JP6783247B2 - ガン予防における短鎖脂肪酸の使用 - Google Patents
ガン予防における短鎖脂肪酸の使用 Download PDFInfo
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- JP6783247B2 JP6783247B2 JP2017557279A JP2017557279A JP6783247B2 JP 6783247 B2 JP6783247 B2 JP 6783247B2 JP 2017557279 A JP2017557279 A JP 2017557279A JP 2017557279 A JP2017557279 A JP 2017557279A JP 6783247 B2 JP6783247 B2 JP 6783247B2
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- 229950003017 zeniplatin Drugs 0.000 description 1
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Description
本出願は、2015年1月23日に出願された米国仮出願第62/106,778号(その内容は、その全体が参照により本明細書に組み入れられる)の優先権を主張する。
肝細胞ガン(HCC)は、世界で5番目に多いガンであり、ガンによる死亡の2番目に多い原因である(Ding J et al., Cancer Lett, 2014; 346(1):17-23)。早期HCCは、治癒的アプローチを適用し得る場合には無症候性であることが多く、進行性疾患が検出される頃には、利用可能な処置選択肢がほとんどない。未処置HCCの生存率は5年間で3%未満であり、マルチキナーゼ阻害剤ソラフェニブを適用した場合であっても、平均余命は平均で3カ月延長されたに過ぎなかった(Peck-Radosavljevic M, Liver Cancer, 2014; 3(2):125-31)。ソラフェニブ+細胞毒性薬を使用した併用療法は、寿命を診断後約1年間延長させた。
本発明は、短鎖脂肪酸が肝臓ガンの化学予防治療アプローチとして有効であるという発見に部分的に基づくものである。本明細書に提示される結果は、肝臓において炎症、肝炎及び前ガン病変を有する被験体への短鎖脂肪酸の投与が、肝細胞ガンへの肝疾患の進行の予防又は遅延に有効であることを実証する。
本発明の図及び説明は、明確性のために、典型的な顕微鏡デバイスに見られる他の多くの要素を排除する一方、本発明の明確な理解に関連する要素を示すために簡略化されていることを理解すべきである。当業者であれば、他の要素及び/又は工程が本発明の実施に望ましい及び/又は必要であることを認識し得る。しかしながら、このような要素及び工程は当技術分野で周知であり、それらは本発明のより良い理解を促すものではないので、このような要素及び工程の議論は、本明細書では提供されない。本明細書の開示は、当業者に公知のこのような要素及び方法に対する全ての変形及び改変を対象とする。
本発明は、SCFAを産生するプロバイオティクス細菌の導入又はSCFAのみの導入が、HCCの病因を遅らせるという発見に部分的に基づくものである。本明細書に提示される結果は、SCFAを産生するプロバイオティクス細菌及び対応するSCFAのみ(プロバイオティクス処置なし)が、HBxトランスジェニックマウスにおける異形成性結節及びHCCの出現を抑制することを実証する。したがって、本発明は、ガン化学予防のための簡便なアプローチとして、SCFAを産生する細菌又はSCFAのみの導入を使用する組成物及び方法を含む。本発明は、肝臓ガン及び解剖学的に大腸から遠位に位置する他の腫瘍型に対する新規適用である。
一実施態様では、本発明は、短鎖脂肪酸又は短鎖脂肪酸の組み合わせを提供する。一実施態様では、本発明は、プロバイオティクス細菌又はプロバイオティクス細菌の組み合わせを提供する。様々な実施態様では、本発明は、それを必要とする被験体、細胞、組織又は器官における肝細胞ガンの発症を予防し又は遅延させるための組成物を含む。本発明の組成物は、肝臓炎、肝疾患、前ガン病変等を治療又は予防するための組成物を含む。
様々な実施態様では、本発明は、肝細胞ガンの発症を予防し又は遅延させる組成物及び方法を含む。様々な実施態様では、本発明は、肝臓炎、肝疾患及び前ガン病変を治療又は予防することによって、肝細胞ガンの発症を予防し又は遅延させる組成物及び方法を含む。一実施態様では、肝細胞ガンの発症を予防し又は遅延させるための組成物は、短鎖脂肪酸又は短鎖脂肪酸の組み合わせを含む。
様々な実施態様では、本発明は、肝細胞ガンの発症を予防し又は遅延させる組成物及び方法を含む。様々な実施態様では、本発明は、肝臓炎、肝疾患及び前ガン病変を治療又は予防することによって、肝細胞ガンの発症を予防し又は遅延させる組成物及び方法を含む。一実施態様では、肝細胞ガンの発症を予防し又は遅延させるための組成物は、プロバイオティクス細菌又はプロバイオティクス細菌の組み合わせを含む。
一実施態様では、本発明は、プロバイオティクス又はプロバイオティクスの組み合わせを使用して、肝細胞ガンを治療、阻害、予防又は軽減するための方法を提供する。別の実施態様では、本発明は、短鎖脂肪酸又は短鎖脂肪酸の組み合わせを使用して、肝細胞ガンを治療、阻害、予防又は軽減するための方法を提供する。
他の抗ガン薬としては、限定されないが、20−エピ−1,25 ジヒドロキシビタミンD3;5−エチニルウラシル;アビラテロン;アクラルビシン;アシルフルベン;アデシペノール;アドゼレシン;アルデスロイキン;ALL−TKアンタゴニスト;アルトレタミン;アンバムスチン;アミドックス;アミフォスチン;アミノレブリン酸;アムルビシン;アムサクリン;アナグレリド;アナストロゾール;アンドログラホリド;血管新生阻害剤;アンタゴニストD;アンタゴニストG;アンタレリクス;抗背側化形態形成タンパク質−1;抗アンドロゲン薬(前立腺ガン);抗エストロゲン薬;抗新生物薬;アンチセンスオリゴヌクレオチド;アフィジコリングリシン塩;アポトーシス遺伝子モデュレーター;アポトーシスレギュレーター;アプリン酸;ara−CDP−DL−PTBA;アルギニンデアミナーゼ;アスラクリン;アタメスタン;アトリムスチン;アキシナスタチン1;アキシナスタチン2;アキシナスタチン3;アザセトロン;アザトキシン;アザチロシン;バッカチンIII誘導体;バラノール;バチマスタット;BCR/ABLアンタゴニスト;ベンゾクロリン;ベンゾイルスタウロスポリン;βラクタム誘導体;β−アレチン;ベタクラマイシンB;ベツリン酸;bFGF阻害剤;ビカルタミド;ビサントレン;ビスアジリジニルスペルミン;ビスナフィド;ビストラテンA;ビゼレシン;ブレフラート;ブロピリミン;ブドチタン;ブチオニンスルホキシイミン;カルシポトリオール;カルフォスチンC;カンプトテシン誘導体;カナリアポックスIL−2;カペシタビン;カルボキサミド−アミノ−トリアゾール;カルボキシアミドトリアゾール;CaRest M3;CARN700;軟骨由来阻害剤;カルゼレシン;カゼインキナーゼ阻害剤(ICOS);カスタノスペルミン;セクロピンB;セトロレリクス;クロリン;クロロキノキサリンスルホンアミド;シカプロスト;cis−ポルフィリン;クラドリビン;クロミフェン類似体;クロトリマゾール;コリスマイシンA;コリスマイシンB;コンブレタスタチンA4;コンブレタスタチン類似体;コナゲニン;クラムベシジン816;クリスナトール;クリプトフィシン8;クリプトフィシンA誘導体;クラシンA;シクロペンタントラキノン;シクロプラタム(cycloplatam);シペマイシン;シタラビンオクホスファート;細胞溶解因子;サイトスタチン;ダクリキシマブ(dacliximab);デシタビン;デヒドロジデムニンB;デスロレリン;デキサメタゾン;デキシホスファミド(dexifosfamide);デクスラゾキサン;デクスベラパミル;ジアジコン;ジデムニンB;ジドックス;ジエチルノルスペルミン;ジヒドロ−5−アザシチジン;9−ジヒドロタキソール(dihydrotaxol, 9-);ジオキサマイシン;ジフェニルスピロムスチン;ドセタキセル;ドコサノール;ドラセトロン;ドキシフルリジン;ドロロキシフェン;ドロナビノール;デュオカルマイシンSA;エブセレン;エコムスチン;エデルホシン;エドレコロマブ;エフロルニチン;エレメン;エミテフール;エピルビシン;エプリステリド;エストラムスチン類似体;エストロゲンアゴニスト;エストロゲンアンタゴニスト;エタニダゾール;エトポシドリン酸塩;エキセメスタン;ファドロゾール;ファザラビン;フェンレチニド;フィルグラスチム;フィナステリド;フラボピリドール;フレゼラスチン;フルアステロン;フルダラビン;塩酸フルオロダウノルニシン(fluorodaunorunicin);ホルフェニメクス;フォルメスタン;フォストリエシン;フォテムスチン;ガドリニウムテキサフィリン;硝酸ガリウム;ガロシタビン;ガニレリックス;ゲラチナーゼ阻害剤;ゲムシタビン;グルタチオン阻害剤;ヘプスルファム;ヘレグリン;ヘキサメチレンビスアセトアミド;ヒペリシン;イバンドロン酸;イダルビシン;イドキシフェン;イドラマントン;イルモホシン;イロマスタット;イミダゾアクリドン(imidazoacridone);イミキモド;免疫刺激ペプチド;インスリン様成長因子−1レセプター阻害剤;インターフェロンアゴニスト;インターフェロン;インターロイキン;イオベングアン;ヨードドキソルビシン;
4−イポメアノール(ipomeanol, 4-);イロプラクト(iroplact);イルソグラジン;イソベンガゾール(isobengazole);イソホモハリコンドリン(isohomohalicondrin)B;イタセトロン;ジャスプラキノライド;カハラリドF;ラメラリン−Nトリアセタート;ランレオチド;レイナマイシン;レノグラスチム;硫酸レンチナン(lentinan sulfate);レプトルスタチン;レトロゾール;白血病阻害因子;白血球αインターフェロン;ロイプロリド+エストロゲン+プロゲステロン;ロイプロレリン;レバミゾール;リアロゾール;直鎖ポリアミン類似体;親油性二糖類ペプチド;親油性白金化合物;リッソクリナミド7;ロバプラチン;ロンブリシン;ロメトレキソール;ロニダミン;ロソキサントロン;ロバスタチン;ロキソリビン;ルルトテカン;ルテチウムテキサフィリン;リソフィリン;溶解ペプチド;メイタンシン(maitansine);マンノスタチンA;マリマスタット;マソプロコール;マスピン;マトリリシン阻害剤;マトリックスメタロプロテイナーゼ阻害剤;メノガリル;メルバロン;メテレリン;メチオニナーゼ;メトクロプラミド;MIF阻害剤;ミフェプリストン;ミルテホシン;ミリモスチム;ミスマッチ二本鎖RNA;ミトグアゾン;ミトラクトール;マイトマイシン類似体;ミトナフィド;マイトトキシン線維芽細胞成長因子−サポリン;ミトキサントロン;モファロテン;モルグラモスチム;モノクローナル抗体;ヒト絨毛性ゴナドトロピン(gonadotrophin);モノホスホリルリピドA+ミオバクテリウム(myobacterium)細胞壁sk;モピダモール;多剤耐性遺伝子阻害剤;多発腫瘍サプレッサー1−ベース療法;マスタード抗ガン剤;ミカペルオキシドB;ミコバクテリア細胞壁抽出物;ミリアポロン;N−アセチルジナリン;N−置換ベンズアミド;ナファレリン;ナグレスチップ(nagrestip);ナロキソン+ペンタゾシン;ナパビン(napavin);ナフテルピン;ナルトグラスチム;ネダプラチン;ネモルビシン;ネリドロン酸;中性エンドペプチダーゼ;ニルタミド;ニサマイシン;一酸化窒素モデュレーター;ニトロキシド抗酸化剤;ニトルリン(nitrullyn);O6−ベンジルグアニン;オクトレオチド;オキセノン;オリゴヌクレオチド;オナプリストン;オンダンセトロン;オンダンセトロン;オラシン;経口サイトカイン誘導因子;オルマプラチン;オサテロン;オキサリプラチン;オキサウノマイシン;パクリタキセル;パクリタキセル類似体;パクリタキセル誘導体;パラウアミン;パルミトイルリゾキシン;パミドロン酸;
パナキシトリオール;パノミフェン;パラバクチン;パゼリプチン;ペグアスパラガーゼ;ペルデシン;ポリ硫酸ペントサンナトリウム;ペントスタチン;ペントロゾール(pentrozole);ペルフルブロン;ペルホスファミド;ペリリルアルコール;フェナジノマイシン;酢酸フェニル;ホスファターゼ阻害剤;ピシバニール;ピロカルピン塩酸塩;ピラルビシン;ピリトレキシム;プラセチン(placetin)A;プラセチン(placetin)B;プラスミノーゲン活性化因子阻害剤;白金錯体;白金化合物;白金−トリアミン錯体;ポルフィマーナトリウム;ポルフィロマイシン;プレドニゾン;プロピルビス−アクリドン;プロスタグランジンJ2;プロテアソーム阻害剤;プロテインAベースの免疫モデュレーター;タンパク質キナーゼC阻害剤;タンパク質キナーゼC阻害剤(微細藻);タンパク質チロシンホスファターゼ阻害剤;プリンヌクレオシドホスホリラーゼ阻害剤;プルプリン;ピラゾロアクリジン;ピリドキシル化ヘモグロビンポリオキシエチレンコンジュゲート;rafアンタゴニスト;ラルチトレキセド;ラモセトロン;rasファルネシルタンパク質トランスフェラーゼ阻害剤;ras阻害剤;ras−GAP阻害剤;脱メチル化レテリプチン;エチドロン酸レニウムRe186(rhenium Re 186 etidronate);リゾキシン;リボザイム;RIIレチンアミド;ログレチミド;ロヒツキン;ロムルチド;ロキニメクス;ルビギノンB1;ルボキシル;サフィンゴール;サイントピン;SarCNU;サルコフィトールA;サルグラモスチム;Sdi1模倣体;セムスチン;老化由来阻害剤1;センスオリゴヌクレオチド;シグナル伝達阻害剤;シグナル伝達モデュレーター;単鎖抗原結合タンパク質;シゾフィラン;ソブゾキサン;ボロカプト酸ナトリウム(sodium borocaptate);フェニル酢酸ナトリウム;ソルベロール(solverol);ソマトメジン結合タンパク質;ソネルミン;スパルホス酸;スピカマイシンD;スピロムスチン;スプレノペンチン;スポンジスタチン1;スクアラミン;幹細胞阻害剤;幹細胞***阻害剤;スチピアミド;ストロメリシン阻害剤;スルフィノシン;超活性血管作動性腸管ペプチドアンタゴニスト;スラジスタ(suradista);スラミン;スワインソニン;合成グリコサミノグリカン;タリムスチン;タモキシフェンメチオジド;タウロムスチン;タザロテン;テコガランナトリウム;テガフール;テルラピリリウム;テロメラーゼ阻害剤;テモポルフィン;テモゾロマイド;テニポシド;テトラクロロデカオキシド;テトラゾミン;タリブラスチン;チオコラリン;トロンボポエチン;トロンボポエチン模倣体;チマルファシン;チモポエチンレセプターアゴニスト;チモトリナン;甲状腺刺激ホルモン;エチルエチオプルプリンスズ;チラパザミン;二塩化チタノセン;トプセンチン;トレミフェン;全能性幹細胞因子;翻訳阻害剤;トレチノイン;トリアセチルウリジン;トリシリビン;トリメトレキサート;トリプトレリン;トロピセトロン;ツロステリド;チロシンキナーゼ阻害剤;チルホスチン;UBC阻害剤;ウベニメクス;尿生殖洞由来成長阻害因子;ウロキナーゼレセプターアンタゴニスト;バプレオチド;バリオリンB;ベクター系、赤血球遺伝子療法;ベラレソール;ベラミン(veramine);ベルジン;ベルテポルフィン;ビノレルビン;ビンキサルチン(vinxaltine);ビタキシン;ボロゾール;ザノテロン;ゼニプラチン;ジラスコルブ及びジノスタチンスチマラマーが挙げられる。一実施態様では、抗ガン薬は、5−フルオロウラシル、タキソール又はロイコボリンである。
本発明は、1つ以上の本発明の組成物を含む医薬組成物を含む。本明細書に記載される医薬組成物の製剤は、薬理学分野で公知の又は将来開発される任意の方法によって調製され得る。一般に、このような調製方法は、有効成分を担体又は1つ以上の他の補助成分と合わせ、次いで、必要な場合又は望ましい場合には、生成物を所望の単回投与単位又は複数回投与単位に成形又はパッケージングする工程を含む。
本発明はまた、本発明の方法に有用な化合物と、例えば、本明細書の他の箇所に記載される短鎖脂肪酸の投与方法又は本明細書の他の箇所に記載されるプロバイオティクス細菌の投与方法を記載する教材とを含むキットを含む。非経口投与に適切な医薬組成物の製剤は、薬学的に許容し得る担体、例えば滅菌水又は滅菌等張生理食塩水と組み合わせた有効成分を含む。このような製剤は、ボーラス投与又は連続投与に適切な形態で調製、パッケージング又は販売され得る。注射用製剤は、単位剤形で、例えばアンプルで、又は保存剤を含有する複数回投与容器で調製、パッケージング又は販売され得る。非経口投与のための製剤としては、限定されないが、懸濁液、溶液、油性又は水性ビヒクル中のエマルション、ペースト、及び埋め込み可能な持続放出性又は生分解性製剤が挙げられる。このような製剤は、限定されないが、懸濁剤、安定化剤又は分散剤を含む1つ以上のさらなる成分をさらに含み得る。非経口投与のための製剤の一実施態様では、有効成分は、再構成組成物の非経口投与前に適切なビヒクル(例えば、発熱物質を含まない滅菌水)で再構成するための乾燥(すなわち、粉末又は顆粒)形態で提供される。
以下の実験例を参照して、本発明をさらに詳細に説明する。これらの実施例は、例示目的でのみ提供されるものであり、特に指定がない限り、限定することを意図するものではない。したがって、本発明は、以下の実施例に限定されると決して解釈されるべきではなく、むしろ、本明細書で提供される教示の結果として明らかになる任意かつ全ての変形を包含すると解釈されるべきである。
B型肝炎ウイルス(HBV)による慢性感染症は、慢性肝疾患(CLD)の進行の発展及び肝細胞ガン(HCC)の出現に関連する。HCCは、世界中で流行しているガンであり、処置選択肢がほとんどない。HCCは感染の数十年後に発症し、慢性炎症のバックグラウンドで出現することが最も多いことを考慮して、炎症を抑制することが公知の選択プロバイオティクス細菌を、HCCの出現を予防し又は遅延させるための簡便で安価な手段として使用し得るという仮説を試験するために、実験を設計した。これを試験するために、HCCに至る進行性肝病変を発症するB型肝炎x(HBx)トランスジェニックマウスを、プロバイオティクス細菌の混合物(Synbiotic 2000(商標))で処置した。その結果、コントロールトランスジェニックマウスと比較して、異形成性結節及びHCC結節の数及びサイズの有意な減少が示された。選択した免疫及びガン関連マーカーのマイクロアレイ分析により、コントロールマウスと比較して、Synbiotic 2000(商標)で処置したマウスの肝臓における強い発現減少が示された。使用した細菌は、複雑な炭水化物を短鎖脂肪酸(SCFA)(これは、他の系で抗炎症特性を有することが公知である)に代謝するので、並行実験では、細菌の非存在下で、Synbiotic 2000(商標)(酢酸塩、プロピオン酸塩(proprionate)、酪酸塩)によって作製したSCFAの組み合わせをHBxトランスジェニックマウスに給餌した。その結果、異形成性結節及びHCC結節の数及びサイズの強い減少が再び示された。これらの結果は、Synbiotic 2000(商標)又はSCFAの形態のそれらの代謝副産物がHCCの病因を弱毒化し、HCCだけではなく、おそらくは慢性炎症のバックグラウンドで発症することが多い他のガンに対するガン化学予防アプローチとして有用であり得ることを示している。
マウス
HCCの病因を研究し、新たな処置アプローチを評価するために、HBxトランスジェニックマウスモデルが作られている(Yu DY et al., J Hepatol, 1999; 31:123-132)。出生時において、これらのHBxトランスジェニックマウスは、肝臓におけるHBx発現がほとんど又は全くなく、病態もない。3〜4カ月齢までに、それらは、肝炎/脂肪症に関連する検出可能なHBxを発症する。6〜7カ月齢までに、肝内HBxの存在、頻度及び分布がかなり高くなり、これは、異形成性結節及び微小HCCの出現に関連する。9〜10カ月齢までに、広範囲のHBx染色が、巨視的なHCC結節の出現に関連する。この一連の事象が慢性ヒト感染症のものと類似することを考慮して、この動物モデルを本研究に使用した。
Synbiotic 2000(商標)は、Medipharm(Des Moines, Iowa)によって提供された。それは、1包み当たり、4つの乳酸産生桿菌(1010のLactobacillus plantarum 2362、1010のLactobacillus paracasei subsp paracasei 19、1010のLeuconostoc mesenteroides 32-77:1e、及び1010のPediococcus pentosaceus 5-33:3)の混合物と、4つの生物活性植物繊維種(イヌリン2.5g;ペクチン2.5g;β−グルカン2.5g及び耐性デンプン2.5g)の混合物とを含有する。それを、強制飼養によって0.05g/用量(2.5g/水30ml、用量0.6ml/マウス)で3カ月間毎日投与した。
テールスニップ分析及びリアルタイムPCR増幅によって、HBx遺伝子の存在について、HBxトランスジェニックマウスを試験した。以前に記載されているように(Arzumanyan A et al., Cancer Res, 2012; 72(22):5912-5920)、ホルマリン固定パラフィン包埋肝臓組織から切断した切片の免疫組織化学染色によって、HBxタンパク質を評価した。この研究のために、3カ月、6カ月及び9カ月の時点の10匹のHBxトランスジェニックマウスの群に、新たに再構成したSynbiotic 2000(商標)を3カ月間毎日強制飼養した。コントロール群には、Synbiotic 2000(商標)に代えてPBSを強制飼養した年齢性別適合HBxトランスジェニックマウスが含まれていた。年齢性別適合HBx陰性同腹仔の群には、Synbiotic 2000(商標)又はPBSを強制飼養した。アラニンアミノトランスフェラーゼ(ALT)測定(ALT/GPT 50, Sigma Chemical Co., St. Louis, MO)のために、全てのマウスを眼窩後から定期的に採血し、処置の3カ月後に安楽死させた。各採血の直前にマウスを計量し、安楽死後に肝臓重量を測定した。各肝臓の表面上に見える腫瘍結節を数えた。次いで、各葉由来の肝臓のサンプルを包埋し、切片をH&Eによって染色した。2人が独立してコード下で光学顕微鏡によって各肝臓のスライドを検査し、様々な病変を記録した。全てのマウスの残りの肝臓組織を液体窒素中で急速凍結し、−80℃で保存した。この研究のための動物プロトコールは全て、Temple University Institutional Animal Care and Use Committeeによって承認された。
オートクレーブ処理された非使い捨てプローブを備えるハンドヘルドローター−ステーターホモジナイザー(TissueRuptor, Qiagen)を使用して、全てのマウスの凍結肝臓組織サンプルを溶解バッファー(RTL)中でホモジナイズした。次いで、製造業者のプロトコールに従ってRNeasy Mini Kit (Qiagen)を使用して、各サンプル由来の全RNAを抽出した。RNase-Free DNase kit(Qiagen)を使用して、夾雑DNAを除去した。Nanodrop UV-Vis Spectrophotometer(Thermo Scientific)によって各サンプル1μlの吸光度を260及び280nmで読み取って、RNA濃度を測定した。初期組織サンプル30mgから、溶出バッファー(最終容量50μl)中50〜1800ng/μlの典型的な収量が得られた。次いで、サンプルを50ng(ηg)/μlの最終濃度に等分し、−80℃で保存した。
マイクロウェルでフォーマットされたCustom RT2 Profiler PCR-array(SA Biosciences, Qiagen, Izasa)は、本研究の特定の研究的興味に合った遺伝子のパネルを含んでいた。RT2 SYBR Green Rox qPCR Mastermix(Qiagen)を使用して、cDNAの増幅を実施した。各逆転写サンプルを1:3希釈し、その51μlをマスターミックス550μlに追加した。この反応混合物から、各混合物10μlを各ウェルにロードした。
コントロールマウスと比較した処置マウスにおける様々な肝病変間の関係を評価するために、カイ二乗検定を使用した。p<0.05の場合に、有意性が得られた。処置マウスとコントロールマウスとの間の腫瘍サイズの差を評価するために、スチューデントt検定を使用した。P<0.05の場合に、有意性が得られた。
HCCの病因は免疫媒介性であること(Feitelson MA et al., Cancer Lett, 2009; 286(1):69-79)、及びSynbiotic 2000(商標)中のプロバイオティクス細菌は抗炎症特性を有し得ることを考慮して、HBxトランスジェニックマウスへのSynbiotic 2000(商標)の給餌がCLDの発症及びそのHCCへの進行を遅らせ又は阻止し得るという仮説を試験するために、実験を設計した。したがって、3カ月、6カ月及び9カ月齢の時点から1群当たり10匹のマウスを3カ月間強制飼養した。処置開始の直前に、及び動物を安楽死させるまで1カ月間隔で、マウスを眼窩後から採血した。次いで、肝臓を摘出し、各葉由来のサンプルをホルマリン固定し、パラフィン包埋した一方、残りの肝臓サンプルを急速凍結した。
HCCは慢性炎症の状況で生じること、及びHBxはこの腫瘍型の発症を促進することを考慮して、Synbiotic 2000(商標)が、HCCの病因に寄与し得る選択腫瘍関連シグナル伝達経路及び/又はサイトカインの発現に対して影響を与えるかを決定するために、限定的なPCRアレイ分析を実施した。3カ月齢マウスをSynbiotic 2000(商標)で3カ月間処置し、選択遺伝子の発現プロファイルをPBS処置動物のものと比較したところ、腫瘍形成に関連するマーカーが1.5〜3倍アップレギュレーションしていた(図4A)。Synbiotic 2000(商標)処置を6カ月齢マウスに3カ月間施した場合、ほとんどの腫瘍関連マーカーの発現は、試験ではコントロールマウスと比較してアップレギュレーションもダウンレギュレーションもしていなかった(図4B)。注目すべき例外はEGFR(これは、成長を刺激する)であり、Synbiotic 2000(商標)では8倍超ダウンレギュレーションしていた。対照的に、Synbiotic 2000(商標)を9カ月齢マウスに3カ月間与えた場合、ほとんどの腫瘍形成に関連するマーカーは、試験ではプラセボ処置動物と比較して強くダウンレギュレーションしていた(図4C)。これらのマーカーには、ヘッジホッグ経路におけるシグナル伝達分子であるGli1及び2、いくつかのNotchレセプター、TGFβ−1及び2並びにTGFβR1(これらは通常、細胞成長を負にレギュレーションする)、Tcf3(これは、β−カテニンシグナル伝達に重要である)、Akt1(これは、発ガンにおいて構成的に活性化されることが多い)並びにMMP−9及び−10(これらは、転移を促進する)が含まれていた(図4C)。6カ月の時点に安楽死させた3カ月齢マウスの免疫媒介マーカーに関して、ほとんどの免疫マーカーは、試験ではプラセボ処置と比較して2倍以内で上昇も抑制もしていなかった(図4A)。9カ月の時点に安楽死させた6カ月齢マウスにおいて、同様の結果が得られた(図4B)。しかしながら、12カ月の時点に安楽死させた9カ月齢マウス間では、免疫応答関連マーカーは全て、試験ではコントロールマウスと比較して低下していたが(図4C)、これは、HBxに対する免疫応答及び/又はHBxの性質のシフトが、疾患進行に伴う肝臓の変化を誘導したことを示唆している。年齢性別適合トランスジェニック(transgene)陰性同腹仔由来の肝臓においてこの分析を実施したところ、これらのマーカーのレベルに統計的な有意差はなかったが、これは、それらの差が、肝臓に対するHBxの影響の増加に関係しており、加齢に伴う変化によるものではなかったことを示唆している(データは示さず)。
Synbiotic 2000(商標)中の乳酸産生菌は、SCFAに代謝される豊富なプレバイオティクス栄養源を供給される。SCFAは抗炎症性であることが公知であり、HCCは炎症性成分を有する慢性肝疾患のバックグラウンドで生じるので、SCFAがHCCの病因に対してSynbiotic 2000(商標)と同じ影響を有するかを決定するために、実験を設計した。Synbiotic 2000(商標)の最大の影響は、HCCを発症していた9〜12カ月齢マウスで観察されたので、9カ月齢マウスにSFCAを強制飼養によって3カ月給餌し、次いで、病変の存在、頻度及び分布について、それらの肝臓を検査した。12カ月齢のSCFA処置マウスから肝臓を摘出したところ、肝臓表面上の腫瘍は、PBS処置マウスでは、SCFA処置マウスと比較して多くかつ大きかった(図5)。各葉から顕微鏡用切片を調製し、腫瘍サイズを再び評価したところ、存在する腫瘍の52%が小さかった一方(直径<0.5cm)、32%は大きかった(直径>1cm)。対照的に、プラセボ処置マウスでは、29%のみが小さかったが、50%は大きいと考えられた(図6;X2=4.59、P<0.05)。これは、2つのマウス群における大きな腫瘍:小さな腫瘍の比にも反映される。プラセボ処置マウスでは、比は1.75であったが、SCFA処置マウスでは、比は0.62にシフトしており、これは、SCFAが大きな腫瘍の発生を部分的に阻止したことを示唆している。全てのマウスにおいて、腫瘍形態は、腫瘍サイズとは無関係に、未分化HCCに特徴的なものであった(図6D)。
Claims (18)
- 被験体における肝細胞ガンの発症を予防し又は遅延させるための医薬組成物であって、酢酸、プロピオン酸、酪酸、及びその薬学的に許容し得る塩からなる群より選択される少なくとも2つの短鎖脂肪酸及び/又はその塩を含む、医薬組成物。
- 短鎖脂肪酸及び/又はその塩が、酢酸又はその薬学的に許容し得る塩、プロピオン酸又はその薬学的に許容し得る塩、及び酪酸又はその薬学的に許容し得る塩を含む、請求項1に記載の医薬組成物。
- 薬学的に許容し得る賦形剤をさらに含む、請求項1又は2に記載の医薬組成物。
- 別の治療剤と組み合わせて投与される、請求項1〜3のいずれか一項に記載の医薬組成物。
- 経口投与される、請求項1〜4のいずれか一項に記載の医薬組成物。
- 食品又は飲料と共に投与される、請求項5に記載の医薬組成物。
- 被験体における肝疾患を処置するための医薬組成物であって、酢酸、プロピオン酸、酪酸、及びその薬学的に許容し得る塩からなる群より選択される少なくとも2つの短鎖脂肪酸及び/又はその塩を含む、医薬組成物。
- 短鎖脂肪酸及び/又はその塩が、酢酸又はその薬学的に許容し得る塩、プロピオン酸又はその薬学的に許容し得る塩、及び酪酸又はその薬学的に許容し得る塩を含む、請求項7に記載の医薬組成物。
- 薬学的に許容し得る賦形剤をさらに含む、請求項7又は8に記載の医薬組成物。
- 別の治療剤と組み合わせて投与される、請求項7〜9のいずれか一項に記載の医薬組成物。
- 経口投与される、請求項7〜10のいずれか一項に記載の医薬組成物。
- 食品又は飲料と共に投与される、請求項11に記載の医薬組成物。
- 被験体における肝細胞ガンの発症を予防し又は遅延させるためのキットであって、酢酸、プロピオン酸、酪酸、及びその薬学的に許容し得る塩からなる群より選択される少なくとも2つの短鎖脂肪酸及び/又はその塩を含む組成物を含有する、キット。
- 短鎖脂肪酸及び/又はその塩が、酢酸又はその薬学的に許容し得る塩、プロピオン酸又はその薬学的に許容し得る塩、及び酪酸又はその薬学的に許容し得る塩を含む、請求項13に記載のキット。
- 被験体における肝疾患の発症を予防し又は遅延させるためのキットであって、酢酸、プロピオン酸、酪酸、及びその薬学的に許容し得る塩からなる群より選択される少なくとも2つの短鎖脂肪酸及び/又はその塩を含む組成物を含有する、キット。
- 短鎖脂肪酸及び/又はその塩が、酢酸又はその薬学的に許容し得る塩、プロピオン酸又はその薬学的に許容し得る塩、及び酪酸又はその薬学的に許容し得る塩を含む、請求項15に記載のキット。
- 肝疾患が肝臓ガンである、請求項7〜12のいずれか一項に記載の医薬組成物。
- 肝疾患が肝臓ガンである、請求項15又は16に記載のキット。
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CN115350171A (zh) | 2022-11-18 |
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AU2020239699A1 (en) | 2020-10-15 |
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