JP2015203014A - Spinal cord tissue targeting peptide and use thereof - Google Patents
Spinal cord tissue targeting peptide and use thereof Download PDFInfo
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- JP2015203014A JP2015203014A JP2014082766A JP2014082766A JP2015203014A JP 2015203014 A JP2015203014 A JP 2015203014A JP 2014082766 A JP2014082766 A JP 2014082766A JP 2014082766 A JP2014082766 A JP 2014082766A JP 2015203014 A JP2015203014 A JP 2015203014A
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Abstract
【課題】本発明は、脊髄組織に被送達物質を効率良く送達できるペプチド等を提供することを目的とする。【解決手段】以下の（１−１）または（１−２）で表される脊髄組織標的化ペプチド、（１−１）配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列の両末端にそれぞれ少なくとも１つのシステイン残基が直接連結してなるペプチド、（１−２）前記（１−１）のペプチドにおいて、配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列が、１または複数のアミノ酸が欠失、置換及び／または付加されたアミノ酸配列からなり、且つ、脊髄組織を標的とするペプチド。【選択図】なしAn object of the present invention is to provide a peptide or the like that can efficiently deliver a substance to be delivered to spinal cord tissue. The spinal cord tissue targeting peptide represented by the following (1-1) or (1-2), (1-1) represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5 A peptide in which at least one cysteine residue is directly linked to both ends of the amino acid sequence, (1-2) In the peptide of (1-1), at least selected from the group consisting of SEQ ID NOs: 1 to 5 A peptide that consists of an amino acid sequence represented by one species, wherein one or more amino acids are deleted, substituted, and / or added, and that targets spinal cord tissue. [Selection figure] None
Description
本発明は、脊髄組織標的化ペプチド及びその利用に関する。 The present invention relates to a spinal cord tissue targeting peptide and use thereof.
これまでのバイオテクノロジーの発展によって、標的遺伝子に対する発現抑制作用を有する核酸をはじめとして、有用作用を備える物質が数多く報告されている（非特許文献１）。また、このような有用作用を備える物質を組織や細胞などへ送達する手段も数多く報告されている。このような送達手段の代表的なものとしてはリポソームが例示でき、リポソームはその内部に種々の被送達物質を封入できることから、核酸や薬物といった様々な被送達物質の送達に利用しやすい。 With the development of biotechnology so far, many substances having useful effects have been reported, including nucleic acids having an expression-inhibiting effect on target genes (Non-patent Document 1). In addition, many means for delivering a substance having such a useful action to tissues or cells have been reported. A typical example of such a delivery means is a liposome, and since the liposome can encapsulate various substances to be delivered, it can be easily used for delivery of various substances to be delivered such as nucleic acids and drugs.
一方、このような送達技術においては、例えば特定の組織や細胞において有用作用を発揮させたい場合には、被送達物質を目的とする組織や細胞に効率良く送達することが求められる。このような送達技術の向上は疾患の診断、予防、治療、更には疾患の原因解明の進展などに大きく寄与することが期待されるため、被送達物質を目的とする組織や細胞に効率良く送達できる新たな技術を提供することは非常に重要である。 On the other hand, in such a delivery technique, for example, when it is desired to exert a useful action in a specific tissue or cell, it is required to efficiently deliver the substance to be delivered to the target tissue or cell. Since such improvements in delivery technology are expected to make a significant contribution to the diagnosis, prevention, treatment of diseases, and further progress in elucidating the cause of diseases, it is possible to efficiently deliver the substance to be delivered to the target tissue or cells. It is very important to provide new technologies that can be used.
このことから、本発明は、目的とする組織や細胞に被送達物質を効率良く送達できる新たな技術を提供することを目的とする。具体的には、本発明は、脊髄組織に被送達物質を効率良く送達できるペプチド等を提供することを目的とする。 Therefore, an object of the present invention is to provide a new technique capable of efficiently delivering a substance to be delivered to a target tissue or cell. Specifically, an object of the present invention is to provide a peptide or the like that can efficiently deliver a substance to be delivered to spinal cord tissue.
本発明者らは、前記課題を解決すべく鋭意検討したところ、脊髄組織を効率良く標的化できるペプチドを見出した。本発明は、かかる知見に基づいて更に検討を重ねることにより完成されたものである。 The present inventors diligently studied to solve the above problems, and found a peptide capable of efficiently targeting spinal cord tissue. The present invention has been completed by further study based on such knowledge.
すなわち、本発明は、以下に掲げる発明を提供する。
項１．以下の（１−１）または（１−２）で表される脊髄組織標的化ペプチド：
（１−１）配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列の両末端にそれぞれ少なくとも１つのシステイン残基が直接連結してなるペプチド、
（１−２）前記（１−１）のペプチドにおいて、配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列が、１または複数のアミノ酸が欠失、置換及び／または付加されたアミノ酸配列からなり、且つ、脊髄組織を標的とするペプチド。
項２．前記両末端のシステイン残基において、片端のシステイン残基が他端のシステイン残基とジスルフィド結合を形成してなる、項１に記載のペプチド。
項３．脊髄組織が、脊髄組織全体、脊髄組織内のアストロサイト及び脊髄組織内のミクログリアからなる群より選択される少なくとも１種である、項１または２に記載のペプチド。
項４．以下の（２−１）または（２−２）で表されるポリヌクレオチド：
（２−１）前記（１−１）及び（１−２）のいずれかに記載のペプチドをコードするポリヌクレオチド、
（２−２）前記（２−１）のポリヌクレオチドの相補鎖に対して、ストリンジェントな条件下でハイブリダイズし、且つ、脊髄組織標的化ペプチドをコードするポリヌクレオチド。
項５．項１〜３のいずれかに記載のペプチドを含有する医薬組成物。
項６．更に被送達物質を含有する、項５に記載の医薬組成物。
項７．項１〜３のいずれかに記載のペプチドまたは項５及び６のいずれかに記載の医薬組成物を対象物に投与する工程を含有する、対象物中の脊髄組織を標的化する方法。
項８．項６に記載の医薬組成物を対象物に投与する工程を含有する、被送達物質の脊髄組織への送達方法。
That is, the present invention provides the following inventions.
Item 1. Spinal cord tissue targeting peptide represented by the following (1-1) or (1-2):
(1-1) a peptide in which at least one cysteine residue is directly linked to both ends of an amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5,
(1-2) In the peptide of (1-1), the amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5 has one or more amino acids deleted, substituted, and / or Alternatively, a peptide consisting of an added amino acid sequence and targeting spinal cord tissue.
Item 2. Item 2. The peptide according to Item 1, wherein, in the cysteine residues at both ends, one cysteine residue forms a disulfide bond with the other cysteine residue.
Item 3. Item 3. The peptide according to Item 1 or 2, wherein the spinal cord tissue is at least one selected from the group consisting of whole spinal cord tissue, astrocytes in spinal cord tissue, and microglia in spinal cord tissue.
Item 4. The polynucleotide represented by the following (2-1) or (2-2):
(2-1) a polynucleotide encoding the peptide according to any one of (1-1) and (1-2),
(2-2) A polynucleotide that hybridizes to a complementary strand of the polynucleotide of (2-1) under stringent conditions and encodes a spinal cord tissue-targeting peptide.
Item 5. Item 4. A pharmaceutical composition comprising the peptide according to any one of Items 1 to 3.
Item 6. Item 6. The pharmaceutical composition according to Item 5, further comprising a substance to be delivered.
Item 7. Item 7. A method for targeting spinal cord tissue in a subject, comprising administering the peptide according to any one of Items 1 to 3 or the pharmaceutical composition according to any one of Items 5 and 6 to the subject.
Item 8. Item 7. A method for delivering a substance to be delivered to spinal cord tissue, comprising a step of administering the pharmaceutical composition according to Item 6 to a subject.
本発明のペプチドによれば脊髄組織を標的化できる。このため、本発明のペプチドと被送達物質とを併用することによって、脊髄組織に被送達物質を効率良く送達することができる。 The peptide of the present invention can target spinal cord tissue. Therefore, by using the peptide of the present invention and the substance to be delivered in combination, the substance to be delivered can be efficiently delivered to the spinal cord tissue.
また、このような本発明によれば被送達物質を脊髄組織へより高効率で送達できることから、被送達物質に起因する有用作用を脊髄組織においてより効率良く発揮させることができる。これによって、脊髄組織の分子イメージング、脊髄疾患の病状観察、被送達物質による疾患治療などを一層効果的に行うことができる。 Further, according to the present invention, since the substance to be delivered can be delivered to the spinal cord tissue with higher efficiency, the useful action resulting from the substance to be delivered can be more efficiently exhibited in the spinal cord tissue. Thereby, molecular imaging of spinal cord tissue, pathological observation of spinal cord disease, disease treatment with a delivered substance, and the like can be performed more effectively.
１．ペプチド
本発明は、以下の（１−１）または（１−２）で表される脊髄組織標的化ペプチドを提供する。すなわち、本発明は、（１−１）配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列の両末端にそれぞれ少なくとも１つのシステイン残基が直接連結してなるペプチド、また、（１−２）前記（１−１）のペプチドにおいて、配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列が、１または複数のアミノ酸が欠失、置換及び／または付加されたアミノ酸配列からなり、且つ、脊髄組織を標的とするペプチドを提供する。
1. Peptide The present invention provides a spinal cord tissue targeting peptide represented by the following (1-1) or (1-2). That is, the present invention relates to (1-1) a peptide in which at least one cysteine residue is directly linked to both ends of an amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5, respectively. (1-2) In the peptide of (1-1), at least one amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5 is deleted, Provided is a peptide consisting of a substituted and / or added amino acid sequence and targeting spinal cord tissue.
前記（１−１）において「配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列」とは、配列番号１〜５で表されるそれぞれのアミノ酸配列が、それぞれその配列を保持しながら１または複数の配列が連結したアミノ酸配列をいう。該連結は、本発明のペプチドが脊髄組織を標的化できることを限度として特に制限されないが、好ましくはペプチド結合によって連結しているものが例示される。該（１−１）において「１または複数の配列」の範囲は本発明のペプチドが脊髄組織を標的化できることを限度として制限されないが、十分に標的化できる観点から、例えば１〜３つの配列、好ましくは１〜２つの配列、より好ましくは１つの配列が挙げられ、また、例えば同種のアミノ酸配列が連結しているものであっても異種のアミノ酸配列が連結しているものであってもよく、脊髄組織を標的化できることを限度としてその種類や順序も制限されない。配列番号１〜５のいずれかで表されるアミノ酸配列は、いずれも７つのアミノ酸残基からなる。 In the above (1-1), “the amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5” means that each amino acid sequence represented by SEQ ID NOs: 1 to 5 is An amino acid sequence in which one or more sequences are linked while retaining the sequence. The linkage is not particularly limited as long as the peptide of the present invention can target spinal cord tissue, but preferably those linked by peptide bonds are exemplified. In (1-1), the range of “one or more sequences” is not limited as long as the peptide of the present invention can target spinal cord tissue. From the viewpoint of sufficient targeting, for example, 1 to 3 sequences, Preferably, there are 1 to 2 sequences, more preferably 1 sequence, and for example, the same kind of amino acid sequences may be linked or heterogeneous amino acid sequences may be linked. As long as it can target spinal cord tissue, its type and order are not limited. Each of the amino acid sequences represented by any of SEQ ID NOs: 1 to 5 consists of 7 amino acid residues.
後述の実施例に示されるように、配列番号１及び２のいずれかで表されるアミノ酸配列は特に脊髄組織全体に対する特異性が高く、配列番号３で表されるアミノ酸配列は特に脊髄組織内のアストロサイトに対する特異性が高く、配列番号４及び５のいずれかで表されるアミノ酸配列は特に脊髄組織内のミクログリアに対する特異性が高い。このことから、特異性をより高める観点から、例えば脊髄組織全体を特に標的化したい場合には、配列番号１及び２からなる群より選択される少なくとも１種で表されるアミノ酸配列が１または複数連結したアミノ酸配列を用いることが好ましい。また、同様の観点から、例えば脊髄組織内のアストロサイトを特に標的化したい場合には配列番号３で表されるアミノ酸配列が１または複数連結したアミノ酸配列を用いることが好ましく、脊髄組織内のミクログリアを特に標的化したい場合には配列番号４及び５からなる群より選択される少なくとも１種で表されるアミノ酸配列が１または複数連結したアミノ酸配列を用いることが好ましい。 As shown in Examples described later, the amino acid sequence represented by any of SEQ ID NOs: 1 and 2 is particularly highly specific to the entire spinal cord tissue, and the amino acid sequence represented by SEQ ID NO: 3 is particularly useful in the spinal cord tissue. The specificity to astrocytes is high, and the amino acid sequence represented by any of SEQ ID NOs: 4 and 5 is particularly highly specific to microglia in the spinal cord tissue. From this, from the viewpoint of further increasing the specificity, for example, when it is desired to specifically target the entire spinal cord tissue, one or more amino acid sequences represented by at least one selected from the group consisting of SEQ ID NOs: 1 and 2 are used. It is preferred to use a linked amino acid sequence. From the same viewpoint, for example, when it is desired to specifically target astrocytes in spinal cord tissue, it is preferable to use an amino acid sequence in which one or a plurality of amino acid sequences represented by SEQ ID NO: 3 are linked, and microglia in spinal cord tissue is used. In particular, it is preferable to use an amino acid sequence in which one or a plurality of amino acid sequences represented by at least one selected from the group consisting of SEQ ID NOs: 4 and 5 are linked.
また、該（１−１）において、前記アミノ酸配列の両末端にはそれぞれ少なくとも１つのシステイン残基が直接連結している。該連結は、本発明のペプチドが脊髄組織を標的化できることを限度として特に制限されないが、好ましくは前記アミノ酸配列の末端にペプチド結合によって直接連結しているものが例示される。両末端のシステイン残基数は、本発明のペプチドが脊髄組織を標的化できることを限度として特に制限されず、また、両末端のシステイン残基数は同じであってもよく異なってもよく、例えば各末端においてそれぞれ１〜３個、好ましくは１〜２個、より好ましくは１個が挙げられる。 In (1-1), at least one cysteine residue is directly linked to both ends of the amino acid sequence. The ligation is not particularly limited as long as the peptide of the present invention can target spinal cord tissue, but preferably ligated directly to the end of the amino acid sequence by a peptide bond. The number of cysteine residues at both ends is not particularly limited as long as the peptide of the present invention can target spinal cord tissue, and the number of cysteine residues at both ends may be the same or different. In each end, 1 to 3, preferably 1 to 2, more preferably 1 is mentioned.
また、本発明のペプチドは、脊髄組織を標的化できることを限度として特に制限されず、直鎖状、ループ状などの任意の形態が挙げられるが、好ましくはループ状であり、より好ましくは前記両末端のシステイン残基において片端のシステイン残基が他端のシステイン残基とジスルフィド結合を形成してなるループ状が例示される。本発明を限定するものではないが、ループ状にあるペプチドのモデル形態は図１のように例示できる。図１は、両末端に１つずつシステイン残基が連結した、合計９つのアミノ酸残基からなるループ状にあるペプチドのモデル形態であり、Ｃはシステイン残基を示し、Ｘ１〜Ｘ７は脊髄組織を標的化できる前述のアミノ酸配列を示す。本発明においてペプチドは、アミノ酸残基の数によってはオリゴペプチド、ポリペプチドと称されるものも含む。 In addition, the peptide of the present invention is not particularly limited as long as it can target spinal cord tissue, and includes any form such as a straight chain or a loop, preferably a loop, and more preferably both Examples of the terminal cysteine residue include a loop shape in which one cysteine residue forms a disulfide bond with the other cysteine residue. Although the present invention is not limited, a model form of a peptide in a loop shape can be illustrated as shown in FIG. FIG. 1 is a model form of a peptide having a loop shape composed of a total of 9 amino acid residues, each having a cysteine residue linked to each end, C is a cysteine residue, and X 1 to X 7 are The amino acid sequences described above that can target spinal cord tissue are shown. In the present invention, the peptide includes what are called oligopeptides and polypeptides depending on the number of amino acid residues.
該（１−１）においてペプチドのアミノ酸残基数は本発明のペプチドが脊髄組織を標的化できることを限度として特に制限されないが、好ましくは９〜２３個、より好ましくは９〜１６個、更に好ましくは９〜１４個、特に好ましくは９個が挙げられる。該（１−１）において９個のアミノ酸残基からなるペプチドの例示としては、配列番号１〜５のいずれか１種で表されるアミノ酸配列の両末端にそれぞれ１つのシステイン残基が直接連結してなるペプチドが挙げられる。 In (1-1), the number of amino acid residues of the peptide is not particularly limited as long as the peptide of the present invention can target spinal cord tissue, preferably 9 to 23, more preferably 9 to 16, and still more preferably. 9 to 14, particularly preferably 9 is mentioned. As an example of a peptide consisting of 9 amino acid residues in (1-1), one cysteine residue is directly linked to both ends of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, respectively. The peptide formed is mentioned.
前記（１−２）において、「１または複数のアミノ酸」の範囲は、該（１−２）に記載されるペプチドが脊髄組織を標的化できることを限度として特に制限されないが、例えば１〜５個、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または２個、特に好ましくは１個が挙げられる。特定のアミノ酸配列において１または複数のアミノ酸を欠失、置換及び／または付加させる技術は公知である。なお、このように欠失、置換及び／または付加されたアミノ酸配列として、「配列番号１〜５からなる群より選択される少なくとも１種のアミノ酸配列」に対して５７％以上の同一性を有するアミノ酸配列からなり、且つ、脊髄組織を標的化できる配列が例示される。また、該アミノ酸配列として、アミノ酸の同一性は好ましくは７０％以上、より好ましくは８０％以上、更に好ましくは９０％以上、特に好ましくは９５％以上、更に特に好ましくは９７％以上、一層好ましくは９８％以上である。 In the above (1-2), the range of “one or more amino acids” is not particularly limited as long as the peptide described in (1-2) can target spinal cord tissue. , Preferably 1 to 4, more preferably 1 to 3, further preferably 1 or 2, particularly preferably 1. Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known. The amino acid sequence thus deleted, substituted and / or added has 57% or more identity to “at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5”. Examples are sequences consisting of amino acid sequences and capable of targeting spinal cord tissue. In addition, the amino acid identity of the amino acid sequence is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more, still more preferably 97% or more, and still more preferably. 98% or more.
本発明のペプチドは、従来公知の化学合成法や遺伝子工学的手法などによって作製できる。例えば、本発明のペプチドをコードするアミノ酸配列やヌクレオチド配列の情報に従って、本発明のペプチドを従来公知の化学合成法により合成、精製して取得してもよい。なお、化学合成法には、液相法や固相法によるペプチド合成法が包含される。また、本発明のペプチドは後述の実施例に基づいて作製できる。このほか、本発明のペプチドをコードするポリヌクレオチドをベクター等に挿入し、次いで、該ベクターが組み込まれた形質転換体を培養したのち、所望のペプチドを取得してもよい。 The peptide of the present invention can be prepared by a conventionally known chemical synthesis method or genetic engineering method. For example, the peptide of the present invention may be obtained by synthesizing and purifying the peptide of the present invention by a conventionally known chemical synthesis method according to the information on the amino acid sequence or nucleotide sequence encoding the peptide of the present invention. The chemical synthesis method includes a peptide synthesis method using a liquid phase method or a solid phase method. Moreover, the peptide of this invention can be produced based on the below-mentioned Example. In addition, a desired peptide may be obtained after inserting a polynucleotide encoding the peptide of the present invention into a vector and then culturing a transformant in which the vector is incorporated.
本発明において「脊髄組織標的化」、「標的とする」とは、本発明のペプチドが他の組織と比較して脊髄組織に高い親和性を有する、あるいは脊髄組織を高効率で認識できることを意味する。特に、本発明のペプチドは、他の組織や細胞と比較して、脊髄組織全体、脊髄組織内のアストロサイト及び脊髄組織内のミクログリアからなる群より選択される少なくとも１種の脊髄組織に高い親和性を有する、あるいは脊髄組織を高効率で認識できることを意味する。 In the present invention, “target spinal cord tissue” and “target” mean that the peptide of the present invention has a higher affinity for spinal cord tissue than other tissues or can recognize spinal cord tissue with high efficiency. To do. In particular, the peptide of the present invention has a higher affinity for at least one spinal cord tissue selected from the group consisting of the entire spinal cord tissue, astrocytes in the spinal cord tissue, and microglia in the spinal cord tissue, as compared with other tissues and cells. It means having a sex or being able to recognize spinal cord tissue with high efficiency.
本発明のペプチドを用いて脊髄組織を標的化するにあたって、その方法は特に制限されず、該ペプチドを脊髄組織に接触させればよい。また、本発明のペプチドによる標的化はin vivo、in vitro及びex vivoのいずれであってもよい。例えば、本発明のペプチドをin vitroやex vivoで標的化させる場合、脊髄組織や脊髄組織を含む対象物に該ペプチドの適当量を接触させ、また、必要に応じてインキュベートすればよい。また、本発明のペプチドをin vivoで標的化させる場合には、組織への直接注入、静脈、皮下、筋肉または腹腔への注射をはじめ、髄腔内（脊髄腔内を含む）投与、経口投与、吸入投与、経粘膜投与等により本発明のペプチドを投与すればよい。本発明のペプチドの適用対象（対象物）は本発明の効果が得られる限り制限されず、ヒトやヒト以外の霊長類をはじめとする哺乳動物、またこれらに由来する脊髄組織に適用される。 In targeting spinal cord tissue using the peptide of the present invention, the method is not particularly limited, and the peptide may be contacted with spinal cord tissue. In addition, targeting with the peptide of the present invention may be any of in vivo, in vitro and ex vivo. For example, when targeting the peptide of the present invention in vitro or ex vivo, an appropriate amount of the peptide may be brought into contact with a spinal cord tissue or an object including spinal cord tissue, and incubated as necessary. In addition, when targeting the peptide of the present invention in vivo, direct injection into tissue, intravenous, subcutaneous, intramuscular or intraperitoneal injection, intrathecal (including intrathecal) administration, oral administration The peptide of the present invention may be administered by inhalation administration, transmucosal administration or the like. The application target (object) of the peptide of the present invention is not limited as long as the effect of the present invention is obtained, and is applied to mammals including humans and non-human primates, and spinal cord tissues derived therefrom.
本発明のペプチドはこのように脊髄組織を標的化できるので、脊髄組織の観察や診断等に有用である。該ペプチドは例えば医薬分野において、脊髄組織の分子イメージング、脊髄組織における疾患の予防、軽減または治療、オーターメード医療、創薬、疾患の原因解明、また、疾患の治療効果を評価するためのツールなど様々な用途に用いることができる。また、本発明のペプチドは、単独で優れた脊髄組織標的化能を示すので、被送達物質の脊髄組織への送達に好適に使用される。また、本発明のペプチドをこのように医薬分野で使用する場合、例えば後述する医薬組成物としても使用され得る。 Since the peptide of the present invention can thus target spinal cord tissue, it is useful for observation and diagnosis of spinal cord tissue. For example, in the pharmaceutical field, the peptide is used for molecular imaging of spinal cord tissue, prevention, alleviation or treatment of diseases in spinal cord tissue, customized medicine, drug discovery, elucidation of the cause of diseases, and tools for evaluating therapeutic effects of diseases, etc. It can be used for various purposes. In addition, since the peptide of the present invention alone exhibits excellent spinal cord tissue targeting ability, it is preferably used for delivery of a substance to be delivered to spinal cord tissue. Moreover, when using the peptide of this invention in the pharmaceutical field | area in this way, it can be used also as a pharmaceutical composition mentioned later, for example.
このように本発明のペプチドによれば脊髄組織を効率良く標的化できる。このため、本発明のペプチドと被送達物質とを併用することによって、脊髄組織に被送達物質をより効率よく送達することができる。また、このような本発明によれば被送達物質の脊髄組織への運搬をより高効率で行うことができることから、被送達物質に起因する有用作用を脊髄組織において効率良く発揮させることができる。また、このような本発明によれば、脊髄組織以外の他組織や他細胞への副作用を軽減あるいは回避することができる。また、本発明のペプチドの利用においては複雑な工程を必要とせず、組み合わせる被送達物質も自由に選択でき、汎用性が非常に高い。従って、本発明によれば、前述のように脊髄組織の分子イメージング、脊髄疾患の病状観察、被送達物質による疾患治療などを一層効果的に行うことができる。また、本発明によって脊髄組織の様子を一層明らかにすることによって、疾患の診断方法、予防方法、治療方法、また、オーダーメード医療、創薬、疾患の原因解明などにおける更なる進展につながる。 Thus, according to the peptide of the present invention, spinal cord tissue can be efficiently targeted. For this reason, by using together the peptide of this invention and a to-be-delivered substance, a to-be-delivered substance can be delivered to spinal cord tissue more efficiently. Further, according to the present invention, since the substance to be delivered can be transported to the spinal cord tissue with higher efficiency, a useful action resulting from the substance to be delivered can be efficiently exhibited in the spinal cord tissue. Moreover, according to the present invention, side effects on other tissues and cells other than spinal cord tissue can be reduced or avoided. In addition, the use of the peptide of the present invention does not require a complicated process, and a substance to be delivered can be freely selected and is very versatile. Therefore, according to the present invention, as described above, molecular imaging of spinal cord tissue, pathological observation of spinal cord disease, disease treatment with a substance to be delivered, and the like can be performed more effectively. Further, by clarifying the state of spinal cord tissue according to the present invention, it will lead to further progress in disease diagnosis methods, prevention methods, treatment methods, customized medicine, drug discovery, disease cause elucidation and the like.
２．ポリヌクレオチド
本発明は、更に脊髄組織標的化ペプチドをコードするポリヌクレオチドを提供する。本発明においてポリヌクレオチドは、前記脊髄組織標的化ペプチドをコードするポリヌクレオチドである限り制限されず、以下のポリヌクレオチドが挙げられる；
（２−１）前記（１−１）及び（１−２）のいずれかに記載のペプチドをコードするポリヌクレオチド、
（２−２）前記（２−１）のポリヌクレオチドの相補鎖に対して、ストリンジェントな条件下でハイブリダイズし、且つ、脊髄組織標的化ペプチドをコードするポリヌクレオチド。
2. Polynucleotide The present invention further provides a polynucleotide encoding a spinal cord tissue targeting peptide. In the present invention, the polynucleotide is not limited as long as it is a polynucleotide encoding the spinal cord tissue targeting peptide, and includes the following polynucleotides;
(2-1) a polynucleotide encoding the peptide according to any one of (1-1) and (1-2),
(2-2) A polynucleotide that hybridizes to a complementary strand of the polynucleotide of (2-1) under stringent conditions and encodes a spinal cord tissue-targeting peptide.
ここで、前記（２−１）のポリヌクレオチドは、当業者であれば前記ペプチドのアミノ酸配列に基づいて、従来公知の手法に基づき容易に解析、入手することができる。本発明を制限するものではないが、配列番号１〜５のいずれかで表されるアミノ酸配列をコードするポリヌクレオチドとしては、配列番号６〜１０のいずれかで表される塩基配列が例示される。配列番号６は、配列番号１で表されるアミノ酸配列をコードする塩基配列を示す。同様に、配列番号７は配列番号２で表されるアミノ酸配列をコードする塩基配列、配列番号８は配列番号３で表されるアミノ酸配列をコードする塩基配列、配列番号９は配列番号４で表されるアミノ酸配列をコードする塩基配列、配列番号１０は配列番号５で表されるアミノ酸配列をコードする塩基配列を示す。そして、これらをはじめとする前述のアミノ酸配列をコードする塩基配列の両末端にそれぞれ少なくとも１つのシステイン残基をコードする塩基配列を設けたものが、前記（１−１）及び（１−２）のいずれかに記載のペプチドをコードするポリヌクレオチドとして例示される。これらの塩基配列に従って前記（２−１）のポリヌクレオチドを従来公知の手法に基づき解析、入手してもよく、また、システイン残基をコードする塩基配列も公知である。 Here, the polynucleotide of (2-1) can be easily analyzed and obtained by a person skilled in the art based on the amino acid sequence of the peptide based on a conventionally known method. Although not limiting the present invention, the polynucleotide encoding the amino acid sequence represented by any of SEQ ID NOs: 1 to 5 is exemplified by the base sequence represented by any of SEQ ID NOs: 6 to 10 . SEQ ID NO: 6 shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. Similarly, SEQ ID NO: 7 is the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 8 is the base sequence encoding the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: 9 is represented by SEQ ID NO: 4. SEQ ID NO: 10 shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 5. And what provided the base sequence which codes each at least 1 cysteine residue in the both ends of the base sequence which codes the above-mentioned amino acid sequences including these is each said (1-1) and (1-2) And a polynucleotide encoding the peptide according to any one of the above. According to these base sequences, the polynucleotide (2-1) may be analyzed and obtained based on a conventionally known technique, and the base sequence encoding a cysteine residue is also known.
前記（２−２）において「ストリンジェントな条件」とは、例えば６５℃で５×ＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は１５０ｍＭ塩化ナトリウム、１５ｍＭクエン酸ナトリウム）中でハイブリダイズさせ、更に０．１％のＳＤＳを含有する０．５×ＳＳＣ溶液で６５℃で洗浄する条件を意味する。ストリンジェントな条件下でのハイブリダイゼーションの各操作は、「Molecular Cloning (Third Edition)」(J. Sambrook & D. W. Russell, Cold Spring Harbor Laboratory Press, 2001)に記載されている方法など、従来公知の方法で行うことができる。通常、温度が高いほど、また、塩濃度が低いほどストリンジェンシーは高くなる。 In the above (2-2), “stringent conditions” means, for example, hybridization at 5 ° C. in a 5 × SSC solution (composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), and It means the condition of washing at 65 ° C. with 0.5 × SSC solution containing 0.1% SDS. Each hybridization operation under stringent conditions is performed by a conventionally known method such as the method described in "Molecular Cloning (Third Edition)" (J. Sambrook & DW Russell, Cold Spring Harbor Laboratory Press, 2001). Can be done. Generally, the higher the temperature and the lower the salt concentration, the higher the stringency.
前記相補鎖に対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドは、通常、前記（２−１）のいずれかのヌクレオチド配列と一定以上の同一性を有し、その同一性は例えば５７％以上が例示され、好ましくは７０％以上、より好ましくは８５％以上、更に好ましくは９０％以上、特に好ましくは９５％以上、更に特に好ましくは９８％以上、一層好ましくは９９％以上である。ヌクレオチド配列の同一性は市販のまたはインターネット等の電気通信回線を通じて利用可能な解析ツールを用いて算出することができ、例えばＦＡＳＴＡ、ＢＬＡＳＴ、ＰＳＩ−ＢＬＡＳＴ、ＳＳＥＡＲＣＨ等のソフトウェアを用いて、計算、決定できる。 The polynucleotide that hybridizes to the complementary strand under stringent conditions usually has a certain level of identity with any one of the nucleotide sequences of (2-1), and the identity is, for example, 57% The above is exemplified, preferably 70% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, still more preferably 98% or more, and still more preferably 99% or more. Nucleotide sequence identity can be calculated using analysis tools that are commercially available or available through telecommunications lines such as the Internet, for example, calculated and determined using software such as FASTA, BLAST, PSI-BLAST, SSEARCH, etc. it can.
ここで「脊髄組織標的化」とは前述と同様に説明され、また、前記ポリヌクレオチドを用いて作製されるペプチドは他の組織と比較して脊髄組織に高い親和性を有し、あるいは脊髄組織を高効率で認識できる。特に、前記ポリヌクレオチドを用いて作製されるペプチドは、他の組織や細胞と比較して、脊髄組織全体、脊髄組織内のアストロサイト及び脊髄組織内のミクログリアからなる群より選択される少なくとも１種の脊髄組織に高い親和性を有し、あるいは脊髄組織を高効率で認識できる。前記ポリヌクレオチドからのペプチドの作製は、当該分野において従来公知の化学合成法や遺伝子工学的手法などを用いて行えばよく、前述の通りこれは当業者にとって容易である。 Here, “targeting spinal cord tissue” is explained in the same manner as described above, and the peptide prepared using the polynucleotide has higher affinity for spinal cord tissue than other tissues, or spinal cord tissue. Can be recognized with high efficiency. In particular, the peptide produced using the polynucleotide is at least one selected from the group consisting of the entire spinal cord tissue, astrocytes in the spinal cord tissue, and microglia in the spinal cord tissue, as compared with other tissues and cells. The spinal cord tissue has high affinity, or the spinal cord tissue can be recognized with high efficiency. The preparation of the peptide from the polynucleotide may be performed using a conventionally known chemical synthesis method or genetic engineering method in the art, and as described above, this is easy for those skilled in the art.
また、本発明のポリヌクレオチドも、従来公知の化学合成法や遺伝子工学的手法などを用いて作製できる（Proc. Natl. Acad. Sci., USA., 78, 6613 (1981)；Science, 222, 778 (1983)；Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press(1989)；続生化学実験講座「遺伝子研究法Ｉ、II、III」、日本生化学会編（1986）等参照）。例えば、配列番号１〜５のいずれかで表されるアミノ酸の配列情報や、配列番号６〜１０のいずれかで表されるヌクレオチドの配列情報に基づいて、従来公知の化学的合成法により作製、取得すればよい。 In addition, the polynucleotide of the present invention can also be prepared using a conventionally known chemical synthesis method or genetic engineering method (Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 222, 778 (1983); Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); secondary biochemistry experiment course "gene research method I, II, III", Japanese Biochemical Society edition (1986) etc.). For example, based on the amino acid sequence information represented by any of SEQ ID NOs: 1 to 5 or the nucleotide sequence information represented by any of SEQ ID NOs: 6 to 10, prepared by a conventionally known chemical synthesis method, Get it.
３．医薬組成物
本発明は、前記ペプチドを含有する医薬組成物を提供する。すなわち、本発明は、前記（１−１）及び（１−２）からなる群より選択される少なくとも１種の脊髄組織標的化ペプチドを含有する医薬組成物を提供する。
3. Pharmaceutical Composition The present invention provides a pharmaceutical composition containing the peptide. That is, the present invention provides a pharmaceutical composition containing at least one spinal cord tissue targeting peptide selected from the group consisting of (1-1) and (1-2).
本発明の医薬組成物は、脊髄組織に対して脊髄組織標的化ペプチドを標的化させるために使用されるものであり、該ペプチドは前述の通りである。本発明の医薬組成物における前記ペプチドの含有量は、該医薬組成物を用いることによって前記ペプチドを脊髄組織に対して標的化できる限り制限されず、該医薬組成物の形態、使用態様、接触される脊髄組織の重量、種類、状態などによって当業者が適宜設定すればよい。例えば、後述の実施例では前記ペプチド１５μｇを静脈投与することで脊髄組織を標的化することが可能であり、該ペプチドの含有量等は該適用量に基づいて、当業者が適宜設定すればよい。 The pharmaceutical composition of the present invention is used for targeting a spinal cord tissue targeting peptide to spinal cord tissue, and the peptide is as described above. The content of the peptide in the pharmaceutical composition of the present invention is not limited as long as the peptide can be targeted to spinal cord tissue by using the pharmaceutical composition, and the form, usage, and contact of the pharmaceutical composition are not limited. A person skilled in the art may set as appropriate according to the weight, type, state, etc. of the spinal cord tissue. For example, in the examples described below, spinal cord tissue can be targeted by intravenous administration of 15 μg of the peptide, and the content of the peptide and the like may be appropriately set by those skilled in the art based on the applied amount. .
本発明の医薬組成物には必要に応じて更に被送達物質が含有されていてもよい。被送達物質としては脊髄組織に送達させたいものであれば何ら制限されず、目的に応じて当業者が適宜決定すればよい。被送達物質として例えば、蛍光物質、ｓｉＲＮＡ、ｍｉＲＮＡ、アンチセンスＲＮＡなどのｓｍａｌｌ ＲＮＡやＤＮＡをはじめとするオリゴヌクレオチドなどの核酸、ペプチド、タンパク質、薬物などが挙げられる。被送達物質を前記ペプチドと共に用いることによって、被送達物質を脊髄組織に効率よく送達できる。 The pharmaceutical composition of the present invention may further contain a substance to be delivered as necessary. The substance to be delivered is not limited as long as it is desired to be delivered to the spinal cord tissue, and may be appropriately determined by those skilled in the art according to the purpose. Examples of the substance to be delivered include nucleic acids such as fluorescent substances, siRNA, miRNA, antisense RNA such as antisense RNA, oligonucleotides such as DNA, peptides, proteins, drugs, and the like. By using the substance to be delivered together with the peptide, the substance to be delivered can be efficiently delivered to the spinal cord tissue.
被送達物質を脊髄組織に更に効率よく送達する観点から、被送達物質は前記ペプチドと連結していることが好ましい。該連結は被送達物質が前記ペプチドと連結しており、且つ、被送達物質の有する有用作用と前記ペプチドの有する標的化能を阻害しないことを限度として制限されない。この限りにおいて、前記ペプチドと被送達物質は、直接またはリンカーを介して連結されていればよく、例えば静電気的結合、塩基性アミノ酸を介した結合、グリシンを介した結合、アミノカプロン酸を介した結合などが例示できる。これらは１種単独で使用してもよく、２種以上を組み合わせて使用してもよい。 From the viewpoint of more efficiently delivering the delivered substance to the spinal cord tissue, the delivered substance is preferably linked to the peptide. The connection is not limited as long as the substance to be delivered is linked to the peptide and does not inhibit the useful action of the substance to be delivered and the targeting ability of the peptide. As long as this is the case, the peptide and the substance to be delivered may be linked directly or via a linker, for example, electrostatic bond, bond via basic amino acid, bond via glycine, bond via aminocaproic acid. Etc. can be exemplified. These may be used alone or in combination of two or more.
本発明を制限するものではないがこれらの連結について例示すると、例えば被送達物質が脊髄組織の細胞内等に働きかけて有用作用を発揮するような場合には、前記ペプチドにより脊髄組織が標的化された後に、被送達物質が前記ペプチドから離れることにより細胞内で効率良く有用作用を発揮することが考えられる。このような場合には、被送達物質は、前記ペプチドの脊髄組織への標的後に被送達物質が前記ペプチドから分離可能な状態で、前記ペプチドに連結されていることが好ましく、例えば静電気的結合が例示される。また、例えば被送達物質が負電荷を帯びているものであれば、被送達物質は正電荷を帯びたリンカーを介して前記ペプチドに連結されていてもよく、正電荷を帯びたリンカーとしてはアルギニン、リジン及び／またはヒスチジンなどの塩基性アミノ酸を含むリンカーが例示される。このようなリンカーの長さは本発明の効果が得られる限り制限されず、当業者が適宜設定すればよい。また、本発明を制限するものではないが、被送達物質の細胞内への導入効率を高める観点から、リンカーは被送達物質の細胞内への導入効率を高める作用を有しているものが好ましく、このようなリンカーとして正電荷を有するリンカーが例示される。 Although the present invention is not limited to these linkages, for example, when the substance to be delivered acts on the cells of the spinal cord tissue and exerts a useful action, the spinal cord tissue is targeted by the peptide. After that, it is conceivable that the substance to be delivered exerts a useful action efficiently in the cell by separating from the peptide. In such a case, the substance to be delivered is preferably linked to the peptide in a state where the substance to be delivered can be separated from the peptide after the peptide is targeted to the spinal cord tissue. Illustrated. In addition, for example, if the substance to be delivered is negatively charged, the substance to be delivered may be linked to the peptide via a positively charged linker, and arginine is used as the positively charged linker. And linkers containing basic amino acids such as lysine and / or histidine. The length of such a linker is not limited as long as the effect of the present invention can be obtained, and may be appropriately set by those skilled in the art. Although not intended to limit the present invention, from the viewpoint of increasing the efficiency of introduction of the substance to be delivered into the cell, it is preferable that the linker has an effect of increasing the efficiency of introduction of the substance to be delivered into the cell. Examples of such a linker include a linker having a positive charge.
また、本発明を制限するものではないが、例えば被送達物質が前記ペプチドから分離されることなくその有用作用を発揮させたい場合には、前記ペプチドの脊髄組織への標的後に被送達物質が前記ペプチドに連結されたままとなっていることが好ましく、この場合には化学的な結合が例示される。また、後述の実施例に示されているように、リンカーは、グリシンリンカー等の別のリンカーを介して更に連結されていてもよい。 Although not intended to limit the present invention, for example, when the substance to be delivered is desired to exert its useful action without being separated from the peptide, the substance to be delivered is the target after the peptide is targeted to the spinal cord tissue. Preferably it remains linked to the peptide, in this case a chemical bond is exemplified. Moreover, as shown in the below-mentioned Example, the linker may be further connected through another linker such as a glycine linker.
また、前記被送達物質または前記リンカーは、被送達物質が有する有用作用と前記ペプチドの有する標的化能を阻害しないことを限度として、前記ペプチドの任意の部分に連結させればよいが、前記ペプチドの前記末端のシステイン残基に連結させることが好ましい。また、本発明の効果が得られる限り制限されないが、被送達物質は片端のシステイン残基のみに連結していてもよく、両端のシステイン残基に連結していてもよい。例えば、後述の実施例のように、被送達物質として蛍光物質と核酸とを用いる場合には、蛍光物質と核酸の有用作用の相違、電荷、物理的な障害等を考慮して、片端のシステイン残基には蛍光物質を、他端のシステイン残基には核酸を連結させてもよい。 In addition, the substance to be delivered or the linker may be linked to any part of the peptide as long as it does not inhibit the useful action of the substance to be delivered and the targeting ability of the peptide. It is preferable to link to the terminal cysteine residue. Moreover, as long as the effect of the present invention is obtained, the substance to be delivered may be linked to only one cysteine residue, or may be linked to cysteine residues at both ends. For example, in the case where a fluorescent substance and a nucleic acid are used as a substance to be delivered, as described later, in consideration of the difference in useful action between the fluorescent substance and the nucleic acid, charge, physical obstacle, etc., one end of cysteine A fluorescent substance may be linked to the residue, and a nucleic acid may be linked to the cysteine residue at the other end.
本発明の医薬組成物における前記被送達物質の含有量は、脊髄組織において被送達物質の有用作用が発揮される限り制限されず、該医薬組成物の形態、使用態様、脊髄組織の重量、種類、状態などによって当業者が適宜設定すればよい。本発明を制限するものではないが、例えば、後述の実施例では前記ペプチドと被送達物質とは重量比３：１で併用することにより脊髄組織を標的化し、また、被送達物質に由来する有用作用を発揮させることが可能であり、該被送達物質の含有量等は、該適用量に基づいて、更に使用する被送達物質を鑑みて当業者が適宜設定すればよい。 The content of the substance to be delivered in the pharmaceutical composition of the present invention is not limited as long as the useful action of the substance to be delivered is exhibited in the spinal cord tissue, the form of the pharmaceutical composition, the mode of use, the weight of the spinal cord tissue, and the type A person skilled in the art may set as appropriate depending on the state. Although not limiting the present invention, for example, in the examples described below, the peptide and the substance to be delivered are used in combination at a weight ratio of 3: 1 to target spinal cord tissue, and useful derived from the substance to be delivered. It is possible to exert the action, and the content of the substance to be delivered and the like may be appropriately set by those skilled in the art based on the amount to be applied and further considering the substance to be delivered.
また、本発明の医薬組成物には必要に応じて薬学的に許容可能な担体が含有されていてもよい。担体としては本発明の効果が得られる限り制限されないが、精製水、緩衝液、生理食塩水、RNase free水、DNAse free水、プロテアーゼフリー水、グルコース水溶液、等張化剤、賦形剤、希釈剤、増粘剤、安定化剤、緩衝剤、保存剤等が例示され、使用形態に応じて当業者が適宜決定すればよい。 In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier as necessary. The carrier is not limited as long as the effect of the present invention is obtained, but purified water, buffer solution, physiological saline, RNase free water, DNAse free water, protease free water, glucose aqueous solution, isotonic agent, excipient, dilution Agents, thickeners, stabilizers, buffers, preservatives and the like are exemplified, and those skilled in the art may appropriately determine them depending on the form of use.
本発明の医薬組成物を脊髄組織に接触させることによって、前記ペプチドによって脊髄組織を標的化でき、また、例えば前記被送達物質を併用する場合には標的化された脊髄組織に該被送達物質が効率良く送達される。本発明の医薬組成物はin vitro、ex vivo及びin vivoのいずれにおいて使用されてもよい。例えば、本発明の医薬組成物をin vitroやex vivoで標的化させる場合、脊髄組織や脊髄組織を含む対象物に該医薬組成物の適当量を接触させ、また、必要に応じてインキュベートすればよい。また、本発明の医薬組成物をin vivoで標的化させる場合には、組織への直接注入、静脈、皮下、筋肉または腹腔への注射をはじめ、髄腔内（脊髄腔内を含む）投与、経口投与、吸入投与、経粘膜投与等により本発明の医薬組成物を投与すればよい。 By bringing the pharmaceutical composition of the present invention into contact with spinal cord tissue, the spinal cord tissue can be targeted by the peptide. For example, when the delivered substance is used in combination, the delivered substance is present in the targeted spinal cord tissue. Delivered efficiently. The pharmaceutical composition of the present invention may be used in any of in vitro, ex vivo and in vivo. For example, when targeting the pharmaceutical composition of the present invention in vitro or ex vivo, an appropriate amount of the pharmaceutical composition is brought into contact with a spinal cord tissue or an object containing the spinal cord tissue, and incubated if necessary. Good. In addition, when targeting the pharmaceutical composition of the present invention in vivo, including intra-tissue direct injection, intravenous, subcutaneous, intramuscular or intraperitoneal injection, intrathecal (including intrathecal) administration, The pharmaceutical composition of the present invention may be administered by oral administration, inhalation administration, transmucosal administration and the like.
このことから本発明の医薬組成物の製剤形態は本発明の効果が得られる限り制限されず、液剤（シロップ等を含む）、点滴剤、注射剤などの液状製剤；ゲル剤などの半固形製剤；凍結乾燥製剤、ドライシロップ剤、錠剤、丸剤、散剤、顆粒剤、カプセル剤（ソフトカプセルを含む）などの固形状製剤が例示される。また、本発明の医薬組成物が固形製剤である場合は、使用時に注射用蒸留水、滅菌水などを加えて再度溶解して使用してもよい。 Accordingly, the pharmaceutical composition of the pharmaceutical composition of the present invention is not limited as long as the effects of the present invention are obtained, and liquid preparations such as liquids (including syrups), drops, injections, etc .; semisolid preparations such as gels Solid preparations such as freeze-dried preparations, dry syrups, tablets, pills, powders, granules, capsules (including soft capsules) are exemplified. Further, when the pharmaceutical composition of the present invention is a solid preparation, it may be used by dissolving again by adding distilled water for injection, sterilized water, etc. at the time of use.
また、本発明の医薬組成物が適用される疾患または症状は脊髄組織が関与しているものである限り制限されない。脊髄組織と疾患または症状の関係については当該技術分野において公知である。また、本発明の医薬組成物の適用対象（対象物）は本発明の効果が得られる限り制限されず、ヒトやヒト以外の霊長類をはじめとする哺乳動物、またこれらに由来する脊髄組織が好ましく例示される。 The disease or symptom to which the pharmaceutical composition of the present invention is applied is not limited as long as spinal cord tissue is involved. The relationship between spinal cord tissue and disease or condition is known in the art. Further, the application target (object) of the pharmaceutical composition of the present invention is not limited as long as the effects of the present invention can be obtained, and mammals including humans and non-human primates, and spinal cord tissues derived therefrom are also included. Preferably exemplified.
本発明の医薬組成物によればこのように脊髄組織を標的化できるので、該医薬組成物は脊髄組織の分子イメージング、脊髄組織における疾患の予防、軽減または治療、オーターメード医療、創薬、疾患の原因解明、また、疾患の治療効果を評価するためのツールなど様々な用途に用いることができる。 Since the spinal cord tissue can be targeted in this way according to the pharmaceutical composition of the present invention, the pharmaceutical composition can be used for molecular imaging of spinal cord tissue, prevention, reduction or treatment of diseases in spinal cord tissue, customized medicine, drug discovery, disease It can be used for various purposes such as elucidation of the cause of the disease and a tool for evaluating the therapeutic effect of the disease.
このように本発明の医薬組成物によれば脊髄組織を効率良く標的化できる。また、本発明の医薬組成物が被送達物質を更に含有する場合には、脊髄組織に被送達物質をより効率よく送達することができる。また、このような本発明によれば被送達物質の脊髄組織への運搬をより高効率で行うことができることから、被送達物質に起因する有用作用を脊髄組織において一層効率良く発揮させることができる。また、このような本発明によれば、脊髄組織以外の他組織や他細胞への副作用を軽減あるいは回避することができる。また、本発明の医薬組成物の利用においては複雑な工程を必要とせず、組み合わせる被送達物質も自由に選択でき、汎用性も非常に高い。従って、本発明によれば、脊髄組織の分子イメージング、脊髄疾患の病状観察、被送達物質による疾患治療などを一層効果的に行うことができる。また、本発明によって脊髄組織の様子を一層明らかにすることによって、疾患の診断方法、予防方法、治療方法、また、オーダーメード医療、創薬、疾患の原因解明などにおける更なる進展につながる。 Thus, according to the pharmaceutical composition of the present invention, spinal cord tissue can be efficiently targeted. In addition, when the pharmaceutical composition of the present invention further contains a substance to be delivered, the substance to be delivered can be delivered to the spinal cord tissue more efficiently. Moreover, according to the present invention, since the substance to be delivered can be transported to the spinal cord tissue with higher efficiency, the useful action resulting from the substance to be delivered can be more efficiently exhibited in the spinal cord tissue. . Moreover, according to the present invention, side effects on other tissues and cells other than spinal cord tissue can be reduced or avoided. In addition, the use of the pharmaceutical composition of the present invention does not require a complicated process, and a substance to be combined can be freely selected, and the versatility is very high. Therefore, according to the present invention, molecular imaging of spinal cord tissue, pathological observation of spinal cord disease, disease treatment with a substance to be delivered, and the like can be performed more effectively. Further, by clarifying the state of spinal cord tissue according to the present invention, it will lead to further progress in disease diagnosis methods, prevention methods, treatment methods, customized medicine, drug discovery, disease cause elucidation and the like.
４．方法
本発明は、更に、前記脊髄組織標的化ペプチドまたは前記医薬組成物を対象物に投与する工程を含有する、対象物中の脊髄組織を標的化する方法を提供する。また、本発明は、更に、前記被送達物質を含有する医薬組成物を対象物に投与する工程を含有する、被送達物質の脊髄組織への送達方法を提供する。
4). Method The present invention further provides a method of targeting spinal cord tissue in a subject comprising the step of administering the spinal cord tissue targeting peptide or the pharmaceutical composition to the subject. The present invention further provides a method for delivering a substance to be delivered to spinal cord tissue, which comprises the step of administering a pharmaceutical composition containing the substance to be delivered to a subject.
これらの方法において、脊髄組織標的化ペプチド、医薬組成物、被送達物質、対象物、投与、標的化、被送達物質の脊髄組織への送達方法等については前述と同様に説明される。本発明の対象物中の脊髄組織を標的化する方法を実施することによって、脊髄組織を効率良く標的化できる。また、該方法において医薬組成物が被送達物質を含有している場合には、あるいは、前記被送達物質の脊髄組織への送達方法によれば、脊髄組織に被送達物質をより効率よく送達することができ、従って、被送達物質に起因する有用作用を脊髄組織において効率良く発揮させることができる。また、これらの方法によれば、脊髄組織以外の他組織や他細胞への副作用を軽減あるいは回避することができる。従って、これらの方法によれば、脊髄組織の分子イメージング、脊髄疾患の病状観察、被送達物質による疾患予防、軽減または治療などを一層効果的に行うことができる。また、これらの方法によって脊髄組織の様子を一層明らかにすることによって、疾患の診断方法、予防方法、治療方法、また、オーダーメード医療、創薬、疾患の原因解明などにおける更なる進展につながる。 In these methods, spinal cord tissue targeting peptide, pharmaceutical composition, delivered substance, subject, administration, targeting, delivery method of delivered substance to spinal cord tissue, etc. are described in the same manner as described above. By implementing the method of targeting spinal cord tissue in the subject of the present invention, spinal cord tissue can be efficiently targeted. In the method, when the pharmaceutical composition contains a substance to be delivered, or according to the method for delivering the substance to be delivered to the spinal cord tissue, the substance to be delivered is more efficiently delivered to the spinal cord tissue. Therefore, a useful action resulting from the substance to be delivered can be efficiently exhibited in the spinal cord tissue. Further, according to these methods, side effects on other tissues other than spinal cord tissues and other cells can be reduced or avoided. Therefore, according to these methods, molecular imaging of spinal cord tissue, pathological observation of spinal cord diseases, disease prevention, mitigation or treatment with a substance to be delivered can be performed more effectively. Further, by clarifying the state of spinal cord tissue by these methods, it will lead to further progress in disease diagnosis methods, prevention methods, treatment methods, customized medicine, drug discovery, and disease cause elucidation.
以下、実施例を挙げて本発明を説明するが、本発明はこれに限定されるものではない。
試験例１：脊髄組織標的化ペプチドの作製
配列番号１で表されるアミノ酸配列（ＬＨＱＳＰＨＩ）の両末端にそれぞれ１つのシステイン残基がペプチド結合により連結し、片端のシステイン残基が他端のシステイン残基とジスルフィド結合することによってループ状を形成してなるペプチドを、化学合成により作製した。これを、ペプチド１とした。すなわち、ペプチド１は、９つのアミノ酸残基（Ｃ−配列番号１−Ｃ；Ｃはシステイン残基）からなる。
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to this.
Test Example 1: Preparation of Spinal Tissue Targeting Peptide One cysteine residue is linked to each end of the amino acid sequence represented by SEQ ID NO: 1 (LHQSPHI) by a peptide bond, and one cysteine residue is the other cysteine A peptide formed by forming a loop by disulfide bond with a residue was prepared by chemical synthesis. This was designated as peptide 1. That is, peptide 1 consists of nine amino acid residues (C-SEQ ID NO: 1-C; C is a cysteine residue).
同様に、配列番号２〜５のそれぞれで表されるアミノ酸配列（それぞれＰＴＮＮＰＲＳ、ＬＮＳＳＱＰＳ、ＨＨＳＳＳＡＲ、ＮＴＧＳＰＹＥ）についても、各アミノ酸配列の両末端にそれぞれ１つのシステイン残基が連結し、片端のシステイン残基が他端のシステイン残基とジスルフィド結合することによってループ状を形成してなる４種のペプチドを、化学合成により作製した。これらを、それぞれペプチド２〜５とした。ペプチド２〜５も、それぞれ９つのアミノ酸残基（それぞれＣ−配列番号２−Ｃ、Ｃ−配列番号３−Ｃ、Ｃ−配列番号４−Ｃ、Ｃ−配列番号５−Ｃ；Ｃはシステイン残基）からなる。 Similarly, in the amino acid sequences represented by SEQ ID NOs: 2 to 5 (PTNPNPRS, LNSSQPS, HHSSSAR, NTGSSPYE, respectively), one cysteine residue is linked to both ends of each amino acid sequence, and the cysteine residue at one end is linked. Four types of peptides were formed by chemical synthesis in which a group formed a loop by disulfide bonding with a cysteine residue at the other end. These were designated as peptides 2 to 5, respectively. Peptides 2-5 also each have 9 amino acid residues (C-SEQ ID NO: 2-C, C-SEQ ID NO: 3-C, C-SEQ ID NO: 4-C, C-SEQ ID NO: 5-C, respectively; Group).
試験例２：脊髄組織標的化ペプチド（ペプチド１及び２）の脊髄組織への標的化評価
１．手順
Ｍ１３ファージのコートタンパク質をコードする遺伝子（ＰＩＩＩ）に前記ペプチド１をコードするＤＮＡ配列（ｔｇｔ−配列番号６−ｔｇｃ）を組み込むことにより、ペプチド１を特異的に発現可能なＭ１３ファージを作製した。次いで、このように作製したＭ１３ファージを１０１２個含有する５０ｍＭ Tris-Buffered Saline（ＴＢＳ）溶液（１００μｌ）を調製した。
Test Example 2: Evaluation of targeting of spinal cord tissue targeting peptides (peptides 1 and 2) to spinal cord tissue
1. Procedure M13 phage capable of specifically expressing peptide 1 was prepared by incorporating the DNA sequence encoding peptide 1 (tgt-SEQ ID NO: 6-tgc) into the gene (PIII) encoding the coat protein of M13 phage. . Next, a 50 mM Tris-Buffered Saline (TBS) solution (100 μl) containing 10 12 M13 phages thus prepared was prepared.
同様に、Ｍ１３ファージのコートタンパク質をコードする遺伝子（ＰＩＩＩ）に前記ペプチド２をコードするＤＮＡ配列（ｔｇｔ−配列番号７−ｔｇｃ）を組み込むことにより、ペプチド２を特異的に発現可能なＭ１３ファージを作製し、次いで、これを１０１２個含有するＴＢＳ溶液（１００μｌ）を作製した。また、同様に、ランダムにペプチドを発現するライブラリー由来のＤＮＡ配列（商品名Ph.D.-C7C Phage Display Peptide Library、ニューイングランドバイオラボ社製）を組み込んだＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）を調製した。 Similarly, by incorporating the DNA sequence encoding the peptide 2 (tgt-SEQ ID NO: 7-tgc) into the gene (PIII) encoding the coat protein of the M13 phage, an M13 phage capable of specifically expressing the peptide 2 is obtained. Then, a TBS solution (100 μl) containing 10 12 of them was prepared. Similarly, the random DNA sequences from libraries expressing peptide (trade name Ph.D.-C7C Phage Display Peptide Library, New England Biolabs, Inc.) TBS solution containing 10 twelve M13 phage incorporating (100 μl) was prepared.
調製した各溶液１００μｌをそれぞれ別々のＣ５７ＢＬ／６マウス（ジャクソンラボラトリー社製、８週齢以上のadultマウス）に尾静脈経由で投与した（ｎ＝３）。投与５分後にマウスを脱血し、ＰＢＳにて経心臓的に還流を行い、臓器を摘出し重量を測定した。次いで、プロテアーゼ阻害剤（商品名protease inhibitor cocktail、シグマ社製）を添加したダルベッコ改変イーグル培地（ＤＭＥＭ）で脊髄、脳、心臓をそれぞれホモゲナイズし、得られた溶液を大腸菌と混合してファージを大腸菌に感染させ、これをＬＢ寒天培地（０．７％アガロース）に混ぜ込み、あらかじめ固めておいたＬＢ寒天培地（１．５％アガロース）に重層し、３７℃で一晩静置培養してファージのプラーク形成を確認した。プラーク数を数えることにより各臓器に含まれていたファージのタイターを求め、臓器重量あたりに計算して比較した。結果を図２に示す。 100 μl of each prepared solution was administered to each of C57BL / 6 mice (Jackson Laboratory, adult mice over 8 weeks old) via the tail vein (n = 3). Five minutes after administration, the mice were bled, and refluxed transcardially with PBS, and the organs were removed and weighed. Next, the spinal cord, brain, and heart were each homogenized with Dulbecco's modified Eagle medium (DMEM) supplemented with protease inhibitors (trade name protease inhibitor cocktail, manufactured by Sigma). This is mixed with LB agar medium (0.7% agarose), layered on LB agar medium (1.5% agarose) that has been hardened in advance, and left to stand overnight at 37 ° C. Plaque formation was confirmed. By counting the number of plaques, the titer of the phages contained in each organ was determined and calculated per organ weight for comparison. The results are shown in FIG.
２．結果
図２から明らかなように、ペプチド１またはペプチド２を用いた場合において、脳や心臓と比較して、脊髄におけるファージ力価が高かった。このことから、ペプチド１またはペプチド２によれば脊髄を非常に高効率で認識できることが分かった。
2. Results As is apparent from FIG. 2, when peptide 1 or peptide 2 was used, the phage titer in the spinal cord was higher than that in the brain or heart. From this, it has been found that peptide 1 or peptide 2 can recognize the spinal cord with very high efficiency.
試験例３：脊髄組織標的化ペプチド（ペプチド１及び２）の脊髄組織への標的化評価
１．手順
前記試験例２で調製した、ペプチド１を特異的に発現可能なＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）を、Ｃ５７ＢＬ／６マウスに尾静脈経由で投与した（ｎ＝３）。投与５分後にマウスを脱血し、パラフォルムアルデヒドにて経心臓的に還流固定を行った。次いで脊髄を取り出して切片を作成し、抗ファージ抗体（商品名rabbit anti-fd Bacteriophage antibody、シグマ社製）及び二次抗体（商品名Anti-Alexa Fluor（登録商標）488 Rabbit IgG Antibody、 モレキュラプローブ社製）にて免疫染色を行った。
Test Example 3: Targeting evaluation of spinal cord tissue targeting peptides (peptides 1 and 2) to spinal cord tissue
1. Procedure A TBS solution (100 μl) prepared in Test Example 2 containing 10 12 M13 phages capable of specifically expressing peptide 1 was administered to C57BL / 6 mice via the tail vein (n = 3). Five minutes after the administration, the mice were bled and refluxed and fixed with paraformaldehyde transcardially. Next, the spinal cord was taken out to prepare a section, and an anti-phage antibody (trade name rabbit anti-fd Bacteriophage antibody, manufactured by Sigma) and a secondary antibody (trade name Anti-Alexa Fluor (registered trademark) 488 Rabbit IgG Antibody, molecular probe) Immunostaining was performed.
前記試験例２で調製した、ペプチド２を特異的に発現可能なＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）、ランダムにペプチドを発現するライブラリー由来のＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）についても同様にしてマウスに投与し、脱血、パラフォルムアルデヒドによる固定を行った後に脊髄の切片を作成し、抗ファージ抗体及び二次抗体にて免疫染色を行った。 TBS solution (100 μl) containing 10 12 M13 phages capable of specifically expressing peptide 2 prepared in Test Example 2 and TBS containing 10 12 M13 phages derived from a library that randomly expresses peptides The solution (100 μl) was similarly administered to mice, and after blood removal and fixation with paraformaldehyde, spinal cord sections were prepared and immunostained with anti-phage antibodies and secondary antibodies.
染色後、各切片を共焦点レーザー顕微鏡（商品名C1si、ニコン社製）にて観察した。結果を図３に示す。 After staining, each section was observed with a confocal laser microscope (trade name C1si, manufactured by Nikon Corporation). The results are shown in FIG.
２．結果
図中、下段の写真はそれぞれ上段の写真を拡大したものである。図３から明らかなように、ペプチド１またはペプチド２を用いた場合において、脊髄部分において抗ファージ抗体由来の蛍光が特異的に観察された。一方、ライブラリーを用いた場合には、このような特異的なシグナルは観察されなかった。このことから、ペプチド１またはペプチド２によれば脊髄を選択的に認識できることが分かった。
2. In the results , the lower photo is an enlarged version of the upper photo. As is clear from FIG. 3, when peptide 1 or peptide 2 was used, fluorescence derived from anti-phage antibody was specifically observed in the spinal cord portion. On the other hand, such a specific signal was not observed when the library was used. From this, it was found that peptide 1 or peptide 2 can selectively recognize the spinal cord.
試験例４：脊髄組織標的化ペプチド（ペプチド３）の脊髄組織への標的化評価
１．手順
前記試験例２と同様にして、ペプチド３をコードするＤＮＡ配列（ｔｇｔ−配列番号８−ｔｇｃ）を用いて調製したペプチド３を特異的に発現可能なＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）を、Ｃ５７ＢＬ／６マウスに尾静脈経由で投与した（ｎ＝3）。投与５分後にマウスを脱血し、パラフォルムアルデヒドにて経心臓的に還流固定を行った。次いで脊髄を取り出し切片を作成し、前述と同様に抗ファージ抗体及び二次抗体にて免疫染色を行った。また、前述と同様にして調製したランダムにペプチドを発現するライブラリー由来のＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）についても同様にしてマウスに投与し、脱血、パラフォルムアルデヒドによる固定を行った後に脊髄の切片を作成し、抗ファージ抗体及び二次抗体にて免疫染色を行った。染色後、各切片を共焦点レーザー顕微鏡にて観察した。結果を図４に示す。
Test Example 4: Targeting evaluation of spinal cord tissue targeting peptide (peptide 3) to spinal cord tissue
1. Procedure In the same manner as in Test Example 2, a TBS solution containing 10 12 M13 phages capable of specifically expressing peptide 3 prepared using a DNA sequence encoding peptide 3 (tgt-SEQ ID NO: 8-tgc) (100 μl) was administered to C57BL / 6 mice via the tail vein (n = 3). Five minutes after the administration, the mice were bled and refluxed and fixed with paraformaldehyde transcardially. Next, the spinal cord was taken out to prepare a section, and immunostaining was performed with an anti-phage antibody and a secondary antibody in the same manner as described above. Further, administered to mice in the same manner for the TBS solution (100 [mu] l) containing 10 12 of M13 phage from libraries expressing peptide randomly prepared in the same manner as described above, fixed by blood removal, paraformaldehyde After that, spinal cord sections were prepared and immunostained with anti-phage antibodies and secondary antibodies. After staining, each section was observed with a confocal laser microscope. The results are shown in FIG.
２．結果
図４から明らかなように、ペプチド３を用いた場合において、脊髄部分において抗ファージ抗体由来のシグナルが特異的に観察された。一方、ライブラリーを用いた場合には、このような特異的なシグナルは観察されなかった。このことから、ペプチド３によれば脊髄を選択的に認識できることが分かった。
2. Results As is clear from FIG. 4, when peptide 3 was used, a signal derived from an anti-phage antibody was specifically observed in the spinal cord portion. On the other hand, such a specific signal was not observed when the library was used. From this, it was found that peptide 3 can selectively recognize the spinal cord.
試験例５：脊髄組織標的化ペプチド（ペプチド３）の脊髄組織への標的化評価
１．手順
末端システイン残基の一方にアミノカプロン酸を介してＦＩＴＣ（fluorescein isothiocyanate）を連結させたペプチド３(１５μｇ) を含有する５％ブドウ糖液（１００μｌ）をＣ５７ＢＬ／６マウスに尾静脈より静脈注射した（ｎ＝３〜５）。投与５分後にマウスを脱血し、パラフォルムアルデヒドにて経心臓的に還流固定を行った。次いで脊髄を取り出し切片を作成し、アストロサイトマーカーである抗ＧＦＡＰ抗体（商品名rabbit anti-GFAP antibody、プロメガ社製）及び二次抗体（商品名Anti-Alexa Fluor（登録商標）555 Rabbit IgG Antibody、 モレキュラプローブ社製）にて免疫染色を行い、共焦点レーザー顕微鏡にて観察した。ＦＩＴＣ由来のシグナルは励起波長４８８ｎｍで観察し、抗ＧＦＡＰ抗体由来のシグナルは励起波長５６１ｎｍで観察した。結果を図５に示す。
Test Example 5: Targeting evaluation of spinal cord tissue targeting peptide (peptide 3) to spinal cord tissue
1. Procedure 5% glucose solution (100 μl) containing peptide 3 (15 μg) in which FITC (fluorescein isothiocyanate) was linked to one of the terminal cysteine residues via aminocaproic acid was intravenously injected into the C57BL / 6 mouse via the tail vein ( n = 3-5). Five minutes after the administration, the mice were bled and refluxed and fixed with paraformaldehyde transcardially. Next, the spinal cord was taken out to prepare a section, and an anti-GFAP antibody (trade name rabbit anti-GFAP antibody, manufactured by Promega) which is an astrocyte marker and a secondary antibody (trade name Anti-Alexa Fluor (registered trademark) 555 Rabbit IgG Antibody, Immunostaining was performed with a molecular probe) and observed with a confocal laser microscope. A signal derived from FITC was observed at an excitation wavelength of 488 nm, and a signal derived from an anti-GFAP antibody was observed at an excitation wavelength of 561 nm. The results are shown in FIG.
２．結果
図中、左の図はＦＩＴＣ由来のシグナルを示し、前記ペプチド３の存在を示す。真ん中の図は抗ＧＦＡＰ抗体由来のシグナルを示し、脊髄組織内のアストロサイトの存在を示す。右の図は、該左の図と真ん中の図を重ね合わせた図である。これらの図から明らかなように、前記ペプチド３は脊髄組織を認識し、特に脊髄組織内のアストロサイトを認識していることから、前記ペプチド３によれば脊髄を認識でき、なかでも脊髄組織内のアストロサイトに対する特異性が高いことが分かった。
2. In the results , the left figure shows a signal derived from FITC, indicating the presence of peptide 3. The middle figure shows the signal from the anti-GFAP antibody, indicating the presence of astrocytes in the spinal cord tissue. The figure on the right is a superposition of the figure on the left and the middle figure. As can be seen from these figures, since the peptide 3 recognizes spinal cord tissue, and in particular, recognizes astrocytes in the spinal cord tissue, the peptide 3 can recognize the spinal cord. Was found to be highly specific for astrocytes.
試験例６：脊髄組織標的化ペプチド（ペプチド４及び５）の脊髄組織への標的化評価
１．手順
前記試験例２と同様にして、ペプチド４をコードするＤＮＡ配列（ｔｇｔ−配列番号９−ｔｇｃ）を用いて調製した、ペプチド４を特異的に発現可能なＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）をマウスに尾静脈経由で投与した。投与５分後にマウスを脱血し、パラフォルムアルデヒドにて経心臓的に還流固定を行った。次いで脊髄を取り出し切片を作成し、前述と同様に抗ファージ抗体及び二次抗体にて免疫染色を行った。また、前記試験例２と同様にして、ペプチド５をコードするＤＮＡ配列（ｔｇｔ−配列番号１０−ｔｇｃ）を用いて調製した、ペプチド５を特異的に発現可能なＭ１３ファージを１０１２個含有するＴＢＳ溶液（１００μｌ）についても同様にしてマウスに投与し、脱血、パラフォルムアルデヒドによる固定を行った後に脊髄切片を作成し、抗ファージ抗体及び二次抗体にて免疫染色を行った。染色後、各切片を共焦点レーザー顕微鏡にて観察した。結果を図６に示す。
Test Example 6: Targeting evaluation of spinal cord tissue targeting peptides (peptides 4 and 5) to spinal cord tissue
1. Procedure In the same manner as in Test Example 2 were prepared using DNA sequence encoding a peptide 4 (TGT- SEQ ID NO: 9-tgc), containing 10 12 specifically expressible M13 phage peptide 4 TBS The solution (100 μl) was administered to mice via the tail vein. Five minutes after the administration, the mice were bled and refluxed and fixed with paraformaldehyde transcardially. Next, the spinal cord was taken out to prepare a section, and immunostaining was performed with an anti-phage antibody and a secondary antibody in the same manner as described above. Further, in the same manner as in Test Example 2, 10 12 M13 phages that can specifically express peptide 5 prepared using a DNA sequence encoding peptide 5 (tgt-SEQ ID NO: 10-tgc) are contained. The TBS solution (100 μl) was similarly administered to mice, and after blood removal and fixation with paraformaldehyde, spinal cord sections were prepared and immunostained with anti-phage antibodies and secondary antibodies. After staining, each section was observed with a confocal laser microscope. The results are shown in FIG.
２．結果
図６から明らかなように、ペプチド４や５を用いた場合においても、脊髄部分に抗ファージ抗体由来のシグナルが特異的に観察された。このことから、ペプチド４や５によれば脊髄を選択的に認識できることが分かった。また、ここには結果は示さないが、ペプチド４や５は、脊髄組織のなかでもミクログリアに対する特異性が高いことが分かった。
2. Results As is clear from FIG. 6, even when peptides 4 and 5 were used, a signal derived from an anti-phage antibody was specifically observed in the spinal cord portion. From this, it was found that the peptides 4 and 5 can selectively recognize the spinal cord. Moreover, although a result is not shown here, it turned out that the peptides 4 and 5 have the high specificity with respect to a microglia also in a spinal cord tissue.
試験例７
本試験は、遺伝的にhuman superoxide dismutase 1（SOD1）の変異型hSOD1(G93A)を発現することにより、運動機能の低下、致死性を示すマウス（hSOD1(G93A)tg筋萎縮性側索硬化症モデルマウス）を用いて行った。具体的には、hSOD1(G93A)tgマウスとして、hSOD1(G93A)1Gur/Jマウス（ジャクソンラボラトリー社製、８週齢のadultマウス）を用いて行った。
Test Example 7
In this study, mice with hSOD1 (G93A) tg amyotrophic lateral sclerosis were expressed by genetically expressing human superoxide dismutase 1 (SOD1) mutant hSOD1 (G93A), resulting in decreased motor function and lethality. Model mouse). Specifically, hSOD1 (G93A) 1 Gur / J mice (manufactured by Jackson Laboratory, 8-week-old adult mice) were used as hSOD1 (G93A) tg mice.
このマウスは、SOD1遺伝子（正常型も変異型も含む）の発現を抑制することにより、症状の進行や致死性が抑制されることが報告されており、siRNA-SOD1-396により、SOD1遺伝子の発現が抑制させることが報告されている。このことから、本試験では被送達物質としてsiRNA-SOD1-396を用い、また、前記ペプチドを用いて脊髄組織を標的化し、その治療効果を検討することにより、その有用性を検証した。なお、siRNA-SOD1-396 は配列番号１１の３’末端にチミン（ＴＴ）を備え、また、配列番号１２の５’ 末端にチミン（ＴＴ）を備え、これら２塩基ＴＴにおいてそれぞれオーバーハングを形成して対になっている。siRNA-SOD1-396のモデルを図７に示す。 It has been reported that this mouse suppresses the progression of symptoms and lethality by suppressing the expression of the SOD1 gene (including normal and mutant forms), and siRNA-SOD1-396 It has been reported that expression is suppressed. Therefore, in this study, siRNA-SOD1-396 was used as a substance to be delivered, and spinal cord tissue was targeted using the peptide, and its usefulness was examined by examining its therapeutic effect. In addition, siRNA-SOD1-396 has thymine (TT) at the 3 ′ end of SEQ ID NO: 11 and thymine (TT) at the 5 ′ end of SEQ ID NO: 12, and forms an overhang at each of these 2-base TTs. And it is paired. A model of siRNA-SOD1-396 is shown in FIG.
本試験例では具体的には、前記ペプチド１のＮ末端側のシステイン残基にアミノカプロン酸を介してＦＩＴＣを連結させ、また、前記ペプチド１のＣ末端側のシステイン残基に、３つのグリシン残基（Ｇ）からなるリンカーを介して、９つのアルギニン残基（Ｒ）からなる陽性に荷電したリンカーを連結させた。これに陰性荷電のsiRNA-SOD1-396を静電的結合作用により連結させて複合体を作製した。前記ペプチド２についても同様にしてsiRNA-SOD1-396との複合体を作製した。このようにして得られた複合体のモデルを図７に示す。図中、Ａ〜Ｇは配列番号１または２で表されるアミノ酸配列を意味する。 Specifically, in this test example, FITC was linked to the N-terminal cysteine residue of Peptide 1 via aminocaproic acid, and three glycine residues were attached to the C-terminal cysteine residue of Peptide 1. A positively charged linker consisting of nine arginine residues (R) was linked via a linker consisting of the group (G). A negatively charged siRNA-SOD1-396 was linked to this by an electrostatic binding action to prepare a complex. A complex with siRNA-SOD1-396 was also prepared in the same manner for peptide 2. A model of the complex thus obtained is shown in FIG. In the figure, AG means the amino acid sequence represented by SEQ ID NO: 1 or 2.
このように連結させた複合体について電気泳動を行ったところ、図８に示すように、ＦＩＴＣやリンカーを連結させた前記ペプチド１または２と、siRNA-SOD1-396との重量比を、例えば３：１とした複合体において電気泳動パターンの変化が認められ、すなわち前記ペプチド１や２にsiRNA-SOD1-396が十分に結合していることが確認された。 When electrophoresis was performed on the complex thus linked, as shown in FIG. 8, the weight ratio between the peptide 1 or 2 linked with FITC or a linker and siRNA-SOD1-396 was, for example, 3 It was confirmed that siRNA-SOD1-396 was sufficiently bound to the peptides 1 and 2 in the electrophoretic pattern change in the complex of 1: 1.
前述のようにしてＦＩＴＣやリンカーを連結させた前記ペプチド１(１５μｇ)とsiRNA-SOD1-396 (５μｇ)とを連結させた複合体（５％ブドウ糖液、１００μｌ）を、週に１または３回尾静脈よりhSOD1(G93A)tgマウスに投与した（ｎ＝３〜５）。該投与は、マウス週齢８週より、運動機能が廃絶するまで継続し、運動機能テストを毎週行い、評価した。運動機能テストは、ロータロッドテスト（5rpm/min-50rpm/min(acceleration 9rpm/min2)、Max 5min、(ウゴバジレ社製)）を用いて行い、マウス１匹あたり３分以上のインターバルで５回施行し、得られた中央値３つを平均することによって評価した。 A complex (5% glucose solution, 100 μl) in which the peptide 1 (15 μg) to which FITC or a linker has been linked as described above and siRNA-SOD1-396 (5 μg) is linked is once or three times a week. HSOD1 (G93A) tg mice were administered from the tail vein (n = 3-5). The administration was continued from 8 weeks of age until the motor function was abolished, and a motor function test was performed every week for evaluation. The motor function test is performed using a rotarod test (5 rpm / min-50 rpm / min (acceleration 9 rpm / min 2 ), Max 5 min, (manufactured by Ugo Basile)), 5 times at intervals of 3 minutes or more per mouse. Enforced and evaluated by averaging the three median values obtained.
前記ペプチド２を用いた複合体についても同様に投与し、評価した。また、コントロール群として、これらの複合体を投与していないhSOD1(G93A)tgマウスについても同様に評価した。 The complex using the peptide 2 was similarly administered and evaluated. In addition, as a control group, hSOD1 (G93A) tg mice not administered with these complexes were also evaluated in the same manner.
その結果、前記ペプチド１、前記ペプチド２のいずれの複合体を用いた場合においても、コントロール群と比較して、１〜２週の運動機能低下の進行遅延が認められた。 As a result, in the case of using either the peptide 1 or the peptide 2 complex, a delay in progression of motor function decline of 1 to 2 weeks was observed as compared with the control group.
また、前記運動機能評価の終了後、各マウスの脊髄組織より抽出したタンパク質を用いて、抗hSOD1抗体によるウエスタンブロットを行った。具体的には、各マウスより深麻酔下に脊髄を摘出した。摘出した脊髄組織を、プロテアーゼ阻害剤（商品名protease inhibitor cocktail、シグマ社製）を添加したRIPA buffer（150 mM NaCl, 2 mM EDTA, 1% nonidet P-40, 1% sodiumdeoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM NaF, 20 mM Tris-HCl buffer, pH 7.4）でホモゲナイズ後、溶液中のタンパク質濃度を測定し、タンパク質濃度をサンプル間で合わせて、sample buffer（×２)（2% SDS, 10% glycerol, 0.1% bromophenol blue, 10% b-mercaptoethanol, 0.5 m Tris-HCl, pH 6.8）を等量加えてボイル（１００℃、５分）した。その後、SDS-PAGEにて二次元展開し（アクリラマイドゲルにてサンプルを泳動）、PVDF膜に転写後、10% milkにてブロッキング後、抗ｈＳＯＤ１抗体（商品名rabbit anti-human SOD1 antibody、ミリポア社製）にて一晩インキュベーションし、その後、HRP標識の二次抗体（ECL Anti-Rabbit IgG, Horseradish Peroxidase linked whole antibody、GEヘルスケアUK社製）、更に使用説明書に従いECL regent （商品名Western Lightning Plus-ECL、パーキンエルマー社製）を用いて発光し、フィルムに感光した。 After the evaluation of the motor function, Western blotting with an anti-hSOD1 antibody was performed using a protein extracted from the spinal cord tissue of each mouse. Specifically, the spinal cord was removed from each mouse under deep anesthesia. RIPA buffer (150 mM NaCl, 2 mM EDTA, 1% nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) to which the extracted spinal cord tissue was added with a protease inhibitor (trade name protease inhibitor cocktail, manufactured by Sigma) (SDS), 50 mM NaF, 20 mM Tris-HCl buffer, pH 7.4), homogenize, measure protein concentration in solution, match protein concentration between samples, sample buffer (× 2) (2% SDS , 10% glycerol, 0.1% bromophenol blue, 10% b-mercaptoethanol, 0.5 m Tris-HCl, pH 6.8), and boiled (100 ° C., 5 minutes). After that, SDS-PAGE is used for two-dimensional development (samples are run on an acrylamide gel), transferred to a PVDF membrane, blocked with 10% milk, and anti-hSOD1 antibody (trade name rabbit anti-human SOD1 antibody, Millipore) And then HRP-labeled secondary antibody (ECL Anti-Rabbit IgG, Horseradish Peroxidase linked whole antibody, manufactured by GE Healthcare UK), followed by ECL regent (trade name Western) Lightning Plus-ECL (manufactured by Perkin Elmer) was used to emit light, and the film was exposed to light.
その結果、図９に示されるように、ペプチド１及び２のいずれにおいても前記複合体を投与したマウスにおいて変異型ｈＳＯＤ１タンパク質の発現が有意に抑制されていることが認められた。 As a result, as shown in FIG. 9, it was confirmed that the expression of the mutant hSOD1 protein was significantly suppressed in both the peptides 1 and 2 in the mice administered with the complex.
また、前記運動機能評価の終了後、各マウスより脊髄を摘出し、脊髄切片を作成した。次いで、抗ｈＳＯＤ１抗体及び二次抗体（商品名Anti-Alexa Fluor（登録商標）555 Rabbit IgG Antibody、モレキュラプローブ社製）、ＤＡＰＩ（4’,6-diamidino-2-phenylindole）にて染色後、Vectashield Mounting Medium with DAPI （ベクターラボラトリーズ社製）を用いて使用説明書に従いマウントし、共焦点レーザー顕微鏡にて観察した。ＦＩＴＣ由来のシグナルは励起波長４８８ｎｍで観察し、抗ｈＳＯＤ１抗体由来のシグナルは励起波長５６１ｎｍで観察し、ＤＡＰＩ由来のシグナルは励起波長４０８ｎｍで観察した。結果を図１０に示す。なお、コントロールとして、前記複合体を与えていないマウスから同様にして作成、染色した切片についても観察した。 In addition, after completion of the motor function evaluation, the spinal cord was removed from each mouse and a spinal cord section was prepared. Subsequently, after staining with an anti-hSOD1 antibody and a secondary antibody (trade name Anti-Alexa Fluor (registered trademark) 555 Rabbit IgG Antibody, manufactured by Molecular Probes), DAPI (4 ′, 6-diamidino-2-phenylindole), The sample was mounted using Vectashield Mounting Medium with DAPI (manufactured by Vector Laboratories) according to the instruction manual and observed with a confocal laser microscope. A signal derived from FITC was observed at an excitation wavelength of 488 nm, a signal derived from an anti-hSOD1 antibody was observed at an excitation wavelength of 561 nm, and a signal derived from DAPI was observed at an excitation wavelength of 408 nm. The results are shown in FIG. As a control, sections prepared and stained in the same manner from mice not given the complex were also observed.
図１０において、上段はコントロール群の結果を、中段はペプチド１を用いた複合体を投与した結果を、下段はペプチド２を用いた複合体を投与した結果を示す。また、図１０において、左の図はＦＩＴＣ由来のシグナルを、真ん中の図は抗ｈＳＯＤ１抗体由来のシグナルを、右の図はＦＩＴＣ、抗ｈＳＯＤ１抗体、ＤＡＰＩ由来のそれぞれのシグナルを重ね合わせた図である。 In FIG. 10, the upper part shows the result of the control group, the middle part shows the result of administering the complex using peptide 1, and the lower part shows the result of administering the complex using peptide 2. In addition, in FIG. 10, the left figure is the FITC-derived signal, the middle figure is the signal derived from the anti-hSOD1 antibody, and the right figure is the figure in which the respective signals derived from FITC, anti-hSOD1 antibody, and DAPI are superimposed. is there.
図１０から明らかなように、コントロール群の切片では抗ｈＳＯＤ１抗体由来のシグナルが明らかに認められ、すなわち、脊髄において変異型hSOD1(G93A)の発現が確認された。これに対して、ペプチド１を用いた複合体やペプチド２を用いた複合体を投与したマウスから作成した切片では、ＦＩＴＣ由来のシグナルやＤＡＰＩ由来のシグナルが認められるものの、抗ｈＳＯＤ１抗体由来のシグナルがほとんど認められなかった。この結果は、前記複合体によって、変異型hSOD1(G93A)の発現が効果的に抑制されていることを示す。 As is clear from FIG. 10, a signal derived from the anti-hSOD1 antibody was clearly observed in the control group section, that is, expression of mutant hSOD1 (G93A) was confirmed in the spinal cord. In contrast, FITC-derived signals and DAPI-derived signals are observed in sections prepared from mice administered with a complex using peptide 1 or a complex using peptide 2, but signals derived from anti-hSOD1 antibodies. Was hardly recognized. This result indicates that the expression of mutant hSOD1 (G93A) is effectively suppressed by the complex.
これらのことから、前記ペプチド１などの脊髄標的化ペプチドによれば脊髄組織を標的化でき、また、siRNAといった被送達物質を脊髄組織に効率よく送達できるとともに、該組織において被送達物質に由来する有用作用を効果的に発揮できることが分かった。このことから、前記脊髄標的化ペプチドは、医薬組成物としても有用であることが確認された。 Based on these facts, spinal cord targeting peptides such as peptide 1 can target spinal cord tissue, and can deliver a delivered substance such as siRNA efficiently to the spinal cord tissue, and is derived from the delivered substance in the tissue. It turned out that a useful effect can be exhibited effectively. From this, it was confirmed that the spinal cord targeting peptide is also useful as a pharmaceutical composition.
Claims (8)
（１−１）配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列の両末端にそれぞれ少なくとも１つのシステイン残基が直接連結してなるペプチド、
（１−２）前記（１−１）のペプチドにおいて、配列番号１〜５からなる群より選択される少なくとも１種で表されるアミノ酸配列が、１または複数のアミノ酸が欠失、置換及び／または付加されたアミノ酸配列からなり、且つ、脊髄組織を標的とするペプチド。 A spinal cord tissue-targeting peptide represented by the following (1-1) or (1-2);
(1-1) a peptide in which at least one cysteine residue is directly linked to both ends of an amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5,
(1-2) In the peptide of (1-1), the amino acid sequence represented by at least one selected from the group consisting of SEQ ID NOs: 1 to 5 has one or more amino acids deleted, substituted, and / or Alternatively, a peptide consisting of an added amino acid sequence and targeting spinal cord tissue.
（２−１）前記（１−１）及び（１−２）のいずれかに記載のペプチドをコードするポリヌクレオチド、
（２−２）前記（２−１）のポリヌクレオチドの相補鎖に対して、ストリンジェントな条件下でハイブリダイズし、且つ、脊髄組織標的化ペプチドをコードするポリヌクレオチド。 The polynucleotide represented by the following (2-1) or (2-2):
(2-1) a polynucleotide encoding the peptide according to any one of (1-1) and (1-2),
(2-2) A polynucleotide that hybridizes to a complementary strand of the polynucleotide of (2-1) under stringent conditions and encodes a spinal cord tissue-targeting peptide.
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| KR102259726B1 (en) | 2016-11-28 | 2021-06-02 | 재단법인 아산사회복지재단 | CCSP-2 Specific Peptide and Use thereof |
| CN106946983A (en) * | 2017-04-01 | 2017-07-14 | 贵州医科大学 | Selectively targeted polypeptide of liver cancer microballoon cell surface and its preparation method and application |
| CN106946983B (en) * | 2017-04-01 | 2021-02-12 | 贵州医科大学 | Liver cancer microsphere cell surface specificity targeting polypeptide and preparation method and application thereof |
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