JP2021514380A - 筋障害を処置するための化合物及び組成物 - Google Patents
筋障害を処置するための化合物及び組成物 Download PDFInfo
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- JP2021514380A JP2021514380A JP2020544632A JP2020544632A JP2021514380A JP 2021514380 A JP2021514380 A JP 2021514380A JP 2020544632 A JP2020544632 A JP 2020544632A JP 2020544632 A JP2020544632 A JP 2020544632A JP 2021514380 A JP2021514380 A JP 2021514380A
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- epicatechin
- bone
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Abstract
Description
a.対象における骨格筋又は心筋の傷害又は機能不全の1つ以上の身体表出を観察すること、
b.エピカテキンの(+)若しくは(−)エナンチオマーのいずれか又は両方の組み合わせ、エピカテキン誘導体、その薬学的に許容される塩若しくはプロドラッグ又はその組み合わせの治療的有効量を、それを必要とする患者に投与すること、及び
c.骨格筋又は心筋の傷害又は機能不全の当該身体表出におけるその変化又は欠如を観察することを含む、方法も提供される。
a)フォリスタチン、ミオスタチンの処置前血漿レベル又はフォリスタチンのミオスタチンに対する比を測定すること、
b)フォリスタチン、ミオスタチンの処置後血漿レベル又はフォリスタチンのミオスタチンに対する比を測定すること、及び
c)フォリスタチン、ミオスタチンの処置前レベル及び処置後レベル又はフォリスタチンのミオスタチンに対する比を比較すること、を含む方法も提供される。
a)フォリスタチン、ミオスタチンの血漿レベル又はフォリスタチンのミオスタチンに対する比を最初に測定すること、
b)第1の量のエピカテキン((+)若しくは(−)エナンチオマーのいずれか又は両方の組み合わせ)、エピカテキン誘導体又はその薬学的に許容される塩若しくはプロドラッグの第1の量を投与すること、
c)フォリスタチン、ミオスタチンの処置後血漿レベル又はフォリスタチンのミオスタチンに対する比を測定すること、
d)フォリスタチン、ミオスタチンの処置前レベル及び処置後レベル又はフォリスタチンのミオスタチンに対する比を比較すること、並びに
e)
i)対象における測定ミオスタチンが低下したとき又は血漿フォリスタチンの血漿ミオスタチンに対する比が上昇したときに、エピカテキン((+)若しくは(−)エナンチオマーのいずれか又は両方の組み合わせ)、エピカテキン誘導体又はその薬学的に許容される塩若しくはプロドラッグの用量を増加すること、又は
ii)対象におけるミオスタチン濃度が上昇したとき又は血漿フォリスタチンの血漿ミオスタチンに対する比が低下したときに、エピカテキン((+)若しくは(−)エナンチオマーのいずれか又は両方の組み合わせ)、エピカテキン誘導体又はその薬学的に許容される塩若しくはプロドラッグのいずれか)の用量を低減若しくは維持することのどちらかを含む方法も提供される。
ウエスタンブロットアッセイ:
細胞又は骨格筋組織サンプルを、1mmol/L PMSF、2mmol/L Na3V04及び1mmol/L NaPを添加したプロテアーゼ及びホスファターゼ阻害剤カクテルを含む、50μLの溶解緩衝剤(1%トリトンX−100、20mmol/Lトリス、140mmol/L NaCI、2mmol/L EDTA及び0.1% SDS)中でホモジナイズした。ホモジネートをインスリン注射器に5回通過させて、4℃にて30分間超音波処理し、10分間遠心分離した(12,000×g)。総タンパク質含量を上清中で測定した。タンパク質合計40μgを5%又は10% SDS−PAGEに投入して、電気泳動転写し、ブロッキング溶液(TBS中の5%の無脂肪乾燥乳及び0.1%ツイン20[TBS−T])中で1時間インキュベートした後、一次抗体を用いて、室温にて3時間インキュベートするか又は4℃にて一晩インキュベートした。一次抗体は、典型的には、TBS−T及び5%ウシ血清アルブミンで1:1000又は1:2000に希釈した。膜をTBS−T中で洗浄して(5分間、3回)、ブロッキング溶液中1:10,000に希釈したHRP標識二次抗体の存在下で、室温にて1時間インキュベートした。膜を再びTBS−Tで3回洗浄し、増強化学発光検出キットを使用して免疫ブロットを発現させた。バンド強度をデジタル定量化した。全ての一次抗体は市販されている。
エピカテキンは、フォリスタチン発現を誘発し、ミオスタチン発現を抑制し、培養マウス筋芽細胞の筋管への分化を促進する。これらの現象は、筋分化のバイオマーカー、例えばミオゲニン及びmyoDの発現上昇に関連している。マウス筋芽細胞株C2C12を6ウェル組織培養プレート内でセミコンフルエントまで成長させ、次いで、文献で標準化されている分化誘導培地:2%ウマ血清を添加したDMEMを用いて、エピカテキン(100nM)に3日間曝露した。タンパク質を抽出し、従来のゲル電気泳動により分離し、筋細胞の成長及び分化のマーカーに特異的な市販の抗体と反応させることによってウエスタンブロットとして染色した。筋細胞に対する(−)及び(+)エピカテキンエナンチオマーの比較効果を調べる実験において、細胞を6ウェルプレート内でセミコンフルエントまで成長させ、ウマ血清を含有する、文献で標準化された分化培地に投入し、次いで(−)又は(+)エピカテキンエナンチオマーを10nM〜1000nMの範囲の濃度で用いて24時間刺激した。次いで、細胞を上記のように採取し、市販の特定の一次抗体を使用してウエスタンブロットを調製して、PGC1α及びフォリスタチンの相対発現を求めた。
2型糖尿病及び心不全の患者5名に、エピカテキンに富むココア製品(1日につきエピカテキン100mgを提供)を3ヶ月間提供した。
筋ジストロフィーを発症する野生型(即ち正常)及びデルタサルコグリカン(8−SG)ヌルマウスに、Sigma−Aldrichから入手したエピカテキン1mg/kgを1日2回又は水のみ(対照)を、30日間強制経口投与した。四頭筋タンパク質サンプルをウエスタンブロットにより分析して、ミトコンドリアタンパク質であるポーリン、マイトジェンフィルム、複合体V(CV)、スーパーオキシドジスムターゼ2(SOD2)及びカタラーゼのタンパク質レベルの変化を評価した。エピカテキン処置によって、野生型マウスではミトコンドリアタンパク質の消失が増大し、8−SGヌルマウスではミトコンドリアタンパク質の消失が防止される。筋ジストロフィーマウスでは、エピカテキンによって、酸化損傷の重症度を低下させることによって酸化損傷の損傷効果に対抗する重要な酵素である、心筋及び四頭筋中のカタラーゼ及びスーパーオキシドジスムターゼ2の両方のタンパク質発現を増加した。
インビトロ及びインビボでのエピカテキンによる、筋成長ホルモンであるフォリスタチンの誘起及び筋成長の阻害剤であるミオスタチンの抑制によって、これらのタンパク質が、インビボでのエピカテキンの効果を監視する際に有用なバイオマーカーであり得ることが示唆されている。心不全の糖尿病患者では、エピカテキンに豊むココアでの処置前後に、フォリスタチンのミオスタチンに対する比を測定及び計算した。処置に関連するフォレスタチン/ミオスタチン比の統計的に有意な上昇が存在し、このことはフォリスタチンの増加及びその天然アンタゴニストであるミオスタチンの減少を示している。
体重50〜60g及び3〜4週齢のSDラットを4グループに分けた。グループAの動物は、ビヒクル対照群の部分であり、グループBの動物にはデキサメタゾンを毎日投与した。グループCの動物には、エピカテキン3mg/kgを投与し、続いてデキサメタゾンを投与した。グループDの動物には、エピカテキン10mg/kgを投与し、続いてデキサメタゾンを投与した。全ての投与は、皮下(SC)投与方式であった。動物への投与を36日間継続した。体重を1日おきに測定し、全長を毎週測定した。動物を定期的に撮影して、更にデータを維持した。飼料摂取、全身健康状態及び運動を日常的に評価した。37日目の動物を犠死させて、大腿骨長及び脛骨長をノギスを使用して測定した。血液を採取して保存した。
Claims (22)
- 必要とする対象において、骨に対する傷害若しくは骨の喪失を反転若しくは改善する、又は骨折を予防する方法であって、(+)エピカテキン及び/若しくは(−)エピカテキン又はエピカテキン誘導体の治療的有効用量を対象に投与することを含む、方法。
- 前記(+)エピカテキン及び/若しくは(−)エピカテキン又はエピカテキン誘導体が1日につき5mg〜2グラム、単回用量又は分割用量で経口投与、静脈内投与又は筋肉内投与される、請求項1に記載の方法。
- 前記(+)エピカテキン及び/若しくは前記(−)エピカテキン又はエピカテキン誘導体が、1日につき0.1mg/kg体重〜1日につき10mg/kg体重の用量にて、単回用量又は分割用量で、経口投与又は静脈内投与又は筋肉内投与される、請求項1に記載の方法。
- 前記方法が、骨折を防止するために、新たな骨形成若しくは追加の骨形成、又はより強度の骨形成若しくは再生を誘起する、請求項1に記載の方法。
- 前記方法が、骨の弱化を反転又は改善し、これにより、骨の弱化又は骨に対する損傷、骨生成の障害、又は骨成長の障害を誘起することが既知である化合物の投与によって引き起こされる骨折を防止し、前記化合物が、コルチコステロイド抗痙攣剤、化学療法薬からなる群から選択される、請求項1に記載の方法。
- 前記骨の弱化が、骨の弱化若しくは骨に対する損傷、骨生成の障害又は骨成長の障害に関連することが既知である医学的状態によって引き起こされる、請求項1に記載の方法。
- 前記(+)エピカテキン及び/又は(−)エピカテキンが、カルシウム、ビタミンD又はカルシトニンと同時投与される、請求項1に記載の方法。
- 前記(+)エピカテキン及び/若しくは(−)エピカテキン又はエピカテキン誘導体が、1日につき5mg〜2グラム、単回用量又は分割用量で経口投与、静脈内投与又は筋肉内投与される、請求項4に記載の方法。
- 前記(+)エピカテキン及び/若しくは(−)エピカテキン又はエピカテキン誘導体が、1日につき5mg〜2グラム、単回用量又は分割用量で経口投与、静脈内投与又は筋肉内投与される、請求項5に記載の方法。
- 前記(+)エピカテキン及び/若しくは(−)エピカテキン又はエピカテキン誘導体が、1日につき5mg〜2グラム、単回用量又は分割用量で経口投与、静脈内投与又は筋肉内投与される、請求項6に記載の方法。
- 前記(+)エピカテキン及び/若しくは(−)エピカテキン又はエピカテキン誘導体が、1日につき5mg〜2グラム、単回用量又は分割用量で経口投与、静脈内投与又は筋肉内投与される、請求項7に記載の方法。
- (+)エピカテキン、(−)エピカテキン又は両方の混合物又はその薬学的に許容される塩若しくはプロドラッグを含む、又は(+)エピカテキン、(−)エピカテキン又は両方の混合物又はその薬学的に許容される塩若しくはプロドラッグからなる組成物であって、
(a)骨に対する傷害若しくは骨の喪失を反転若しくは改善する、又は骨折を防止する、及び/又は
(b)骨粗鬆症を予防、処置若しくは治癒する、薬剤として使用するための、組成物。 - 前記(+)エピカテキン、(−)エピカテキン若しくはその両方の混合物又はその薬学的に許容される塩若しくはプロドラッグが、経口投与又は静脈内投与又は筋肉内投与されることを特徴とする、請求項12に記載の組成物。
- 前記(+)エピカテキン、(−)エピカテキン若しくはその両方の混合物又はその薬学的に許容される塩若しくはプロドラッグが、1日につき5mg〜2グラム、単回用量又は分割用量で投与されることを特徴とする、請求項12及び/又は13に記載の組成物。
- 前記(+)エピカテキン、(−)エピカテキン若しくはその両方の混合物又はその薬学的に許容される塩若しくはプロドラッグが、1日につき0.1mg/kg体重〜1日につき10mg/kg体重の用量にて、単回用量又は分割用量で、1日につき10mg/kg体重の用量にて、経口投与又は静脈内投与又は筋肉内投与されることを特徴とする、請求項12〜14のいずれかに記載の組成物。
- 前記組成物が、骨折を防止するために、新たな骨形成若しくは追加の骨形成、又はより強度の骨形成若しくは再生を誘起することを特徴とする、請求項12〜15のいずれか一項に記載の組成物。
- 前記骨の弱化又は喪失が、骨の弱化又は骨に対する損傷、骨生成の障害、又は骨成長の障害を誘起することが既知である化合物の投与によって引き起こされ、前記化合物が、コルチコステロイド抗痙攣剤及び化学療法薬又はその混合物からなる群から選択されることを特徴とする、請求項12〜16のいずれかに記載の組成物。
- 前記骨の弱化が、骨の弱化若しくは骨に対する損傷、骨生成の障害又は骨成長の障害に関連することが既知である医学的状態によって引き起こされることを特徴とする、請求項12〜17のいずれかに記載の組成物。
- 前記組成物が、カルシウム、ビタミンD若しくはカルシトニン又はその混合物を更に含むことを特徴とする、請求項12〜18のいずれかに記載の組成物。
- 前記組成物が、コルチコステロイド抗痙攣剤、化学療法薬又はその混合物を更に含むことを特徴とする、請求項12〜19のいずれか一項に記載の組成物。
- 前記組成物が、フォリスタチン及びフォリスタチン様タンパク質の細胞産生又は筋産生又は体内産生を増加させることを特徴とする、請求項12〜20のいずれかに記載の組成物。
- 前記治療用量が、フォリスタチン及びフォリスタチン様タンパク質の細胞産生又は筋肉産生又は身体産生を増加させる、請求項1に記載の方法。
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