JP2021503477A - 細胞膜結合シグナル伝達因子の使用 - Google Patents
細胞膜結合シグナル伝達因子の使用 Download PDFInfo
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Abstract
Description
本出願は、2017年11月16日に出願された米国仮出願第62/587338号の優先権の利益を主張する。当該出願の内容全体を参照により本明細書で援用する。
天然由来のシクロデキストリンには、シクロデキストリン−α、シクロデキストリン−β、シクロデキストリン−γの三種がある。シクロデキストリンは、他の多くの化学薬品とともに安定な水性複合体を形成する。典型的なシクロデキストリンは、6−8グルコピラノシド単位を含み、溶媒に二級水酸基を晒している大きな開口と、溶媒に一級水酸基を晒している小さな開口とを有する環状体としてトポロジー的には表現できる。この配置により、環状体の内部は、疎水性ではないものの水性環境よりも大幅に親水性が低いため、他の疎水性分子を収容することが可能である。一方、その外部は、シクロデキストリン(又はそれらの複合体)に水溶性を付与するほど十分に親水性である(図1)。
コスモトロープは水分子を好適に相互作用させ、これにより(事実上)タンパク質などの高分子の分子内相互作用を安定化する。コスモトロープの例としては、これらに限定されるわけではないが、プロピレングリコール、プロリン、トレハロース、エクトイン、及びトリメチルアミン−N−オキシドなどが挙げられる。トレハロース(ミコース、トレマロース)は、2つのグルコース分子からなる二糖である。いくつかの実施の形態は、上記で開示された1種又は複数種のコスモトロープを特異的に含む。いくつかの実施の形態は、上記で開示された1種又は複数種のコスモトロープを特異的に除外する。
哺乳類は、三種のヘッジホッグホモローグ、デザート(DHh)、インディアン(IHh)、及びソニック(SHh)ヘッジホッグを有しており、このなかでも、ソニックが最も良く研究されている。このシグナル伝達経路は、ノックアウトマウスで研究され、脳、骨格、筋肉組織、胃腸管、肺、及び心臓での細胞特異性が示された。最近の研究は、成体組織の維持と再生に関係する成体幹細胞の調節におけるヘッジホッグシグナル伝達の役割を指摘している。また、この経路はいくつかのがんの発生に関与している。ヘッジホッグシグナル伝達を特異的に標的としがんと戦う薬剤が活発に開発されている。
そこで、本明細書は、脂質修飾されたHedgehog(Hh)及び/又はWingless(Wnt)タンパク質及び少なくとも1種のシクロデキストリンを含有する組成物を開示する。脂質修飾Hh及び/又はWntタンパク質は本明細書で記載するようにヒト幹細胞から単離される。特定の実施形態では、幹細胞は、多能性幹細胞、複能性幹細胞、単系統分裂中前駆細胞、又は不死化細胞系列である。
シクロデキストリン/脂質修飾タンパク質複合体含有組成物の治療用途としては、これらに限定されるわけではないが、組織再生を含む。
本明細書に開示された組成物を、神経変性疾患の治療を必要とする対象へ投与することを含む。いくつかの実施の形態では、神経変性疾患は、アルツハイマー病、パーキンソン病、脊髄損傷、脳損傷、末梢神経損傷、末梢神経障害、多発性硬化症、筋萎縮性側索硬化症又は認知症である。
胚性幹細胞は、MATRIGEL(登録商標)の薄い層からなる接着基質上にbFGF(10ng/mL)及びactivin A(5ng/mL)を補充した無血清培地を用いて現在刊行法に従って増殖した。コンフルエントになった後、培養物の半分に成長因子であるbFGF及びActivin Aを含まない以外は同じ培地を与えた。細胞培養上清サンプルを、フォリスタチン濃度について分析した。
細胞の分化状態とベータ−シクロデキストリン化学修飾との違いを検証するために、我々は部分的に分化した、及び多能性幹細胞の培養物から膜結合シグナルを採取し、2つの異なるベータ−シクロデキストリン:メチル及びヒドロキシプロピル修飾したものを用いた。
部分的分化型胚性幹細胞の培養物を、1mL/106細胞の体積で注入した10mMのメチル−β−シクロデキストリン及び20%のトレハロースを含有する採取水溶液に室温で3時間暴露して、シクロデキストリン/脂質修飾タンパク質複合体(以降、“有効成分”とする)を得た。
毛球区域が明確に存在する、毟り取ったヒトの毛を、採取直後に細胞増殖培地に浸けた。毛サンプルのいくつかは、実施例2に記載の胚性幹細胞培養物由来の脂質修飾タンパク質/MBCD複合体(封入済みMBCD)(有効成分と呼ぶ)、又は、対照としての未封入MBCDに、シクロデキストリン成分の0.25mMの同一濃度で、暴露した。他の成長因子(例えば、EGF、KGFなど)は毛包培養物中で用いなかった。培養2日後(図14A−C)、細胞の接着とわずかな増殖が、位相コントラスト顕微鏡下で、MBCD含有培養物及び対照毛包培養物の両方で観察されたが、MBCD封入因子を含有する毛包培養物で増殖はより多かった。暴露5日後(図14D−G)、対照毛包は老化をむかえ基質から剥がれたが、MBCDに暴露した毛包培養物は約80%の直径の増大と細胞の増殖を続けた。
シクロデキストリン/脂質修飾タンパク質複合体からなる有効成分を部分的分化型ヒト胚性幹細胞の培養物を1mL/106細胞の体積で注入した10mMのメチル−β−シクロデキストリン及び20%のトレハロースを含有する採取水溶液に室温で3時間暴露することで産生した。組成物を、片手の手関節の背側区域上に外用し、もう片手は未処置のままにした。手のこの部分は、末梢腕産毛に覆われており、左と右でそのパターンや密度が同じだが、機械的摩耗により休止期の明確な兆候を示している。適用は、約1滴(30μL)を皮膚に広げて、ベタつくまで乾燥し、その後、ベタつきがなくなるまで擦った。当該区域を、5日間にわたって毎日処置し、2週間後に評価を行った。末梢腕毛の成長は、処置区域で明確に目立っていた(図16C−D)。さらに、老化に関連するしわ及び斑点状色素沈着が明らかに減っており、肌のきめも改善した。2点識別能力の増大に基づき、触感の改善が対象から報告された。観察結果は、皮膚における再活性化効果を示唆している(図16A−B)。
25%又は50%の濃度の幹細胞膜抽出物を含む蒸留水と微生物阻害剤(フェノキシエタノール及びソルビン酸、1%)とを混合することで局所用調製物が調製された。
・8週の退行期に続く4、8、12、16週及び24週での共通の包括的な写真の治験責任医師による格付け
・8週の退行期に続く4、8、12、16及び24週での毛髪成長パラメーターの対象による格付け
・基準に加え、8週の退行期に続く4、8、12、16及び24週で撮影された頭皮の共通の包括的な写真
・基準、16及び24週で撮られた頭頂の直径1cm領域の超接写写真と、基準、16及び24週の画像を用いた16及び24週後に行われるか毛髪密度及び直径に関する画像解析
・基準に加え、8週の退行期に続く4、8、12、16及び24週でのすべて対象による自己評価アンケート
・基準に加え、8及び16週で採取された対象ごとの3mm生検(対象あたり計3生検)。試料は頭の皮膚及び毛包構造の組織学的改善の分析のために賛助者に送られる。
・耐性の臨床的格付け
・有害事象(AE)に関する安全性の検査
・研究期間全体のAEの監視
・1週間あたり少なくとも3回の提供された毛髪洗浄製品の使用
・適用:
−1日1回、毛根付近に溶体を2〜3滴落とす。頭皮の領域全体に均一に拡げ、約1分間、製品を皮膚に揉み込む。
−洗髪の日に、毛髪を洗浄した後(湿り気又は乾燥)、製品を適用する。1日に2回以上洗髪する場合、各洗浄後に製品を再度適用する。
−製品をつけたままにしておく。ドライクリーンで残余がなくなる。
−いつもどおりスタイリングする。スタイリング製品の使用は避ける(ヘアースプレイ、ジェル等)。
結果:
8週後、対象の多数が前向きな結果供述に同意又は中立を報告した(表7参照)。治験責任医師の評価は、表8に示されたようにすべてのパラメーターに関して改善であった。
対象によって報告された、又は治験責任医師によって観察された有害事象はなかった。
胚性幹細胞から分化させた14日目の凍結保存された神経前駆細胞が解凍され、ラミニン被覆されたイメージング細胞培養スライドに同一の密度で撒かれた。対照のために、シクロデキストリン膜抽出物が、同一の培地での終濃度10μL/mL v/vでいくつかのスライドに添加された。培地を交換し、2週間で1週間あたり3回、膜抽出物が添加されて培養が継続された。
a)ストレス因子(本実験での凍結/解凍サイクル)に曝された後の神経細胞の生存への貢献
b)神経フィラメントが陽性で、頑丈な神経突起伸長で観察されたような形態的発展の増強
c)ダブルコルチンが陽性で、培養中の初期の神経前駆細胞からの移動性の若いニューロンの持続又は拡大
d)他の細胞の種類に対する増殖増強効果は見られない
(付記1)
脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物に組織を暴露することを含み、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、組織再生の促進を必要とする組織における組織再生の促進方法。
前記シクロデキストリンは、α−シクロデキストリン、β−シクロデキストリン、γ−シクロデキストリン、ヒドロキシプロピル−β−シクロデキストリン、又はメチル−β−シクロデキストリンの1種以上である、付記1に記載の方法。
前記シクロデキストリンは、水素添加反応、ヒドロホルミル化反応、メチル化反応、酸化反応、還元化反応、又は炭素−炭素カップリング反応により化学修飾された、化学修飾シクロデキストリンである、付記1又は2に記載の方法。
前記シクロデキストリンは、メチル−β−シクロデキストリン又はヒドロキシプロピル−β−シクロデキストリンである、付記1から3のいずれか一つに記載の方法。
前記脂質修飾タンパク質は、細胞膜脂質と結合したWingless(Wnt)タンパク質又はHedgehog(Hh)タンパク質の1種以上を含む、付記1から4のいずれか一つに記載の方法。
前記Hhタンパク質は、ソニックヘッジホッグ(SHh)タンパク質、デザートヘッジホッグ(DHh)タンパク質、又はインディアンヘッジホッグ(IHh)タンパク質の1種以上である、付記5に記載の方法。
前記Wntタンパク質は、Wnt3a、Wnt7b、又はWnt10bの1種以上である、付記5に記載の方法。
前記脂質修飾タンパク質は、Wingless(Wnt)又はHedgehog(Hh)ファミリーの属するタンパク質以外の他のタンパク質から構成される、付記1から4のいずれか一つに記載の方法。
前記脂質修飾タンパク質の採取時に細胞から分泌される可溶性タンパク質を含む、付記1から4のいずれか一つに記載の方法。
前記脂質修飾タンパク質は、幹細胞集団から採取される、付記1から9のいずれか一つに記載の方法。
前記幹細胞は、胚性幹細胞、単為生殖幹細胞、成体幹細胞、胎児幹細胞、又は人工多能性幹細胞である、付記10に記載の方法。
前記幹細胞は、動物の幹細胞である、付記10又は11に記載の方法。
前記幹細胞は、ヒト幹細胞である、付記12に記載の方法。
前記幹細胞は、Wntタンパク質又はHhタンパク質を過剰発現するように遺伝子操作されている、付記10から13のいずれか一つに記載の方法。
前記幹細胞は、遺伝子操作により不死化されている、付記10から14のいずれか一つに記載の方法。
前記幹細胞は、テロメア逆転写酵素(hTERT)を発現するように遺伝子操作されている、付記15に記載の方法。
少なくとも1種のコスモトロープをさらに含む、付記1から16のいずれか一つに記載の方法。
前記少なくとも1種のコスモトロープは、プロピレングリコール、プロリン、トレハロース、エクトイン、又はトリメチルアミンN−オキシドである、付記17に記載の方法。
前記少なくとも1種のコスモトロープは、トレハロースである、付記17に記載の方法。
前記組成物が脂質修飾タンパク質とシクロデキストリンとの前記複合体を含む局所用組成物である、付記1から19のいずれか一つに記載の方法。
前記局所用組成物は、水性製剤である、付記20に記載の方法。
前記局所用組成物が少なくとも1種のコスモトロープと、任意に抗菌剤をさらに含む、付記20に記載の方法。
前記少なくとも1種のコスモトロープは、トレハロースである、付記22に記載の方法。
前記局所用組成物のpHが約4.5から約8.0の間である、付記20から23のいずれか一つに記載の方法。
組織再生の促進を必要とする組織での組織再生の促進における使用のための脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物であって、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、組成物。
脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物の、組織再生の促進を必要とする組織での組織再生の促進における使用であって、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、使用。
組織再生の促進を必要とする組織における組織再生の促進のための薬剤の製造における脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物の使用であって、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、使用。
Claims (27)
- 脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物に組織を暴露することを含み、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、組織再生の促進を必要とする組織における組織再生の促進方法。
- 前記シクロデキストリンは、α−シクロデキストリン、β−シクロデキストリン、γ−シクロデキストリン、ヒドロキシプロピル−β−シクロデキストリン、又はメチル−β−シクロデキストリンの1種以上である、請求項1に記載の方法。
- 前記シクロデキストリンは、水素添加反応、ヒドロホルミル化反応、メチル化反応、酸化反応、還元化反応、又は炭素−炭素カップリング反応により化学修飾された、化学修飾シクロデキストリンである、請求項1又は2に記載の方法。
- 前記シクロデキストリンは、メチル−β−シクロデキストリン又はヒドロキシプロピル−β−シクロデキストリンである、請求項1から3のいずれか一項に記載の方法。
- 前記脂質修飾タンパク質は、細胞膜脂質と結合したWingless(Wnt)タンパク質又はHedgehog(Hh)タンパク質の1種以上を含む、請求項1から4のいずれか一項に記載の方法。
- 前記Hhタンパク質は、ソニックヘッジホッグ(SHh)タンパク質、デザートヘッジホッグ(DHh)タンパク質、又はインディアンヘッジホッグ(IHh)タンパク質の1種以上である、請求項5に記載の方法。
- 前記Wntタンパク質は、Wnt3a、Wnt7b、又はWnt10bの1種以上である、請求項5に記載の方法。
- 前記脂質修飾タンパク質は、Wingless(Wnt)又はHedgehog(Hh)ファミリーの属するタンパク質以外の他のタンパク質から構成される、請求項1から4のいずれか一項に記載の方法。
- 前記脂質修飾タンパク質の採取時に細胞から分泌される可溶性タンパク質を含む、請求項1から4のいずれか一項に記載の方法。
- 前記脂質修飾タンパク質は、幹細胞集団から採取される、請求項1から9のいずれか一項に記載の方法。
- 前記幹細胞は、胚性幹細胞、単為生殖幹細胞、成体幹細胞、胎児幹細胞、又は人工多能性幹細胞である、請求項10に記載の方法。
- 前記幹細胞は、動物の幹細胞である、請求項10又は11に記載の方法。
- 前記幹細胞は、ヒト幹細胞である、請求項12に記載の方法。
- 前記幹細胞は、Wntタンパク質又はHhタンパク質を過剰発現するように遺伝子操作されている、請求項10から13のいずれか一項に記載の方法。
- 前記幹細胞は、遺伝子操作により不死化されている、請求項10から14のいずれか一項に記載の方法。
- 前記幹細胞は、テロメア逆転写酵素(hTERT)を発現するように遺伝子操作されている、請求項15に記載の方法。
- 少なくとも1種のコスモトロープをさらに含む、請求項1から16のいずれか一項に記載の方法。
- 前記少なくとも1種のコスモトロープは、プロピレングリコール、プロリン、トレハロース、エクトイン、又はトリメチルアミンN−オキシドである、請求項17に記載の方法。
- 前記少なくとも1種のコスモトロープは、トレハロースである、請求項17に記載の方法。
- 前記組成物が脂質修飾タンパク質とシクロデキストリンとの前記複合体を含む局所用組成物である、請求項1から19のいずれか一項に記載の方法。
- 前記局所用組成物は、水性製剤である、請求項20に記載の方法。
- 前記局所用組成物が少なくとも1種のコスモトロープと、任意に抗菌剤をさらに含む、請求項20に記載の方法。
- 前記少なくとも1種のコスモトロープは、トレハロースである、請求項22に記載の方法。
- 前記局所用組成物のpHが約4.5から約8.0の間である、請求項20から23のいずれか一項に記載の方法。
- 組織再生の促進を必要とする組織での組織再生の促進における使用のための脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物であって、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、組成物。
- 脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物の、組織再生の促進を必要とする組織での組織再生の促進における使用であって、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、使用。
- 組織再生の促進を必要とする組織における組織再生の促進のための薬剤の製造における脂質修飾タンパク質とシクロデキストリンとの複合体を含む組成物の使用であって、前記組織は、表皮、真皮、皮膚感覚受容体、皮膚付属器、毛髪、爪、皮脂腺、汗腺又は立毛筋である、使用。
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EP3710060A1 (en) | 2020-09-23 |
EP3970695A1 (en) | 2022-03-23 |
CN111655293A (zh) | 2020-09-11 |
CA3082896A1 (en) | 2019-05-23 |
KR20200103660A (ko) | 2020-09-02 |
US11666522B2 (en) | 2023-06-06 |
US20200390678A1 (en) | 2020-12-17 |
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AU2018368440A1 (en) | 2020-05-28 |
IL274731A (en) | 2020-07-30 |
KR20200116906A (ko) | 2020-10-13 |
JP2021503473A (ja) | 2021-02-12 |
AU2022203819A1 (en) | 2022-06-23 |
WO2019099861A1 (en) | 2019-05-23 |
EP3710060A4 (en) | 2021-05-19 |
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AU2018370144B2 (en) | 2022-02-03 |
IL274730A (en) | 2020-07-30 |
EP3709975A4 (en) | 2021-09-22 |
WO2019099850A1 (en) | 2019-05-23 |
US20240299282A1 (en) | 2024-09-12 |
CA3140941C (en) | 2024-01-02 |
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