JP2009506069A - ムスカリン性受容体調節による神経発生 - Google Patents
ムスカリン性受容体調節による神経発生 Download PDFInfo
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Abstract
【選択図】図1
Description
(CH3O)2−PO−CHOH−OCl3
DOV 102,677(Popik et al. "Pharmacological Profile of the "Triple" Monoamine Neurotransmitter Uptake Inhibitor, DOV 102,677." Cell Mol Neurobiol. 2006 Apr 25;プリント版に先立つ電子ジャーナル版を参照)、
DOV 216,303(Beer et al. "DOV 216,303, a "triple" reuptake inhibitor: safety, tolerability, and pharmacokinetic profile," J Clin Pharmacol. 2004 44(12): 1360-7を参照)、
DOV 21,947((+)-1-(3, 4-ジクロロフェニル)-3-アザビシクロ-(3.1.0)ヘキサン・ハイドロクロライド)、Skolnick et al. "Antidepressant-like actions of DOV 21,947: a "triple" reuptake inhibitor." Eur J Pharmacol. 2003 461(2-3):99-104を参照)、
NS-2330又はテソフェンシン(CAS RN 402856-42-2)、又はNS 2359(CAS RN 843660-54-8)など;並びに
デヒドロエピアンドロステロン(DHEA)、及びDHEA硫酸(DHEAS)、CP-122,721(CAS RN 145742-28-5)等の薬剤
などが挙げられる。
[1−(β−メルカプト−β,β−シクロペンタメチレンプロピオン酸)、2−O−エチルチロシン,4−バリン]アルギニン・バソプレシン(d(CH2)5[Tyr(Et2)]VAVP(WK1−1)、
9−デスグリシン[1−(β−メルカプト−β,β−シクロペンタメチレンプロピオン酸)、2−O−エチルチロシン,4−バリン]アルギニン・バソプレシン・デスGly9d(CH2)5[Tyr(Et2)]−VAVP(WK3−6)、又は
9−デスグリシン[1−(β−メルカプト−β,β−シクロペンタメチレンプロピオン酸),2−D−(O−エチル)チロシン,4−バリン]アルギニン・バソプレシン・デスGly9d(CH2)5[D−Tyr(Et2)]VAVP(AO3−21)など;コルチコトロピン放出因子(CRF)R拮抗薬、例えば、CP−154,526など(構造は、Schulz et el. "CP-154,526: a potent and selective nonpeptide antagonist of corticotropin releasing factor receptors." Proc Natl Acad Sci USA, 1996 93(19):10477-82 に開示されている)、NBI30775(別名R121919又は2,5−ジメチル−3−(6−ジメチル−4−メチルピリジン−3−イル)−7−ジプロピルアミノピラゾール[1,5−a]ピリミジン)、アストレシン(CAS RN 170809-51-5)又はBonk et al. "Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF)" Eur. J. Biochem. 267:3017-3024 (2000) に記載のその光励起性類似体、又は(Novartisからの)AAG561;メラニン濃縮ホルモン(MCH)拮抗薬、例えば3,5−ジメトキシ−N−(1−(ナフタレン−2−イルメチル)ピペリジン−4−イル)ベンズアミド又は(R)−3,5−ジメトキシ−N−(1−(ナフタレン−2−イルメチル)−ピロリジン−3−イル)ベンズアミドなど(Kim et al. "Identification of substituted 4-aminopiperidines and 3-aminopyrrolidines as potent MCH-R1 antagonists for the treatment of obesity." Bioorg Med Chem Lett. 2006 Jul 29;両方とも[プリント版に先立つ電子ジャーナル版]を参照)、或いは、米国特許番号第7,045,636号又は公開された米国特許出願US2005/0171098で開示された何れかのMCH拮抗薬が挙げられる。
ヒト神経幹細胞(hNSC)を、米国特許番仮出願番号第60/697,905号(援用する)に記載のとおり、単層培養で単離及び培養し、植え付け、様々な濃度のサブコメリン(試験化合物)で処理し、TUJ−1抗体で染色した。5μMのDHEAを含んでなる分裂促進因子不含試験培地が神経分化についての陽性対照としての役目を果たし、増殖因子不含基礎培地が陰性対照としての役目を果たした。結果を図1に示す。
アストロサイトの分化ついての陽性対照が50ng/mlのBMP−2、50ng/ml:LIF、及び0.5%のFBSを含んでなる分裂促進因子不含試験培地を含み、並びに細胞をGFAP抗体で染色したことを除いて、実験を実施例1に記載のとおり実施した。結果を図2に示す。
陽性対照が基礎培地だけを含み、並びに細胞を核色素(Hoechst 33342)で染色したことを除いて、実験を実施例1に記載のとおり実施した。結果を図3に示す。
ニューロスフィア培養におけるhNSCの増殖速度に対するサブコメリンの効果を、米国特許仮出願番号第60/697,905号(援用する)に記載の通り、時間の関数として個々のニューロスフィアの面積を計測することによって測定した。結果を図4に示す。
免疫組織化学を、米国特許仮出願番号第60/697,905号(援用する)に記載のとおり実施した。
雄Fischer F344ラットを、0、0.01、及び0.05mg/kgのサブコメリンで28日間、処理した。図12は、歯状回の顆粒細胞層下帯内の成熟ニューロンへの神経始原細胞の分化に対するサブコメリンのラット長期間投与の効果を示す。処置ラットにおける様々な特徴及び行動応答を、以下で記載するように計測した。
概日周期の昼間相の間のオープンフィールド活動を、Plexiglas立方体オープンフィールド・アリーナ(赤外線(I/R)アレイを備えた45cm×45cm×高さ50cm、Hamilton Kinder San Diego, CA)において観察した光電セルにより定量化される。測定値:中心と周縁の歩行距離;中心と周縁の歩行時間;中心と周縁の合計時間;中心と周縁の立ち上がり;区間入場の回数;及び総距離を、30分間(5分間の6ブロック)採集した。試験を、サブコメリン注射の30分後に開始した。
毎日、ラットの体重を計測した。
活性な運動行動を、水泳タンク内で計測したが、この試験はPorsolt, R.D., Bertin, A., Jalfree, M, Arch. Int. Pharmacodyn Ther. 229 (1977) 327-336によって記載されている変法である。動物を、水泳タンク(深さ38cm)の中に入れる。水泳試験は、2段階:15分間の予備試験と24時間後の5分間の試験、から成る。活動を、行動の3つの態様:(1)浮いたままでいるのに必要とされるものを除いた動きの不在であると定義される、不動状態、(2)浮いたままでいるのに必要とされるものより大きな水平運動であると定義される、遊泳、(3)浮いたままでいるのに必要とされるより大きな上下運動、クライミングを計測することによって定量化する。主な行動を、訓練された観察者によって5秒毎に合計5分間、スコア化する。
行動試験の24時間前に、全ての飼料をホーム・ケージから取り除く。試験の時点で、1つのペレットを、新奇アリーナの中心に置く。動物を、そのアリーナの隅に置き、上記ペレットを食べるまでの潜時を記録する。通常、化合物を、試験の30分前に投与する。動物には21日間、毎日、化合物を与え、試験を21日目に実施する。短縮された飼料ペレットを食べるまでの潜時は、神経発生及び抗鬱活性の両方を示唆している。
装置は、ポリカーボネート製のオープンフィールド(45×45×高さ50cm)で構成された。3つの複製物を、識別されるべき物体のために使用した。それらが非常に異なった外観を持っているとはいえ、嗅覚の手掛りによって容易に識別できないように、必ず試験される物体の組を同じ材料から作製するようにすることに配慮した。各試験セションは、2つの段階から構成された。最初の習熟段階において、2つの同一物体(A1とA2)を、ボックス・アリーナの遠い隅に置いた。次に、ラットをアリーナの中央に置き、15分間、両方の物体を探らせる。物体の探索を、2cm未満の距離にて物体に鼻を向けること及び/又は鼻でそれに触れることと規定した。中休みの後に、ラットを、アリーナを再び導き入れた(「試験段階」)。その時、ボックスには、見慣れた物体の3つ目の同一複製物(A3)と新奇物体(B)が含まれた。これらを、サンプル刺激と同じ位置に置き、それによって、試験段階における新奇物体の位置(左や右)をラットの間で平均化した。半数のラットに関して、物体Aをサンプルとし、物体Bを新奇代替物とした。試験段階は、好ましいスコアを見つけ出すために使用される最初の30秒の物体交流(object interaction)を伴った15分の継続時間であった。15秒未満の物体探索しかない全ての動物を分析から除外した。図10には、新奇物体認知アッセイにおける認知能力に対するサブコメリンのラット長期間投与の効果を示す。
雄Fischer F344ラットに、5日間、毎日1回、様々な用量の試験化合物+溶媒、又は溶媒のみ(陰性対照)を注射し、それに続いて、100mg/kgのBrdUの腹腔内注射を行った。次に、28日目に、ラットを麻酔し、4%のパラホルムアルデヒドの経心臓潅流によって屠殺した。脳を、迅速に摘出し、4%のパラホルムアルデヒド中に24時間保存し、その後、リン酸緩衝化30%ショ糖中で平衡化した。フリーフローティング40μm切片を、凍結ミクロトームにより回収し、抗凍結剤中に保存した。BrdU、及び着目の細胞型(例えばニューロン、アストロサイト、乏突起膠細胞、内皮細胞)に対する抗体もまた、細胞分化の検出に使用される。要するに、組織を、洗浄し(0.01MのPBS)、1%の過酸化水素で内在性ペルオキシダーゼを阻害し、PBS(0.01M、pH7.4、10%の正常ヤギ血清、0.5%のTriton X−100)中、室温にて2時間インキューベートした。次に、組織を、一次抗体と共に4℃にて一晩インキューベートした。その組織を、PBSによりすすぎ、それに続いて、ビオチン化二次抗体と共にインキュベーションを行った(室温にて1時間)。組織を、PBSで更に洗浄し、アビジン−ビオチン複合体キット溶液中、室温にて1時間インキューベートした。ストレプトアビジンに連結した様々な蛍光発色団を、視覚化に使用した。組織を、PBSで洗浄し、dH2Oで簡単にすすぎ、連続的に脱水し、カバースリップで覆った。
雄Fischer F344系ラットに、試験化合物+溶媒、又は溶媒のみ(陰性対照)を毎日1回、長期的に注射し、実施例6において先に記載のとおり、新奇さにより抑制される摂食のアッセイによりアッセイした。図13Aに示された結果は、サブコメリンとフルオキセチンの併用が、何れかの化合物単独に比べて有意に大きな抗鬱作用を非常に低い用量にて生じ得ることを示している。
雄Fischer F344ラットに、試験化合物+溶媒、又は溶媒のみ(陰性対照)を28日間、毎日1回注射する。100mg/kgのBrdUの腹腔内投与が、7日目の後に5日間、毎日行われる。次に、ラットを、28日目に、麻酔し、4%のパラホルムアルデヒドの経心臓潅流によって屠殺し、実施例7において先に記載されたとおり、増殖型のNCSsを計測する。図13Bに示された結果は、サブコメリンとフルオキセチンの併用が化合物の非常に低い用量にて有意な神経原性効果をもたらすことを示している。
ヒト神経幹細胞(hNSC)を、実施例1に記載のとおり、単層培養において単離及び培養し、植え付け、様々な濃度のドネペジル及び/又はカプトプリル(試験化合物)で処理し、TUJ−1抗体で染色した。バックグラウンド培地値の差し引き後の神経分化の濃度依存的反応曲線を示している結果を、図14に示す。ドネペジルとカプトプリルの併用の濃度反応曲線を、ドネペジル又はカプトプリル単独の濃度反応曲線と共に示す。データを、ニューロンの陽性対照に対する百分率として提示する。データは、ドネペジルとカプトプリルの併用が、何れかの薬剤単独に比べて神経分化の優れた促進をもたらすことを示している。
ヒト神経幹細胞(hNSC)を、米国特許仮出願番号第60/697,905号(援用する)に記載のとおり、単層培養において単離及び培養し、植え付け、様々な濃度のドネペジル及び/又はブスピロン(試験化合物)で処理し、GFAP抗体で染色した。神経分化の陽性対照を含んでなる分裂促進因子不含試験培地を、陰性対照としての増殖因子不含基礎培地と共に使用した。
相乗効果の存在を、併用指数(CI)の使用によって測定した。併用する時、1組の化合物が相加、相乗(相加より高い)、又は拮抗効果を生じるかどうかを測定するのに使用される場合、CIはEC50に基づく。CIは、それぞれを単独でアッセイした時の、2つの化合物のEC50を、併用状態でアッセイした時のそれぞれの化合物のEC50と比較した、薬物相互作用の性質の定量的尺度である。併用指数(CI)は、以下の式に相当する:
Claims (26)
- 対象又は患者の、細胞変性、精神状態、細胞の外傷及び/又は傷害に関連する神経系障害、或いは他の神経学的に関連する状態を治療するための方法であって、
ムスカリン様作用剤を、任意により他のムスカリン様作用剤、及び/又は、他の神経原性剤との併用で、上記の対象又は患者に投与し、上記障害の改善を生じさせる工程を含んでなる方法。 - 前記の細胞変性に関連する神経系障害が、神経変性障害、神経幹細胞障害、神経始原細胞障害、網膜の変性疾患、虚血性障害、及び、それらの組合せから選択される、請求項1記載の方法。
- 前記の精神状態に関連する神経系障害が、神経精神疾患、情動障害、鬱病、軽躁病、不安発作、不安症、過度の高揚感、双極性鬱病、双極性障害(躁鬱病)、季節的な気分(又は情動)障害、統合失調症及び他の精神病、脳回欠損症候群、不安症候群、不安障害、恐怖症、ストレス及び関連症候群、認識機能障害、攻撃性、薬物及びアルコール乱用、強迫的行動症候群、境界性人格障害、非老年性認知症、疼痛後鬱病、産後鬱病、脳性麻痺、及びそれらの組合せから選択される、請求項1記載の方法。
- 前記の細胞の外傷及び/又は傷害に関連する神経系障害が、神経学的な外傷及び傷害、外科手術に関連する外傷及び/又は傷害、網膜の傷害及び外傷、癲癇に関連する傷害、脊髄傷害、脳傷害、脳の外科手術、外傷に関連する脳傷害、外傷に関連する脊髄傷害、癌治療に関連する脳傷害、癌治療に関連する脊髄傷害、感染に関連する脳傷害、炎症に関連する脳傷害、感染に関連する脊髄傷害、炎症に関連する脊髄傷害、環境有害物質に関連する脳傷害、環境有害物質に関連する脊髄傷害、並びにそれらの組合せから選択される、請求項1記載の方法。
- 前記の神経学的に関連する状態が、学習障害、記憶障害、自閉症、注意欠陥障害、ナルコレプシー、睡眠障害、認知障害、癲癇、側頭葉癲癇、及びそれらの組合せから選択される、請求項1記載の方法。
- 前記精神状態が鬱病を含んでなる、請求項3に記載の方法。
- 前記ムスカリン様作用剤がサブコメリンである、請求項1〜6の何れか一項に記載の方法。
- 前記鬱病が、対象又は患者によるモルヒネ、アルコール、又は薬物の使用に起因する、請求項6記載の方法。
- 前記ムスカリン様作用剤がサブコメリンである、請求項8記載の方法。
- 前記ムスカリン様作用剤が、AChE阻害剤、例えば有機リン酸、アミノアクリジン、カルバマート、フェナントリン誘導体、ピペリジン誘導体、イトプリド、(−)−ヒューペルジンA、又はフェンセリン等である、請求項1〜6の何れか一項に記載の方法。
- 前記AChE阻害剤が、メトリホナート、エコチオファート、タクリン、フィゾスチグミン、ネオスチグミン、リバスチグミン、ガランタミン、ドネペジル、イトプリド、リバスチグミン、臭化水素酸ガランタミン、(−)−ヒューペルジンA、又はフェンセリンである、請求項9記載の方法。
- 対象又は患者への移植用の細胞又は組織を調製する方法であって、
上記の細胞又は組織を、任意により他のムスカリン様作用剤、及び/又は、他の神経原性剤との併用で、ムスカリン様作用剤と接触させ、上記の細胞又は組織の神経発生を刺激又は増強する工程を含んでなる方法。 - 細胞又は組織における神経発生の刺激又は増強するための方法であって、
上記の細胞又は組織を、任意により他のムスカリン様作用剤、及び/又は、他の神経原性剤との併用で、ムスカリン様作用剤と接触させる工程を含んでなり、
上記ムスカリン様作用剤又は併用が、上記の細胞又は組織において神経発生を引き起こすのに有効である、方法。 - 前記の細胞又は組織が、動物対象又はヒト患者由来である、請求項13記載の方法。
- 前記患者が、神経発生を必要とする患者、又は、中枢又は末梢神経系の疾患、状態又は傷害と診断された患者である、請求項14記載の方法。
- 前記神経原性剤が、オピオイド又は非オピオイド神経原性剤である、請求項13〜15の何れか一項に記載の方法。
- 前記非オピオイド神経原性剤がドーパミンである、請求項16記載の方法。
- 前記神経発生が、ニューロンの系統に沿った神経幹細胞(NSCs)の分化を含んでなる、請求項13記載の方法。
- 前記神経発生が、グリア細胞系統に沿った神経幹細胞(NSCs)の分化を含んでなる、請求項14記載の方法。
- 前記のオピオイドが、κオピオイド受容体拮抗薬である、請求項16記載の方法。
- 前記オピオイドが、κオピオイド受容体選択的拮抗薬である、請求項20記載の方法。
- 前記オピオイドが、JDTic、ノル−ビナルトルフィミン及びブプレノルフィンから選択される、請求項21記載の方法。
- 前記の細胞又は組織が、神経発生の減弱を示す細胞又は組織、或いは、神経発生を減弱又は阻害する薬剤に晒されている細胞又は組織である、請求項14又は請求項15に記載の方法。
- 前記の神経発生を減弱又は阻害する薬剤が、オピオイド受容体作動薬である、請求項23記載の方法。
- 前記オピオイド受容体作動薬が、モルヒネ又は他のアヘン剤である、請求項24記載の方法。
- 前記の対象又は患者が、1又は2以上の薬物中毒又は依存状態を患っている、請求項14又は請求項15に記載の方法。
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CA2620333A1 (en) | 2007-03-01 |
EP2275096A2 (en) | 2011-01-19 |
EP1928437A2 (en) | 2008-06-11 |
US7678363B2 (en) | 2010-03-16 |
US20070049576A1 (en) | 2007-03-01 |
US20100120842A1 (en) | 2010-05-13 |
WO2007025177A3 (en) | 2007-10-04 |
EP2275095A3 (en) | 2011-08-17 |
AU2006282896A1 (en) | 2007-03-01 |
EP2275095A2 (en) | 2011-01-19 |
WO2007025177A2 (en) | 2007-03-01 |
EP2275096A3 (en) | 2011-07-13 |
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