JP2022541724A - 新規なフィーカリバクテリウムプラウスニッツィイ菌株eb-fpdk11およびその用途 - Google Patents
新規なフィーカリバクテリウムプラウスニッツィイ菌株eb-fpdk11およびその用途 Download PDFInfo
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- JP2022541724A JP2022541724A JP2021574236A JP2021574236A JP2022541724A JP 2022541724 A JP2022541724 A JP 2022541724A JP 2021574236 A JP2021574236 A JP 2021574236A JP 2021574236 A JP2021574236 A JP 2021574236A JP 2022541724 A JP2022541724 A JP 2022541724A
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- fpdk11
- prausnitzii
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- faecalibacterium prausnitzii
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Abstract
Description
本発明の他の態様は、フィーカリバクテリウムプラウスニッツィイ(F.prausnitzii)EB-FPDK11、前記フィーカリバクテリウムプラウスニッツィイ(F.prausnitzii)EB-FPDK11菌株の培養液、破砕物または抽出物からなる群より1つ以上を含む代謝性疾患の予防または治療用薬学的組成物を提供する。
したがって、本発明の薬学的組成物は、方法によって、散剤、顆粒剤、錠剤、カプセル剤、懸濁液、エマルジョン、シロップ、エアロゾルなどの経口型剤形、外用剤およびパッチの形態に剤形化して使用可能であり、組成物の製造に通常使用する適切な担体、賦形剤または希釈剤を追加的に含むことができる。
前記プロバイオティクス製剤は、当業界にて公知の方法により多様な剤形と方法で製造および投与可能である。例えば、本発明のフィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株、その培養液、前記培養液の濃縮液または乾燥物は、薬剤学的分野にて通常使用される担体と混合して、散剤(powder)、液剤(liquids and solutions)、錠剤(tablet)、カプセル(capsule)、シロップ(syrup)、懸濁剤(suspension)または顆粒剤(granule)などの形態に製造されて投与可能である。前記担体としては、例えば、結合剤、滑沢剤、崩壊剤、賦形剤、可溶化剤、分散剤、安定化剤、懸濁化剤、色素および香料などであってもよいが、これに限定されない。また、投与用量は、体内での活性成分の吸収度、不活性率、排泄速度、被投与者の年齢、性別、体重、状態および疾病の重度程度などによって適宜選択可能である。
1.1.フィーカリバクテリウムプラウスニッツィイ試料の確保および分離
健康な韓国人(女性、9歳、BMI15.5)の糞便からフィーカリバクテリウムプラウスニッツィイを分離するために、Martinの方法により、YBHI培地[Brain-heart infusion medium supplemented with 0.5%w/v yeast extract(Difco)、0.1%w/v D-cellobiose、0.1%w/v D-maltose]を用いて培養した後、EOS(Extremely Oxygen Sensitivity)菌種を選別した後に分離した。
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ィイ菌株であるか否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。その結果、図1に示された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ィイDSM 17677T菌株であるAとフィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株を1000倍の倍率で拡大したBとも、形状が棒状細胞で真っ直ぐなまたは曲がっている形態で類似の形態を示すことを確認した。
フィーカリバクテリウムプラウスニッツィイ菌株であるか否かを確認するために、分離された菌株を下記表1のFP-特異性プライマー(配列番号2および配列番号3)を用いてPCR分析を実施した。その結果、図2のように、陽性対照群菌株であるフィーカリバクテリウムプラウスニッツィイDSM 17677Tと類似のバンドで結果値が出たことを確認することができた。
前記のように分離された菌株がすでに報告された同種の標準菌株と比較して異なるか否かを確認するために、分子タイピングの一種であるRAPD(Random Amplified Polymorphic DNA)分析を実施した。このために、菌体から抽出したgDNA(genomic DNA)を対象に下記表2のuniversal primer(配列番号4~配列番号6)を用いてDNAを増幅した後、1%アガロースゲルで90分間電気泳動し、図3のように、UV transilluminatorを用いてDNA分節パターンを比較した。
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ィイ菌株であるか否かを確認するために、16S rRNA塩基配列を分析した後、BLASTして確認した結果、フィーカリバクテリウムプラウスニッツィイ種と99%以上一致しており、このような結果に基づいて分離された菌株をフィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株と名付け、韓国微生物保存センター(KCCM)に寄託して受託番号KCCM12621Pを受けた。
前記菌株の同定結果、現在知られている菌株に対して類似の菌株は存在するものの、正確に一致する結果は得られず、系統樹分析を行った。分離されたフィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株の全長16S rRNA遺伝子の塩基配列分析のために、下記表3の27F(配列番号7)および1492R(配列番号8)プライマーを用いて16S rRNA遺伝子を増幅した後、3730xl DNA Analyzer(Thermo Fisher Scientific、USA)を用いて塩基配列を決定した。このように得たEB-FPDK11菌株および標準菌株を含めてすで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、Maximum Likelihood methodにより図4のような系統樹を作成した。
2.1.抗菌剤感受性の確認
フィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株の抗菌剤感受性を把握するために、Clinical&Laboratory Standard Institute(CLSI)ガイドラインの液体培地微量希釈法によって嫌気性菌用抗菌剤(piperacillin-tazobactam、ceftizoxime、chloramphenicol、clindamycin、meropenem、moxifloxacin、metronidazole、ciprofloxacin)に対する最小阻害濃度(minimum inhibitory concentration、MIC)を確認した。
フィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株の安全性検証のために、溶血活性を保有するか否かを評価した。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 D-cellobiose、0.1%w/v D-maltose)に1.5%w/v bacto-agarと5%w/v defbrinated 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た後、コロニー周辺の溶血現象の有無を観察した。陽性対照群としてβ-hemolysisを起こすStreptococcus pyogenes ATCC 19615と比較した。
分離されたフィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株の機能性代謝物確認のために、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 D-cellobiose、0.1%w/v D-maltose)に菌株を24時間培養した後、12,000×gで5分間遠心分離して上澄液を回収し、上澄液は0.2μmのシリンジフィルタを用いて濾過した後、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)を用いており、条件は表5のように設定した。
3.1.HT-29腸上皮細胞における抗炎症効能評価
炎症性腸疾患において炎症反応調節にサイトカインが関与するので、フィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株を投与してサイトカインの遺伝子発現の変化を確認した。実験管実験で抗炎症効能評価実験をするために、ヒト由来大腸上皮細胞であるHT-29細胞(ATCCR HTB-38TM、USA)を培養した。10%FBS(fetal bovine serum、Hyclone、USA)、10μg/mlのゲンタマイシンを添加したMcCoy’s 5A modified media(Gibco、USA)を基本培養培地として用いて、37℃、5%CO2インキュベータ(NUAIRE、USA)で培養した。フィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株がHT-29細胞においてLPSで誘導された炎症性サイトカインであるIL-8遺伝子の発現を抑制するかを確認するために、下記表6のプライマー(配列番号9~12)を用いてリアルタイムPCRを進行させた。
抗炎症反応を観察するために樹状細胞(Dendritic cells、DC)のサイトカインの分泌を確認した。マウス骨髄由来樹状細胞(bone marrow derived dendritic cells、BMDC)を用いて菌株の抗炎症効能を評価するために、BMDC細胞を分離した。0.5mlのマイクロチューブに18Gニードルを用いて穴を開けた後、6週齢C57BL/6マウスの大腿骨と硬骨を分離して1.5mlの沈殿管に入れた後、10,000×gに15秒間遠心分離をした。PBSを用いて1.5mlの沈殿管に入ったペレットを3回洗浄した後、ペレットをRPMI-1640(10%FBS、1%P/S、media、1X mercaptoethanol、20μg GM-CSF)培地を入れて、150mmの培養皿に培養した。翌日に100mlのペトリ皿にBMDCを移して培養をし、5日目になる日に培養液10mlを15mlのコニカルチューブ(conical tube)に移して入れた後、1,000×gに15分間遠心分離して、上澄液を除去し、BMDC media10mlを添加してペトリ皿に入れた。6~7日目にBMDCを実験に使用した。フィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株の抗炎症効能分析のために、代表的な抗炎症サイトカインであるIL-10の分泌をmIL-10 ELISA(Invitrogen、USA)で分析した。
脂肪蓄積と肥満関連バイオマーカーの発現が本発明の菌株の投与によって影響を受けるか否かを調べた。
3T3-L1細胞において脂肪細胞の分化および脂肪の生成に本発明のフィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株が及ぼす影響を調べるために、Oil Red-O(ORO)染色実験を行った。まず、3T3-L1脂肪前駆細胞を脂肪細胞に分化させるために、24well plateに2×104/wellの細胞を分注した。10%BSを含むDMEM培地で4日間培養した後、細胞がプレートで飽和状態になると、分化用培地[DMEM、10%Fetal Bovine Serum、0.5mM IBMX(3-isobutyl-1-methylxanthine、Sigma I5879)1μMデキサメタゾン(Sigma D4902、FW392.5)、10mg/ml insulin]に切り替え、試料(フィーカリバクテリウムプラウスニッツィイの菌株または培養液)50μl(1×107/well)を処理した後、37℃、5%CO2条件で2日間培養した。以後、インスリン培地(10%FBS、10mg/ml insulin)に2日ごとに切り替えて、8日間同じ条件で培養した。フィーカリバクテリウムプラウスニッツィイの菌株と培養液を培地を切り替えるたびに同時に処理した。菌株と菌株培養液(107cfu/ml)は10%v/vで細胞に処理した。
菌株の脂肪細胞分化抑制に対する効果を確認するために、下記表7に記載の遺伝子特異的プライマー(配列番号13~24)を用いてリアルタイムPCRを進行させて、脂肪細胞への分化段階で脂肪細胞の分化と成熟に関与する転写因子であるC/EBPα(CCAAT/enhancer binding protein alpha)、脂肪合成遺伝子aP2(adipocyte protein2)、FAS(fatty acid synthase)、ACC1(acetyl-coenzyme A-carboxylase)、LPL(lipoprotein lipase)遺伝子のmRNA発現を分析した。
5.1.非アルコール性脂肪肝炎動物モデルの作製
動物実験はInstitutional Animal Care and Use Committee(IACUC)のAnimal use and Care Protocolを守って進行させた。実験動物は8週齢雄マウスC57BL/6(グループあたり9匹)を購入して、1週間適応期間を持った後、12週間飼育が行われ、飼育環境は一定の温度(22℃)と相対湿度(40~60%)を維持し、12時間の周期で明暗を調節しながら1週間適応させた。
非アルコール性脂肪肝炎誘導実験を行って16週後、実験群の体重の変化を測定して、その結果を図12に示した。
フィーカリバクテリウムプラウスニッツィイEB-FPDK11菌株の投与が耐糖能に及ぼす効果を確認するために、実験を行って16週後、マウスを18時間絶食にした状態でブドウ糖(glucose2g/kg)を経口投与した。ブドウ糖の投与直前と投与後30、60、90、および120分後に尾静脈から血液を採取して血糖を血糖測定器で測定して、その結果を図13に示した。
実験終了時にCO2で痲酔した状態で肝と脾臓を摘出して生理食塩水で洗浄し、水分を除去した後、重量を測定し、それらの大きさと色を肉眼で観察した。
18時間絶食にした後、各実験動物から血液を採取した後、分離した血清(serum)で脂質含有量の指標である中性脂肪(triglyceride、TG)、総コレステロール(total cholesterol、TC)、肝機能の指標であるGOT(glutamic oxaloacetic transaminase)、GPT(glutamic pyruvic transaminase)を測定して、図16に示した。脂質組成の指標であるTG、TC、GOT、GPT濃度はいずれも、ASAN製薬から購入した個別測定キットを用いて定量した。
EB-FPDK11菌株の投与が非アルコール性脂肪肝炎の緩和に及ぼす影響を観察するために、肝組織切断面のhematoxylin&eosin(H&E)染色と、肝線維化を測定可能なSirius red染色、肝損傷時に発現するalpha-smooth muscle actin(α-SMA)の発現を染色して観察した。マウスから分離した肝組織を約5μmに組織切片にした後、パラフィンに包埋し、それぞれの染色により形態学的変化の差を観察した。それぞれの染色による肝損傷の程度はImage Jプログラムによりpositive areaを百分率(%)に換算して示した。
マウスの肝組織内脂質抽出物により中性脂肪と総コレステロールを分析した。肝組織30mgにPBSを120μl入れて、ホモゲナイザーを用いて粉砕後、Chloroform320μlとMeOH160μlを入れて混合物を作った。前記混合物を常温で1日間シェイキングインキュベータで混合した後、2000rpmで遠心分離し、上澄液のみ別途に分離して溶媒を蒸発させた。その後、前記溶媒を蒸発させた上澄液を1mlのisopropanolに溶かした後、TG、TC測定キット(ASAN製薬、韓国)を用いてマウスの全体肝重量対比で換算して定量した。
Claims (7)
- 受託番号KCCM12621Pであるフィーカリバクテリウムプラウスニッツィイ(Faecalibacterium prausnitzii)EB-FPDK11菌株。
- 請求項1に記載の菌株、前記菌株の培養液、前記菌株の破砕物および前記菌株の抽出物からなる群より1つ以上を含む炎症性疾患の予防または治療用薬学的組成物。
- 請求項1に記載の菌株、前記菌株の培養液、前記菌株の破砕物および前記菌株の抽出物からなる群より1つ以上を含む肝疾患の予防または治療用薬学的組成物。
- 請求項1に記載の菌株、前記菌株の培養液、前記菌株の破砕物および前記菌株の抽出物からなる群より1つ以上を含む代謝性疾患の予防または治療用薬学的組成物。
- 請求項1に記載の菌株、前記菌株の培養液、前記菌株の破砕物および前記菌株の抽出物からなる群より1つ以上を含む炎症性疾患の予防または改善用食品組成物。
- 請求項1に記載の菌株、前記菌株の培養液、前記菌株の破砕物および前記菌株の抽出物からなる群より1つ以上を含む肝疾患の予防または改善用食品組成物。
- 請求項1に記載の菌株、前記菌株の培養液、前記菌株の破砕物および前記菌株の抽出物からなる群より1つ以上を含む代謝性疾患の予防または改善用食品組成物。
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PCT/KR2020/008348 WO2021261632A1 (ko) | 2020-06-24 | 2020-06-26 | 신규한 피칼리박테리움 프로스니치 균주 eb-fpdk11 및 그의 용도 |
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US20220323514A1 (en) | 2022-10-13 |
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