JP2019099578A - Ophthalmic pharmaceutical composition containing cell membrane-permeable peptide - Google Patents
Ophthalmic pharmaceutical composition containing cell membrane-permeable peptide Download PDFInfo
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- JP2019099578A JP2019099578A JP2018227946A JP2018227946A JP2019099578A JP 2019099578 A JP2019099578 A JP 2019099578A JP 2018227946 A JP2018227946 A JP 2018227946A JP 2018227946 A JP2018227946 A JP 2018227946A JP 2019099578 A JP2019099578 A JP 2019099578A
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- Prior art keywords
- drug
- solution
- cell membrane
- permeable peptide
- salt
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- 239000003814 drug Substances 0.000 claims abstract description 95
- 150000003839 salts Chemical class 0.000 claims abstract description 65
- 210000000170 cell membrane Anatomy 0.000 claims description 63
- 239000000203 mixture Substances 0.000 claims description 57
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Abstract
Description
本発明は、細胞膜透過性ペプチド（以下、「ＣＰＰ」ともいう。）又はその塩と薬物を含有する眼科用医薬組成物に関する。 The present invention relates to an ophthalmic pharmaceutical composition containing a cell membrane permeable peptide (hereinafter also referred to as "CPP") or a salt thereof and a drug.
眼科用の薬物を投与する方法として、点眼剤による点眼投与が汎用されている。しかし、点眼投与された薬物が角膜に移行する割合は決して高くなく、さらに内部にある前房、虹彩、毛様体、水晶体、硝子体、網膜等に到達する割合はわずかである。このような点眼剤の低いバイオアベイラビリティを改善する目的で、リポソーム、エマルジョン、マイクロスフェア、デポ製剤、徐放デバイス等、種々のドラッグデリバリーシステムが開発されている。アルコール類や界面活性剤等は角膜移行性を向上させる目的で使用されるが、毒性の問題等が懸念されるために、高濃度での使用には適さない。 Ophthalmic administration by means of eye drops is widely used as a method for administering ophthalmic drugs. However, the rate at which the drop-administered drug migrates to the cornea is never high, and the rate at which the drug reaches the internal anterior chamber, iris, ciliary body, lens, vitreous body, retina, etc. is small. In order to improve the low bioavailability of such eye drops, various drug delivery systems such as liposomes, emulsions, microspheres, depot preparations, sustained release devices and the like have been developed. Alcohols, surfactants and the like are used for the purpose of improving corneal migration, but are not suitable for use at high concentrations because of the concern of toxicity and the like.
また、細胞膜透過性ペプチドを利用した眼科用ドラッグデリバリーシステムも報告されている。例えば、非特許文献１には、ＰＯＤ−ＧＦＰ ｆｕｓｉｏｎ ｐｒｏｔｅｉｎの点眼投与により、角膜上皮への取り込みが改善することが報告されている。また、非特許文献２には、ＴＡＴ−ｃｏｎｊｕｇａｔｅｄ ａＦＧＦ−Ｈｉｓの点眼投与により、網膜への移行が向上することが報告されている。しかし、これらは薬物と細胞膜透過性ペプチドが直接又はリンカー等を介して共有結合した複合体を利用する。 In addition, ophthalmic drug delivery systems using cell membrane permeable peptides have also been reported. For example, Non-Patent Document 1 reports that eye drop administration of POD-GFP fusion protein improves uptake into the corneal epithelium. In addition, Non-Patent Document 2 reports that the transition to the retina is improved by eye drop administration of TAT-conjugated aFGF-His. However, these utilize a complex in which a drug and a cell membrane permeable peptide are covalently linked directly or via a linker or the like.
本発明の課題は、薬物のターゲット部位への移行性を改善できる新たな眼科用医薬組成物を提供することである。 An object of the present invention is to provide a new ophthalmic pharmaceutical composition capable of improving the transferability of a drug to a target site.
本発明者らは、鋭意研究の結果、細胞膜透過性ペプチドは薬物と共有結合させなくとも、薬物と共存させて点眼投与することにより、薬物の角膜上皮細胞への移行性を改善させることができ、しかも、角膜への顕著な毒性を生じることなく安全に利用できることを見出して、本発明を完成させた。具体的に、本発明は以下を提供する。 The present inventors, as a result of earnest studies, can improve the transferability of a drug to corneal epithelial cells by coadministration with a drug, even if the cell membrane permeable peptide is not covalently linked to the drug. Furthermore, the present invention has been completed by finding that it can be safely used without causing significant toxicity to the cornea. Specifically, the present invention provides the following.
（１）薬物と、前記薬物と共有結合せずに存在する細胞膜透過性ペプチド又はその塩とを含有する眼科用医薬組成物。
（２）細胞膜透過性ペプチドがＲ８、Ｒ８−ＮＨ２、Ｐｅｎｅｔｒａｔｉｎ ＲＱＩＫＩＷＦＱＮＲＲＭＫＷＫＫ、ＨＩＶ−ＴＡＴ （４７−５７） ＹＧＲＫＫＲＲＱＲＲＲ、ＨＩＶ−ＴＡＴ （４８−６０） ＧＲＫＫＲＲＱＲＲＲＰＰＱ、ｐＶＥＣ ＬＬＩＩＬＲＲＲＩＲＫＱＡＨＡＨＳＫ−ＮＨ２、 Ｔｒａｎｓｐｏｒｔａｎ （ＴＰ１０） ＡＧＹＬＬＧＫＩＮＬＫＡＬＡＡＬＡＫＫＩＬ−ＮＨ２、Ｐｒｏｔｅｉｎ ｔｒａｎｓｄｕｃｔｉｏｎ ｄｏｍａｉｎｓ ｓｅｑｕｅｎｃｅ ＡＲＫＫＡＡＫＡ、２８９−Ｗ ＲＷＩＫＩＷＦＷＷＲＲＭＫＷＫＫ、Ｌ１７Ｅ ＩＷＬＴＡＬＫＦＬＧＫＨＡＡＫＨＥＡＫＱＱＬＳＫＬ、及びＬ−Ｋ６ ＩＫＩＬＳＫＩＫＫＬＬＫからなる群より選択される一又は複数の細胞膜透過性ペプチドである、（１）に記載の組成物。
（３）細胞膜透過性ペプチド又はその塩を０．００００１〜１０％（ｗ／ｖ）含有する、（１）又は（２）に記載の組成物。
（４）細胞透過性ペプチド又はその塩と、薬物とのモル比が、１０００：１〜１：１０００である、（１）〜（３）のいずれかに記載の組成物。
（５）細胞膜透過性ペプチド又はその塩を含有し、送達対象の薬物と共有結合させずに用いられる眼科用ドラッグデリバリーシステム。
（６）眼科用医薬組成物に細胞膜透過性ペプチド又はその塩を薬物と共有結合させずに含有させることによる、前記薬物の眼組織への移行性を向上させる方法。
(1) An ophthalmic pharmaceutical composition containing a drug and a cell membrane-permeable peptide or a salt thereof present without being covalently linked to the drug.
(2) a cell membrane permeable peptide is R8, R8-NH 2, Penetratin RQIKIWFQNRRMKWKK, HIV-TAT (47-57) YGRKKRRQRRR, HIV-TAT (48-60) GRKKRRQRRRPPQ, pVEC LLIILRRRIRKQAHAHSK-NH 2, Transportan (TP10) AGYLLGKINLKALAALAKKIL- It is described in (1), which is one or more cell membrane permeable peptides selected from the group consisting of NH 2 , Protein transduction domains sequence ARKKAAKA, 289-W RWI KIWF WWRRM KWKK, L17E IWLTALKLFLGKHAAKHEAKQQLSKL, and L-K6 IKILSKIKKLLK Composition of
(3) The composition according to (1) or (2), containing 0.00001 to 10% (w / v) of a cell membrane permeable peptide or a salt thereof.
(4) The composition according to any one of (1) to (3), wherein the molar ratio of the cell permeable peptide or the salt thereof to the drug is 1000: 1 to 1: 1000.
(5) An ophthalmic drug delivery system containing a cell membrane permeable peptide or a salt thereof and used without being covalently linked to a drug to be delivered.
(6) A method for improving the transferability of the drug to ocular tissues by containing a cell membrane permeable peptide or a salt thereof in a pharmaceutical composition for ophthalmic use without covalently bonding the drug to the drug.
なお、前記（１）から（６）の各構成は、任意に２以上を選択して組み合わせることができる。 In addition, each structure of said (1) to (6) can select 2 or more arbitrarily, and can combine them.
本発明によれば、細胞膜透過性ペプチドを薬物と共存させることにより、眼組織中への薬物の移行性を改善した眼科用医薬組成物を提供することができる。細胞膜透過性ペプチドと薬物とを共有結合させた複合体を形成させる必要がないため、細胞膜透過性ペプチドと薬物を別個に配合するだけで簡便に調製することができる。また、アルコール類や界面活性剤等ではなく細胞膜透過性ペプチドを利用したものであることから、医薬品として安全に眼に投与することができる。 According to the present invention, it is possible to provide an ophthalmic pharmaceutical composition in which the migration of the drug into the ocular tissue is improved by making the cell membrane permeable peptide coexist with the drug. Since it is not necessary to form a complex in which the cell membrane permeable peptide and the drug are covalently bound, it can be conveniently prepared only by separately blending the cell membrane permeable peptide and the drug. In addition, since the cell membrane-permeable peptide is used instead of alcohols, surfactants and the like, it can be safely administered to the eye as a pharmaceutical.
以下に、本発明の実施形態を詳細に説明するが、本発明はこれに特に限定されない。 Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not particularly limited thereto.
本発明の医薬組成物は、薬物と、前記薬物と共有結合せずに存在する細胞膜透過性ペプチドとを含有する眼科用医薬組成物である。なお、細胞膜透過性ペプチドは、薬物と非共有結合的相互作用（金属イオンを用いた架橋以外の静電的相互作用、水素結合、疎水結合、π−スタッキング相互作用、ファンデルワールス引力による相互作用など）を介した結合をしてもよいし、していなくてもよい。 The pharmaceutical composition of the present invention is an ophthalmic pharmaceutical composition containing a drug and a cell membrane-permeable peptide which is present without being covalently linked to the drug. In addition, the cell membrane permeable peptide has noncovalent interaction with drug (electrostatic interaction other than crosslinking with metal ion, hydrogen bond, hydrophobic bond, π-stacking interaction, interaction by van der Waals attraction) Etc.) may or may not be linked.
本発明の組成物に含有される細胞膜透過性ペプチドは、細胞膜を透過する性質を有するペプチドであり、具体的には、所望の薬物と共存した状態で投与した際、その薬物単独での投与時に比べ、薬物の透過性を向上（例えば、透過速度の増加）できる任意のペプチドを指す。従って、細胞膜透過性ペプチドは、眼組織への移行を促進すべき薬物の種類に応じて適宜選択されてよい。 The cell membrane-permeabilizing peptide contained in the composition of the present invention is a peptide having a property of permeating the cell membrane, and specifically, when administered in the state of coexistence with a desired drug, the drug alone is administered In comparison, it refers to any peptide that can improve drug permeability (eg, increase permeation rate). Therefore, the cell membrane permeable peptide may be appropriately selected according to the type of drug to be promoted to ocular tissue.
本発明の組成物に含有される細胞膜透過性ペプチドのアミノ酸残基の数は、特に制限されないが、６〜５０が好ましく、６〜４０がより好ましく、６〜３０であることが特に好ましい。ペプチドは、アルギニン残基に富むことが好ましく、具体的にはペプチド中に、３以上、より好ましくは４以上、特に好ましくは５以上のアルギニン残基を含んでよい。６〜５０のアミノ酸残基を含み、そのうち３以上の残基がアルギニン残基であるペプチドが好ましく、６〜４０のアミノ酸残基を含み、そのうち４以上の残基がアルギニン残基であるペプチドがより好ましく、６〜３０のアミノ酸残基を含み、そのうち５以上の残基がアルギニン残基であるペプチドが特に好ましい。アルギニンは、各々独立してＬ体でもＤ体でもよい。また、細胞膜透過性ペプチドには、Ｃ末端及び／又はＮ末端及び／又は側鎖が修飾されたペプチドも含まれる。カルボキシル基の修飾の具体例としては、アミド化（ＣＯＮＨ２）、メチルアミド化等のアミド化；メチルエステル化、エチルエステル化等のエステル化等が挙げられ、アミノ基の修飾の具体例としては、アセチル化、ホルミル化等のアミド化；メチル化、ジメチル化等のアルキル化等が挙げられ、水酸基の修飾の具体例としては、アセチル化等のエステル化；リン酸化等が挙げられ、チオール基の修飾の具体例としては、メチル化等のアルキル化等が挙げられる。細胞膜透過性ペプチドは、具体的には、Ｒ８（アミノ酸配列；ＲＲＲＲＲＲＲＲ）、配列番号１）、Ｒ８−ＮＨ２（アミノ酸配列；ＲＲＲＲＲＲＲＲ−ＮＨ２）、配列番号２）、ＨＩＶ−ＴＡＴ （４８−６０）（アミノ酸配列；ＧＲＫＫＲＲＱＲＲＲＰＰＱ、配列番号３）、Ｐｅｎｅｔｒａｔｉｎ ＲＱＩＫＩＷＦＱＮＲＲＭＫＷＫＫ（配列番号４）， ＨＩＶ−ＴＡＴ （４７−５７） ＹＧＲＫＫＲＲＱＲＲＲ（配列番号５）、ｐＶＥＣ ＬＬＩＩＬＲＲＲＩＲＫＱＡＨＡＨＳＫ（配列番号６）−ＮＨ２、Ｔｒａｎｓｐｏｒｔａｎ （ＴＰ１０） ＡＧＹＬＬＧＫＩＮＬＫＡＬＡＡＬＡＫＫＩＬ（配列番号７）−ＮＨ２，Ｐｒｏｔｅｉｎ ｔｒａｎｓｄｕｃｔｉｏｎ ｄｏｍａｉｎｓ ｓｅｑｕｅｎｃｅ ＡＲＫＫＡＡＫＡ（配列番号８）、ＨＩＶ−１ Ｒｅｖ （３４−５０） ＫＱＡＩＰＶＡＫ（配列番号９）−ａｍｉｄｅ， ＦＨＶ ｃｏａｔ（３５−４９） ＲＲＲＲＮＲＴＲＲＮＲＲＲＶＲ（配列番号１０）−ａｍｉｄｅ、 Ｏｌｉｇｏａｒｇｉｎｉｎｅｓ （Ｒ９−Ｒ１２） ＲＲＲＲＲＲＲＲＲ（配列番号１１）／ ＲＲＲＲＲＲＲＲＲＲＲＲ（配列番号１２）， ＣＣＭＶ Ｇａｇ （７−２５） ＫＬＴＲＡＱＲＲＡＡＡＲＫＮＫＲＮＴＲＧＣ（配列番号１３）， Ｃｈｉｍｅｒｉｃ ｄｅｒｍａｓｅｐｔｉｎ Ｓ４ ａｎｄ ＳＶ４０ ‘Ｓ４１３−ＰＶ’ ＡＬＷＫＴＬＬＫＫＶＬＫＡＰＫＫＫＲＫＶＣ（配列番号１４）， Ｈｅｒｐｅｓ ｓｉｍｐｌｅｘ ｖｉｒｕｓ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ （２６７−３００） ＶＰ２２ ＤＡＡＴＡＴＲＧＲＳＡＡＳＲＰＴＥＲＰＲＡＰＡＲＳＡＳＲＰＲＲＰＶＥ（配列番号１５）， （Ｃｈｉｍｅｒｉｃ ｇａｌａｎｉｎ／ｍａｓｔｏｐａｒａｎ） Ｔｒａｎｓｐｏｒｔａｎ ＧＷＴＬＮＳＡＧＹＬＬＧＫＩＮＬＫＡＬＡＡＬＡＫＫＩＬ（配列番号１６）−ａｍｉｄｅ， ＣＰＰ ｗｉｔｈ ｐｒｏｔｅａｓｅ ｃｌｅａｖａｇｅ ｓｉｄｅ ＹＴＡ２ ＹＴＡＩＡＷＶＫＡＦＩＲＫＬＲＫ（配列番号１７）−ａｍｉｄｅ， Ｐｅｐ−１ Ａｃ−ＫＥＴＷＷＥＴＷＷＴＥＷＳＱＰＫＫＫＲＫＶ（配列番号１８）−ＮＨ−ＣＨ２−ＣＨ２−ＳＨ， ＭＰＧα Ａｃ−ＧＡＬＦＬＡＦＬＡＡＡＬＳＬＭＧＬＷＳＱＰＫＫＫＲＫＶ（配列番号１９）−ＮＨ−ＣＨ２−ＣＨ２−ＳＨ， ＭＰＧβ Ａｃ−ＧＡＬＦＬＧＦＬＧＡＡＧＳＴＭＧＡＷＳＱＰＫＫＫＲＫＶ（配列番号２０）−ＮＨ−ＣＨ２−ＣＨ２−ＳＨ， ＭＰＧ８β ＡＦＬＧＷＬＧＡＷＧＴＭＧＷＳＰＫＫＲＫ（配列番号２１）−ＮＨ−ＣＨ２−ＣＨ２−ＳＨ， ＣＡＤＹ Ａｃ−ＧＬＷＲＡＬＷＲＬＬＲＳＬＷＲＬＬＷＫＡ（配列番号２２）−ＮＨ−ＣＨ２−ＣＨ２−ＳＨ， Ｐｅｐｆｅｃｔ６ Ｓｔｅａｒｙｌ−ＡＧＹＬＬＧＫ（ε−ＴＭＱ）ＩＮＬＫＡＬＡＡＬＡＫＫＩＬ（配列番号２３）， ＰｅｐＦｅｃｔ１４ Ｓｔｅａｒｙｌ−ＡＧＹＬＬＧＫＬＬＯＯＬＡＡＡＡＬＯＯＬＬ（配列番号２４）−ａｍｉｄｅ， ＰｅｐＦｅｃｔ １５ Ｓｔｅａｒｙｌ−ＡＧＹＬＬＧＫ（Ｋ３ＱＮ４）ＬＬＯＯＬＡＡＡＡＬＯＯＬＬ（配列番号２５）−ａｍｉｄｅ， ＮｉｃｋＦｅｃｔ ＮＦ６１： Ｓｔｅａｒｙｌ−ＡＧＹＬＬＧＯＩＮＬＫＡＬＡＡＬＡＫＫＩＬ（配列番号２６）−ａｍｉｄｅ， Ｈｅｌ １１−２７ ＫＬＬＫＬＬＬＫＬＷＫＬＬＬＫＬＬＫ（配列番号２７）， ＩｎｆｌｕｅｎｃａＨＡ−２ （１−２０） ＫＡＬＡ ｓｅｑｕｅｎｃｅ ＷＥＡＫＬＡＫＡＬＡＫＡＬＡＨＬＡＫＡＬＡＫＡＬＫＡＣＥＡ（配列番号２８）， ＫＬＡ ｓｅｑｕｅｎｃｅ Ａｃｅｔｙｌ−ＫＬＡＬＫＬＡＬＫＡＬＫＡＡＬＫＬＡ（配列番号２９）−ａｍｉｄｅ， ｓＣ１８ ＧＬＲＫＲＬＲＫＦＲＮＫＩＫＥＫ（配列番号３０）， （Ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｃａｄｈｅｒｉｎ） ｐＶＥＣ ＬＬＩＩＬＲＲＲＩＲＫＱＡＨＡＨＳＫ（配列番号３１）−ａｍｉｄｅ， Ｋａｐｏｓｉｓ ｓａｒｃｏｍａ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ Ｋａｐｏｓｉ ＦＧＦ ＡＡＶＡＬＬＰＡＶＬＬＡＬＬＡＰ（配列番号３２）， Ｓｉｇｎａｌ ｓｅｑｕｅｎｃｅ ｏｆ Ｉｇ ｌｉｇｈｔ ｃｈａｉｎ ｆｒｏｍ Ｃａｉｉｍａｎ ｃｒｏｃｏｄｙｌｕｓ ＭＧＬＧＬＨＬＬＶＬＡＡＡＬＱＧＡＭＧＬＧＬＨＬＬＬＡＡＡＬＱＧＡ（配列番号３３）， Ｉｎｔｅｇｒｉｎ β３−ｆｒａｇｍｅｎｔ ＶＴＶＬＡＧＡＬＡＧＶＧＶＧ（配列番号３４）， Ｇｒｂ２ （ＳＨ２ ｄｏｍａｉｎ） ＡＡＶＬＬＰＶＬＬＡＡＰ（配列番号３５）， Ｆｕｓｉｏｎ ｓｅｑｕｅｎｃｅ ＨＩＶ−１ ｇｐ４１（１−２３） ＧＡＬＦＬＧＦＬＧＡＡＧＳＴＭＧＡ（配列番号３６）， Ｈｅｐａｔｉｔｉｓ Ｂ ｖｉｒｕｓ ｔｒａｎｓｌｏｃａｔｉｏｎ ｍｏｔｉｆ ＰＬＳＳＩＦＳＲＩＧＤＰ（配列番号３７）， Ｓｐｅｒｍ−ｅｇｇ ｆｕｓｉｏｎ ｐｒｏｔｅｉｎ （８９−１１１） ＳＦＰ２３： Ａｃ−ＫＬＩＡＴＧＩＳＳＩＰＰＩＲＡＬＦＡＡＩＱＩＰ（配列番号３８）−ａｍｉｄｅ， Ｈｕｍａｎ ｃａｌｃｉｔｏｎｉｎ ｐａｒｔｉａｌ ｓｅｑｕｅｎｃｅ ９−３２， ｈＣＴ（９−３２）−ｂｒ， ＬＧＴＹＴＱＤＦＮＫ（Ｘ）ＦＨＴＦＰＱＴＡＩＧＶＧＡＰ（配列番号３９）−ａｍｉｄｅ Ｘ： ＰＫＫＫＲＫＶＥＤＰＧＶＧＦＡ（配列番号４０）， ｈＣＴ（１８−３２）−ｂｒ， Ｃａｐｒ−Ｋ（Ｘ）ＦＨＴＦＰＱＴＡＩＧＶＧＡＰ（配列番号４１）−ａｍｉｄｅ Ｘ：ＫＫＲＫＡＰＫＫＫＲＫＦＡ（配列番号４２）， Ｓｕｂｓｔａｎｃｅ Ｐ ａｎｄ ａｎａｌｏｇｓ ＲＰＫＰＱＱＦＧＬＭ（配列番号４３）−ａｍｉｄｅ， ＲＧＤ ｐｅｐｔｉｄｅｓ αｖβ３ ＲＧＤ−Ｔｅｍｐｏｒｉｎ−ＬＡ， ＲＧＤ−Ｄｙｅ ＲＧＤ−ｂｅａｒｉｎｇ ＰＡＭＡＭ ｄｅｎｄｒｉｍｅｒｓ， ＲＧＤ ｐｅｐｔｉｄｅｓ αｖβ３， αｖβ５ Ｃｉｌｅｎｇｉｔｉｄｅ： Ｃｙｃｌｏ［ＲＧＤｆＮｍｅＶ］， ＲＧＤ ｐｅｐｔｉｄｅ Ｃｙｃｌｏ（ＲＧＤｆＫ）； ｃｙｃｌｏ（ＲＧＤｙＫ）， ＲＧＤ ｐｅｐｔｉｄｅｓ α５β１ ＲＧＤ ｍｉｍｉｃ， ＡＧＲ （ｐｒｏｓｔａｔａ ｃａｒｃｉｎｏｍａ） ＣＡＧＲＲＳＡＹＣ（配列番号４４）， ＬｙＰ−２ （ｓｋｉｎ ａｎｄ ｃｅｒｖｉｘ ｔｕｍｏｒ） ＣＮＲＲＴＫＡＧＣ（配列番号４５）， ＲＥＡ （ｐｒｏｓｔａｔａ， ｃｅｒｖｉｘ， ｂｒｅａｓｔ ｃａｒｃｉｎｏｍａ） ＣＲＥＡＧＲＫＡＣ（配列番号４６）， ＬＳＤ （ｍｅｌａｎｏｍａ， ｏｓｔｅｏｓａｒｃｏｍａ） ＣＬＳＤＧＫＲＫＣ（配列番号４７）， Ｍａｓｔｏｐａｒａｎ ＩＮＬＫＡＬＡＡＬＡＫＫＩＬ（配列番号４８）−ａｍｉｄｅ， Ｍｅｌｌｉｔｉｎ ＧＩＧＡＶＬＫＶＬＴＴＧＬＰＡＬＩＳＷＩＫＲＫＲＱＱ（配列番号４９）−ａｍｉｄｅ， Ｓｃｏｒｐｉｏｎ ｔｏｘｉｎ ＭＡＵＲＯＣＡＬＣＩＮＥ ｗｉｔｈ ｒｅｐｌａｃｅｍｅｎｔ ｏｆ Ｃ ｂｙ Ａｂｕ ＧＤＡｂｕＬＰＨＬＫＬＡｂｕＫＥＮＫＤＡｂｕＡｂｕＳＫＫＡｂｕＫＲＲＧＴＮＩＥＫＲＡｂｕＲ（配列番号５０）， Ｍｉｎｉ ｍａｕｒｏｃａｌｃｉｎｅ ｐｅｐｔｉｄｅｓ ＭＣａＵＦ１−９： ＧＤＡｂｕＬＰＨＬＫＬ（配列番号５１）， Ｒａｔｔｌｅ ｓｎａｋｅ ｔｏｘｉｎ （Ｃｒｏｔａｍｉｎｅ） ｄｅｒｉｖｅｄ ＮｒＴＰ６ ＹＫＱＳＨＫＫＧＧＫＫＧＳＧ（配列番号５２）， ＣＰＰ５ ＶＰＴＬＫ， ＣＰＰ５ ＫＬＰＶＭ， Ｂａｃ７ （１−３５） ＲＲＩＲＰＲＰＲＬＰＲＰＲＰＲＰＬＰＦＰＲＧＰＲＰＩＰＲＰＬＰＦＰ（配列番号５３）， Ｂａｃ７ （５−３５） ＰＲＰＲＬＰＲＰＲＰＲＰＲＰＬＰＦＰＲＧＰＲＰＩＰＲＰＬＰＦＰ（配列番号５４）， Ｐｒｏｔｅｇｒｉｎ−１ ＲＧＧＲＬＣＹＣＲＲＲＦＣＶＣＶＧＲ（配列番号５５）， Ｌａｃｔｏｆｅｒｒｉｎ ｓｅｑｕｅｎｃｅｓ ＶＳＱＰＥＡＴＫＣＦＱＷＱＲＮＭＲＫＶＲＧＰＰＶＳＣＩＫＲＤＳＰＩＱＩ（配列番号５６）， Ｐａｒｔｉａｌ ｓｅｑｕｅｎｃｅｓ １−２４ Ｂｏｖｉｎｅ ＰｒＰ １−２４： ＭＶＫＳＫＩＧＳＷＩＬＶＬＦＶＡＭＷＳＤＶＧＬＣ（配列番号５７）， Ｐａｒｔｉａｌ ｓｅｑｕｅｎｃｅｓ １−３０ Ｂｏｖｉｎｅ ＰｒＰ １−３０： ＭＶＫＳＫＩＧＳＷＩＬＶＬＦＶＡＭＷＳＤＶＧＬＣＫＫＲＰＫＰ（配列番号５８）， Ｈｉｓｔｉｄｉｎｅ−ｒｉｃｈ ＣＰＰ ＨＲ９： Ｃ−ＨＨＨＨＨ−ＲＲＲＲＲＲＲＲＲ−ＨＨＨＨＨ−Ｃ（配列番号５９）， Ｂｒａｎｃｈｅｄ ｃｏｐｏｌｙｍｅｒｓ Ｐｏｌｙ（Ｌｙｓ）， ｐｏｌｙ（Ｏｒｎ）， ｐｏｌｙ［Ｌｙｓ（Ｇｌｕｎ，Ａｌａｍ）］， Ｓｗｅｅｔ ａｒｒｏｗ ｐｅｐｔｉｄｅ （ＳＡＰ） （ＶＲＬＰＰＰ（配列番号６０））３， Ａｍｐｈｉｐａｔｈｉｃ ｎｅｇａｔｉｖｅ ＣＰＰ ＳＡＰ（Ｅ）： （ＶＥＬＰＰＰ（配列番号６１））３， Ｎｅｇａｔｉｖｅ ｃｈａｒｇｅｄ ｐｏｌｙｍｅｒ Ｐｏｌｙｇｌｕｔａｍｉｃ ａｃｉｄ， ＭＭＰ （２，９）−ａｃｔｉｖａｔａｂｌｅ ＣＰＰ Ｓｕｃ−（Ｄ−Ｇｌｕ）８−ＰＬＧＣ（Ｍｅ）ＡＧ（配列番号６２）−（Ｄ−Ａｒｇ）９−ａｍｉｄｅ， Ｔｈｒｏｍｂｉｎ−ａｃｔｉｖａｔａｂｌｅ ＣＰＰ Ｓｕｃ−（Ｄ−Ｇｌｕ）８−ｘＰＬＧＬＡＧ（配列番号６３）−（Ｄ−Ａｒｇ）９−Ｄ−Ｃ−Ｃｙ５ Ｓｕｃ−（Ｄ−Ｇｌｕ）８−Ｏｘ−ＤＰＲＳＦＬ（配列番号６４）−（Ｄ−Ａｒｇ）９−ａｍｉｄｅ， ｐＨ−ｓｅｎｓｉｔｉｖｅ （ａｃｔｉｖａｔａｂｌｅ） ｃｅｌｌ−ｐｅｎｅｔｒａｔｉｎｇ ｐｅｐｔｉｄｅｓ ｐＨＬＩＰ： ＡＥＱＮＰＩＹＷＡＲＹＡＤＷＬＦＴＴＰＬＬＬＬＥＬＡＬＬＶＤＡＤＥＧＴ（配列番号６５）， ＫＫＬＡＤａｐ（Ｍｅ２）ＡＬ Ｄａｐ（Ｍｅ２）ＬＬＡＬＬＷＬＤａｐ（Ｍｅ２）ＬＡＤａｐ（Ｍｅ２）ＡＬＫＫＡ−ａｍｉｄｅ， ε−Ｌｙｓ ｓｕｃｃｉｎｙｌ ａｍｉｄｅ， Ｖｉｒａｌ ｆｕｓｉｏｎ ｐｒｏｔｅｉｎ ｓｅｑｕｅｎｃｅ Ｒｅｐｅａｔ−ｓｅｑｕｅｎｃｅ： ＥＡＬＡＷＥＡＡＬＡＥＡＬＡＥＡＬＡＥＨＬＡＥＡＬＡＥＡＬＡＥＡＬＥＡＬＡＡ（配列番号６６）， ｐＨ−ｓｅｎｓｉｔｉｖｅ Ｒ８−ｃｏｎｓｔｒｕｃｔ Ｓｔｅａｒｏｙｌ−ＲＲＲＲＲＲＲＲ−（ＥＡＬＡ）ｎ， Ｒｅｄｏｘ ｐｒｏｔｅｉｎ ａｚｕｒｉｎ ‘ｐ１８’ Ａｚｕｒｉｎ Ｌｅｕ５０−Ｇｌｙ６７： ＬＳＴＡＡＤＭＱＧＶＶＴＤＭＧＡＳＧ（配列番号６７）， Ｒｅｄｏｘ ｐｒｏｔｅｉｎ ａｚｕｒｉｎ ‘ｐ２８’ Ａｚｕｒｉｎ Ｌｅｕ５０−Ａｓｐ７７： ＬＳＴＡＡＤＭＱＧＶＶＴＤＭＧＡＳＧＬＤＫＤＹＬＫＰＤＤ（配列番号６８）， Ｉｎｆｌｕｅｎｃａ ＨＡ−２ （１−２０） ＫＡＬＡ ｓｅｑｕｅｎｃｅ ＷＥＡＫＬＡＫＡＬＡＫＡＬＡＨＬＡＫＡＬＡＫＡＬＫＡＣＥＡ（配列番号６９）， Ｎ
ｉｃｋＦｅｃｔｓ ＮＦ６１： Ｓｔｅａｒｙｌ−ＡＧＹＬＬＧＯＩＮＬＫＡＬＡＡＬＡＫＫＩＬ（配列番号７０）−ａｍｉｄｅ， Ｎｏｎａａｒｇｉｎｉｎｅ （Ｒ９） ＲＲＲＲＲＲＲＲＲ， Ｓｔｅａｒｙｌａｔｅｄ ａｒｇｉｎｉｎｅ−ｒｉｃｈ ｐｅｐｔｉｄｅｓ Ｓｔｅａｒｙｌ−ＲＲＲＲＲＲＲＲ， Ａｍｐｈｉｐａｔｈｉｃ ｐｅｐｔｉｄｅ Ｔｄ３７０１ ＴＲＹＬＲＩＨＰＲＳＷＶＨＱＩＡＬＲＬＲＹＬＲＩＨＰＲＳＷＶＨＱＩＡＬＲＳ（配列番号７１）， Ａｍｐｈｉｐａｔｈｉｃ ｈｅｌｉｃａｌ ｐｅｐｔｉｄｅ ｐｐＴＧ１ ＧＬＰＫＡＬＬＫＬＬＫＳＬＷＫＬＬＬＫＡ（配列番号７２）， Ｈｕｍａｎ ｃａｌｃｉｔｏｎｉｎ ｐａｒｔｉａｌ ｓｅｑｕｅｎｃｅ ９−３２， ｈＣＴ（９−３２）−ｂｒ ＬＧＴＹＴＱＤＦＮＫ（Ｘ）ＦＨＴＦＰＱＴＡＩＧＶＧＡＰ（配列番号７３）−ａｍｉｄｅ Ｘ： ＰＫＫＫＲＫＶＥＤＰＧＶＧＦＡ（配列番号７４）， Ｃｅｌｌ−ｐｅｎｅｔｒａｔｉｎｇ ＤＮＡ−ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ ＹＡＲＶＲＲＲＧＰＲＲ（配列番号７５）、 （Ｈｐｈ−１）−ＧＡＬ４（ＤＮＡ−ｂｉｎｄｉｎｇ ｄｏｍａｉｎ）， Ｓｉｍｉａｎ ｖｉｒｕｓ ４０ ｌａｒｇｅ Ｔ−ａｎｔｉｇｅｎ （ＮＬＳ） ＰＫＫＫＲＫ（配列番号７６）， Ｓｔｅａｒｙｌａｔｅｄ ＮＬＳ Ｓｔｅａｒｙｌ−ＰＫＫＫＲＫＶ（配列番号７７）， Ｈｏｍｏｄｉｍｅｒｉｃ ＮＬＳ （Ｃ−ｔｅｒｍｉｎａｌ） （ＧＹＧＰＫＫＫＲＫＶＧＧＣ（配列番号７８））２， Ｎｕｃｌｅｏｐｌａｓｍｉｎ ＮＬＳ， ｂｉｐａｒｔｉｔｅ ＮＬＳ ＫＲＰＡＡＴＫＫＡＧＱＡＫＫＫＫ（配列番号７９）， Ｋｕ７０２−ＮＬＳ Ｃ−ＧＳＫＧＡＲＰＡＫＫＲＫＰＫＲＧＡＡＨＫ−ＨＡＧＡＫＶＲＫＴＶＴＧＡＫＫ（配列番号８０）， ＡＤＡＲ１ ＮＬＳ ＭＭＰＮＶＫＲＫＩＧＥＬＶＲＹＬＮＴＮＰＶＧ（配列番号８１）， ＧｌｕＯｃｔ６ ＥＥＥＡＡＧＲＫＲＫＫＲＴ（配列番号８２）， ＲｅＩＢ ４０５ＹＧＶＤＫＫＲＫＲＧＭＰＤＶＬＧＥＬＮＳ４２４−４２５ＳＤＰＭＧＩＥＳＫＲＲＫＫＫＰＡＩＬＤＨＦＬ４４６（配列番号８３）， Ｄｅｒｍａｓｅｐｔｉｎ−ｐｅｐｔｉｄｅ Ｋ４−ＮＬＳ ＰＶＫ： ＰＫＫＫＲＬＫＶＡＫＷＫＴＬＬＫＫＶＬＫＡ（配列番号８４）−ａｍｉｄｅ， Ｎｕｃｌｅｏｌａｒ ｔａｒｇｅｔｉｎｇ ｐｅｐｔｉｄｅ ＹＫＱＣＨＫＫＧＧＸＫＫＧＳＧ（配列番号８５）， Ｏｐｉｏｉｄ ｐｅｐｔｉｄｅｓ Ｄｉｍｅｔｈｙｔｙｒｏｓｉｎｅ−ｒＦＫＦ−ＮＨ２， Ｍｉｔｏｃｈｏｎｄｒｉａ−ｐｅｎｅｔｒａｔｉｎｇ ｐｅｐｔｉｄｅｓ Ｆ−ｒ−Ｆ−Ｋ−Ｆ−ｒ−Ｆ−Ｋ， Ｍｉｔｏｐａｒａｎ ＮＬＫＫＬＡＫＬ（Ａｉｂ）ＫＫＩＬ（配列番号８６）， Ｌｙｓｏｓｏｍａｌ ｓｏｒｔｉｎｇ ｓｅｑｕｅｎｃｅｓ Ｌ１ ＹＱＲＬＣ（配列番号８７）， Ｌｙｓｏｓｏｍａｌ ｓｏｒｔｉｎｇ ｓｅｑｕｅｎｃｅｓ Ｌ２ ＣＮＰＧＹ（配列番号８８），２８９−Ｗ： ＲＷＩＫＩＷＦＷＷＲＲＭＫＷＫＫ（配列番号８９），Ｌ１７Ｅ： ＩＷＬＴＡＬＫＦＬＧＫＨＡＡＫＨＥＡＫＱＱＬＳＫＬ（配列番号９０），Ｌ−Ｋ６： ＩＫＩＬＳＫＩＫＫＬＬＫ（配列番号９１）等が挙げられ、Ｒ８、Ｒ８−ＮＨ２、Ｐｅｎｅｔｒａｔｉｎ ＲＱＩＫＩＷＦＱＮＲＲＭＫＷＫＫ， ＨＩＶ−ＴＡＴ （４７−５７） ＹＧＲＫＫＲＲＱＲＲＲ， ＨＩＶ−ＴＡＴ （４８−６０） ＧＲＫＫＲＲＱＲＲＲＰＰＱ， ｐＶＥＣ ＬＬＩＩＬＲＲＲＩＲＫＱＡＨＡＨＳＫ−ＮＨ２， Ｔｒａｎｓｐｏｒｔａｎ （ＴＰ１０） ＡＧＹＬＬＧＫＩＮＬＫＡＬＡＡＬＡＫＫＩＬ−ＮＨ２，Ｐｒｏｔｅｉｎ ｔｒａｎｓｄｕｃｔｉｏｎ ｄｏｍａｉｎｓ ｓｅｑｕｅｎｃｅ ＡＲＫＫＡＡＫＡ、２８９−Ｗ、Ｌ１７Ｅ、Ｌ−Ｋ６が好ましく、Ｐｅｎｅｔｒａｔｉｎ、Ｒ８、Ｒ８−ＮＨ２、ｐＶＥＣ、２８９−Ｗ、Ｌ１７Ｅ、Ｌ−Ｋ６がより好ましく、Ｐｅｎｅｔｒａｔｉｎ、Ｒ８−ＮＨ２が特に好ましい。なお、これら細胞膜透過性ペプチドを構成するアミノ酸の立体配置は、特に断りのない限りＬ体であり、Ｄ体のアミノ酸から構成される場合は、例えば、Ｄ−Ｒ８−ＮＨ２のように立体を明記することとする。Ｄ体のアミノ酸から構成される細胞膜透過性ペプチドとしては、Ｄ−Ｒ８−ＮＨ２が好ましい。
The number of amino acid residues of the cell membrane permeable peptide contained in the composition of the present invention is not particularly limited, but is preferably 6 to 50, more preferably 6 to 40, and particularly preferably 6 to 30. The peptide is preferably enriched in arginine residues, in particular it may comprise 3 or more, more preferably 4 or more, particularly preferably 5 or more arginine residues in the peptide. A peptide comprising 6 to 50 amino acid residues, of which 3 or more residues are arginine residues is preferred, and a peptide comprising 6 to 40 amino acid residues, 4 or more of which residues are arginine residues More preferred is a peptide comprising 6 to 30 amino acid residues, of which 5 or more residues are arginine residues. The arginines may each independently be L-form or D-form. In addition, cell membrane permeable peptides also include C-terminal and / or N-terminal and / or side chain modified peptides. Specific examples of modification of carboxyl group include amidification (CONH 2 ), amidification such as methyl amidification; esterification such as methyl esterification, ethyl esterification and the like, and specific examples of modification of an amino group are Amidation such as acetylation and formylation; alkylation such as methylation and dimethylation; and the like. Specific examples of modification of hydroxyl group include esterification such as acetylation; phosphorylation and the like; Specific examples of the modification include alkylation such as methylation and the like. Specifically, the cell membrane permeable peptide is R8 (amino acid sequence; RRRRRRRR), SEQ ID NO: 1), R8-NH 2 (amino acid sequence: RRRRRRRR-NH 2 ), SEQ ID NO: 2), HIV-TAT (48-60) (Amino acid sequence; GRKKRRQRRRPPQ, SEQ ID NO: 3), Penetratin RQIKIWFQ NRRM KWKK (SEQ ID NO: 4), HIV-TAT (47-57) YGRKKRRQRRR (SEQ ID NO: 5), pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 6)-NH 2 , Transportan (TP10) AGYLLGKINLKALAALAKKIL (SEQ ID NO: 7) -NH 2, Protein transduction domains sequence ARKKAAKA ( SEQ ID NO: 8), HI -1 Rev (34-50) KQAIPVAK (SEQ ID NO: 9) -amide, FHV coat (35-49) RRRRNRTRRNRRRVR (SEQ ID NO: 10)-amide, Oligoarginines (R9-R12) RRRRRRRRR (SEQ ID NO: 11) / RRRRRRRRRRRR (SEQ ID NO: 11) 12), CCMV Gag (7-25) KLTRAQRRAARKNKRNTRGC (SEQ ID NO: 13), Chimeric dermaseptin S4 and SV40 'S413-PV' ALWKTLLKKVLKAPKKKRKVC (SEQ ID NO: 14), Herpes simplex virus transcription factor (267-300) VP22 DAATATRGRSARPTER SRPRRPVE (SEQ ID NO: 15), (Chimeric galanin / mastoparan) Transportan GWTLNS AGYLLGKINLCALAAKKIL (SEQ ID NO: 16)-amide, CPP with protease cleavage side YTA2 YTAIAWVKAFIRKLRK (SEQ ID NO: 17)-amide, Pep-1 Ac-KITKW (SEQ ID NO: 19) -NH-CH2-CH2-SH, MPGβ Ac-GALFLGFLGAGSTMGWS QPKKKRKV (SEQ ID NO: 20)-NH-CH2-CH2-SH, M NH-CH2-CH2-SH, MPGα Ac-GALFLAFLAALSLMGLWSQPKKKRKV (SEQ ID NO: 19) G8β AFLGWLGAWGTM GWSPKKRK (SEQ ID NO: 21)-NH-CH2-CH2-SH, CADY Ac-GLWRALWRLLRSLWRLWKA (SEQ ID NO: 22)-NH-CH2-CH2-SH, Pepfect 6 Stearyl-AGYLLGK (ε-TMQ) INLKALALAKA L (SEQ ID NO: 23), PepFect14 Stearyl-AGYLLGKLOLOAAAOLOLL (SEQ ID NO: 24) -amide, PepFect 15 Stearyl-AGYLLGK (K3QN4) LLOOLAAAALOOLL (SEQ ID NO: 25) -amide, NickFect NF61: Stearyl-AGYLLGINLKALALALK LIL (SEQ ID NO: 26) LKLLLKLW KLLLKLLK (SEQ ID NO: 27), Influenca HA-2 (1-20) KALA sequence WEAK LAKA LKA HLA KAL AK LACE A (SEQ ID NO: 28), KLA sequence Acetyl-KLAL K LALK ALKA AL KLA (SEQ ID NO: 29) pVEC LLIILRRRIRKQAHHASK (SEQ ID NO: 31) -amide, Kaposis sarcoma fibroblast growth factor Kaposi FGF AAVALL PAVLLALLAP (SEQ ID NO: 32), Signal sequence of Ig light chain from Caiiman crocodylus MGLGLHLLVLAALQ QGAMGLGLHLLLAAALQGA (SEQ ID NO: 33), Integrin β3-fragment VTGLAGALAGVGVG (SEQ ID NO: 34), Grb2 (SH2 domain) AAVLLPLVLAAP (SEQ ID NO: 35), Fusion sequence SEQ ID NO: 36), Hepatitis B virus translocation motif PLSSIFSRIGDP (SEQ ID NO: 37), Sperm-egg fusion protein (89-111) SFP23: Ac-KLIAGSISIPPIRALFAAI IP (SEQ ID NO: 38) -amide, Human calcitonin partial sequence 9-32, hCT (9-32)-br, LGTYTQDFNK (X) FHTFPQ TAIGVGAP (SEQ ID NO: 39)-amide X: PKKKRKVEDPGVGFA (SEQ ID NO: 40), hCT (18) -32) -br, Capr-K (X) FHTFPQTAIGVGAP (SEQ ID NO: 41)-amide X: KKRKAPKKKRKFA (SEQ ID NO: 42), Substance P and analogues RPKPQQFGLM (SEQ ID NO: 43)-amide, RGD peptides αvβ3 RGD-Temporin-LA , RGD-Dye RGD-bearing PAMAM dendrimers, RGD pept des αvβ3, αvβ5 Cilengitide: Cyclo [RGDfNmeV], RGD peptide Cyclo (RGDfK); cyclo (RGDyK), RGD peptides α5β1 RGD mimic, AGR (prostata carcinoma) CAGRRSAYC (SEQ ID NO: 44), LyPi-2 CNRRTKAGC (SEQ ID NO: 45), REA (prostata, cervix, breast carcinoma) CREAGRKAC (SEQ ID NO: 46), LSD (melanoma, osteosarcoma) CLSDGKRKC (SEQ ID NO: 47), Mastoparan INLKALALAKAKIL (SEQ ID NO: 48)-amide, Meide Liin GIGAVLKVLTTGLPALISWK RQQ (SEQ ID NO: 49)-Abi, Scorpion toxin MAUROCALC INE with replacement of C by Abu GDA bu L H L Abu Abu KEN K D Abu Abu SK K BU T R K N RU T b, (Sequence No. 50), ma uro uro SEQ ID NO: 52), CPP5 VPTLK, CPP5 KLPVM, Bac7 (1-35) RRIRPRPRLPRPRPFPRPPRGPRPIPLPL PFP (SEQ ID NO: 53), Bac7 (5-35) PRPRLPRPRPRPLPRPPRGPRPIPRPLPFP (SEQ ID NO: 54); MVKSKIGSWILVLFVAMWSDVGLC (SEQ ID NO: 57), Partial sequences 1-30 Bovine PrP 1-30: MVKSKIGSWILVLF VAMWSD VGLCKKRPKP (SEQ ID NO: 58), Histidine-rich CPP HR9: C-HHHHH-RRRRRRRRRR-HHHHH-C (SEQ ID NO: 59), Branched copolymer poly (Lys), poly (Orn), poly [Lys (Glun, Alam)], Sweet arrow peptide (SAP) (VRLPPP (SEQ ID NO: 60) ), 3, Amphipathic negative CPP SAP (E): (VELPPP (SEQ ID NO: 61)) 3, Negative charged polymer Polyglutamic acid, MMP (2, 9)-activatable CPP Suc-(D-Glu) 8- PLGC (Me) AG (SEQ ID NO: 62)-(D-Arg) 9-amide, Thrombin-activatable CPP Suc-(D-Glu) 8 -XPLGLAG (SEQ ID NO: 63)-(D-Arg) 9-DCCy 5 Suc-(D-Glu) 8- Ox-DPRSFL (SEQ ID NO: 64)-(D-Arg) 9-amide, pH-sensitive (Activatable) cell-penetrating peptides pHLIP: AEQNPIYWERYADWLFTTPLLLLALLDVADEGT (SEQ ID NO: 65), KKLADap (Me2) AL Dap (Me2) LLALLWLDap (Me2) LADap (Me2) ALKKA-amide, ε-Lys s : EALAWEAALAEALAEALAEHLAEALAEAL EALEALAA (SEQ ID NO: 66), pH-sensitive R8-construct Stearoyl-RRRRRRRR- (EALA) n, Redox protein azurin 'p18' Azurin Leu 50-Gly 67: LSTAADMQGVVTDMGASG (SEQ ID NO: 67), Redox protein azurin 'p 28' Azurin Leu 50-Asp 77 : LSTAADMQGVVTDMGASGLDKDYLKPD (SEQ ID NO: 68), Influenca HA-2 (1-20) KALA sequence WEAK LAKA LAKA HLA KAL KAL KACEA (SEQ ID NO 69), N
ickFects NF61: Stearyl-AGYLLGOINLKALAAKKIL (SEQ ID NO: 70)-amide, Nonaarginine (R9) RRRRRRRRR, Stearylated arginine-rich peptides Staryl-RRRRR, Amphipathic peptides Td3701 TRYLRI HRSHH IlH IlH Ih, a helhi Human calcitonin partial sequence 9-32, hCT (9-32)-br LGTYTQDFNK (X) FHTF PQTAIGVGAP (SEQ ID NO: 73) -amide X: PKKKRKVEDPGVGFA (SEQ ID NO: 74), Cell-penetrating DNA-binding protein YARVRRRGPRR (SEQ ID NO: 75), (Hph-1) -GAL4 (DNA-binding domain), Simian virus 40 large T -Antigen (NLS) PKKKRK (SEQ ID NO: 76), Stearylated NLS Stearyl-PKKKRKV (SEQ ID NO: 77), Homodimeric NLS (C-terminal) (GYGPKKKRKVGGC (SEQ ID NO: 78)) 2, Nucleoplasmin NLS, bipartite NLS KRPAATKKAGQAKKKK (SEQ ID NO: 79) ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Or,,,,,,,,,,,,,,,,,,,,,,,,,, or. PKKKRLKVAKWKTLLKKVLKA (SEQ ID NO: 84) -amide, Nucleolar targeting peptide YKQCHKKGGXKKGSG (SEQ ID NO: 85), Iopoid peptides Dimet hytyrosine-rFKF-NH 2, Mitochondria -penetrating peptides F-r-F-K-F-r-F-K, Mitoparan NLKKLAKL (Aib) KKIL ( SEQ ID NO: 86), Lysosomal sorting sequences L1 YQRLC ( SEQ ID NO: 87), Lysosomal sorting sequences L2 CNPGY (SEQ ID NO: 88), 289-W: RWIKIWFWWRRMKWKK (SEQ ID NO: 89), L17E: IWLTALKLFLGKHAAKHEAKQQLSKL (SEQ ID NO: 90), L-K6: IKILSKIKKLLK (SEQ ID NO: 91), etc., R8, R8- NH 2, Penetratin RQIKIWFQNRRMKWKK, H V-TAT (47-57) YGRKKRRQRRR, HIV-TAT (48-60) GRKKRRQRRRPPQ, pVEC LLIILRRRIRKQAHAHSK-NH 2, Transportan (TP10) AGYLLGKINLKALAALAKKIL-NH 2, Protein transduction domains sequence ARKKAAKA, 289-W, L17E, L-K6 preferably, Penetratin, R8, R8-NH 2, pVEC, 289-W, L17E, more preferably L-K6, Penetratin, R8- NH 2 are especially preferred. In addition, the configuration of the amino acids constituting these cell membrane permeable peptides is L form unless otherwise specified, and when it is composed of D form amino acids, for example, it is steric like D-R8-NH 2 It shall be stated clearly. The cell membrane-permeable peptide consists of D-amino acids, D-R8-NH 2 are preferred.
本発明の組成物に含有される細胞膜透過性ペプチドの分子量としては、特に制限はないが、８００〜８０００が好ましく、９００〜６０００がより好ましく、１０００〜４０００が特に好ましい。 The molecular weight of the cell membrane permeable peptide contained in the composition of the present invention is not particularly limited, but is preferably 800 to 8000, more preferably 900 to 6000, and particularly preferably 1000 to 4000.
本発明の組成物に含有される細胞膜透過性ペプチドを構成するアミノ酸は、多くのアルギニン残基を含むことが好ましく、それ以外に特に制限はなく、天然アミノ酸でも非天然アミノ酸でもよい。 The amino acids constituting the cell membrane-permeable peptide contained in the composition of the present invention preferably contain a large number of arginine residues, and are not particularly limited, and may be natural amino acids or non-natural amino acids.
本発明の組成物に含有される細胞膜透過性ペプチドは、細胞膜透過性ペプチドの塩であってもよく、医薬として許容される塩であれば特に制限はない。細胞膜透過性ペプチドの塩としては無機酸との塩、有機酸との塩等が挙げられる。 The cell membrane permeable peptide contained in the composition of the present invention may be a salt of a cell membrane permeable peptide, and is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples of salts of cell membrane permeable peptides include salts with inorganic acids and salts with organic acids.
無機酸との塩の例としては、塩酸、臭化水素酸、ヨウ化水素酸、硝酸、硫酸、リン酸等との塩が挙げられ、塩酸塩が好ましい。 Examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid and the like, with hydrochloride being preferred.
有機酸との塩の例としては、酢酸、シュウ酸、フマル酸、マレイン酸、コハク酸、リンゴ酸、クエン酸、酒石酸、アジピン酸、グルコン酸、グルコヘプト酸、グルクロン酸、テレフタル酸、メタンスルホン酸、アラニン、乳酸、馬尿酸、１，２−エタンジスルホン酸、イセチオン酸、ラクトビオン酸、オレイン酸、没食子酸、パモ酸、ポリガラクツロン酸、ステアリン酸、タンニン酸、トリフルオロメタンスルホン酸、ベンゼンスルホン酸、ｐ−トルエンスルホン酸、硫酸ラウリル、硫酸メチル、ナフタレンスルホン酸、スルホサリチル酸、トリフルオロ酢酸等との塩が挙げられる。 Examples of salts with organic acids are acetic acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, tartaric acid, adipic acid, gluconic acid, glucoheptic acid, glucuronic acid, terephthalic acid, methanesulfonic acid Alanine, lactic acid, hippuric acid, 1,2-ethanedisulfonic acid, isethionic acid, lacteionic acid, lactobionic acid, oleic acid, gallic acid, pamoic acid, polygalacturonic acid, stearic acid, tannic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, Examples thereof include salts with p-toluenesulfonic acid, lauryl sulfate, methyl sulfate, naphthalenesulfonic acid, sulfosalicylic acid, trifluoroacetic acid and the like.
本発明の組成物において、細胞膜透過性ペプチド及びその塩は、水和物又は溶媒和物の形態をとってもよい。 In the compositions of the present invention, cell membrane permeable peptides and salts thereof may be in the form of hydrates or solvates.
本発明の組成物において、細胞膜透過性ペプチド又はその塩の含有量は、医薬として許容される量であれば、特に制限されず、その下限が、例えば０．００１％（ｗ／ｖ）、好ましくは０．０１％（ｗ／ｖ）、より好ましくは０．１％（ｗ／ｖ）、更に好ましくは１％（ｗ／ｖ）、特に好ましくは５％（ｗ／ｖ）であり、その上限が、例えば３５％（ｗ／ｖ）、好ましくは３０％（ｗ／ｖ）、より好ましくは２５％（ｗ／ｖ）、更に好ましくは２０％（ｗ／ｖ）、特に好ましくは１５％（ｗ／ｖ）である。さらに、それらの上限と下限は適宜組み合わせて使用することができる。例えば、下限の５％（ｗ／ｖ）と上限の３５％（ｗ／ｖ）、３０％（ｗ／ｖ）、２５％（ｗ／ｖ）、２０％（ｗ／ｖ）及び１５％（ｗ／ｖ）を組み合わせて、５〜３５％（ｗ／ｖ）、５〜３０％（ｗ／ｖ）、５〜２５％（ｗ／ｖ）、５〜２０％（ｗ／ｖ）、５〜１５％（ｗ／ｖ）のように範囲を設定することができる。より具体的には、本発明の組成物において、細胞膜透過性ペプチド又はその塩の含有量は、例えば、０．００１〜３５％（ｗ／ｖ）が好ましく、０．０１〜３０％（ｗ／ｖ）がより好ましく、０．１〜２５％（ｗ／ｖ）がさらにより好ましく、１〜２０％（ｗ／ｖ）が特に好ましく、５〜１５％（ｗ／ｖ）が最も好ましい。 In the composition of the present invention, the content of the cell membrane permeable peptide or a salt thereof is not particularly limited as long as it is a pharmaceutically acceptable amount, and the lower limit thereof is, for example, 0.001% (w / v), preferably Is 0.01% (w / v), more preferably 0.1% (w / v), still more preferably 1% (w / v), particularly preferably 5% (w / v), and the upper limit thereof For example, it is 35% (w / v), preferably 30% (w / v), more preferably 25% (w / v), still more preferably 20% (w / v), particularly preferably 15% (w) / V). Furthermore, those upper limits and lower limits can be used in combination as appropriate. For example, the lower limit 5% (w / v) and the upper limit 35% (w / v), 30% (w / v), 25% (w / v), 20% (w / v) and 15% (w) / V) in combination, 5 to 35% (w / v), 5 to 30% (w / v), 5 to 25% (w / v), 5 to 20% (w / v), 5 to 15 The range can be set as% (w / v). More specifically, in the composition of the present invention, the content of cell membrane-permeable peptide or a salt thereof is, for example, preferably 0.001 to 35% (w / v), and 0.01 to 30% (w / v). v) is more preferred, 0.1 to 25% (w / v) is even more preferred, 1 to 20% (w / v) is particularly preferred, and 5 to 15% (w / v) is most preferred.
本発明の組成物では、細胞透過性ペプチド又はその塩と、薬物とのモル比を自由に設定することができ、細胞透過性ペプチド又はその塩：薬物のモル比は、特に制限されないが、例えば１：１０００、１：２００、１：１００、１：１０、１：２、１：１、２：１、１０：１、１００：１、２００：１または１０００：１の任意の組合せの間であってよい。具体的には、細胞透過性ペプチド又はその塩と、薬物とのモル比は、例えば、１：１０００〜１０００：１、１：２００〜２００：１、１：１００〜１００：１、１：１０〜１０：１、１：２〜２：１の範囲内であってよい。別の観点で、細胞透過性ペプチド又はその塩は、薬物に対して当量未満であっても眼組織中への薬物の移行性を改善可能なことから、上記モル比は１：１．１以上であってよい。具体的には、細胞透過性ペプチド又はその塩と、薬物とのモル比は、例えば、１：１０００〜１：１．１、１：２００〜１：１．１、１：１００〜１：１．１、１：１０〜１：１．１、１：２〜１：１．１の範囲内であってよい。また、薬物の量が少なくても眼組織へ十分に移行し得るから、上記モル比は１．１以上：１であってよい。具体的には、細胞透過性ペプチド又はその塩と、薬物とのモル比は、例えば、１０００：１〜１．１：１、２００：１〜１．１：１、１００：１〜１．１：１、１０：１〜１．１：１、２：１〜１．１：１の範囲内であってよい。 In the composition of the present invention, the molar ratio of the cell permeable peptide or the salt thereof to the drug can be freely set, and the molar ratio of the cell permeable peptide or the salt thereof to the drug is not particularly limited. Between any combination of 1: 1000, 1: 200, 1: 100, 1:10, 1: 2, 1: 1, 2: 1, 10: 1, 100: 1, 200: 1 or 1000: 1 May be there. Specifically, the molar ratio of the cell permeable peptide or the salt thereof to the drug is, for example, 1: 1000 to 1000: 1, 1: 200 to 200: 1, 1: 100 to 100: 1, 1:10. It may be in the range of ̃10: 1, 1: 2 to 2: 1. In another aspect, the above-mentioned molar ratio is at least 1: 1.1, since the cell permeable peptide or the salt thereof can improve the transferability of the drug into the ocular tissue even if it is less than the equivalent to the drug. It may be. Specifically, the molar ratio of the cell penetrating peptide or the salt thereof to the drug is, for example, 1: 1000 to 1: 1.1, 1: 200 to 1: 1.1, 1: 100 to 1: 1. .1, 1:10 to 1: 1.1, 1: 2 to 1: 1.1. In addition, the molar ratio may be 1.1 or more: 1 because the drug can be sufficiently transferred to ocular tissues even with a small amount of drug. Specifically, the molar ratio of the cell penetrating peptide or a salt thereof to the drug is, for example, 1000: 1 to 1.1: 1, 200: 1 to 1.1: 1, 100: 1 to 1.1. It may be in the range of 1: 1, 10: 1 to 1.1: 1, 2: 1 to 1.1: 1.
なお、本発明の組成物において細胞膜透過性ペプチドの塩が含有される場合、これらの値は塩の質量を基準にした含有量である。なお、「％（ｗ／ｖ）」は、本発明の組成物１００ｍＬ中に含まれる対象成分（ここでは、細胞膜透過性ペプチド）の質量（ｇ）を意味する。対象成分が界面活性剤等の添加剤等である場合も同様であり、またその塩や水和物が含有される場合は、これらの値は塩や水和物の質量を基準にした含有量である。 In addition, when the salt of a cell membrane permeable peptide is contained in the composition of this invention, these values are content based on the mass of a salt. In addition, "% (w / v)" means the mass (g) of the object component (here, cell membrane permeable peptide) contained in 100 mL of the composition of the present invention. The same applies to the case where the target component is an additive such as surfactant, etc. Also, when the salt or hydrate thereof is contained, these values are the content based on the mass of the salt or hydrate It is.
本発明の組成物は、必要に応じて添加剤を含有することができる。添加剤の例としては、界面活性剤、緩衝化剤、等張化剤、安定化剤、防腐剤、抗酸化剤、粘稠化剤、ｐＨ調整剤等が挙げられる。 The composition of the present invention can optionally contain an additive. Examples of additives include surfactants, buffering agents, tonicity agents, stabilizers, preservatives, antioxidants, thickening agents, pH adjusters and the like.
本発明の組成物には、医薬品の添加物として使用可能な界面活性剤を適宜配合することができる。ただし、本発明では細胞膜透過性ペプチドによって薬の眼組織への移行性が向上しているため、界面活性剤の使用は必須ではない。この観点で、組成物中の界面活性剤の含有量は、好ましくは０．２％（ｗ／ｖ）未満、より好ましくは０．１％（ｗ／ｖ）未満、さらに好ましくは０．０１％（ｗ／ｖ）未満、最も好ましくは０．００１％（ｗ／ｖ）未満である。 In the composition of the present invention, a surfactant which can be used as an additive of a pharmaceutical can be appropriately blended. However, in the present invention, the use of a surfactant is not essential since the transferability of the drug to ocular tissues is improved by the cell membrane permeable peptide. In this respect, the content of surfactant in the composition is preferably less than 0.2% (w / v), more preferably less than 0.1% (w / v), still more preferably 0.01% Less than (w / v), most preferably less than 0.001% (w / v).
本発明の組成物には、医薬品の添加物として使用可能な緩衝剤を配合することができる。緩衝剤の例としては、リン酸又はその塩、或いはそれらの水和物等が挙げられる。 The composition of the present invention can be incorporated with a buffer that can be used as an additive for pharmaceutical products. Examples of the buffer include phosphoric acid or a salt thereof, or a hydrate thereof.
本発明の組成物は、１つ又は複数の、好ましくは１〜３つの、より好ましくは１つ又は２つの薬物を含有することができる。薬物としては、フルオレセイン、６−カルボキシフルオレセイン、ＦＤ（ＦＩＴＣ−ｄｅｘｔｒａｎ）４、ＦＤ１０、リン酸ブレドニゾロン、ペミロラスト、ホスミドマイシン、リン酸ベタメサゾン、ジクロフェナク、リン酸ヒドロコルチゾン、ローダミン、ブリンゾラミド、ドルゾラミド塩酸塩、オロパタジン塩酸塩、プロプラノロール塩酸塩、チモロールマレイン酸塩、ブリモニジン酒石酸塩等の低分子医薬のほか、核酸医薬、抗体医薬、タンパク質医薬等の高分子医薬が挙げられる。好ましい薬物の具体的な例としては、分子内にカルボキシル基（ＣＯ２Ｈ）、スルホン酸基（ＳＯ３Ｈ）、リン酸基（ＯＰＯ３Ｈ）及び水酸基（ＯＨ）からなる群より選択される置換基を１又は複数個、好ましくは１〜２０個、より好ましくは１〜１０個有する薬物が挙げられ、分子内にカルボキシル基及び水酸基からなる群より選択される置換基を１又は複数個、好ましくは１〜２０個、より好ましくは１〜１０個有する薬物がより好ましく、分子内にカルボキシル基及びフェノール性水酸基からなる群より選択される置換基を１又は複数個、好ましくは１〜２０個、より好ましくは１〜１０個有する薬物が特に好ましい。 The compositions of the invention may contain one or more, preferably one to three, more preferably one or two drugs. As the drug, fluorescein, 6-carboxyfluorescein, FD (FITC-dextran) 4, FD10, bredonizolone phosphate, pemirolast, phosmidomycin, betamethasone phosphate, diclofenac, hydrocortisone phosphate, rhodamine, brinzolamide, dorzolamide hydrochloride, olopatadine In addition to low molecular weight drugs such as hydrochloride, propranolol hydrochloride, timolol maleate and brimonidine tartrate, high molecular weight drugs such as nucleic acid drugs, antibody drugs and protein drugs can be mentioned. Specific examples of preferred drugs are selected from the group consisting of carboxyl group (CO 2 H), sulfonic acid group (SO 3 H), phosphoric acid group (OPO 3 H) and hydroxyl group (OH) in the molecule. And drugs having one or more, preferably 1 to 20, more preferably 1 to 10, substituents, and one or more substituents selected from the group consisting of a carboxyl group and a hydroxyl group in the molecule, The drug preferably has 1 to 20, more preferably 1 to 10, and one or more, preferably 1 to 20, substituents selected from the group consisting of a carboxyl group and a phenolic hydroxyl group in the molecule. Particularly preferred is a drug having 1 to 10, more preferably 1 to 10.
また、表面プラズモン共鳴（ＳＰＲ；Ｓｕｒｆａｃｅ Ｐｌａｓｍｏｎ Ｒｅｓｏｎａｎｃｅ）を利用して細胞膜透過性ペプチドと薬物の間の相互作用を測定する評価系により、本発明の組成物に含まれる好ましい薬物を、使用する細胞膜透過性ペプチドに応じて選択することができる。 In addition, cell membrane permeation using a preferred drug contained in the composition of the present invention according to an evaluation system that measures the interaction between cell membrane permeable peptide and the drug using surface plasmon resonance (SPR; Surface Plasmon Resonance) It can be selected according to the sex peptide.
表面プラズモン共鳴（ＳＰＲ；Ｓｕｒｆａｃｅ Ｐｌａｓｍｏｎ Ｒｅｓｏｎａｎｃｅ）測定を利用した評価系は、表面プラズモン共鳴分析装置、好ましくは、例えばＢｉａｃｏｒｅＴ２００（ＧＥヘルスケア）を使用して、レゾナンスユニット（ＲＵ）値を測定することにより細胞膜透過性ペプチドと薬物の相互作用の強度を評価する系である。細胞膜透過性ペプチドは、センサーチップにアミド結合やジスルフィド結合を介して直接固定化されてもよいし、ビオチン−アビジン相互作用を介して固定化されてもよく、例えば細胞膜透過性ペプチドをビオチンと共有結合させた分子をアビジンを介してセンサーチップ（例えば、Ｓｅｒｉｅｓ Ｓ Ｓｅｎｓｏｒ Ｃｈｉｐ ＳＡ／ＧＥヘルスケア）に固定化させるのが好ましい。細胞膜透過性ペプチドをセンサーチップに固定化した後、薬物を０．０００１〜１％（ｗ／ｖ）、好ましくは０．０００５〜０．１％（ｗ／ｖ）、より好ましくは０．００１〜０．０１％（ｗ／ｖ）含有する溶液を０．５〜１０分間、好ましくは１〜７分間、より好ましくは２〜５分間かけてインジェクトする。この時、移動相（バッファー）は、好ましくはＨＢＳ−Ｐ、ＨＢＳ−Ｎ、ＰＢＳ、ＨＢＳ−ＥＰなどが用いられ、５〜５０μＬ／分、好ましくは１０〜４０μＬ／分、より好ましくは２０〜３０μＬ／分の流速で流すとよい。薬物のインジェクション開始前のＲＵ値とインジェクション終了１〜１０分後、好ましくは３〜７分後、より好ましくは５分後のＲＵ値の差（ΔＲＵ）を求めることにより、本発明の組成物に含まれる好ましい薬物を、使用する細胞膜透過性ペプチドに応じて選択することができる。 The evaluation system using surface plasmon resonance (SPR) measurement is performed by measuring the resonance unit (RU) value using a surface plasmon resonance analyzer, preferably, for example, Biacore T200 (GE Healthcare). It is a system to evaluate the strength of the interaction between cell membrane permeable peptide and drug. The cell membrane permeable peptide may be directly immobilized on the sensor chip via an amide bond or disulfide bond, or may be immobilized via a biotin-avidin interaction, for example, the cell membrane permeable peptide is shared with biotin It is preferable to immobilize the bound molecule via avidin on a sensor chip (eg, Series S Sensor Chip SA / GE Healthcare). After immobilizing the cell membrane permeable peptide on the sensor chip, the drug is preferably 0.0001 to 1% (w / v), preferably 0.0005 to 0.1% (w / v), more preferably 0.001 to 1%. The solution containing 0.01% (w / v) is injected for 0.5 to 10 minutes, preferably 1 to 7 minutes, more preferably 2 to 5 minutes. At this time, the mobile phase (buffer) is preferably HBS-P, HBS-N, PBS, HBS-EP, etc., and is 5 to 50 μL / min, preferably 10 to 40 μL / min, more preferably 20 to 30 μL. It is good to flow at a flow rate of / min. The composition of the present invention can be obtained by determining the difference (ΔRU) between the RU value before the start of injection of the drug and the RU value 1 to 10 minutes after the end of injection, preferably 3 to 7 minutes, more preferably 5 minutes. Preferred drugs to be included can be selected depending on the cell membrane permeable peptide used.
本発明の組成物に含まれる好ましい薬物は、表面プラズモン共鳴分析試験を利用した評価系において測定に用いた薬物を含有する溶液の薬物濃度によっても異なるが、ΔＲＵ×１００／Ｍｗ（ここで、Ｍｗは薬物の分子量（Ｄａ））の値が０．０１以上、好ましくは０．１以上、より好ましくは０．２以上、特に好ましくは０．４以上である薬物である。本発明の組成物に含まれる好ましい薬物として、ΔＲＵ×１００／Ｍｗの上限に特に制限はないが、好ましくは３００以下、より好ましくは２００以下、特に好ましくは１５０以下、最も好ましくは１００以下である。本発明の組成物に含まれる好ましい薬物は、ΔＲＵ×１００／Ｍｗが０．０１〜３００、より好ましくは０．１〜２００、特に好ましくは０．２〜１５０、最も好ましくは０．４〜１００である薬物である。これらΔＲＵは、使用する細胞膜透過性ペプチド毎に測定できる。 Although the preferable drug contained in the composition of the present invention varies depending on the drug concentration of the solution containing the drug used for measurement in the evaluation system using surface plasmon resonance analysis test, ΔRU × 100 / Mw (where, Mw Is a drug having a molecular weight (Da) value of 0.01 or more, preferably 0.1 or more, more preferably 0.2 or more, particularly preferably 0.4 or more. The preferred drug contained in the composition of the present invention is not particularly limited to the upper limit of ΔRU × 100 / Mw, but is preferably 300 or less, more preferably 200 or less, particularly preferably 150 or less, most preferably 100 or less . The preferred drug contained in the composition of the present invention has a ΔRU × 100 / Mw of 0.01 to 300, more preferably 0.1 to 200, particularly preferably 0.2 to 150, most preferably 0.4 to 100 Is a drug. These ΔRU can be measured for each cell membrane permeable peptide used.
本発明の組成物に薬物を配合する場合、その含有量は、薬物の種類等により適宜調整することができ、０．００００１〜１０％（ｗ／ｖ）が好ましく、０．０００５〜５％（ｗ／ｖ）がより好ましく、０．００１〜３％（ｗ／ｖ）がさらに好ましく、０．００１〜２％（ｗ／ｖ）が最も好ましい。 When a drug is added to the composition of the present invention, the content thereof can be appropriately adjusted according to the type of drug etc., and 0.00001 to 10% (w / v) is preferable, 0.0005 to 5% (w / v) w / v) is more preferable, 0.001 to 3% (w / v) is more preferable, and 0.001 to 2% (w / v) is the most preferable.
本発明の医薬組成物は、例えば、溶液のほか懸濁剤、エマルジョン、リポソーム、マイクロスフェア、デポ剤などであってもよく、溶媒又は分散媒は水であることが好ましく、水溶液であることが最も好ましい。 The pharmaceutical composition of the present invention may be, for example, a solution, a suspension, an emulsion, a liposome, a microsphere, a depot and the like, and the solvent or dispersion medium is preferably water, and an aqueous solution. Most preferred.
本発明の組成物の剤形は、眼科用医薬品として使用可能なものであれば特に制限されないが、例えば、点眼剤、眼軟膏、注射剤が挙げられ、点眼剤が好ましい。注射剤としては、硝子体内投与、前房内投与、結膜嚢内投与、前房内投与、結膜下投与又はテノン嚢下投与用の注射剤が挙げられる。これらの剤形は、当該技術分野における通常の方法に従って製造することができる。 The dosage form of the composition of the present invention is not particularly limited as long as it can be used as an ophthalmic drug, and examples include eye drops, eye ointments, and injections, and eye drops are preferable. Injections include injections for intravitreal administration, administration in the anterior chamber, administration in the conjunctival sac, administration in the anterior chamber, administration under the conjunctiva, or subtenon administration. These dosage forms can be manufactured according to a method conventional in the art.
本発明の組成物は、薬物の眼組織への移行性に優れる。薬物が移行される眼組織は、角膜のほか、結膜、ブドウ膜、眼瞼、前房、毛様体、虹彩、水晶体、硝子体、網膜、脈絡膜等が挙げられる。 The composition of the present invention is excellent in transferability of the drug to ocular tissues. Eye tissues to which a drug is transferred include conjunctiva, uvea, eyelid, anterior chamber, ciliary body, iris, lens, vitreous body, retina, choroid, etc. in addition to cornea.
上記の本発明の組成物の詳細な説明は、本発明のドラッグデリバリーシステム、薬物の眼組織への移行性を向上させる方法にも適用される。 The above detailed description of the composition of the present invention also applies to the drug delivery system of the present invention, a method of improving the transferability of a drug to ocular tissue.
本発明のドラッグデリバリーシステムは、細胞膜透過性ペプチド又はその塩を含有し、送達対象の薬物と共有結合させずに用いられる眼科用ドラッグデリバリーシステムである。 The drug delivery system of the present invention is an ophthalmic drug delivery system containing a cell membrane permeable peptide or a salt thereof and used without being covalently linked to a drug to be delivered.
本発明の薬物の眼組織への移行性を向上させる方法は、眼科用医薬組成物に細胞膜透過性ペプチド又はその塩を薬物と共有結合させずに含有させることを含む。 The method for improving the transferability of the drug of the present invention to ocular tissues comprises the inclusion of a cell membrane permeable peptide or a salt thereof in the ophthalmic pharmaceutical composition without being covalently linked to the drug.
本発明の方法によれば、薬物の眼組織への移行性が向上し、より低用量の薬物で薬効を発揮することができる。 According to the method of the present invention, the transferability of the drug to ocular tissues is improved, and the drug effect can be exerted with a lower dose of drug.
以下に製剤例、薬物透過性試験、安全性試験及び表面プラズモン共鳴分析試験の結果を示すが、これらは本発明をより良く理解するためのものであり、本発明の範囲を限定するものではない。 The following shows the results of formulation examples, drug permeability tests, safety tests and surface plasmon resonance analysis tests, but these are for the purpose of better understanding the present invention and do not limit the scope of the present invention. .
製剤例
以下に本発明の代表的な製剤例を示す。なお、下記製剤例において各成分の配合量は製剤１ｍＬ中の含量である。
Formulation Examples Representative formulation examples of the present invention are shown below. In addition, the compounding quantity of each component is a content in 1 mL of formulation in the following formulation example.
製剤例１
Ｒ８−ＮＨ２ １０ｍｇ
リン酸緩衝液 適量
精製水 適量
Formulation example 1
R8-NH 2 10mg
Phosphate buffer solution appropriate amount Purified water appropriate amount
製剤例２
Ｒ８ １０ｍｇ
リン酸緩衝液 適量
精製水 適量
Formulation example 2
R8 10 mg
Phosphate buffer solution appropriate amount Purified water appropriate amount
製剤例３
ＨＩＶ−ＴＡＴ （４８−６０） １０ｍｇ
リン酸緩衝液 適量
精製水 適量
Formulation example 3
HIV-TAT (48-60) 10 mg
Phosphate buffer solution appropriate amount Purified water appropriate amount
なお、前記製剤例１〜３における細胞膜透過性ペプチド及び添加剤の種類や配合量、並びに、ｐＨを適宜調整し所望の組成物を得ることができる。 In addition, the kind and compounding quantity of the cell membrane permeable peptide in the said preparation examples 1-3, and a compounding quantity, and pH can be adjusted suitably, and a desired composition can be obtained.
１．薬物移行性試験１
１−１．被験製剤の調製
Ｒ８−ＮＨ２・９ＴＦＡ塩（９．０９ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、１０００μＭの溶液を調製した（溶液Ａ）。６−カルボキシフルオレセイン（６−ＦＡＭ）（７．５２ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、２０００μＭの溶液を調製した（溶液Ｂ）。
溶液Ｂを０．９６％ＰＢＳ溶液で希釈して、１０００μＭの６−ＦＡＭ溶液を調製し比較例１とした。
溶液Ｂと０．９６％ＰＢＳ溶液で５０倍希釈した溶液Ａを１：１で混合したものを実施例１とした。溶液Ａと溶液Ｂを１：１で混合したものを実施例２とした。
1. Drug transferability test 1
1-1. Preparation of Test Preparation The R8-NH 2 · 9 TFA salt (9.09 mg) was dissolved in 0.96% PBS solution to prepare a solution of 1000 μM (solution A). 6-Carboxyfluorescein (6-FAM) (7.52 mg) was dissolved in 0.96% PBS solution to prepare a 2000 μM solution (solution B).
Solution B was diluted with a 0.96% PBS solution to prepare a 1000 μM 6-FAM solution, which was referred to as Comparative Example 1.
A mixture of solution B and solution A diluted 50-fold with a 0.96% PBS solution at 1: 1 was used as Example 1. A mixture of solution A and solution B at 1: 1 was designated as Example 2.
１−２．試験方法
ヒト３次元角膜上皮（ＬａｂＣｙｔｅ ＣＯＲＮＥＡ−ＭＯＤＥＬ，Ｊ−ＴＥＣ社製）を付属の培地中でＣＯ２インキュベータ内にて１時間培養後、各ウェルの培地を除去し上記サンプルを５０μＬずつ添加し、ＣＯ２インキュベータ内で５分間暴露した。上清を除去し、細胞表面にＰＢＳを５００ｕＬずつ添加し、吸引除去する操作を３回繰り返した。ＰＢＳを５０ｕＬずつ添加し、蛍光顕微鏡（ＢＺ−Ｘ７００）で各ウェルのフルオレセインの蛍光顕微鏡写真を撮影した（露光時間；５０ｍｓ）。顕微鏡写真は画像解析ソフトＩｍａｇｅＪのＡｎａｌｙｚｅメニューのＭｅａｓｕｒｅモードを用いて解析した。
1-2. Test method After culturing human three-dimensional corneal epithelium (LabCyte CORNEA-MODEL, manufactured by J-TEC Co., Ltd.) for 1 hour in a CO 2 incubator in the attached medium, the medium in each well is removed and 50 μL of the above sample is added, Exposed for 5 minutes in a CO2 incubator. The supernatant was removed, and 500 uL each of PBS was added to the cell surface, and the operation of aspiration and removal was repeated three times. 50 uL of PBS was added, and a fluorescence micrograph of fluorescein in each well was taken with a fluorescence microscope (BZ-X700) (exposure time; 50 ms). Photomicrographs were analyzed using the Measure mode of the Analyze menu of the image analysis software ImageJ.
１−３．試験結果及び考察
試験結果を図１に示す。図１から分かるように、Ｒ８−ＮＨ２非共存下に比べて、Ｒ８−ＮＨ２共存下で６−ＦＡＭの角膜上皮細胞への移行率が向上した。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、薬物の眼組織への移行性を向上させることが分かった。
1-3. Test Results and Discussion The test results are shown in FIG. As it can be seen from Figure 1, compared to the R8-NH 2 non coexistence, and improved transfer rate to the corneal epithelial cells of 6-FAM at 2 coexistence R8-NH. From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof improves the transferability of the drug to ocular tissues.
２．薬物移行性試験２
２−１．被験製剤の調製
Ｒ８−ＮＨ２・９ＴＦＡ塩（４．５４ｍｇ）を０．９６％ＰＢＳ溶液に溶解させ、１０００μＭの溶液を調製した（溶液Ｃ）。フルオレセイン（３．３４ｇ）を０．９６％ＰＢＳ溶液に溶解させ、５００μＭの溶液を調製した（溶液Ｄ）。
溶液Ｄを０．９６％ＰＢＳ溶液で希釈して、２５０μＭの フルオレセイン溶液を調製し比較例２とした。
また、溶液Ｃと溶液Ｄを１：１で混合したものを実施例３とした。
2. Drug transferability test 2
2-1. Preparation of Test Preparation The R8-NH 2 · 9 TFA salt (4.54 mg) was dissolved in 0.96% PBS solution to prepare a solution of 1000 μM (solution C). Fluorescein (3.34 g) was dissolved in 0.96% PBS solution to prepare a 500 μM solution (solution D).
Solution D was diluted with a 0.96% PBS solution to prepare a 250 μM fluorescein solution, which was referred to as Comparative Example 2.
Moreover, what mixed the solution C and the solution D by 1: 1 was made into Example 3.
２−２．試験方法
薬物移行性試験１と同様の方法により試験を行った。蛍光顕微鏡写真の撮影の露光時間は１０ｍｓとした。
2-2. Test Method A test was conducted in the same manner as in the drug transferability test 1. The exposure time for taking a fluorescence micrograph was 10 ms.
２−３．試験結果及び考察
試験結果を図２に示す。図２から分かるように、Ｒ８−ＮＨ２非共存下に比べて、Ｒ８−ＮＨ２共存下でフルオレセインの角膜上皮細胞への移行率が向上した。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、薬物の眼組織への移行性を向上させることが分かった。
2-3. Test Results and Discussion The test results are shown in FIG. As can be seen from Figure 2, as compared with R8-NH 2 non coexistence, migration rates of fluorescein to the corneal epithelial cells was improved in two coexistence R8-NH. From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof improves the transferability of the drug to ocular tissues.
３．薬物移行性試験３
３−１．被験製剤の調製
ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩（０．３ｍｇ）を０．９６％ＰＢＳ溶液に溶解させ、１０００μＭの溶液を調製した（溶液Ｅ）。
溶液Ｂと溶液Ｅを１：１で混合したものを実施例４とした。
3. Drug transferability test 3
3-1. Preparation of test preparation The penetratin-NH 2 · 7 TFA salt (0.3 mg) was dissolved in a 0.96% PBS solution to prepare a 1000 μM solution (solution E).
A mixture of solution B and solution E at 1: 1 was named Example 4.
３−２．試験方法
薬物移行性試験１と同様の方法により試験を行った。蛍光顕微鏡写真の撮影の露光時間は５０ｍｓとした。
3-2. Test Method A test was conducted in the same manner as in the drug transferability test 1. The exposure time for taking a fluorescence micrograph was 50 ms.
３−３．試験結果及び考察
試験結果を図３に示す。図３から分かるように、ｐｅｎｅｔｒｔｉｎ−ＮＨ２非共存下に比べて、ｐｅｎｅｔｒａｔｉｎ−ＮＨ２共存下で６−カルボキシフルレセイン（６ＦＡＭ）の角膜上皮細胞への移行率が向上した。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、薬物の眼組織への移行性を向上させることが分かった。
3-3. Test Results and Discussion The test results are shown in FIG. As can be seen from FIG. 3, the rate of transfer of 6-carboxyflurescein (6FAM) to corneal epithelial cells was improved in the presence of penetratin-NH 2 as compared to the absence of penetrtin-NH 2 . From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof improves the transferability of the drug to ocular tissues.
４．安全性試験１
４−１．被験製剤の調製
Ｒ８−ＮＨ２・９ＴＦＡ塩（９．０９ｍｇ）を０．９６％ＰＢＳ溶液に溶解させ、５００μＭの溶液を調製した（溶液Ｆ）。溶液Ｆを０．９６％ＰＢＳ溶液で希釈して、１０μＭ、１００μＭ、５００μＭの溶液を調製し、それぞれ実施例５、実施例６、実施例７とした。
4. Safety test 1
4-1. Preparation of Test Preparation The R8-NH 2 · 9 TFA salt (9.09 mg) was dissolved in 0.96% PBS solution to prepare a 500 μM solution (solution F). The solution F was diluted with a 0.96% PBS solution to prepare solutions of 10 μM, 100 μM, and 500 μM, respectively, which were referred to as Example 5, Example 6, and Example 7, respectively.
４−２．試験方法
ＭＴＴ試薬（６．７４ｍｇ）を１３．４８ｍｌのアッセイ培地で希釈して調製したＭＴＴ培地を０．５ｍＬずつ新たな２４穴プレートに分注し、０．９６％ＰＢＳ溶液（陰性対照）、実施例５、実施例６または実施例７に２時間暴露したヒト３次元角膜上皮（ＬａｂＣｙｔｅ ＣＯＲＮＥＡ−ＭＯＤＥＬ，Ｊ−ＴＥＣ社製）の入ったウェルを上述のプレートに設置した。ＣＯ２インキュベータ内で３時間静置し、各ウェルを取り外し、細胞部分を切り取った。切り取った細胞を１．５ｍＬのＰＰチューブに入れ３００ ｕＬのイソプロパノールに２時間以上浸漬した。このイソプロパノールを９６ウェルプレートに２００μＬずつ注入し、吸光度（５４０ｎｍ−６５５ｎｍの吸光度の差を使用）をプレートリーダーで測定し以下の計算式で生細胞率を算出した。
４−３．試験結果及び考察
試験結果を図４に示す。図４から分かるように、１０〜５００μＭのＲ８−ＮＨ２を２時間暴露しても角膜細胞に対して細胞障害性を示さなかった。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、眼科適用できる安全性を示すことが分かった。
4-3. Test Results and Discussion The test results are shown in FIG. As can be seen from FIG. 4, exposure to 10 to 500 μM of R8-NH 2 for 2 hours did not show any cytotoxicity on corneal cells. From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof exhibits ophthalmologically applicable safety.
５．安全性試験２
５−１．被験製剤の調製
上記１−１．で調製した比較例１及び３−１．で調製した実施例４を用いた。
5. Safety test 2
5-1. Preparation of test preparation 1-1. Comparative Examples 1 and 3-1 prepared by Example 4 prepared in the above was used.
５−２．試験方法
ＭＴＴ試薬（６．７４ｍｇ）を１３．４８ｍｌのアッセイ培地で希釈して調製したＭＴＴ培地を０．５ｍＬずつ新たな２４穴プレートに分注し、０．９６％ＰＢＳ溶液（陰性対照）、実施例４または比較例１に５分間暴露したヒト３次元角膜上皮（ＬａｂＣｙｔｅ ＣＯＲＮＥＡ−ＭＯＤＥＬ，Ｊ−ＴＥＣ社製）の入ったウェルを上述のプレートに設置した。ＣＯ２インキュベータ内で２時間静置し、各ウェルを取り外し、細胞部分を切り取った。切り取った細胞を１．５ｍＬのＰＰチューブに入れ３００ ｕＬのイソプロパノールに２時間以上浸漬した。このイソプロパノールを９６ウェルプレートに２００μＬずつ注入し、吸光度（５４０ｎｍ−６５５ｎｍの吸光度の差を使用）をプレートリーダーで測定し「数１」の計算式で生細胞率を算出した。
5-2. Test method 0.5 ml of MTT medium prepared by diluting MTT reagent (6.74 mg) in 13.48 ml of assay medium is aliquoted into fresh 24-well plates, 0.96% PBS solution (negative control), A well containing human three-dimensional corneal epithelium (LabCyte CORNEA-MODEL, manufactured by J-TEC) exposed for 5 minutes to Example 4 or Comparative Example 1 was placed on the above-mentioned plate. After standing for 2 hours in a CO2 incubator, each well was removed and the cell portion was cut off. The cut cells were placed in a 1.5 mL PP tube and immersed in 300 uL of isopropanol for 2 hours or more. 200 μL each of this isopropanol was injected into a 96-well plate, and the absorbance (using the difference in absorbance at 540 nm-655 nm) was measured with a plate reader, and the viable cell rate was calculated by the formula of "Number 1."
５−３．試験結果及び考察
試験結果を図５に示す。図５から分かるように、５００μＭのｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩も角膜細胞に対して細胞障害性を示さなかった。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、眼科適用できる安全性を示すことが分かった。
5-3. Test Results and Discussion The test results are shown in FIG. As can be seen from FIG. 5, 500 μM of penetratin-NH 2 · 7 TFA salt also showed no cytotoxicity to corneal cells. From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof exhibits ophthalmologically applicable safety.
６．表面プラズモン共鳴分析試験
６−１ ビオチン化細胞膜透過性ペプチドの合成
Ｆｍｏｃ−Ｌ−アルギニンとＲｉｎｋ ａｍｉｄｅ ＰＥＧＡ ｒｅｓｉｎ（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒｅ，Ｎｏｖａｂｉｏｃｈｅｍ（登録商標））を用いて固相合成したＦｍｏｃ−Ｒ８−レジン（０．１２４ｍｍｏｌ）を２０％ Ｐｉｐｅｒｉｄｉｎｅ／ＤＭＦ８ｍｌ中で１時間常温で撹拌し脱Ｆｍｏｃを行った。
レジンをＤＭＦ、ジクロロメタン、メタノールで洗浄後、ビオチン（ｂｉｏｔｉｎ） ９１．３ ｍｇ，縮合剤（ＨＢＴＵ）１４２．４ ｍｇ，ジイソプロピルエチルアミン ６４．５ μＬ，ＤＭＦ １１ｍｌを加え終夜室温で撹拌した。反応後、レジンを洗浄し乾燥した。
6. Surface Plasmon Resonance Analysis Test 6-1 Synthesis of Biotinylated Cell Membrane Permeable Peptide Fmoc-R8-Resin (0) solid phase synthesized using Fmoc-L-arginine and Rinkamide PEGA resin (Merck Millipore, Novabiochem®) .24 mmol) was stirred in 8 ml of 20% Piperidine / DMF for 1 hour at room temperature to remove Fmoc.
After washing the resin with DMF, dichloromethane and methanol, 91.3 mg of biotin (biotin), 142.4 mg of condensing agent (HBTU), 64.5 μL of diisopropylethylamine and 11 ml of DMF were added and stirred overnight at room temperature. After the reaction, the resin was washed and dried.
ビオチン化Ｒ８−レジン（７６４．３ ｍｇ）をＴＦＡ、フェノール、チオアニソール、水、エタンジチオール（８３：６．３：４．３：４．３：２．１）混合溶液を用いて室温で４時間撹拌した。反応後レジンを濾過にて除去した。濾液を減圧濃縮し溶媒を留去したのち分取ＨＰＬＣ（０．１％ＴＦＡ／アセトニトリル系）により精製を行い、目的のビオチン化Ｒ８−ＮＨ２・８ＴＦＡ塩を得た（４６．７ｍｇ）。また、ビオチン化Ｐｅｎｅｔｒａｔｉｎ−ＮＨ２、ビオチン化Ｄ−Ｒ８−ＮＨ２、ビオチン化ＡＲＫＫＡＡＫＡ−ＮＨ２ビオチン化ＴＡＴ（４７−５７）−ＮＨ２、ビオチン化ＨＩＶ−ＴＡＴ （４８−６０）−ＮＨ２は、ビオチン化Ｒ８−ＮＨ２と同様の方法により合成した。 Biotinylated R8-Resin (764.3 mg) in TFA, phenol, thioanisole, water, ethanedithiol (83: 6.3: 4.3: 4.3: 2.1) mixed solution 4 at room temperature Stir for hours. After the reaction, the resin was removed by filtration. The filtrate was purified by preparative HPLC Chi was distilled off and the solvent was concentrated under reduced pressure (0.1% TFA / acetonitrile system) to obtain biotinylated R8-NH 2 · 8TFA salt of target (46.7 mg). Furthermore, biotinylated Penetratin-NH 2, biotinylated D-R8-NH 2, biotinylated ARKKAAKA-NH 2 biotinylated TAT (47-57) -NH 2, biotinylated HIV-TAT (48-60) -NH 2 has It was synthesized in the same manner as biotinylated R8-NH 2.
６−２．ビオチン化細胞膜透過性ペプチド溶液の調製
ビオチン化Ｒ８−ＮＨ２・８ＴＦＡ塩（１．４９ｍｇ）を４．９ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア） に溶解させ１０倍希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し３０μｇ／ｍｌの溶液を調製した。
6-2. Preparation of biotinylated cell membrane permeable peptide solution Biotinylated R8-NH 2 · 8 TFA salt (1.49 mg) is dissolved in 4.9 ml of buffer (HBS-EP buffer / GE Healthcare) and diluted 10 times and centrifuged ( 12000 rpm, 10 minutes) and a solution of 30 μg / ml was prepared.
また、同様にビオチン化Ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩（１．３３ｍｇ）を４．４ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア） に溶解させ１０倍希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し３０μｇ／ｍｌのビオチン化Ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩溶液を調製した。
同様にビオチン化Ｄ−Ｒ８−ＮＨ２・８ＴＦＡ塩（２．１３ｍｇ）を７．１ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア） に溶解させ１０倍希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し３０μｇ／ｍｌの溶液を調製した。
ビオチン化ＡＲＫＫＡＡＫＡ−ＮＨ２・４ＴＦＡ塩（０．４ｍｇ）を１．３ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア） に溶解させ１０倍希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し３０μｇ／ｍｌの溶液を調製した。
ビオチン化ＴＡＴ（４７−５７）−ＮＨ２・８ＴＦＡ塩（１．６ｍｇ）を５．４ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア） に溶解させ１０倍希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し３０μｇ／ｍｌの溶液を調製した。
ビオチン化ＨＩＶ−ＴＡＴ （４８−６０）−ＮＨ２・８ＴＦＡ塩（４．２ｍｇ）を１３．９ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア） に溶解させ１０倍希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し３０μｇ／ｍｌの溶液を調製した。
Similarly, biotinylated Penetratin-NH 2 · 7 TFA salt (1.33 mg) is dissolved in 4.4 ml of buffer (HBS-EP buffer / GE healthcare) and diluted 10-fold, and centrifuged (12000 rpm, 10 minutes) ) 30 μg / ml of biotinylated Penetratin-NH 2 · 7 TFA salt solution was prepared.
Similarly 10-fold diluted dissolved biotinylated D-R8-NH 2 · 8TFA salt (2.13 mg) in a buffer (HBS-EP buffer / GE Healthcare) in 7.1 ml, centrifuged (12000 rpm, 10 minutes ) A solution of 30 μg / ml was prepared.
Biotinylated ARKKAAKA-NH 2 · 4TFA salt (0.4 mg) and post-buffer 10x dissolved in (HBS-EP buffer / GE Healthcare) diluted in 1.3 ml, centrifuged (12000 rpm, 10 minutes) and 30 [mu] g / A solution of ml was prepared.
Biotinylated TAT (47-57) -NH 2 · 8 TFA salt (1.6 mg) is dissolved in 5.4 ml of buffer (HBS-EP buffer / GE Healthcare) and diluted 10-fold and centrifuged (12000 rpm, 10) The solution was prepared at 30 μg / ml.
Biotinylated HIV-TAT (48-60) -NH 2 · 8TFA salt (4.2 mg) is dissolved in 13.9 ml of buffer (HBS-EP buffer / GE healthcare) and diluted 10-fold and centrifuged (12000 rpm) , 10 minutes) and a solution of 30 μg / ml was prepared.
６−３．被験溶液の調製
６−カルボキシフルオレセイン（６−ＦＡＭ）（３．７５ｍｇ）を１０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液１とした。
ローダミン１２３（１．９５ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解させ遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液２とした。
フルオレセイン（３．３２ｍｇ）を ２０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解させ遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液３とした。
ＦＤ４（８．１９ｍｇ）を ２ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液４とした。
ＦＤ１０（１９．６３ｍｇ）を ２ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液５とした。
リン酸プレドニゾロン（４．８４ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製被験溶液６とした。
ペミロラストカリウム（２．６７ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液７とした。
ホスミドマイシン（１．１０ｍｇ）を ２ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液８とした。
リン酸ベタメサゾン（１．８ｍｇ）を ３．５ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液９とした。
ジクロフェナクナトリウム（１．１ｍｇ）を ３．５ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液１０とした。
リン酸ヒドロコルチゾン（０．５ｍｇ）を １．２ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し５００μＭの溶液を調製し被験溶液１１とした。
ブリンゾラミド（３．８ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、１０００μＭの溶液を調製した。本溶液をさらにバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）で５００μＭに希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し、被験溶液１２とした。
ドルゾラミド塩酸塩（３．６ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、遠心分離（１２０００ ｒｐｍ，１０分）し１０００μＭの溶液を調製した。本溶液をさらにバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）で５００μＭに希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し、被験溶液１３とした。
オロパタジン塩酸塩（３．７ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、１０００μＭの溶液を調製した。本溶液をさらにバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）で５００μＭに希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し、被験溶液１４とした。
プロプラノロール塩酸塩（３．０ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、１０００μＭの溶液を調製した（溶液Ｄ）。本溶液をさらにバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）で５００μＭに希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し、被験溶液１５とした。
チモロールマレイン酸塩（４．３ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、１０００μＭの溶液を調製した。本溶液をさらにバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）で５００μＭに希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し、被験溶液１６とした。
ブリモニジン酒石酸塩（４．４ｍｇ）を １０ｍｌのバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）に溶解し、１０００μＭの溶液を調製した（溶液Ｇ）。本溶液をさらにバッファー（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）で５００μＭに希釈後、遠心分離（１２０００ ｒｐｍ，１０分）し、被験溶液１７とした。
6-3. Preparation of test solution 6-Carboxyfluorescein (6-FAM) (3.75 mg) is dissolved in 10 ml of buffer (HBS-EP buffer / GE Healthcare), centrifuged (12000 rpm, 10 minutes) and 500 μM solution It was prepared as a test solution 1.
Rhodamine 123 (1.95 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was designated as test solution 2.
Fluorescein (3.32 mg) was dissolved in 20 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was designated as test solution 3.
FD4 (8.19 mg) was dissolved in 2 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was designated as test solution 4.
FD10 (19.63 mg) was dissolved in 2 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was designated as test solution 5.
Prednisolone phosphate (4.84 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution as prepared test solution 6.
Pemirolast potassium (2.67 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was used as test solution 7.
Phosmidomycin (1.10 mg) was dissolved in 2 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was designated as test solution 8.
Betamethasone phosphate (1.8 mg) was dissolved in 3.5 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was used as a test solution 9.
Diclofenac sodium (1.1 mg) was dissolved in 3.5 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was used as a test solution 10.
Hydrocortisone phosphate (0.5 mg) was dissolved in 1.2 ml of buffer (HBS-EP buffer / GE Healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 500 μM solution, which was used as a test solution 11.
Brinzolamide (3.8 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE Healthcare) to prepare a 1000 μM solution. This solution was further diluted to 500 μM with buffer (HBS-EP buffer / GE healthcare), and centrifuged (12000 rpm, 10 minutes) to obtain a test solution 12.
Dorzolamide hydrochloride (3.6 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE healthcare) and centrifuged (12000 rpm, 10 minutes) to prepare a 1000 μM solution. The solution was further diluted with buffer (HBS-EP buffer / GE healthcare) to 500 μM and centrifuged (12000 rpm, 10 minutes) to obtain a test solution 13.
Olopatadine hydrochloride (3.7 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE healthcare) to prepare a 1000 μM solution. The solution was further diluted with buffer (HBS-EP buffer / GE healthcare) to 500 μM and centrifuged (12000 rpm, 10 minutes) to obtain a test solution 14.
Propranolol hydrochloride (3.0 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE healthcare) to prepare a solution of 1000 μM (solution D). The solution was further diluted with buffer (HBS-EP buffer / GE healthcare) to 500 μM and centrifuged (12000 rpm, 10 minutes) to obtain a test solution 15.
Timolol maleate (4.3 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE Healthcare) to prepare a 1000 μM solution. The solution was further diluted with buffer (HBS-EP buffer / GE healthcare) to 500 μM and centrifuged (12000 rpm, 10 minutes) to obtain a test solution 16.
Brimonidine tartrate (4.4 mg) was dissolved in 10 ml of buffer (HBS-EP buffer / GE Healthcare) to prepare a solution of 1000 μM (solution G). The solution was further diluted with buffer (HBS-EP buffer / GE healthcare) to 500 μM and centrifuged (12000 rpm, 10 minutes) to obtain a test solution 17.
６−４．試験方法
表面プラズモン共鳴分析装置（ＢｉａｃｏｒｅＴ２００／ＧＥヘルスケア）を用いて、ビオチン化Ｒ８−ＮＨ２溶液又はビオチン化Ｐｅｎｅｔｒａｔｉｎ−ＮＨ２、ビオチン化Ｄ−Ｒ８−ＮＨ２、ビオチン化ＡＲＫＫＡＡＫＡ−ＮＨ２、ビオチン化ＴＡＴ（４７−５７）−ＮＨ２、ビオチン化ＨＩＶ−ＴＡＴ （４８−６０）−ＮＨ２をそれぞれセンサーチップ（Ｓｅｒｉｅｓ Ｓ Ｓｅｎｓｏｒ Ｃｈｉｐ ＳＡ／ＧＥヘルスケア）に固定化（ｆｌｏｗ ｒａｔｅ ： ５ μＬ／ｍｉｎ、ｉｎｊｅｃｔｉｏｎ ｔｉｍｅ：５ｍｉｎ）した後、被験溶液を１００μＬ注入した（ｆｌｏｗ ｒａｔｅ ： ２０μＬ／ｍｉｎ、ｉｎｊｅｃｔｉｏｎ ｔｉｍｅ：５ｍｉｎ）。バッファーは（ＨＢＳ−ＥＰ ｂｕｆｆｅｒ ／ＧＥヘルスケア）を用いた。被験溶液のインジェクション開始前のＲＵ値（ｂａｓｅｌｉｎｅ）とインジェクション５分後のＲＵ値（ｂｉｎｄｉｎｇ）を測定してその差（ΔＲＵ）を求め、ΔＲＵ×１００／Ｍｗ（ここで、Ｍｗは薬物の分子量（Ｄａ））を相互作用の指標とした。なお、被験溶液のインジェクション前後のＲＵ値は、２流路使用により、Ｒ８−ＮＨ２ センサーチップ固定化面のＲＵ値からセンサーチップの非固定化面のＲＵ値をブランクとして引いた値を測定した。また、次の被験溶液を注入する前には、１ＭＮａＣｌ水溶液を３０秒流しセンサーチップを再生した。
6-4. Test method Biotinylated R8-NH 2 solution or biotinylated Penetratin-NH 2 , biotinylated D-R8-NH 2 , biotinylated ARKKAAKA-NH 2 , biotin using a surface plasmon resonance analyzer (Biacore T200 / GE healthcare) Immobilized TAT (47-57) -NH 2 and biotinylated HIV-TAT (48-60) -NH 2 on the sensor chip (Series S Sensor Chip SA / GE Healthcare) (flow rate: 5 μL / min) , Injection time: 5 min), 100 μL of test solution was injected (flow rate: 20 μL / min, injection time: 5 min). The buffer used was (HBS-EP buffer / GE healthcare). Measure the RU value (baseline) before the start of injection of the test solution and the RU value (binding) after 5 minutes of injection to determine the difference (ΔRU), ΔRU × 100 / Mw (where Mw is the molecular weight of the drug ( Da)) was used as an indicator of interaction. The RU value before and after injection of the test solution was determined by subtracting the RU value of the non-immobilized surface of the sensor chip from the RU value of the R8-NH 2 sensor chip-immobilized surface as a blank by using two channels. . In addition, before injecting the next test solution, the sensor chip was regenerated by flowing 1 M NaCl aqueous solution for 30 seconds.
６−５．試験結果及び考察
試験結果を図６から図１３に示す。Ｒ８−ＮＨ２の共存により角膜上皮細胞への移行率の向上が確認された６−カルボキシフルオレセイン（６−ＦＡＭ）及びフルオレセインは、表面プラズモン共鳴分析試験においてＲ８−ＮＨ２への高いアフィニティを示している（図６）。この事実は、細胞膜透過性ペプチドと薬物のアフィニティが、細胞膜透過性ペプチドを、眼組織への移行を促進すべき薬物の種類に応じて選択する際の指標となりうることを示唆している。
6-5. Test Results and Discussion The test results are shown in FIGS. R8-NH transfer rate improvement in the coexistence of the two corneal epithelial cells was confirmed 6-carboxyfluorescein (6-FAM) and fluorescein, show high affinity to R8-NH 2 in the surface plasmon resonance analysis test (Figure 6). This fact suggests that the affinity between the cell membrane permeable peptide and the drug can be an index in selecting the cell membrane permeable peptide depending on the type of drug to be promoted to the ocular tissue.
７．角膜上皮細胞による細胞膜透過性ペプチドの移行性試験１
７−１．６−ＦＡＭ結合細胞膜透過性ペプチドの合成
Ｆｍｏｃ−Ｌ−アルギニンとＲｉｎｋ ａｍｉｄｅ ＰＥＧＡ ｒｅｓｉｎ（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒｅ，Ｎｏｖａｂｉｏｃｈｅｍ（登録商標））を用いて固相合成したＦｍｏｃ−Ｒ８−レジン（０．１４０ｍｍｏｌ）を２０％ Ｐｉｐｅｒｉｄｉｎｅ／ＤＭＦ８ｍｌ中で１時間常温で撹拌し脱Ｆｍｏｃを行った。
レジンをＤＭＦ、ジクロロメタン、メタノールで洗浄後、６−カルボキシフルオレセイン（６−ＦＡＭ）１０５ｍｇ，縮合剤（ＨＢＴＵ）１６０．０ ｍｇ，ジイソプロピルエチルアミン ７３．０ μＬ，ＤＭＦ １０ｍｌを加え終夜室温で撹拌した。反応後、レジンを洗浄し乾燥した。
7. Transferability test of cell membrane permeable peptide by corneal epithelial cells 1
Synthesis of 7-1.6-FAM coupled cell membrane permeable peptide Fmoc-R8-resin (0.140 mmol) solid phase synthesized using Fmoc-L-arginine and Rink amide PEGA resin (Merck Millipore, Novabiochem (R)) ) Was stirred at room temperature for 1 hour in 8 ml of 20% Piperidine / DMF to carry out removal of Fmoc.
The resin was washed with DMF, dichloromethane, and methanol, then, 105 mg of 6-carboxyfluorescein (6-FAM), 160.0 mg of condensing agent (HBTU), 73.0 μL of diisopropylethylamine, and 10 ml of DMF were added and stirred overnight at room temperature. After the reaction, the resin was washed and dried.
６−ＦＡＭ−Ｒ８−レジン（６００．０ ｍｇ）をＴＦＡ、トリイソプロピルシラン、水、（９５：２．５：２．５）混合溶液を用いて室温で６時間撹拌した。反応後レジンを濾過にて除去した。濾液を減圧濃縮し溶媒を留去したのち分取ＨＰＬＣ（０．１％ＴＦＡ／アセトニトリル系）により精製を行い、目的の６−ＦＡＭ−Ｒ８−ＮＨ２・８ＴＦＡ塩を得た（３０．０ｍｇ）。また、６−ＦＡＭ−Ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩、６−ＦＡＭ−ＴＡＴ（４７−５７）−ＮＨ２・８ＴＦＡ塩、６−ＦＡＭ−ＨＩＶ−ＴＡＴ （４８−６０）−ＮＨ２・８ＴＦＡ塩、６−ＦＡＭ−ｐＶｅｃ−ＮＨ２・６ＴＦＡ塩は、６−ＦＡＭ−Ｒ８−ＮＨ２・８ＴＦＡ塩と同様の方法により合成した。 The 6-FAM-R8-resin (600.0 mg) was stirred at room temperature for 6 hours using a mixed solution of TFA, triisopropylsilane, water and (95: 2.5: 2.5). After the reaction, the resin was removed by filtration. The filtrate was purified by preparative HPLC Chi was distilled off and the solvent was concentrated under reduced pressure (0.1% TFA / acetonitrile system), to give the 6-FAM-R8-NH 2 · 8TFA salt of target (30.0 mg) . Also, 6-FAM-Penetratin-NH 2 · 7 TFA salt, 6-FAM-TAT (47-57) -NH 2 · 8 TFA salt, 6-FAM-HIV-TAT (48-60) -NH 2 · 8 TFA salt, 6-FAM-pVec-NH 2 · 6 TFA salt was synthesized by the same method as 6-FAM-R 8 -NH 2 · 8 TFA salt.
７−２．被験製剤の調製
６−ＦＡＭ−Ｒ８−ＮＨ２・８ＴＦＡ塩（１．２６ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１０μＭの６−ＦＡＭ−Ｒ８−ＮＨ２・８ＴＦＡ塩 溶液を調製し、実施例８とした。
６−ＦＡＭ−ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩（３．３９ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、１００μＭの溶液を調製し、実施例１３とした。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１０μＭの６−ＦＡＭ−ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ 塩溶液を調製し、実施例９とした。
６−ＦＡＭ−ＨＩＶ−ＴＡＴ（４７−５７）−ＮＨ２・８ＴＦＡ塩（１．４３ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１０μＭの６−ＦＡＭ−ＨＩＶ−ＴＡＴ（４７−５７）−ＮＨ２・８ＴＦＡ塩 溶液を調製し、実施例１０とした。
６−ＦＡＭ−ＨＩＶ−ＴＡＴ（４８−６０）−ＮＨ２・８ＴＦＡ塩（１．４５ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１０μＭの６−ＦＡＭ−ＨＩＶ−ＴＡＴ（４８−６０）−ＮＨ２・８ＴＦＡ塩 溶液を調製し、実施例１１とした。
６−ＦＡＭ−ｐＶｅｃ−ＮＨ２・６ＴＦＡ塩（１．３２ｍｇ） を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１０μＭの６−ＦＡＭ−ｐＶｅｃ−ＮＨ２・６ＴＦＡ塩 溶液を調製し、実施例１２とした。
６−カルボキシフルレセイン（６ＦＡＭ）（１．８９ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１０μＭの６−カルボキシフルレセイン（６ＦＡＭ）溶液を調製し、比較例３とした。
６−カルボキシフルレセイン（６ＦＡＭ）（３．７６ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、１００μＭの溶液に調製し、比較例４とした。
7-2. Preparation of test formulation 6-FAM-R8-NH 2 · 8TFA salt (1.26 mg) was dissolved in 0.96% PBS solution was prepared in a solution of 50 [mu] M. This solution was further diluted with a 0.96% PBS solution to prepare 10 μM of 6-FAM-R8-NH 2 · 8 TFA salt solution, which was named Example 8.
6-FAM-penetratin-NH 2 · 7 TFA salt (3.39 mg) was dissolved in 0.96% PBS solution to prepare a 100 μM solution, which is referred to as Example 13. This solution was further diluted with a 0.96% PBS solution to prepare 10 μM of 6-FAM-penetratin-NH 2 · 7TFA salt solution, which was named Example 9.
6-FAM-HIV-TAT (47-57) -NH 2 · 8 TFA salt (1.43 mg) was dissolved in 0.96% PBS solution to prepare a 50 μM solution. This solution was further diluted with 0.96% PBS solution to prepare 10 μM of 6-FAM-HIV-TAT (47-57) -NH 2 · 8 TFA salt solution, which was named Example 10.
6-FAM-HIV-TAT ( 48-60) -NH 2 · 8TFA salt (1.45 mg) was dissolved in 0.96% PBS solution was prepared in a solution of 50 [mu] M. This solution was further diluted with a 0.96% PBS solution to prepare 10 μM of 6-FAM-HIV-TAT (48-60) -NH 2 · 8 TFA solution, which was named Example 11.
6-FAM-pVec-NH 2 · 6 TFA salt (1.32 mg) was dissolved in 0.96% PBS solution to prepare a 50 μM solution. This solution was further diluted with 0.96% PBS solution to prepare 10 μM of 6-FAM-pVec-NH 2 .6TFA salt solution, which was named Example 12.
6-Carboxyflurescein (6FAM) (1.89 mg) was dissolved in 0.96% PBS solution to prepare a 50 μM solution. This solution was further diluted with a 0.96% PBS solution to prepare a 10 μM solution of 6-carboxyflurescein (6FAM), which was referred to as Comparative Example 3.
6-Carboxyflurescein (6FAM) (3.76 mg) was dissolved in 0.96% PBS solution to prepare a 100 μM solution, which was referred to as Comparative Example 4.
７−３．試験方法
ヒト３次元角膜上皮（ＬａｂＣｙｔｅ ＣＯＲＮＥＡ−ＭＯＤＥＬ，Ｊ−ＴＥＣ社製）を付属の培地中でＣＯ２インキュベータ内にて１時間培養後、各ウェルの培地を除去し上記サンプルを５０μＬずつ添加し、ＣＯ２インキュベータ内で２時間暴露した。上清を除去し、細胞表面にＰＢＳ溶液を５００ｕＬずつ添加し、吸引除去する操作を３回繰り返した。ＰＢＳ溶液を５０ｕＬずつ添加し、蛍光顕微鏡（ＢＺ−Ｘ７００）で各ウェルのフルオレセインの蛍光顕微鏡写真を撮影した（露光時間；５０ｍｓ）。顕微鏡写真は画像解析ソフトＩｍａｇｅＪのＡｎａｌｙｚｅメニューのＭｅａｓｕｒｅモードを用いて解析した。
7-3. Test method After culturing human three-dimensional corneal epithelium (LabCyte CORNEA-MODEL, manufactured by J-TEC Co., Ltd.) for 1 hour in a CO 2 incubator in the attached medium, the medium in each well is removed and 50 μL of the above sample is added, It was exposed for 2 hours in a CO2 incubator. The supernatant was removed, and 500 μL each of PBS solution was added to the cell surface, and the operation of removing by suction was repeated three times. 50 uL of PBS solution was added, and a fluorescence micrograph of fluorescein in each well was taken with a fluorescence microscope (BZ-X700) (exposure time; 50 ms). Photomicrographs were analyzed using the Measure mode of the Analyze menu of the image analysis software ImageJ.
７−４．試験結果及び考察
試験結果を図１４及び図１５に示す。図１４及び図１５から分かるように、Ｐｅｎｅｔｒａｔｉｎ、Ｒ８−ＮＨ２、ＨＩＶ−ＴＡＴ （４７−５７）、ＨＩＶ−ＴＡＴ （４８−６０）及びｐＶＥＣから誘導した蛍光性分子による蛍光が観測された。以上の結果により、細胞膜透過性ペプチド又はその塩は、角膜移行性を示すことが分かった。
7-4. Test Results and Discussion The test results are shown in FIG. 14 and FIG. As it can be seen from FIGS. 14 and 15, Penetratin, R8-NH 2 , HIV-TAT (47-57), fluorescence was observed by fluorescent molecules derived from HIV-TAT (48-60) and pVEC. From the above results, it was found that the cell membrane permeable peptide or a salt thereof exhibits corneal migration.
８．角膜上皮細胞による細胞膜透過性ペプチドの移行性試験２
８−１．６−ＦＡＭ結合細胞膜透過性ペプチドの合成
上記７−１．と同様の手法により、目的の６−ＦＡＭ−Ｌ１７Ｅ−ＮＨ２・１０ＴＦＡ塩、６−ＦＡＭ−２８９−Ｗ−ＮＨ２・７ＴＦＡ塩、６−ＦＡＭ−Ｌ−Ｋ６−ＮＨ２・８ＴＦＡ塩を得た。
8. Transferability test of cell membrane permeable peptide by corneal epithelial cells 2
Synthesis of 8-1.6-FAM linked cell membrane permeable peptide 7-1 above. In the same manner as to give 6-FAM-L17E-NH 2 · 10TFA salts purposes, 6-FAM-289-W -NH 2 · 7TFA salt, a 6-FAM-L-K6- NH 2 · 8TFA salt .
８−２．被験製剤の調製
６−ＦＡＭ−Ｌ１７Ｅ−ＮＨ２・１０ＴＦＡ塩（１．５８ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１００μＭの６−ＦＡＭ−Ｌ１７Ｅ−ＮＨ２・１０ＴＦＡ塩 溶液を調製し、実施例１４とした。
６−ＦＡＭ−２８９−Ｗ−ＮＨ２・７ＴＦＡ塩（０．３６ｍｇ）を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１００μＭの６−ＦＡＭ−２８９−Ｗ−ＮＨ２・７ＴＦＡ塩 溶液を調製し、実施例１５とした。
６−ＦＡＭ−Ｌ−Ｋ６−ＮＨ２・８ＴＦＡ塩（２．５１ｍｇ） を０．９６％ＰＢＳ溶液に溶解し、５０μＭの溶液に調製した。この溶液をさらに０．９６％ＰＢＳ溶液で希釈して、１００μＭの６−ＦＡＭ−Ｌ−Ｋ６−ＮＨ２・８ＴＦＡ塩 溶液を調製し、実施例１６とした。
8-2. Preparation of test formulation 6-FAM-L17E-NH 2 · 10TFA salt (1.58 mg) was dissolved in 0.96% PBS solution was prepared in a solution of 50 [mu] M. This solution was further diluted with a 0.96% PBS solution to prepare a 100 μM 6-FAM-L17E-NH 2 · 10 TFA salt solution, which was named Example 14.
6-FAM-289-W-NH 2 · 7 TFA salt (0.36 mg) was dissolved in 0.96% PBS solution to prepare a 50 μM solution. This solution was further diluted with 0.96% PBS solution to prepare 100 μM of 6-FAM-289-W-NH 2 · 7TFA salt solution, which was named Example 15.
6-FAM-L-K6-NH 2. 8TFA salt (2.51 mg) was dissolved in 0.96% PBS solution to make a 50 μM solution. This solution was further diluted with 0.96% PBS solution to prepare 100 μM of 6-FAM-L-K6-NH 2 · 8TFA salt solution, which was named Example 16.
８−３．試験方法
ヒト３次元角膜上皮（ＬａｂＣｙｔｅ ＣＯＲＮＥＡ−ＭＯＤＥＬ，Ｊ−ＴＥＣ社製）を付属の培地中でＣＯ２インキュベータ内にて１時間培養後、各ウェルの培地を除去し上記サンプルを５０μＬずつ添加し、ＣＯ２インキュベータ内で２時間暴露した。上清を除去し、細胞表面にＰＢＳ溶液を５００ｕＬずつ添加し、吸引除去する操作を３回繰り返した。ＰＢＳ溶液を５０ｕＬずつ添加し、蛍光顕微鏡（ＢＺ−Ｘ７００）で各ウェルのフルオレセインの蛍光顕微鏡写真を撮影した（露光時間；１０ｍｓ）。顕微鏡写真は画像解析ソフトＩｍａｇｅＪのＡｎａｌｙｚｅメニューのＭｅａｓｕｒｅモードを用いて解析した。
8-3. Test method After culturing human three-dimensional corneal epithelium (LabCyte CORNEA-MODEL, manufactured by J-TEC Co., Ltd.) for 1 hour in a CO 2 incubator in the attached medium, the medium in each well is removed and 50 μL of the above sample is added, It was exposed for 2 hours in a CO2 incubator. The supernatant was removed, and 500 μL each of PBS solution was added to the cell surface, and the operation of removing by suction was repeated three times. 50 uL of PBS solution was added, and a fluorescence micrograph of fluorescein in each well was taken with a fluorescence microscope (BZ-X700) (exposure time; 10 ms). Photomicrographs were analyzed using the Measure mode of the Analyze menu of the image analysis software ImageJ.
８−４．試験結果及び考察
試験結果を図１６に示す。図１６から分かるように、Ｐｅｎｅｔｒａｔｉｎ、Ｌ１７Ｅ、２８９−Ｗ及びＬ−Ｋ６から誘導した蛍光性分子による蛍光が観測された。以上の結果により、細胞膜透過性ペプチド又はその塩は、角膜移行性を示すことが分かった。
8-4. Test Results and Discussion The test results are shown in FIG. As can be seen from FIG. 16, fluorescence by fluorescent molecules derived from Penetratin, L17E, 289-W and L-K6 was observed. From the above results, it was found that the cell membrane permeable peptide or a salt thereof exhibits corneal migration.
９．摘出眼球による細胞膜透過性ペプチドの移行性試験１
９−１．被験製剤の調製
上記７−２．と同様の方法で調製した、１０μＭの６−ＦＡＭ−Ｒ８−ＮＨ２・８ＴＦＡ塩 溶液（実施例８）、１０μＭの６−ＦＡＭ−ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ 塩溶液（実施例９）及び１０μＭの６−ＦＡＭ−ＨＩＶ−ＴＡＴ（４７−５７）−ＮＨ２・８ＴＦＡ塩 溶液（実施例１０）を用いた。
9. Transferability test of cell membrane permeable peptide by isolated eye 1
9-1. Preparation of test preparation: 7-2. Prepared in the same manner as above, 10 μM 6-FAM-R8-NH2 • TFA salt solution (Example 8), 10 μM 6-FAM-Penetratin-NH2 · 7 TFA salt solution (Example 9) and 10 μM 6- FAM-HIV-TAT (47-57) -NH2 8 TFA solution (Example 10) was used.
９−２．試験方法
１２ウェルプレートの各ウェルの上記サンプルを２ｍｌずつ添加したのち、日本白色家兎の摘出眼球を角膜が下になるように各ウェル中のサンプル溶液に浸漬させ、１時間および４時間暴露した。暴露後、各眼球内の房水をシリンジで採取し、蛍光プレートリーダーで房水の蛍光強度を測定した。また、各眼球の角膜を切除し、プレパラート上にのせ、蛍光顕微鏡で各角膜の蛍光顕微鏡写真を撮影した。撮影した顕微鏡写真は画像解析ソフトＩｍａｇｅＪのＡｎａｌｙｚｅメニューのＭｅａｓｕｒｅモードを用いて解析した。
9-2. Test method 2 ml of the above sample from each well of the 12-well plate was added, and then the excised eye of a Japanese white rabbit was immersed in the sample solution in each well with the cornea facing down, and exposed for 1 hour and 4 hours . After exposure, the aqueous humor in each eye was collected by a syringe and the fluorescence intensity of the aqueous humor was measured with a fluorescence plate reader. In addition, the cornea of each eyeball was excised, placed on a slide, and a fluorescence microscope photograph of each cornea was taken with a fluorescence microscope. The photomicrographs taken were analyzed using the Measure mode of the Analyze menu of the image analysis software ImageJ.
９−３．試験結果及び考察（角膜移行性）
試験結果を図１７（１時間暴露）及び図１８（４時間暴露）に示す。図１７及び図１８から分かるように、Ｐｅｎｅｔｒａｔｉｎ、Ｒ８−ＮＨ２及びＨＩＶ−ＴＡＴ （４７−５７）から誘導した蛍光性分子による蛍光が観測された。以上の結果により、細胞膜透過性ペプチド又はその塩は、摘出眼球においても角膜移行性を示すことが分かった。
9-3. Test results and discussion (corneal migration)
The test results are shown in FIG. 17 (1 hour exposure) and FIG. 18 (4 hour exposure). As it can be seen from FIGS. 17 and 18, fluorescence by fluorescent molecules derived from Penetratin, R8-NH 2 and HIV-TAT (47-57) was observed. From the above results, it was found that the cell membrane permeable peptide or a salt thereof exhibits corneal migration even in the isolated eye.
９−４．試験結果及び考察（房水移行性）
試験結果を表４に示す。
9-4. Test results and discussion (transferability to aqueous humor)
The test results are shown in Table 4.
表４から分かるように、Ｐｅｎｅｔｒａｔｉｎ、Ｒ８−ＮＨ２及びＨＩＶ−ＴＡＴ （４７−５７）から誘導した蛍光性分子は、いずれも房水中への移行が確認された。特に、Ｐｅｎｅｔｒａｔｉｎから誘導した蛍光性分子は、投与４時間後において、高い房水移行を示した。以上の結果により、細胞膜透過性ペプチド又はその塩は、摘出眼球においても房水移行性を示すことが分かった。 As can be seen from Table 4, a fluorescent molecule derived from Penetratin, R8-NH 2 and HIV-TAT (47-57) are both transition to aqueous humor was confirmed. In particular, fluorescent molecules derived from Penetratin showed high aqueous humor transfer 4 hours after administration. From the above results, it was found that the cell membrane permeable peptide or a salt thereof exhibits aqueous humor transferability also in the isolated eye.
１０．安全性試験３
１０−１．被験製剤の調製
上記７−２．で調製した実施例８、実施例９、実施例１０、実施例１１、実施例１２及び実施例１３を用いた。
10. Safety test 3
10-1. Preparation of test preparation: 7-2. Example 8, Example 9, Example 10, Example 11, Example 12, and Example 13 which were prepared by this were used.
１０−２．試験方法
上記３−１．と同様の方法により、角膜上皮の生細胞率を算出した。
１０−３．試験結果及び考察
試験結果を図１９及び図２０に示す。図１９及び図２０から分かるように、６−ＦＡＭ−Ｒ８−ＮＨ２・８ＴＦＡ塩のほか６−ＦＡＭ−ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩、６−ＦＡＭ−ＨＩＶ−ＴＡＴ （４７−５７）−ＮＨ２・８ＴＦＡ塩、６−ＦＡＭ−ＨＩＶ−ＴＡＴ （４８−６０）−ＮＨ２・８ＴＦＡ塩及び６−ＦＡＭ−ｐＶＥＣ−ＮＨ２・６ＴＦＡ塩も角膜細胞に対して細胞障害性を示さなかった。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、眼科適用できる安全性を示すことが分かった。
10-2. Test method 3-1 above. The viable cell rate of the corneal epithelium was calculated by the same method as in.
10-3. Test Results and Discussion The test results are shown in FIG. 19 and FIG. As can be seen from FIG. 19 and FIG. 20, in addition to the 6-FAM-R8-NH 2. 8TFA salt, 6-FAM-penetratin-NH 2 7TFA salt, 6-FAM-HIV-TAT (47-57) -NH 2 The 8TFA salt, 6-FAM-HIV-TAT (48-60) -NH 2 · 8TFA salt and 6-FAM-pVEC-NH 2 · 6TFA salt also showed no cytotoxicity on corneal cells. From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof exhibits ophthalmologically applicable safety.
１１．安全性試験４
１１−１．被験製剤の調製
上記７−２．で調製した比較例３及び実施例４、並びに、上記８−２．で調製した実施例１４、実施例１５及び実施例１６を用いた。
11. Safety test 4
11-1. Preparation of test preparation: 7-2. Comparative Example 3 and Example 4 prepared in the above, and 8-2. Example 14, Example 15, and Example 16 which were prepared by this were used.
１１−２．試験方法
上記４−２．と同様の方法により、角膜上皮の生細胞率を算出した。
11-2. Test method 4-2. The viable cell rate of the corneal epithelium was calculated by the same method as in.
１１−３．試験結果及び考察
試験結果を図２１に示す。図２１から分かるように、６−ＦＡＭ−ｐｅｎｅｔｒａｔｉｎ−ＮＨ２・７ＴＦＡ塩のほか６−ＦＡＭ−Ｌ１７Ｅ−ＮＨ２・１０ＴＦＡ塩、６−ＦＡＭ−２８９−Ｗ−ＮＨ２・７ＴＦＡ塩及び６−ＦＡＭ−Ｌ−Ｋ６−ＮＨ２・８ＴＦＡ塩も角膜細胞に対して細胞障害性を示さなかった。以上の結果より、細胞膜透過性ペプチド又はその塩を含有する医薬組成物は、眼科適用できる安全性を示すことが分かった。
11-3. Test Results and Discussion The test results are shown in FIG. As can be seen from FIG. 21, 6-FAM-Penetratin-NH 2 · 7 TFA salt and 6-FAM-L 17 E-NH 2 · 10 TFA salt, 6-FAM-289-W-NH 2 · 7 TFA salt and 6-FAM- L-K6-NH 2 · 8 TFA salt also showed no cytotoxicity to corneal cells. From the above results, it has been found that a pharmaceutical composition containing a cell membrane permeable peptide or a salt thereof exhibits ophthalmologically applicable safety.
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