JP5801716B2 - 椎間板の修復および/または再構成 - Google Patents
椎間板の修復および/または再構成 Download PDFInfo
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Description
別段特に定義されなければ、本明細書で使用される技術用語および科学用語はすべて、技術分野(たとえば、細胞培養、幹細胞生物学、分子遺伝学、免疫学、免疫組織化学、タンパク質化学、および生化学)の当業者により一般に理解されているのと同じ意味を有すると解釈されることとする。
本明細書で使用されるように、語句「STRO-1+多分化能細胞」は、多分化能細胞コロニーを形成することができるSTRO-1+および/またはTNAP+前駆細胞を意味すると解釈されることとする。
投与されるSTRO-1+多分化能細胞またはその子孫の投与量は、個人の病状、年齢、性別および体重などの要因に従って変化してもよい。投与計画は、最適治療応答を与えるように調整してもよい。たとえば、単回ボーラスを施してもよく、数回に分けた用量を時間をかけて投与してもよく、または用量は治療状況の緊急事態により示されるのに比例して減らしても増やしてもよい。投与のしやすさおよび投与量の均一性のために非経口組成物を投与量単位形で処方するのは有利である可能性がある。本明細書で使用される「投与量単位形」とは、治療される対象のための単位投与量として適した物理的に別々の単位のことであり、各単位は必要な医薬担体と関連して所望の治療効果が生じるように計算された所定の量の活性化合物を含有する。
一実施形態では、STRO-1+多分化能細胞および/またはその子孫細胞は、たとえば、対象のタンパク質、たとえば、治療的および/または予防的利点をもたらすタンパク質、たとえば、インスリン、グルカゴン、ソマトスタチン、トリプシノーゲン、キモトリプシノーゲン、エラスターゼ、カルボキシペプチダーゼ、膵臓リパーゼまたはアミラーゼまたは増強血管新生に関連するもしくは原因となるポリペプチドまたは細胞の膵臓細胞もしくは血管細胞への分化に関連するポリペプチドを発現するかつ/または分泌するように遺伝子改変される。
実験計画
進行性椎間板変性を惹起するために、24匹のヒツジが1.0 IUコンドロイチナーゼABC(cABC) (Seikagaku Corporation、日本)の注射を3つの隣接する腰部椎間板(名目上L3-L4、L4-L5、およびL5-L6)内に受けた。残りの腰部椎間板(名目上L1-L2およびL2-L3)にはcABCを注入せず、正常対照と見なされた。cABC投与に続く15(±3週間)週目、高用量もしくは低用量(それぞれ、4×106または0.5×106細胞)のSTRO-l+多分化能細胞または等量のEuflexxa(登録商標)ヒアルロン酸(Ferring Pharmaceuticals)と混合したProFreeze(商標) NOA Freezing Medium (Lonza Walkersville Md.)の注射を椎間板の髄核内に直接投与した(Table 1)(表1)。動物は、注入の3カ月または6カ月後に死体解剖した。図1は、実験のために使用された脊髄レベルおよび処置のためのプロトコルの模式図である。
免疫選択されたSTRO-l+多分化能細胞の増殖
これらの実験に使用されるSTRO-l+多分化能細胞は、フランス産ヒツジに由来し、Lonza (USA)により調製された。骨髄(BM)吸引液は、そのヒツジから得られ、BM単核細胞(BMMNC)は、基本的に以前記載された通り(US 2005-0158289)に調製された。
放射線学
動物は、以下の時点:0日目(cABCの注入)、被験物質投与の日(腰椎間板変性導入の15±3週間後)、被験物質埋め込みの3カ月および6カ月後に、導入麻酔下で腰椎の側面単純X線写真を撮られた。すでに記載された通り(24)に、IVDの前方、中央および後方部分の測定値を平均し、それを隣接する椎間本体の高さの平均で割ることにより計算される椎間高(DHI)の指標を使用して盲検観察者により、X線写真の評価は行われた。
磁気共鳴画像法(MRI)
MRIは、コンドロイチナーゼABCの注入による椎間板変性導入のほぼ15週間後に、HAまたは高用量および低用量HA+STRO-l+細胞の円板内注入を用いた処置(Tx)に先立って、導入麻酔下で全ヒツジの腰椎について行われた。処置の3カ月および6カ月後、死体解剖(Nx)の直前に、脊髄MRIは全ヒツジに行われた。
病理組織学的解析
組織学的および生化学的解析のために処理される椎間板ユニットは、それぞれ成長板に近い隣接頭蓋および仙骨椎体を骨ノコギリで切り開くことにより、分離した。これらの脊髄セグメントは56時間Histochoice(登録商標)にひとまとめにして固定し、Faxitron HP43855A X線キャビネット(Hewlett Packard、McMinnville、米国)を使用して完全脱灰が確認されるまで、2週間絶えず攪拌しながら5%中性緩衝ホルマリン中10%ギ酸を数回変えて脱灰した。
固定化された椎間板組織の生化学的解析
線維輪(AF)および髄核(NP)は、中央矢状方向スライスを組織学的研究のために取り除いた後に残る処理した脱灰椎間板組織から慎重に解体された。この過程は、NPとAF間の分界境界が失われている甚だしく変性した椎間板ではより困難であった。細かいさいの目状に切ったNPとAF組織のアリコットは恒量まで凍結乾燥させ、乾燥組織の三つ組部分(1〜2mg)は、110℃で6時間、6M HCl中で加水分解された。すでに記載されている(4)通りに、中和消化物のアリコットは、組織コラーゲン含有量の尺度としてヒドロキシプロリンについてアッセイされた。凍結乾燥組織の三つ組部分(〜2mg)も、すでに記載されている(27)通りにパパインで消化され、アリコットは異染色色素の1,9-ジメチルメチレンブルーを使用して硫酸化グリコサミノグリカン(GAG)についてアッセイされた。
データの統計的解析
3カ月および6カ月処置群すべてのDHIおよび生化学的データの統計学的比較は、p<0.05が有意と見なされるスチューデント無対応t-検定を使用して行われた。組織学的およびMRI集計変性およびNPスコア(MRI aggregate degeneration and NP scores)では、様々な椎間板処置間の比較は、ダン多重比較ポストホック検定と一緒にクラスカルワオリスまたはフリードマン検定(ノンパラメトリック反復測定ANOVA)を使用して行われた。群間の統計的有意性は、p<0.05として解釈された。
免疫選択されたSTRO-1+多分化能細胞を使用するウシ椎間板再生実験の結果
高用量(4.0×106 STRO-1+細胞)(群2および4)ならびに低用量(0.5×106 STRO-1+細胞)(群1および3)注入群の動物はすべて、実験期間にわたり正常体重を維持し有害な副作用の証拠は全く示さなかった。
本実験の結果によれば、変性椎間板への低用量STRO-1+細胞+HAの椎間板内投与は、HAを注入した椎間板よりも大きな程度に構造回復を改善することが明らかになった。この結論は、椎間板完全性および正常椎間板高指標の50%に相当する基線変性状況からのマトリックス回復の3つの独立した評価により得られた実験データにより支持された。
(参考文献)
Claims (7)
- STRO-1 bright 多分化能細胞について濃縮した細胞集団を含む、対象において椎間板を再構成または修復するための組成物であって、椎間板の髄核に投与用の組成物。
- STRO-1 bright 多分化能細胞が、投与前にin vitroで培養されるものである、請求項1に記載の組成物。
- STRO-1 bright 多分化能細胞が同種供給源に由来する、請求項1に記載の組成物。
- グリコサミノグリカン(GAG)をさらに含む、請求項1に記載の組成物。
- GAGが、ヒアルロン酸(HA)、コンドロイチン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパリン、ヘパリン硫酸、およびガラクトサミノグリクロングリカン硫酸(GGGS)からなる群から選択される、請求項4に記載の組成物。
- 対象が椎間板の変性により特徴づけられる脊髄状態を有する、請求項1に記載の組成物。
- 脊髄状態が、腰痛、椎間板の加齢性変化または脊椎分離症である、請求項6に記載の組成物。
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US13311108P | 2008-06-25 | 2008-06-25 | |
US61/133,111 | 2008-06-25 | ||
PCT/AU2009/000817 WO2009155656A1 (en) | 2008-06-25 | 2009-06-25 | Repair and/or reconstitution of invertebral discs |
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JP2011525493A JP2011525493A (ja) | 2011-09-22 |
JP5801716B2 true JP5801716B2 (ja) | 2015-10-28 |
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JP2015079034A Active JP6058729B2 (ja) | 2008-06-25 | 2015-04-08 | 椎間板の修復および/または再構成 |
JP2016236490A Active JP6745713B2 (ja) | 2008-06-25 | 2016-12-06 | 椎間板の修復および/または再構成 |
JP2018150268A Pending JP2018167098A (ja) | 2008-06-25 | 2018-08-09 | 椎間板の修復および/または再構成 |
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JP2015079034A Active JP6058729B2 (ja) | 2008-06-25 | 2015-04-08 | 椎間板の修復および/または再構成 |
JP2016236490A Active JP6745713B2 (ja) | 2008-06-25 | 2016-12-06 | 椎間板の修復および/または再構成 |
JP2018150268A Pending JP2018167098A (ja) | 2008-06-25 | 2018-08-09 | 椎間板の修復および/または再構成 |
JP2020092260A Pending JP2020125361A (ja) | 2007-08-06 | 2020-05-27 | 椎間板の修復および/または再構成 |
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EP (2) | EP2303290B1 (ja) |
JP (5) | JP5801716B2 (ja) |
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CN (2) | CN102099043B (ja) |
AU (1) | AU2009262358B2 (ja) |
CA (1) | CA2728102C (ja) |
ES (2) | ES2872337T3 (ja) |
HK (1) | HK1217179A1 (ja) |
SG (1) | SG10201507149RA (ja) |
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TR201809635T4 (tr) | 2011-09-09 | 2018-07-23 | Mesoblast Inc | Osteoblastik fonksiyonun arttırılmasına yönelik yöntemler. |
WO2015084972A1 (en) * | 2013-12-03 | 2015-06-11 | Cornell University | Method of repairing an annulus and collagen gel composition |
SG10201906601TA (en) * | 2015-01-16 | 2019-09-27 | Spineovations Inc | Method of treating spinal disk |
JP6984829B2 (ja) * | 2018-01-31 | 2021-12-22 | 国立大学法人神戸大学 | 椎間板変性の治療剤および椎間板細胞培養材 |
CN108523978B (zh) * | 2018-03-29 | 2020-09-04 | 中南大学湘雅三医院 | 脊柱全椎板切除后的后方韧带复合体重建系统 |
JP2022515916A (ja) * | 2019-01-02 | 2022-02-22 | メゾブラスト・インターナショナル・エスアーエールエル | 腰痛を治療するための方法 |
US20220160834A1 (en) * | 2019-03-29 | 2022-05-26 | Kolon Tissuegene, Inc. | Treatment Of Intervertebral Disc Degeneration |
CN111973807B (zh) * | 2020-08-31 | 2021-08-31 | 四川大学 | 一种仿生人工颞下颌关节盘及其制备方法 |
KR20230134514A (ko) * | 2021-01-22 | 2023-09-21 | 메조블라스트 인터내셔널 에스에이알엘 | 오피오이드 스페어링 조성물 및 이를 사용하는 방법 |
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