JP2020513007A - 皮膚損傷の処置に使用するための細菌セクレトーム - Google Patents
皮膚損傷の処置に使用するための細菌セクレトーム Download PDFInfo
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Abstract
Description
a)前記細菌をその生育に適切な培地及び条件下で培養し、
b)固液分離し、かつ液相を回収し、
c)液相中の前記細菌により分泌された生体分子を含む前記セクレトームを得る。
・第一の接種は、この前培養に適切なミネラル、タンパク質、及び糖質を含む適切な培地が入った三角フラスコの中で調製することができる。
・その後、バッチ方式で、第一の培地(もしくは相当する培地)と類似、もしくは同一の第二の培地で予備発酵培養が調製される。
・最後に、高い細胞濃度を得るため、流加培養(fed-batch mode)で、第二の培地と類似、もしくは同一の培地で発酵する。
・よく知られた治療もしくは修復成分:パンテノール、マデカソサイド、アラントイン、アロエベラ、はちみつ;
・抗感染成分:銅−亜鉛−型微量元素、はちみつ;
・保湿及び栄養成分:グリセリン、シアバター、天然ワックス、ヒアルロン酸、脂肪酸;
・無痛化剤:アラントイン、ビサボロール、パンテノール、エノキソロン;
・UVフィルター。
非限定の例示として、好ましい培養培地は塩化アンモニウム、硫酸マグネシウム、酵母エキスを含む。出願WO2012/085182号より生じるように、とりわけ、他の類似の培地は使用されるかもしれず、そのようにして、本詳細な説明に不可欠な要素を形成するとみなさなければならないこともまた、明記されるべきである。当業者によるいかなる改変もまた、本発明の開示の一部であるとみなさなければならない。
・無菌の50%グルコース溶液を含むフラスコ。この溶液(グルコース前培養バッチ)は、最初のグルコース濃度である20g/Lを達成するため、すぐに培養培地に移される。
・上記の接種工程で開示された接種液を含む三角フラスコは、前発酵槽において接種される。
・培養:それから、発酵槽はpH7.0に調整された127Lの培地で満たされ、それから完全に殺菌される。3つのサテライトフラスコが使用される。
・無菌のグルコース溶液が含まれるフラスコ。この溶液は最初のグルコース濃度である20g/Lを達成するため、すぐに培養溶媒に移される。
・アンチフォームのフラスコ。このアンチフォームは、槽中のフォームの水位を調整するために、培養中、自動的に加えられるだろう。
・流加回分のグルコースのフラスコ。この溶液は細胞の生育を促進するため、培養中、加えられるだろう。
好ましい実施態様の説明として、以下の例により示されるが、本発明を限定するものとみなしてはならない。
Bis−TrisSDS−PAGEゲル(4〜12%)、変性及び還元条件下で、アルギニンバッファー前及び後の培養上澄み液の様々な量のロード量(それぞれ10、15、20、30μl)のクマシーブルーによる検出。いずれの場合においても、14〜100kDaの間で、約38kDaの主要なバンドを含む、様々なタンパク質のバンドが観察された。
使用した技術は、37℃での、S期の細胞のDNAへの、ヌクレオチドである、チミジンアナログである5−ブロモ−2’−デオキシウリジン(BrdU)の取り込みである。この技術は、細胞周期における増殖細胞(SもしくはDNA合成期)の進行状態の特徴を持つ細胞の定量化を可能にする。
本研究は、Oris Cell Migration Assay(Platypus Technologies)を用いて、メーカーにより推奨されている基本的な試験法で行った。主に、HaCaT(ヒト角化細胞)細胞は96ウェルプレートに播種されている。Oris(商標)ストッパーを取り外し、評価される化合物が培養培地に添加される。細胞走化が可能なように24時間、培養が続く。ストッパーにより区切られた区域における細胞の可視可を可能にするため、細胞侵襲性蛍光トレーサー、カルセインが、それから、添加される。
ブタ耳の皮膚移植片は生存条件で維持される。丸い3ミリの傷が作製されている中心において、6ミリメートルの生検が実行される。傷は全体の上皮及び上方の真皮を損傷する。評価のための生成物は局所的に適用される。48時間後、生検は凍結され、部分ごとにヘマトキシリン・エオジン染色が実行される。上皮再形成の速度論は以下の工程によって評価される。
1)J.Brandnerにより開発された試験法(Pollok et al., 2010, J Cell Mol Med)により、0〜3まで規定した評点を帰属。傷の端から上皮再形成の区域の範囲のμmでの正確な測定。有意性のために、スチューデントのt検定を適用する。
2)組織保存の質を評価するために、傷の端における形態学的な外見もまた評価される。
例2によるセクレトーム: 0.1−5%
グリセリン: 1−30%
ヘキシレングリコール: 0.1−7%
セテアリルグリコシド: 0.1−1%
セテアリルアルコール: 0.9−4%
ステアリン酸: 0.5−4%
ステアリン酸グリセリル: 0.1−4%
植物油: 0.1−10%
水: 適量100%
Claims (15)
- 配列番号1又は配列番号1と少なくとも80%の同一性を有する配列を含む16S rRNAを含み、皮膚損傷の処置に使用するための、ベータプロテオバクテリア鋼、サブファミリー ナイセリア科に属する非病原性のグラム陰性細菌の細菌セクレトームであって、以下の工程を含む方法によって得られる、セクレトーム:
a)前記細菌をその生育に適切な培地及び条件下で培養し、
b)固液分離し、かつ液相を回収し、
c)液相中の前記細菌により分泌された生体分子を含む前記セクレトームを得る。 - 工程b)において得られる液相に基本バッファーが加えられ、かつ好ましくは1〜6℃の温度で、得られた緩衝液相が場合により1〜7時間接触して静置することを特徴とする、請求項1に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 基本バッファーがトリスバッファー、アルギニンバッファーもしくはその混合物から選択されることを特徴とする、請求項2に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 固液分離の工程b)の後、工程c)より前に、液相又は緩衝液相を濾過により清澄化する工程b’)を含むことを特徴とする、請求項1〜3のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 濾過がカットオフ0.2μmで行われることを特徴とする、請求項4に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 生体分子が、前記細菌から分泌されるペプチド、タンパク質、及び二次代謝物からなることを特徴とする、請求項1〜5のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 細菌が、配列番号2又は配列番号2と少なくとも80%の同一性を有する配列を含む少なくとも1つのプラスミドを含むことを特徴とする、請求項1〜6のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 細菌が、2010年4月8日にI−4920の番号でCNCMに寄託された細菌であることを特徴とする、請求項1〜7のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 皮膚損傷が、擦過傷、火傷、日焼け、かき傷、切り傷、すり傷、縫合からなる群から選択されることを特徴とする、請求項1〜8のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 皮膚損傷が、外傷、手術、皮膚科学的処置、又は美容医療的処置の後に生じるものであることを特徴とする、請求項1〜9のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 皮膚損傷の処置が治療及び皮膚修復の改善からなることを特徴とする、請求項1〜10のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 銅塩及び/又は亜鉛塩と組み合わせることを特徴とする、請求項1〜11のいずれか一項に記載の皮膚損傷の処置に使用するための細菌セクレトーム。
- 皮膚損傷の処置に使用するための、請求項1〜8のいずれか一項に記載の細菌セクレトームと、局所投与に適する皮膚科学的な賦形剤とを含む、皮膚科学的組成物。
- 銅塩及び/又は亜鉛塩と、局所投与に適する皮膚科学的な賦形剤と、請求項1〜8のいずれか一項に定義された細菌セクレトームとを含む、皮膚科学的組成物。
- 皮膚損傷の処置に使用するための、請求項14に記載の皮膚科学的組成物。
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FR1753012A FR3065009B1 (fr) | 2017-04-06 | 2017-04-06 | Secretome bacterien pour son utilisation dans le traitement des lesions cutanees |
PCT/EP2018/058929 WO2018185324A1 (fr) | 2017-04-06 | 2018-04-06 | Sécrétome bactérien pour son utilisation dans le traitement des lésions cutanées |
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"Improvement of atopic dermatitis skin symptoms by Vitreoscilla filiformis bacterial extract", EUR. J. DERMATOL., vol. 16, no. 4, JPN6022011922, 2006, pages 380 - 384, ISSN: 0004915088 * |
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US11484555B2 (en) | 2022-11-01 |
CN111630152A (zh) | 2020-09-04 |
CA3059380A1 (en) | 2018-10-11 |
RU2019134658A (ru) | 2021-05-06 |
AU2018248899A1 (en) | 2019-11-28 |
JP7334119B2 (ja) | 2023-08-28 |
FR3065009B1 (fr) | 2020-11-27 |
SG11201909144VA (en) | 2019-11-28 |
KR20190138640A (ko) | 2019-12-13 |
KR102585934B1 (ko) | 2023-10-06 |
EP3607049A1 (fr) | 2020-02-12 |
CN111630152B (zh) | 2023-07-04 |
WO2018185324A1 (fr) | 2018-10-11 |
US20200030387A1 (en) | 2020-01-30 |
FR3065009A1 (fr) | 2018-10-12 |
MX2019011951A (es) | 2019-11-07 |
BR112019020954A2 (pt) | 2020-05-05 |
RU2019134658A3 (ja) | 2021-07-28 |
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