対して低い活性が観察された(上記の表45)。
要約すると、SIBO病原体の中でインビトロでメロペネムまたはセフトリアキソンに対して自然に発症する耐性の傾向はほとんどなかった。コロニーがメロペネムまたはセフトリアキソン選択プレートに現れたまれな実例では、さらなる調査に基づいて、それらは典型的に、薬物含有寒天上で継代培養したときに増殖せず、親分離株のMIC値と一致するMIC値を有していたため、真の変異体ではなかった。対照的に、MIC値が上昇した自然変異体は、E.coliおよびStreptococcus spp.に対してリファンピンを用いて高率(約10-8)で容易に選択され、Bacteroides spp.の場合はそれほど頻繁ではなかった(10-9~10-10)。この結果は、この抗菌クラスの細菌で観察された高い突然変異頻度と一致している。
Claims (15)
- 小腸細菌異常増殖(SIBO)の治療をそれを必要とする対象において行うための方法における使用のための医薬製剤であって、
前記方法が、抗菌剤を含む有効量の医薬製剤を前記対象に経口投与し、それにより前記対象のSIBOを治療することを含む、および/または
前記抗菌剤がSIBOの病因に関与する細菌に対して抗菌活性を示し、前記抗菌剤がメロペネムである、医薬製剤。 - 前記抗菌剤が約0.001μg/mL~約64μg/mL、約0.004μg/mL~約32μg/mL、約0.015μg/mL~約16μg/mL、約0.03μg/mL~約8μg/mL、または約0.5μg/mL~約2μg/mLのMIC50値またはMIC90値で前記細菌に対して抗菌活性を示す、請求項1に記載の使用のための医薬製剤。
- 前記抗菌剤が約0.001μg/mL~約128μg/mL、約0.001μg/mL~約64μg/mL、約0.004μg/mL~約32μg/mL、約0.015μg/mL~約16μg/mL、約0.03μg/mL~約8μg/mL、または約0.5μg/mL~約2μg/mLのMIC範囲で前記細菌に対して抗菌活性を示す、および/または
前記細菌がグラム陽性細菌、グラム陰性細菌、嫌気性細菌、およびそれらの組み合わせからなる群から選択される、請求項1または2に記載の使用のための医薬製剤。 - 前記細菌がEnterobacter aerogenes、Escherichia coli、Klebsiella spp.、K.oxytoca、K.pneumonia、Proteus mirabilis、Pseudomonas aeruginosa、Staphylococcus aureus、Enterococcus faecalis、Streptococcus spp.、Streptococcus pyogenes、Streptococcus agalactiae、Viridans group Streptococcus、Clostridium spp.、C.sporogenes、C.ramosum、C.innocuum、Prevotella spp.、P.melanogenica、P.bivia、P.buccae、P.nanceiensis、P.intermedia、P.denticola、P.nigrescens、P.corporis、P.bergensis、P.disiens、Veillonella spp.、V.parvula、Veillonella dispr、V.atypica、Bacteroides fragilis、Bacteroides non-fragilis、B.caccae、B.thetaiotaomicron、B.ovatus、B.vulgatus、B.uniformis、B.stercoris、B.xylanisolvens、B salyersiae、B.intestinalis、およびB.faecisからなる群から選択される、および/または
前記抗菌剤が前記細菌に対して殺菌効果を示す、請求項2または3に記載の使用のための医薬製剤。 - 前記抗菌剤が約0.5倍のMIC~約8倍のMIC、約1倍のMIC~約6倍のMIC、または約2倍のMIC~約4倍のMICの濃度で、前記細菌に対して殺菌効果を示す、および/または
前記抗菌剤が約2時間、約6時間、約24時間、または約48時間で、mLあたりコロニー形成単位(CFU)の少なくとも3log減少の殺菌効果を示す、請求項4に記載の使用のための医薬製剤。 - 前記抗菌剤が前記抗菌剤への約24時間の曝露後にE.coli、Streptococcus spp.、Bacteroides spp、またはそれらの任意の組み合わせのCFU/mLの最小3log減少を示す、および/または
前記抗菌剤への前記細菌の曝露が前記細菌の再増殖を防止する、請求項4または5に記載の使用のための医薬製剤。 - 前記細菌が約7.45×10-9、約5.75×10-9、約5.15×10-9、約9.55×10-10、約1.85×10-10、約1.75×10-10、約1.50×10-10、または約1.05×10-10未満の自然突然変異の頻度を有する、および/または
前記抗菌剤が前記細菌に対して約0.01μg/mL~約32μg/mL、約0.05μg/mL~約1μg/mL、または約0.1μg/mL~約0.25μg/mLの突然変異防止濃度を示す、請求項2~6の何れか一項に記載の使用のための医薬製剤。 - 前記細菌がEscherichia coli、Streptococcus spp.、Bacteroides spp.、およびそれらの組み合わせからなる群から選択され、任意には、前記細菌が、E.coli、B.fragilis、B.vulgatus、B.ovatus、S.pneumonia、S.pyogenesおよびS.agalactiae、ならびにそれらの組み合わせからなる群から選択される、請求項7に記載の使用のための医薬製剤。
- SIBOの病因に関与する細菌の再増殖の防止を、それを必要とする対象において行うための方法における使用のための医薬製剤であって、
前記方法が抗菌剤を含む有効量の医薬製剤を前記対象に経口投与することであって、前記抗菌剤がSIBOの病因に関与する細菌に対して抗菌活性を示す、経口投与することと、前記製剤の投与開始時の前記細菌の量と比較して、CFU/mLの少なくとも3log減少で前記細菌の量を低下させることと、を含む、
前記抗菌剤への前記細菌の曝露が、前記細菌の再増殖を防止する、
任意には前記細菌が、E.coli、Streptococcus spp.、Bacteroides spp、またはそれらの任意の組み合わせからなる群から選択される、および
前記抗菌剤がメロペネムである、医薬製剤。 - SIBOの病因に関与する細菌の抗菌剤による治療に対する耐性の発現の防止を、それを必要とする対象において行うための方法における使用のための医薬製剤であって、
前記方法が抗菌剤を含む有効量の医薬製剤を前記対象に経口投与することを含む、
前記抗菌剤がメロペネムである、
任意には前記細菌が約7.45×10-9、約5.75×10-9、約5.15×10-9、約9.55×10-10、約1.85×10-10、約1.75×10-10、約1.50×10-10、または約1.05×10-10未満の自然突然変異の頻度を有する、医薬製剤。 - 前記抗菌剤が前記細菌に対して約0.01μg/mL~約32μg/mL、約0.05μg/mL~約1μg/mL、または約0.1μg/mL~約0.25μg/mLの突然変異防止濃度を示す、請求項10に記載の使用のための医薬製剤。
- 前記細菌がEscherichia coli、Streptococcus spp.、Bacteroides spp.、およびそれらの組み合わせからなる群から選択される、請求項10または11に記載の使用のための医薬製剤。
- 前記細菌がE.coli、B.fragilis、B.vulgatus、B.ovatus、S.pneumonia、S.pyogenesおよびS.agalactiae、およびそれらの組み合わせからなる群から選択される、請求項12に記載の使用のための医薬製剤。
- 前記細菌が摂取可能なデバイスによって対象の胃腸(GI)管から回収されたサンプル中において同定される、請求項1~13のいずれか一項に記載の使用のための医薬製剤。
- 前記細菌が摂取可能なデバイスによって対象のGI管から回収されたサンプル中において定量される、請求項1~14のいずれか一項に記載の使用のための医薬製剤。
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