JPWO2016148213A1 - Blood brain barrier permeability peptide - Google Patents
Blood brain barrier permeability peptide Download PDFInfo
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- JPWO2016148213A1 JPWO2016148213A1 JP2017506596A JP2017506596A JPWO2016148213A1 JP WO2016148213 A1 JPWO2016148213 A1 JP WO2016148213A1 JP 2017506596 A JP2017506596 A JP 2017506596A JP 2017506596 A JP2017506596 A JP 2017506596A JP WO2016148213 A1 JPWO2016148213 A1 JP WO2016148213A1
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- polypeptide
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Abstract
開示されているのは、以下の(a)、(b)又は(c)のいずれかのポリペプチド:(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、該ポリペプチドを含む脳内送達用キャリア分子、該ポリペプチド、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、該複合体を含む医薬組成物、該複合体を用いる脳疾患の予防及び/又は治療方法、並びに脳疾患の診断方法である。What is disclosed is any of the following polypeptides (a), (b) or (c): (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 (b) SEQ ID NO: 1 A polypeptide (c) (a) or (b) consisting of an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented and having brain translocation activity A polypeptide comprising an amino acid sequence having 1 to 5 arbitrary amino acids added to the C-terminal side and / or the N-terminal side of the polypeptide shown and having a brain transition activity, and delivery in the brain containing the polypeptide Carrier molecule, polypeptide, and protein, polypeptide, oligopeptide, low molecular compound, or nucleic acid complex containing the complex, pharmaceutical composition containing the complex, and prevention of brain disease using the complex And / or treatment method, line It is a diagnostic method of brain disease.
Description
本発明は、血液脳関門(Blood-Brain Barrier, BBB)透過性及び脳移行活性を有するポリペプチド、及び該ポリペプチドを含む脳内送達用キャリア分子に関する。さらに、本発明は、上記ポリペプチドを含有する複合体、及び該複合体を含む医薬組成物に関する。また、本発明は、上記複合体を用いる脳疾患の予防及び/又は治療方法、並びに脳疾患の診断方法に関する。 The present invention relates to a polypeptide having blood-brain barrier (BBB) permeability and brain translocation activity, and a carrier molecule for intracerebral delivery containing the polypeptide. Furthermore, the present invention relates to a complex containing the above polypeptide and a pharmaceutical composition comprising the complex. The present invention also relates to a method for preventing and / or treating a brain disease using the complex, and a method for diagnosing a brain disease.
末梢組織に投与した水溶性薬物及びタンパク質は、脳内にほとんど移行しないことが知られている。これは、BBBが脳と末梢組織とを隔てる物理的及び生理的障壁として機能し、脳内への物質の受動拡散を厳密に制限しているからである。実際、高い脂溶性を備えた小分子化合物を除くほぼすべての物質において、血流を介して脳内に移行することは極めて困難である。そのため、末梢組織に投与した目的物質を脳内に送達するには、BBBを効率的に透過するキャリア分子の開発が必要不可欠である。折しも人口高齢化社会の到来に伴い、アルツハイマー病及びパーキンソン病といった中枢神経系疾患患者の著しい増大が予想されているものの、これら疾患の多くは未だ有効な治療法に乏しい難病である。中枢神経系疾患に対する治療薬開発という観点からも、BBBを透過する脳内送達用キャリア分子の開発が求められている。 It is known that water-soluble drugs and proteins administered to peripheral tissues hardly migrate into the brain. This is because the BBB functions as a physical and physiological barrier that separates the brain from peripheral tissues and strictly restricts the passive diffusion of substances into the brain. In fact, almost all substances except small molecule compounds with high fat solubility are very difficult to move into the brain via the bloodstream. Therefore, in order to deliver a target substance administered to peripheral tissues into the brain, it is essential to develop a carrier molecule that efficiently permeates the BBB. With the advent of an aging society, the number of patients with central nervous system diseases such as Alzheimer's disease and Parkinson's disease is expected to increase significantly, but many of these diseases are intractable diseases that still lack effective treatment. From the viewpoint of developing therapeutic agents for central nervous system diseases, there is a need for the development of carrier molecules for intracerebral delivery that penetrate the BBB.
タンパク質、水溶性薬物などの本来脳内に移行しない物質を脳内にデリバリーするためのペプチド性キャリア分子として、過去にTatペプチドが報告されている。Tatペプチドは、細胞移行性ペプチド(Protein Transduction Domain, PTDs; Cell-Penetrating Peptides, CPPs)の一種として、タンパク質などの細胞内デリバリー分子として広く利用されているペプチドであるが、一部の研究グループによりこれが脳内へも移行することが報告されている。例えば、SchwarzeらはTatペプチドとβ-ガラクトシダーゼとを融合したタンパク質(Tat-β-ガラクトシダーゼ)をマウスに皮下注射すると、脳を含む多くの臓器でβ-Gal活性が確認されたことを報告している(Science 285, 1569-1572 (1999).)。また、Caoらは、アポトーシス抑制因子Bcl-xLをTatペプチドと融合させ、脳虚血モデルマウスに皮下注射すると、脳内の神経細胞死が抑制されたことを報告している(J. Neurosci. 22, 5423-5431 (2002).)。 Tat peptides have been reported in the past as peptide carrier molecules for delivering substances that do not naturally enter the brain, such as proteins and water-soluble drugs, into the brain. Tat peptide is a peptide that is widely used as an intracellular delivery molecule such as protein as a kind of cell translocation domain (PTDs; Cell-Penetrating Peptides, CPPs). It has been reported that this also moves into the brain. For example, Schwarze et al. Reported that β-Gal activity was confirmed in many organs including the brain when a mouse was injected subcutaneously with a protein fused with Tat peptide and β-galactosidase (Tat-β-galactosidase). (Science 285, 1569-1572 (1999).) Also, Cao et al. Reported that neuronal cell death in the brain was suppressed when the apoptosis inhibitor Bcl-xL was fused with a Tat peptide and injected subcutaneously into cerebral ischemia model mice (J. Neurosci. 22, 5423-5431 (2002).).
上記Tatペプチドの脳内移行性に関し、近年複数のグループから再評価が行われている。そもそもTatペプチドを用いたタンパク質の脳内導入例は、そのほぼすべてが脳虚血あるいは脳卒中モデル動物という、BBBを構成する脳血管内皮細胞の部分的な傷害が否定できないモデルを用いて行われたものであり、厳密な意味でのBBB透過性を示すものではない。これに関連して、Simonらは、TatペプチドのBBB透過性を評価するため、血管内皮細胞層を用いて検討を行った結果、これを全く透過しないことを明らかにし、Tatを用いた脳内デリバリー法は一般的に有効な手法ではないと指摘している(Ann. Biomed. Eng. 39, 394-401 (2010).)。 In recent years, the Tat peptide has been re-evaluated by several groups regarding its ability to migrate into the brain. In the first place, almost all cases of introducing proteins using the Tat peptide into the brain were performed using a model in which partial damage to the cerebral vascular endothelial cells constituting the BBB cannot be ruled out, which is a model animal of cerebral ischemia or stroke. It does not show BBB permeability in a strict sense. In connection with this, Simon et al. Examined the vascular endothelial cell layer to evaluate the BBB permeability of the Tat peptide, and found that it did not penetrate at all. It is pointed out that the delivery method is not generally effective (Ann. Biomed. Eng. 39, 394-401 (2010)).
また、Caiらは、Tat-β-ガラクトシダーゼを用いて追試実験を行ったところ、皮下注射、静脈内注射及び経口投与のいずれにおいても脳内におけるβ-Gal活性がほとんど検出できなかったことを、上記Science誌に掲載された論文の責任著者とともに報告している(Eur. J. Pharm. Sci. 27, 311-319 (2006).)。現時点では、Tatペプチドを含め、正常なBBBを備えたモデル動物に対して確実に脳内移行性が証明されたBBB透過性ペプチドは報告されていない。 In addition, Cai et al. Conducted a follow-up experiment using Tat-β-galactosidase and found that β-Gal activity in the brain was hardly detectable in any of subcutaneous injection, intravenous injection and oral administration. It is reported with the author responsible for the paper published in the above Science magazine (Eur. J. Pharm. Sci. 27, 311-319 (2006)). At present, no BBB-penetrating peptide has been reported that has been demonstrated to reliably enter the brain, including Tat peptide, in model animals with normal BBB.
非特許文献1−3では、培養細胞へオリゴヌクレオチドを効率的にデリバリーするためのペプチド性キャリア分子であるMPGペプチド(Ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-Cya (Ac: アセチル, Cya: システアミド))について報告されている。MPGとオリゴヌクレオチドとは静電的相互作用で結合し、また複数のMPG分子がオリゴヌクレオチドをコートする形で結合するので、オリゴヌクレオチドは安定化され、分解から保護されている。 Non-Patent Documents 1-3 report an MPG peptide (Ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-Cya (Ac: acetyl, Cya: cysteamide)), which is a peptide carrier molecule for efficiently delivering oligonucleotides to cultured cells. . Since the MPG and the oligonucleotide are bound by electrostatic interaction, and since a plurality of MPG molecules are bound to coat the oligonucleotide, the oligonucleotide is stabilized and protected from degradation.
非特許文献4では、MPGを改良したMPG-8 (βAla-FLGWLGAWGTMGWSPKKKRK-Cya)について報告されており、MPG-8はsiRNAとナノパーティクルを形成し、培養細胞及びインビボでのsiRNAの効率的なデリバリーを促進することが記載されている。 Non-Patent Document 4 reports on MPG-8 (βAla-FLGWLGAWGTMGWSPKKKRK-Cya) with improved MPG. MPG-8 forms nanoparticles with siRNA and efficiently delivers siRNA in cultured cells and in vivo. It is described to promote.
しかしながら、非特許文献1−4には、MPG及びMPG-8の脳移行性についての言及はなく、更に脳移行性を示す明確なデータも存在しない。 However, Non-Patent Documents 1-4 do not mention the brain transferability of MPG and MPG-8, and there is no clear data indicating the brain transferability.
そこで、本発明は、高いBBB透過性及び脳移行活性を有するポリペプチド、及び該ポリペプチドを含む脳内送達用キャリア分子を提供することを目的とする。さらに、本発明は、上記ポリペプチドを含有する複合体、及び該複合体を含む医薬組成物を提供することを目的とする。また、本発明は、上記複合体を用いる脳疾患の予防及び/又は治療方法、並びに脳疾患の診断方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a polypeptide having high BBB permeability and brain translocation activity, and a carrier molecule for intracerebral delivery containing the polypeptide. Furthermore, an object of this invention is to provide the composite_body | complex containing the said polypeptide, and the pharmaceutical composition containing this composite_body | complex. Another object of the present invention is to provide a method for preventing and / or treating a brain disease using the complex, and a method for diagnosing a brain disease.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、mMPG8 ((βAla)-FLGWLGAWGTMGWSPKKKRK-CONH2)が高いBBB透過性及び脳移行性を有しており、また、mMPG8は共有結合により結合させた小分子の水溶性薬物モデルであるフルオレセインを脳内に高効率且つ迅速に移行させることができるという知見を得た。As a result of intensive studies to achieve the above object, the present inventors have found that mMPG8 ((βAla) -FLGWLGAWGTMGWSPKKKRK-CONH 2 ) has high BBB permeability and brain migration, and mMPG8 is shared The present inventors have found that fluorescein, which is a small molecule water-soluble drug model bound by binding, can be rapidly and efficiently transferred into the brain.
本発明は、これら知見に基づき、更に検討を重ねて完成されたものであり、次のポリペプチド、脳内送達用キャリア分子、複合体、医薬組成物、予防及び/又は治療方法、並びに診断方法を提供するものである。 The present invention has been completed based on these findings, and has been completed. The following polypeptide, carrier molecule for delivery in the brain, complex, pharmaceutical composition, prevention and / or treatment method, and diagnosis method are as follows. Is to provide.
項1.以下の(a)、(b)又は(c)のいずれかのポリペプチド:
(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。
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having brain transition activity
(c) It consists of an amino acid sequence in which 1 to 5 arbitrary amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (a) or (b), and has a brain transition activity Polypeptide.
(d) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2
(e) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having brain translocation activity
(f) It consists of an amino acid sequence in which 1 to 5 arbitrary amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (d) or (e), and has a brain transition activity Polypeptide.
Item 4.
Item 7.
Item 8.
Item 9. A method for diagnosing a brain disease, comprising a step of administering an effective amount of the complex according to Item 4 to a patient.
Item 11.
本発明のポリペプチドは高いBBB透過性及び脳移行性を有しており、本来脳内に移行しないタンパク質、ペプチド、低分子化合物などを本発明のポリペプチドと結合させることによりこれらを効率的に脳内に移行させることが可能となる。 The polypeptide of the present invention has high BBB permeability and brain transportability, and efficiently binds proteins, peptides, low-molecular compounds, etc. that do not inherently enter the brain by binding them to the polypeptide of the present invention. It can be transferred into the brain.
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
なお、本明細書において「含む(comprise)」とは、「本質的にからなる(essentially consist of)」という意味と、「からなる(consist of)」という意味をも包含する。 In the present specification, “comprise” includes the meaning of “essentially consist of” and the meaning of “consist of”.
ポリペプチド
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。 Polypeptide The polypeptide of the present invention is any of the following (a), (b) or (c).
(a) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 (FLGWLGAWGTMGWSPKKKRK)
(b) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having brain transition activity
(c) It consists of an amino acid sequence in which 1 to 5 arbitrary amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (a) or (b), and has a brain transition activity Polypeptide.
以下、本明細書において「本発明のポリペプチド」と称する場合は、上記(a)、(b)又は(c)のポリペプチドを意味するものとする。 Hereinafter, the term “polypeptide of the present invention” in the present specification means the above-mentioned polypeptide (a), (b) or (c).
本発明において「脳移行活性」とは、ポリペプチドなどの分子が体内へ静脈投与などにより投与された場合に、当該分子が脳組織内へ移行する活性を意味する。 In the present invention, “brain transfer activity” means an activity of transferring a molecule such as a polypeptide into the brain tissue when the molecule is administered intravenously into the body.
本発明のポリペプチドは、高いBBB透過性及び脳移行活性を有している。いかなる理論にも拘束されることを望むものではないが、後述する実施例で示しているように、本発明のポリペプチドによるBBB透過は、BBBに存在する何らかの輸送担体を介した取り込み機構が関与するものではないと推測される。また、本発明のポリペプチドは、他の分子と結合することにより、他の分子を脳内に移行させることが可能である。 The polypeptide of the present invention has high BBB permeability and brain translocation activity. While not wishing to be bound by any theory, as shown in the examples below, BBB permeation by the polypeptides of the present invention involves an uptake mechanism via some transport carrier present in BBB. Presumed not to do. In addition, the polypeptide of the present invention can transfer other molecules into the brain by binding to the other molecules.
本発明のポリペプチドが脳移行活性を示す対象となり得る動物は、BBBを有する動物であれば特に限定されない。動物の中でも、好ましくは哺乳動物であり、哺乳動物としては例えば、ラット、マウス、ウサギ、イヌ、ネコ、ヤギ、ウシ、サル、ヒトなどが挙げられる。 The animal that can be a subject to which the polypeptide of the present invention exhibits brain translocation activity is not particularly limited as long as it is an animal having BBB. Among the animals, mammals are preferred, and examples of mammals include rats, mice, rabbits, dogs, cats, goats, cows, monkeys, humans, and the like.
本発明のポリペプチドには、その塩も含まれる。ここで「塩」とは、ポリペプチドの薬理学的に許容される任意の塩であり、例えば、ポリペプチドのナトリウム塩、カリウム塩、カルシウム塩、塩酸塩、硫酸塩、硝酸塩、マグネシウム塩、アンモニウム塩、リン酸塩、有機酸塩(酢酸塩、トリフルオロ酢酸塩、クエン酸塩、マレイン酸塩、シュウ酸塩、リンゴ酸塩、乳酸塩、コハク酸塩、プロピオン酸塩、フマル酸塩、ギ酸塩、ピクリン酸塩、安息香酸塩、ベンゼンスルホン酸塩など)などが挙げられる。 The polypeptide of the present invention includes a salt thereof. Here, the “salt” is any pharmacologically acceptable salt of a polypeptide, for example, sodium salt, potassium salt, calcium salt, hydrochloride, sulfate, nitrate, magnesium salt, ammonium of the polypeptide. Salt, phosphate, organic acid salt (acetate, trifluoroacetate, citrate, maleate, oxalate, malate, lactate, succinate, propionate, fumarate, formic acid Salt, picrate, benzoate, benzenesulfonate, etc.).
また、本発明のポリペプチドには、その誘導体も含まれる。ここで「誘導体」とは、本発明のポリペプチドの官能基を公知の方法により修飾、付加、変異、置換、削除などにより改変されたものをいう。例えば、本発明のポリペプチドのN末端、C末端、又はアミノ酸の側鎖が保護基などによって修飾されているものが挙げられる。誘導体としては、例えば、アセチル化、パルミトイル化、アミド化、ミリスチル化、ダンシル化、アクリル化、ビオチン化、リン酸化、アニリド化、サクシニル化、ベンジルオキシカルボニル化、ホルミル化、ニトロ化、スルフォン化、アルデヒド化、グリコシル化、環状化、モノメチル化、ジメチル化、トリメチル化、グアニジル化、マレイル化、トリフルオロアセチル化、トリニトロフェニル化、カルバミル化、ポリエチレングリコール化、アセトアセチル化、標識化(例えば、PET用放射性核種、蛍光色素など)されたものなどが挙げられる。中でもN末端のアセチル化、C末端のアミド化は、末端からポリペプチドを分解するエキソペプチダーゼに対する抵抗性が付与されるため好ましい。 The polypeptide of the present invention also includes derivatives thereof. Here, the “derivative” refers to a derivative obtained by modifying the functional group of the polypeptide of the present invention by modification, addition, mutation, substitution, deletion or the like by a known method. For example, the N-terminal, C-terminal, or amino acid side chain of the polypeptide of the present invention is modified with a protecting group. Examples of the derivatives include acetylation, palmitoylation, amidation, myristylation, dansylation, acrylation, biotinylation, phosphorylation, anilide, succinylation, benzyloxycarbonylation, formylation, nitration, sulfonation, Aldehydation, glycosylation, cyclization, monomethylation, dimethylation, trimethylation, guanidylation, maleylation, trifluoroacetylation, trinitrophenylation, carbamylation, polyethylene glycolation, acetoacetylation, labeling (e.g. PET radionuclides, fluorescent dyes, etc.). Of these, acetylation at the N-terminus and amidation at the C-terminus are preferred because they impart resistance to exopeptidases that degrade the polypeptide from the terminus.
ただし、本発明のポリペプチドには、C末端がシステアミドにより修飾されたものは含まれない。この点から、本発明のポリペプチドは、C末端がシステアミドにより修飾されている非特許文献4に記載のMPG-8と相違する。 However, the polypeptide of the present invention does not include those in which the C-terminus is modified with cysteamide. From this point, the polypeptide of the present invention is different from MPG-8 described in Non-Patent Document 4 in which the C-terminus is modified with cysteamide.
本発明のポリペプチドを構成するアミノ酸は、L体又はD体のいずれであってもよい。また、本発明のポリペプチドを構成するアミノ酸は、天然のアミノ酸に限定されず、非天然のアミノ酸であってもよい。 The amino acid constituting the polypeptide of the present invention may be either L-form or D-form. Moreover, the amino acid which comprises the polypeptide of this invention is not limited to a natural amino acid, A non-natural amino acid may be sufficient.
上記(b)のポリペプチドにおいて、欠失、置換、挿入及び/又は付加されるアミノ酸の個数は、好ましくは1〜5個、より好ましくは1〜4個、更に好ましくは1〜3個、特に好ましくは1又は2個、より特に好ましくは1個である。アミノ酸を置換する場合、性質の似たアミノ酸に置換すれば、もとのポリペプチドの活性が維持されやすいと考えられる。 In the polypeptide (b), the number of amino acids to be deleted, substituted, inserted and / or added is preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably Preferably 1 or 2, more particularly preferably 1. When an amino acid is substituted, it is considered that the activity of the original polypeptide is easily maintained if the amino acid is substituted with an amino acid having similar properties.
上記(c)のポリペプチドにおいて、C末端側及び/又はN末端側に付加される任意のアミノ酸の個数は、好ましくは1〜4個、より好ましくは1〜3個、更に好ましくは1又は2個、特に好ましくは1個である。 In the polypeptide (c) above, the number of arbitrary amino acids added to the C-terminal side and / or N-terminal side is preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 or 2. One, particularly preferably one.
任意のアミノ酸は特に限定されない。アミノ酸の中でもシステインが望ましい。システイン残基を付加させた場合、システインのSH基を利用して、本発明のポリペプチドに化合物を結合させることができるため望ましい。システインを付加する場合、直接システインを付加するのではなく、他のアミノ酸(例えば、グリシン)を間に介在させた状態でシステインを付加させることもできる。 Any amino acid is not particularly limited. Of the amino acids, cysteine is preferred. The addition of a cysteine residue is desirable because the compound can be bound to the polypeptide of the present invention using the SH group of cysteine. When cysteine is added, cysteine can be added in a state where other amino acids (for example, glycine) are interposed, instead of directly adding cysteine.
特定のアミノ酸配列において、1若しくは2個以上のアミノ酸を欠失、置換、挿入及び/又は付加させる技術は公知である。 Techniques for deleting, substituting, inserting and / or adding one or more amino acids in a specific amino acid sequence are known.
本発明のポリペプチドは、固相合成法、液相合成などの公知の合成手法を利用すること、該ポリペプチドをコードする遺伝子を導入した形質転換体を培養することなどにより製造することができる。形質転換体を作製するための宿主としては、例えば、大腸菌、酵母、哺乳動物細胞、植物細胞、昆虫細胞などが挙げられる。 The polypeptide of the present invention can be produced by using a known synthesis method such as solid phase synthesis or liquid phase synthesis, or by culturing a transformant introduced with a gene encoding the polypeptide. . Examples of the host for producing the transformant include Escherichia coli, yeast, mammalian cells, plant cells, and insect cells.
生産したポリペプチドの精製は、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、ハイドロキシアパタイトカラムクロマトグラフィー、硫酸アンモニウム塩析法などにより行うことができる。 The produced polypeptide can be purified by affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, ammonium sulfate salting-out method, or the like.
後述する実施例で示されているように、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以外にも、配列番号2(RQIKIWFQNRRMKWKK)で表されるアミノ酸配列からなるポリペプチドも高いBBB透過性を示す。そのため、上記の本発明のポリペプチドの説明において、配列番号1を配列番号2に置き換えることもできる。 As shown in Examples described later, in addition to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (RQIKIWFQNRRMKWKK) is also highly BBB permeable. Indicates. Therefore, in the above description of the polypeptide of the present invention, SEQ ID NO: 1 can be replaced with SEQ ID NO: 2.
脳内送達用キャリア分子
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。 Carrier molecule for intracerebral delivery The carrier molecule for intracerebral delivery of the present invention is characterized by comprising the polypeptide of (a), (b) or (c) above.
上記(a)、(b)又は(c)のポリペプチドは、高いBBB透過性及び脳移行活性を有しているため、本発明のポリペプチドは、他の分子及び物質を脳内に送達するためのキャリア分子として利用することが可能である。例えば、本発明のキャリア分子を他の分子及び物質と結合させることにより、他の分子及び物質を脳内に移行させることが可能である。 Since the polypeptide of (a), (b) or (c) has high BBB permeability and brain translocation activity, the polypeptide of the present invention delivers other molecules and substances into the brain. Can be used as a carrier molecule. For example, by binding the carrier molecule of the present invention to other molecules and substances, it is possible to transfer other molecules and substances into the brain.
また、本発明のポリペプチドにリポソーム、ミセル又はマイクロカプセルを結合させることによっても脳内送達用キャリア分子として利用することができる。この場合、リポソーム、ミセル又はマイクロカプセル内に目的の分子及び物質を封入することによって、これらを脳内に移行させることができる。 It can also be used as a carrier molecule for delivery in the brain by binding liposomes, micelles or microcapsules to the polypeptide of the present invention. In this case, these can be transferred into the brain by encapsulating target molecules and substances in liposomes, micelles or microcapsules.
複合体
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。 Complex The complex of the present invention comprises the above-mentioned polypeptide (a), (b) or (c), and a protein, polypeptide, oligopeptide, low molecular compound, or nucleic acid (hereinafter referred to as `` compound A '') bound thereto. It is characterized by containing.
本発明のポリペプチドは脳移行活性を有することから、本発明のポリペプチドに結合させる化合物Aは脳移行活性が付与される。本発明のポリペプチドは高いBBB透過性及び脳移行性を有しており、本来脳内に移行しない化合物Aを効率的に脳内に移行させることが可能となる。 Since the polypeptide of the present invention has brain translocation activity, Compound A to be bound to the polypeptide of the present invention is imparted with brain translocation activity. The polypeptide of the present invention has high BBB permeability and brain transportability, and it is possible to efficiently transport Compound A, which does not naturally travel into the brain, into the brain.
化合物Aとしては、脳疾患の予防又は治療のために脳組織へ移行させることが望ましい化合物などを挙げることができる。化合物Aを本発明のポリペプチドに結合させることにより効率的に脳組織に移行して、治療効果を発揮することが期待される。 Examples of compound A include compounds that are desirably transferred to brain tissue for the prevention or treatment of brain diseases. By binding Compound A to the polypeptide of the present invention, it is expected to efficiently migrate to brain tissue and exert a therapeutic effect.
上記タンパク質としては、例えば、抗体、抗体断片、アンタゴニスト、アゴニストなどのタンパク質系薬剤、抗癌剤などが挙げられる。 Examples of the protein include protein drugs such as antibodies, antibody fragments, antagonists and agonists, and anticancer agents.
上記核酸としては、例えば、プラスミド、疾患関連遺伝子に関するsiRNA及びアンチセンスDNAが挙げられる。 Examples of the nucleic acid include plasmids, siRNA related to disease-related genes, and antisense DNA.
化合物Aの大きさは特に制限されず、通常は、物理的にBBBを通過し得る程度の大きさを上限とする。 The size of compound A is not particularly limited, and usually the upper limit is a size that can physically pass through BBB.
本発明のポリペプチドと化合物Aとは、適宜、公知の方法を利用して結合させることができる。 The polypeptide of the present invention and compound A can be appropriately combined using a known method.
本発明のポリペプチドがシステイン残基を有する場合には、本発明のポリペプチドのシステイン残基と化合物Aとを-SS-結合を介して結合させることができ、適当な架橋剤を介して結合させることもできる。また、本発明のポリペプチドと化合物A(タンパク質、ポリペプチド又はオリゴペプチド)とをそれぞれコードするDNAを連結したDNAをベクターに導入して、大腸菌などの宿主細胞内で発現させるなどの常法により、複合体を得ることもできる。 When the polypeptide of the present invention has a cysteine residue, the cysteine residue of the polypeptide of the present invention and compound A can be bound via an -SS- bond, and bound via an appropriate crosslinking agent. It can also be made. In addition, by a conventional method such as introducing a DNA ligated with the DNA of the present invention and a compound A (protein, polypeptide or oligopeptide) into a vector and expressing it in a host cell such as E. coli. A complex can also be obtained.
架橋剤としては、本発明のポリペプチドと化合物Aとを結合できる少なくとも2価の架橋剤であれば特に限定されず、例えばN-(6-マレイミドカプロイルオキシ)コハク酸イミドエステル(EMCS)などが挙げられる。 The cross-linking agent is not particularly limited as long as it is an at least divalent cross-linking agent capable of binding the polypeptide of the present invention and compound A. For example, N- (6-maleimidocaproyloxy) succinimide ester (EMCS) and the like Is mentioned.
医薬組成物
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。 Pharmaceutical composition The pharmaceutical composition of the present invention is characterized by comprising the above complex.
医薬組成物として調製する場合、上記の複合体をそのまま使用するか、又は医薬品において許容される無毒性の担体、希釈剤若しくは賦形剤とともに、タブレット(素錠、糖衣錠、発泡錠、フィルムコート錠、チュアブル錠、トローチ剤などを含む)、カプセル剤、丸剤、粉末剤(散剤)、細粒剤、顆粒剤、液剤、懸濁液、乳濁液、シロップ、ペースト、注射剤(使用時に、蒸留水又はアミノ酸輸液、電解質輸液などの輸液に配合して液剤として調製する場合を含む)などの形態に調製して、医薬用の製剤にすることが可能である。 When preparing as a pharmaceutical composition, the above complex is used as it is, or together with a non-toxic carrier, diluent or excipient that is acceptable in pharmaceuticals, tablets (plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets). , Capsules, pills, powders (powder), fine granules, granules, solutions, suspensions, emulsions, syrups, pastes, injections (when used, It is possible to prepare a pharmaceutical preparation by preparing it in a form such as distilled water, amino acid infusion, electrolyte infusion, etc.
本発明の医薬組成物における上記の複合体の含量は、医薬組成物全量中0.0001〜100重量%、好ましくは0.001〜99.9重量%、より好ましくは0.01〜99重量%の範囲から適宜選択することが可能である。 The content of the complex in the pharmaceutical composition of the present invention may be appropriately selected from the range of 0.0001 to 100% by weight, preferably 0.001 to 99.9% by weight, more preferably 0.01 to 99% by weight, based on the total amount of the pharmaceutical composition. Is possible.
本発明の医薬組成物の投与方法は特に限定されず、例えば、動脈内投与、静脈内投与、口腔内投与、直腸投与、経腸投与、経皮投与、経口投与などにより行うことができる。 The administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be performed by, for example, intraarterial administration, intravenous administration, buccal administration, rectal administration, enteral administration, transdermal administration, oral administration, and the like.
本発明の医薬組成物は、ヒトを含む哺乳動物に対して投与される。 The pharmaceutical composition of the present invention is administered to mammals including humans.
本発明の医薬組成物の投与量は、患者の体重、年齢、性別、症状などの種々の条件に応じて適宜決定することができる。 The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to various conditions such as the body weight, age, sex, and symptoms of the patient.
本発明の医薬組成物は、脳疾患の予防及び/又は治療に有用である。また、本発明の医薬組成物は、脳疾患の診断にも有用である。 The pharmaceutical composition of the present invention is useful for the prevention and / or treatment of brain diseases. The pharmaceutical composition of the present invention is also useful for diagnosing brain diseases.
本発明における脳疾患とは、例えば、脳腫瘍、転移性脳腫瘍、統合失調症、てんかん、アルツハイマー病、パーキンソン病、ハンチントン病、脳卒中などの脳血管障害、もやもや病、脳炎及び脳膿瘍など脳感染症、多発性硬化症、ダウン症候群、前頭側頭型認知症、ピック病、進行核上性麻痺(PSP)、プリオン病、筋萎縮性側索硬化症、脊髄小脳変性症、多系統委縮症、代謝性脳疾患、頭痛、自閉症など脳発達障害、躁鬱病などが挙げられる。 The brain diseases in the present invention include, for example, brain tumors, metastatic brain tumors, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebrovascular disorders such as stroke, brain infections such as moyamoya disease, encephalitis and brain abscess, Multiple sclerosis, Down's syndrome, frontotemporal dementia, Pick's disease, progressive supranuclear palsy (PSP), prion disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, multisystem atrophy, metabolic Examples include brain disorders, headaches, brain development disorders such as autism, and manic depression.
本発明のポリペプチドは脳移行活性を有することから、ポリペプチドに結合させた脳疾患に対する予防及び/又は治療効果を有する化合物を効率的に脳組織に移行させて、脳疾患の予防及び/又は治療効果を発揮することが期待される。 Since the polypeptide of the present invention has a brain transition activity, the compound having a preventive and / or therapeutic effect on brain disease bound to the polypeptide is efficiently transferred to brain tissue, and thus brain disease can be prevented and / or Expected to exert therapeutic effects.
また、本発明のポリペプチドをPET用放射性核種、蛍光色素などで標識化した上で、脳疾患の原因物質に特異的に結合する抗体などと結合させた複合体を体内に投与することで、当該複合体を効率的に脳組織に移行でき、脳疾患の診断が可能となると考えられる。 In addition, by labeling the polypeptide of the present invention with a PET radionuclide, a fluorescent dye, etc., and then administering a complex bound with an antibody that specifically binds to a causative agent of brain disease, into the body, It is considered that the complex can be efficiently transferred to the brain tissue and the diagnosis of the brain disease becomes possible.
以下に実施例を挙げて、本発明の内容を更に詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
製造例
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。 Production Examples Table 1 shows the amino acid sequences of the peptides used in the experiments in this study. Each peptide has a Gly-Cys sequence for introducing fluorescein on the carboxy terminus (C terminus) side. The amino terminal (N terminal) is acetylated and the C terminal is an amide. However, as an exception, C6R8 and mMPG8 each have hexanoic acid or β-alanine at the N-terminal instead of an acetyl group.
ペプチドは、すべてFmoc式固相合成法により手動合成した。ペプチド合成には、Novabiochem社製の固相担体(Rink Amide Resin)、ペプチド研究所社製のFmocアミノ酸及び縮合剤(1-ヒドロキシベンゾトリアゾール)を用い、ジイソプロピルエチルアミン、無水酢酸、及びジメチルホルムアミドは和光純薬工業社製のものを用いた。固相担体上に合成した保護ペプチドは、エタンジチオール(和光純薬工業株式会社)を5%(v/v)含有するトリフルオロ酢酸(渡辺化学工業株式会社)で脱保護した。脱保護後のペプチドは、逆相HPLC (JASCO社製HPLCにナカライテスク社製カラム5C4AR-300を接続したもの)により主生成物として精製した。精製したペプチドは、エレクトロスプレーイオン化質量分析(ESI-MS、Thermo Fischer Scientific社製LCQ Deca XP)により分子量の確認を行った(これを非標識ペプチドとする)。 All peptides were manually synthesized by the Fmoc solid phase synthesis method. For peptide synthesis, a solid phase carrier (Rink Amide Resin) manufactured by Novabiochem, an Fmoc amino acid and a condensing agent (1-hydroxybenzotriazole) manufactured by Peptide Laboratories were used, and diisopropylethylamine, acetic anhydride, and dimethylformamide were combined. A product made by Kojun Pharmaceutical Co., Ltd. was used. The protected peptide synthesized on the solid support was deprotected with trifluoroacetic acid (Watanabe Chemical Co., Ltd.) containing 5% (v / v) of ethanedithiol (Wako Pure Chemical Industries, Ltd.). The peptide after deprotection was purified as a main product by reversed-phase HPLC (JASCO HPLC column connected to Nacalai Tesque column 5C4AR-300). The purified peptide was confirmed for molecular weight by electrospray ionization mass spectrometry (ESI-MS, LCQ Deca XP manufactured by Thermo Fischer Scientific) (this is referred to as unlabeled peptide).
次に、非標識ペプチドと5-(ヨードアセトアミド)フルオレセイン(シグマアルドリッチ社)とを、ジメチルホルムアミド/メタノール(N-メチルモルホリンを0.1%(v/v)含有する1:1混合溶媒、いずれも和光純薬工業社製)中で1.5時間反応させることにより、C末端のCys側鎖にフルオレセインが導入されたペプチドを合成した(これを標識ペプチドとする)。標識ペプチドはすべて逆相HPLCにより主生成物として精製した後、ESI-MSにより分子量の確認を行った。ESI-MSによる物性値として、分子量実測値とその理論値を表2に示す。 Next, a 1: 1 mixed solvent containing unlabeled peptide and 5- (iodoacetamido) fluorescein (Sigma Aldrich), dimethylformamide / methanol (0.1% (v / v) N-methylmorpholine), both The peptide in which fluorescein was introduced into the C-terminal Cys side chain was synthesized by reacting for 1.5 hours in Kojunkaku Kogyo Co., Ltd. (this is referred to as a labeled peptide). All of the labeled peptides were purified as main products by reverse phase HPLC, and the molecular weight was confirmed by ESI-MS. Table 2 shows the measured molecular weights and the theoretical values as physical properties by ESI-MS.
試験例1:一次スクリーニング
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)。 Test Example 1: Primary screening It was examined how efficiently the peptide (1)-(13) shown in Table 1 can deliver a substance that does not originally migrate into the cell. Fluorescein, a fluorescent dye, was used as a water-soluble model substance that does not inherently migrate into cells. Two days before the experiment, Dulbecco's modified Eagle's medium (DMEM, Gibco) was used for mouse neuroblastoma Neuro2A cells seeded in a 4-well chamber slide (Lab-Tek II chamber slide, Nunc) at 50,000 cells / well. After the labeled peptide (1)-(13) diluted to 10 μM was administered and cultured for 1 hour, the cell nucleus was stained with Hoechst33342 (Molecular Probes) and then observed with a confocal microscope (Olympus FV1000). Performed (FIG. 1).
その結果、SAP(8)以外の標識ペプチドを投与した場合において、顕著なフルオレセイン蛍光が細胞内で観察された。一方、SAP(8)を投与した細胞においては、蛍光シグナルはほとんど観察されなかった。このことから、少なくともSAP(8)以外のペプチドは、フルオレセインを細胞内へと送達する能力を有することが示唆された。 As a result, when a labeled peptide other than SAP (8) was administered, significant fluorescein fluorescence was observed in the cells. On the other hand, almost no fluorescence signal was observed in the cells administered with SAP (8). This suggested that at least peptides other than SAP (8) have the ability to deliver fluorescein into cells.
次に、10μMの標識ペプチド(1)-(13)をラット由来脳血管内皮細胞に1時間投与し、細胞内におけるフルオレセインの蛍光強度を測定した。この測定値から標識ペプチドの細胞内移行量を算出した(図2)。本試験例では、細胞内移行性をほとんど示さない化合物であるフルオレセインナトリウム塩(Na-F)を、ネガティブコントロールとして用いた。 Next, 10 μM of the labeled peptide (1)-(13) was administered to rat cerebral vascular endothelial cells for 1 hour, and the fluorescence intensity of fluorescein in the cells was measured. From this measured value, the intracellular migration amount of the labeled peptide was calculated (FIG. 2). In this test example, fluorescein sodium salt (Na-F), which is a compound that hardly shows intracellular migration, was used as a negative control.
実験の3日前に初代培養ラット脳毛細血管内皮細胞を15,000 cells/wellで96 well culture plate (Iwaki社)に播種し、DMEM/F12 (和光純薬工業株式会社)に10% Plasma derived serum (Animal technology社)、bFGF (1.5 ng/mL; 和光純薬工業株式会社)、heparin (100μg/mL; シグマアルドリッチ社)、及びITS supplement (シグマアルドリッチ社)を加えた培養液で培養した。培養液で10μMとなるように希釈した標識ペプチド(1)-(13)とNa-Fを投与し、1時間培養した。培養液を除き、リン酸緩衝液で3回洗浄後、0.2N NaOHで細胞を溶解した。細胞溶解液の蛍光強度を蛍光プレートリーダー(Wallac 1420 ARVO Multilabel Counter, Perkin Elmer社)で測定し(Ex:485 nm, Em: 535 nm)、各標識ペプチドとNa-Fの検量線から細胞内の濃度を算出した。 Three days before the experiment, primary cultured rat brain capillary endothelial cells were seeded at 15,000 cells / well in a 96-well culture plate (Iwaki) and 10% Plasma derived serum (Animal) on DMEM / F12 (Wako Pure Chemical Industries, Ltd.). technology), bFGF (1.5 ng / mL; Wako Pure Chemical Industries, Ltd.), heparin (100 μg / mL; Sigma-Aldrich), and ITS supplement (Sigma-Aldrich). Labeled peptides (1)-(13) and Na-F diluted to 10 μM in the culture solution were administered and cultured for 1 hour. The culture solution was removed, and the cells were lysed with 0.2N NaOH after washing 3 times with phosphate buffer. The fluorescence intensity of the cell lysate was measured with a fluorescence plate reader (Wallac 1420 ARVO Multilabel Counter, Perkin Elmer) (Ex: 485 nm, Em: 535 nm). Concentration was calculated.
実験の結果、Penetratin(2), pVEC(4), mMPG(8)などにおいて高い細胞内移行性が確認された。一方、SAP(8)を投与した細胞においてはフルオレセイン蛍光がほとんど検出されず、その細胞内移行量はNa-Fとほぼ同程度であった。このことから、各ペプチドが有する細胞内送達能力は、それぞれ大きく異なることが示唆された。 As a result of the experiment, high intracellular migration was confirmed in Penetratin (2), pVEC (4), mMPG (8) and the like. On the other hand, almost no fluorescein fluorescence was detected in the cells to which SAP (8) was administered, and the intracellular transfer amount was almost the same as that of Na-F. From this, it was suggested that the intracellular delivery capability of each peptide is greatly different.
以上の検討から、高い細胞内送達性が示唆された6種類のペプチド(Tat(1), Penetratin(2), pVEC(4), Rev(7), C6R8(10), mMPG8(13))について、以後の検討を進めた。 Based on the above examinations, six types of peptides (Tat (1), Penetratin (2), pVEC (4), Rev (7), C6R8 (10), mMPG8 (13)) that have been suggested to have high intracellular delivery The following examination was advanced.
試験例2:二次スクリーニング
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)。 Test Example 2: BBB permeability was evaluated for the six peptides selected in Secondary Screening Test Example 1. BBB permeability was evaluated using an in vitro BBB reconstitution system (BBB kit, Cell. Mol. Neurobiol. 27, 687-694 (2007).) Developed by Nakagawa et al. In this test example, fluorescein-labeled dextran (FD4), which is a compound having almost the same molecular weight as that of the labeled peptide (approximately 4000) and hardly showing BBB permeability, and a peptide that showed almost no intracellular delivery in Test Example 1 were used. A SAP (8) was used as a negative control. 1 μM labeled peptide was administered to the blood vessel side of the BBB kit, and the fluorescein fluorescence intensity in the brain side culture solution was measured over time. The fluorescence intensity was measured with a fluorescence plate reader, and the concentration of the labeled peptide in the brain side culture solution was calculated from the measured value (FIG. 3).
その結果、pVEC(4)を除く5種類のペプチド(Tat(1), Penetratin(2), Rev(7), C6R8(10), mMPG8(13))は、FD4及びSAP(8)と比較して、脳側培養液中に有意に高い濃度で検出された。一方、pVEC(4)は脳側培養液中にほとんど検出されず、その濃度はFD4及びSAP(8)と同程度であった。 As a result, 5 types of peptides (Tat (1), Penetratin (2), Rev (7), C6R8 (10), mMPG8 (13)) except for pVEC (4) were compared with FD4 and SAP (8). And was detected at a significantly higher concentration in the brain-side culture medium. On the other hand, pVEC (4) was hardly detected in the brain side culture solution, and its concentration was similar to FD4 and SAP (8).
また、これと同時に、細胞層の緊密性の指標である経内皮電気抵抗値(TEER)を測定した(図4)。TEERは電気抵抗測定器(EVOM、World Precision Instruments社)とカップ型電極(ENDOHM、WPI社)とを用いて測定した。BBBキットから得られた電気抵抗値から、細胞を播種していないインサート膜のみの電気抵抗値を差し引き、インサート膜の培養面積を掛けることで算出した。その結果、いずれのペプチド及びFD4においても、TEER値の変化に差は見られず、かつ、その顕著な減少は確認されなかった。 At the same time, transendothelial electrical resistance (TEER), which is an index of cell layer tightness, was measured (FIG. 4). TEER was measured using an electrical resistance measuring instrument (EVOM, World Precision Instruments) and a cup-type electrode (ENDOHM, WPI). The electric resistance value obtained from the BBB kit was subtracted from the electric resistance value of only the insert membrane on which cells were not seeded, and was calculated by multiplying the culture area of the insert membrane. As a result, there was no difference in the change of TEER value in any peptide and FD4, and no significant decrease was confirmed.
これらの結果から、上記5種類のペプチドがBBBを透過し、フルオレセインを血管側から脳側へと送達する能力を有すること、また、この送達にBBB細胞層のバリア機能の低下が関与する可能性は低いことが示唆された。このうち、3種類のペプチド(Penetratin(2), C6R8(10), mMPG8(13))に関しては、過去に一部の研究者により脳内移行性が示唆されているTat(1)よりも高いBBB透過性を示した。 From these results, the above five peptides have the ability to permeate the BBB and deliver fluorescein from the blood vessel side to the brain side, and this delivery may involve a decrease in the barrier function of the BBB cell layer Was suggested to be low. Of these, the three peptides (Penetratin (2), C6R8 (10), and mMPG8 (13)) are higher than Tat (1), which has been suggested by some researchers in the past to enter the brain. BBB permeability was shown.
試験例3:インビボ実験
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。 Test Example 3: In Vivo Experiment In Test Example 2, mMPG8 (13), which showed the highest BBB permeability, was evaluated for its ability to enter the brain using an animal model. In this test example, Na-F, which is a compound that hardly shows the ability to enter the brain, and pVEC (4), which is a peptide that hardly shows BBB permeability in Test example 2, were used as negative controls.
雄性ICRマウス(8週齢)の左心室から標識ペプチド(100μg/ml)を2 ml/minの流速で灌流し、0.5, 1, 1.5, 2分後に脳を採取した。採取した脳は湿重量を測定後、2倍容の0.5Mホウ酸水溶液(pH 10.0)を加え、ホモジナイズした。1000gで15分遠心後、更に2倍容の99.5%エタノールを加えて撹拌した。15,000gで15分遠心後、上清中の蛍光強度を蛍光プレートリーダー(CytoFluor, PerSpective Biosystems社)で測定し、検量線から脳中濃度を求めた。灌流液中の標識ペプチド濃度も同様に求め、その脳移行量を経時的に脳中濃度/灌流液中濃度比として算出した。また、脳中濃度/灌流液中濃度比を縦軸に、灌流時間を横軸にプロットし(図5)、その傾きを血液側から脳実質側への透過クリアランスとして算出した(表3)。 A labeled peptide (100 μg / ml) was perfused from the left ventricle of male ICR mice (8 weeks old) at a flow rate of 2 ml / min, and brains were collected 0.5, 1, 1.5, and 2 minutes later. The collected brain was weighed and then homogenized with 2 volumes of 0.5 M aqueous boric acid (pH 10.0). After centrifugation at 1000 g for 15 minutes, 2 volumes of 99.5% ethanol was further added and stirred. After centrifugation at 15,000 g for 15 minutes, the fluorescence intensity in the supernatant was measured with a fluorescence plate reader (CytoFluor, PerSpective Biosystems), and the brain concentration was determined from the calibration curve. The labeled peptide concentration in the perfusate was also determined in the same manner, and the amount of transferred brain was calculated as the brain concentration / perfusate concentration ratio over time. The brain concentration / perfusate concentration ratio was plotted on the vertical axis and the perfusion time on the horizontal axis (FIG. 5), and the slope was calculated as the permeation clearance from the blood side to the brain parenchyma side (Table 3).
その結果、mMPG8(13)は、Na-F及びpVEC(4)と比較して、有意に高い濃度で脳中に検出され、透過クリアランスも有意に高い値を示した。これは、正常なBBBをほとんど通過しないタンパク質であるアルブミンのフルオレセイン標識体(FITC-Alb, シグマアルドリッチ社)を同一条件下で灌流した結果と比較すると、およそ18倍に相当する脳移行量であった(図6)。これらの結果から、mMPG8(13)が高い脳内移行性を有することが示唆された。 As a result, mMPG8 (13) was detected in the brain at a significantly higher concentration than Na-F and pVEC (4), and the permeation clearance also showed a significantly higher value. Compared to the result of perfusion of fluorescein labeled albumin (FITC-Alb, Sigma-Aldrich), which is a protein that hardly passes through normal BBB, under the same conditions, it was about 18 times the amount of brain migration. (FIG. 6). From these results, it was suggested that mMPG8 (13) has a high ability to migrate into the brain.
次に、mMPG8(13)の脳内移行において、BBBに存在する何らかの輸送担体が関与する可能性について検討を行った。一般に、物質の膜輸送が輸送担体に依存する場合、物質を過剰量投与すると、担体の飽和により輸送効率が低下する。そこで、上記と同様の灌流実験を異なる投与濃度で行い、その脳移行量の変化を調べた(図7)。その結果、mMPG8(13)の脳移行量は、投与濃度に依存して増大した。 Next, we investigated the possibility that some transport carrier present in BBB might be involved in the migration of mMPG8 (13) into the brain. In general, when membrane transport of a substance depends on the transport carrier, if an excessive amount of the substance is administered, the transport efficiency decreases due to carrier saturation. Therefore, perfusion experiments similar to those described above were performed at different administration concentrations, and changes in the amount of brain transfer were examined (FIG. 7). As a result, the amount of mMPG8 (13) transferred to the brain increased depending on the administration concentration.
また、一定量の標識mMPG8(13)とともに、過剰量の非標識mMPG8(13)を投与し、標識体の脳移行量の変化を調べた(図8)。その結果、標識体の脳移行量は、非標識体の投与濃度の増加に伴って顕著に増大した。 In addition, an excessive amount of unlabeled mMPG8 (13) was administered together with a certain amount of labeled mMPG8 (13), and the change in the amount of labeled substance transferred to the brain was examined (FIG. 8). As a result, the amount of labeled substance transferred to the brain markedly increased as the concentration of unlabeled substance increased.
いずれの実験においても投与濃度の増加に伴う輸送効率の低下が見られなかったことから、mMPG8(13)の脳内への移行には、BBBに存在する何らかの輸送担体を介した取り込み機構ではなく、他の未知機構が関与する可能性が示唆された。 In all experiments, there was no decrease in transport efficiency with increasing dose concentration, so mMPG8 (13) translocation into the brain was not an uptake mechanism via any transport carrier present in the BBB. This suggests that other unknown mechanisms may be involved.
試験例4:毒性試験
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)。 Test Example 4: Toxicity Test The cytotoxicity of mMPG8 (13) under the test conditions of Test Examples 1 to 3 was evaluated. In this test example, pVEC (4), which is a peptide that showed high intracellular transferability in Test Example 1 but hardly showed BBB permeability and brain transfer in Test Example 2 and Test Example 3, was negative. Used as a control. To the mouse brain vascular endothelial cell line MBEC4 seeded in a 96-well plate (Iwaki) at 5,000 cells / well two days before the experiment, an unlabeled peptide (5 μM, 10 μM, or 30 μM) diluted with DMEM was administered, Cultured for 1 hour, 6 hours, or 24 hours. After culture, cell proliferation reagent WST-1 (Roche) was added, and after 1 hour, the absorbance at 450 nm was measured with an absorption microplate reader (Multiskan FC, Thermo Fisher Scientific) to evaluate the cell viability (FIG. 9). ).
その結果、mMPG8(13)においては30μMの濃度(およそ100μg/mlに相当)で24時間投与した場合にのみ、細胞生存率のわずかな低下が見られたが(生存率87.0±7.8%)、他の条件下では細胞生存率の低下は確認されなかった。一方、pVEC(4)においては10μM、又は30μMの濃度で24時間投与した場合において、細胞生存率の低下が見られた(それぞれ94.9±2.1%、又は68.1±3.2%)。これらの結果から、いずれのペプチドにおいても細胞傷害性は極めて低いものの、pVEC(4)がより高い細胞傷害性を有すると考えられる。 As a result, in mMPG8 (13), a slight decrease in cell viability was observed only when administered at a concentration of 30 μM (corresponding to approximately 100 μg / ml) for 24 hours (viability 87.0 ± 7.8%). No decrease in cell viability was observed under other conditions. On the other hand, when pVEC (4) was administered at a concentration of 10 μM or 30 μM for 24 hours, a decrease in cell viability was observed (94.9 ± 2.1% or 68.1 ± 3.2%, respectively). From these results, it is considered that pVEC (4) has higher cytotoxicity although cytotoxicity is extremely low in any peptide.
以上から、試験例1から3における実験条件(投与濃度、又は投与時間)において、mMPG8(13)は細胞傷害性をほとんど持たないこと、また、mMPG8(13)のBBB透過性及び脳内移行性に細胞傷害性が関与する可能性は低いことが示唆された。 From the above, under the experimental conditions (administration concentration or administration time) in Test Examples 1 to 3, mMPG8 (13) has almost no cytotoxicity, and BMP permeability and translocation into the brain of mMPG8 (13). This suggests that it is unlikely that cytotoxicity is involved.
Claims (10)
(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。The following polypeptide (a), (b) or (c):
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having brain transition activity
(c) It consists of an amino acid sequence in which 1 to 5 arbitrary amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (a) or (b), and has a brain transition activity Polypeptide.
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