JP6817188B2 - 血液脳関門透過性ペプチド - Google Patents
血液脳関門透過性ペプチド Download PDFInfo
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- JP6817188B2 JP6817188B2 JP2017506596A JP2017506596A JP6817188B2 JP 6817188 B2 JP6817188 B2 JP 6817188B2 JP 2017506596 A JP2017506596 A JP 2017506596A JP 2017506596 A JP2017506596 A JP 2017506596A JP 6817188 B2 JP6817188 B2 JP 6817188B2
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Description
(a) 配列番号1で表されるアミノ酸配列からなるポリペプチド
(b) 配列番号1で表されるアミノ酸配列において、1〜6個のアミノ酸が欠失、置換、挿入及び/又は付加されたアミノ酸配列からなり、且つ脳移行活性を有するポリペプチド
(c) (a)又は(b)に示されるポリペプチドのC末端側及び/又はN末端側に、1〜5個の任意のアミノ酸が付加されたアミノ酸配列からなり、且つ脳移行活性を有するポリペプチド。
項2.以下の(d)、(e)又は(f)のいずれかのポリペプチド:
(d) 配列番号2で表されるアミノ酸配列からなるポリペプチド
(e) 配列番号2で表されるアミノ酸配列において、1〜6個のアミノ酸が欠失、置換、挿入及び/又は付加されたアミノ酸配列からなり、且つ脳移行活性を有するポリペプチド
(f) (d)又は(e)に示されるポリペプチドのC末端側及び/又はN末端側に、1〜5個の任意のアミノ酸が付加されたアミノ酸配列からなり、且つ脳移行活性を有するポリペプチド。
項3.項1又は2に記載のポリペプチドを含む脳内送達用キャリア分子。
項4.項1又は2に記載のポリペプチド、及びそれに結合したタンパク質、ポリペプチド、オリゴペプチド、低分子化合物、又は核酸を含有する複合体。
項5.項4に記載の複合体を含む医薬組成物。
項6.脳疾患の予防及び/又は治療用である、項5に記載の医薬組成物。
項7.脳疾患の診断用である、項5に記載の医薬組成物。
項8.項4に記載の複合体の有効量を患者に投与する工程を含む脳疾患の予防及び/又は治療方法。
項9.項4に記載の複合体の有効量を患者に投与する工程を含む脳疾患の診断方法。
項10.脳疾患の予防及び/又は治療用の医薬組成物の製造における、項4に記載の複合体の使用。
項11.脳疾患の診断用の医薬組成物の製造における、項4に記載の複合体の使用。
本発明のポリペプチドは、以下の(a)、(b)又は(c)のいずれかであることを特徴とする。
(a) 配列番号1(FLGWLGAWGTMGWSPKKKRK)で表されるアミノ酸配列からなるポリペプチド
(b) 配列番号1で表されるアミノ酸配列において、1〜6個のアミノ酸が欠失、置換、挿入及び/又は付加されたアミノ酸配列からなり、且つ脳移行活性を有するポリペプチド
(c) (a)又は(b)に示されるポリペプチドのC末端側及び/又はN末端側に、1〜5個の任意のアミノ酸が付加されたアミノ酸配列からなり、且つ脳移行活性を有するポリペプチド。
本発明の脳内送達用キャリア分子は、上記(a)、(b)又は(c)のポリペプチドを含むことを特徴とする。
本発明の複合体は、上記(a)、(b)又は(c)のポリペプチド、及びそれに結合したタンパク質、ポリペプチド、オリゴペプチド、低分子化合物、又は核酸(以下、「化合物A」と称する)を含有することを特徴とする。
本発明の医薬組成物は、上記の複合体を含むことを特徴とする。
本研究で実験に使用したペプチドのアミノ酸配列を表1に示す。各ペプチドは、カルボキシ末端(C末端)側に、フルオレセインを導入するためのGly-Cys配列を有する。また、アミノ末端(N末端)はアセチル化されており、C末端はアミドである。ただし例外として、C6R8及びmMPG8は、そのN末端に、アセチル基ではなく、それぞれヘキサン酸又はβアラニンを有している。
表1に示すペプチド(1)-(13)が、本来細胞内に移行しない物質を、どの程度効率よく細胞内に送達することが可能であるかを調べた。本来細胞内に移行しない水溶性モデル物質として、蛍光色素であるフルオレセインを用いた。実験の2日前に50,000 cells/wellで4-well chamber slide (Lab-Tek II chamber slide、Nunc社)に播種したマウス神経芽細胞腫Neuro2A細胞に対し、ダルベッコ改変イーグル培地(DMEM、ギブコ社)で10μMとなるように希釈した標識ペプチド(1)-(13)を投与し、1時間培養した後、細胞核をHoechst33342 (モレキュラープローブ社)で染色してから共焦点顕微鏡(オリンパス社製FV1000)観察を行った(図1)。
試験例1で選択された6種類のペプチドについて、BBB透過性を評価した。BBB透過性の評価は、共同発明者の中川らが開発したin vitro BBB再構成系(BBBキット、Cell. Mol. Neurobiol. 27, 687-694 (2007).)を用いて行った。本試験例では、標識ペプチドと同程度の分子量(およそ4000)でBBB透過性をほとんど示さない化合物であるフルオレセイン標識デキストラン(FD4)、及び試験例1においてほとんど細胞内送達性を示さなかったペプチドであるSAP(8)を、ネガティブコントロールとして用いた。BBBキットの血管側に1μMの標識ペプチドを投与し、脳側培養液中のフルオレセイン蛍光強度を経時的に測定した。蛍光強度は蛍光プレートリーダーで測定し、この測定値から脳側培養液中の標識ペプチド濃度を算出した(図3)。
試験例2において、最も高いBBB透過性を示したmMPG8(13)について、動物モデルを用いて脳内移行性を評価した。本試験例では、脳内移行性をほとんど示さない化合物であるNa-F、及び試験例2においてほとんどBBB透過性を示さなかったペプチドであるpVEC(4)を、ネガティブコントロールとして用いた。
試験例1から3の実験条件におけるmMPG8(13)の細胞傷害性を評価した。本試験例では、試験例1において高い細胞内移行性を示したものの、試験例2及び試験例3においてほとんどBBB透過性及び脳内移行性を示さなかったペプチドであるpVEC(4)を、ネガティブコントロールとして用いた。実験の2日前に5,000 cells/wellで96-well plate (Iwaki社)に播種したマウス脳血管内皮細胞株MBEC4に対し、DMEMで希釈した非標識ペプチド(5μM、10μM、又は30μM)を投与し、1時間、6時間、又は24時間培養した。培養後、細胞増殖試薬WST-1 (ロシュ社)を添加し、1時間後に450 nmの吸光度を吸光マイクロプレートリーダ(Multiskan FC、Thermo Fisher Scientific社)で測定し細胞生存率を評価した(図9)。
Claims (3)
- 以下の(a)又は(b)のポリペプチドを含む脳移行性付与剤:
(a) 配列番号1で表されるアミノ酸配列のN末端にβアラニンを有するアミノ酸配列からなるポリペプチド
(b) (a)に示されるポリペプチドのC末端側に、Cys又はGly-Cysのアミノ酸が付加されたアミノ酸配列からなるポリペプチド。 - 以下の(a)又は(b)のポリペプチド、及びそれに結合したタンパク質、ポリペプチド、オリゴペプチド、低分子化合物、又は核酸を含有する複合体を含む、脳疾患の予防及び/又は治療用医薬組成物:
(a) 配列番号1で表されるアミノ酸配列のN末端にβアラニンを有するアミノ酸配列からなるポリペプチド
(b) (a)に示されるポリペプチドのC末端側に、Cys又はGly-Cysのアミノ酸が付加されたアミノ酸配列からなるポリペプチド。 - 以下の(a)又は(b)のいずれかのポリペプチド、及びそれに結合したタンパク質、ポリペプチド、オリゴペプチド、低分子化合物、又は核酸を含有する複合体を含む、脳疾患の診断用医薬組成物:
(a) 配列番号1で表されるアミノ酸配列のN末端にβアラニンを有するアミノ酸配列からなるポリペプチド
(b) (a)に示されるポリペプチドのC末端側に、Cys又はGly-Cysのアミノ酸が付加されたアミノ酸配列からなるポリペプチド。
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