Pharmaceutical composition

Abstract

The present invention provides a pharmaceutical composition which contains a complex comprising: a peptide having a helical structure; and a pharmacologically active ingredient. The peptide has three or more amino acids joined by peptide bonds, and at least one amino acid residue constituting the peptide has a side chain containing a group represented by general formula (1) [wherein Z1 represents a single bond or a di-, tri-, or tetra-valent linking group (excluding an alkylene group); Rf represents a C1-30 alkyl group that is substituted with at least two fluorine atoms, a C2-30 alkyl group that has one to five ether-bonding oxygen atoms between carbon atoms and is substituted with at least two fluorine atoms, -SF5, -SF4-CR101R102-CR103R104Cl (wherein R101, R102, R103, and R104 each independently represent a hydrogen atom, a fluorine atom, or a chlorine atom, provided that two or more of R101, R102, R103, and R104 are fluorine atoms), or a C8-30 alkyl group that is not substituted with a fluorine atom; n3 represents 1, 2, or 3; and the solid circle denotes an atomic bond].

Description

Pharmaceutical composition

[0001] The present invention relates to a pharmaceutical composition comprising a peptide containing an amino acid residue in which a fluorine atom is introduced into the side chain, and a pharmacoactive ingredient. This application claims priority pursuant to Japanese Patent Application No. 2025-009484 filed in Japan on January 22, 2025, and Japanese Patent Application No. 2025-110893 filed in Japan on June 30, 2025, the contents of which are herein by reference.

[0002] Antibody drugs, peptide drugs, and nucleic acid drugs have advantages such as high specificity for target molecules and fewer side effects. However, they all have the problem of being difficult to deliver to target molecules present inside cells. Various methods are being investigated to solve this problem. Among them, cell-penetrating peptides (CPPs) are considered promising. Representative examples of CPPs include peptides derived from the HIV virus TAT ​​protein (Patent Document 1) and peptides with polyargon sequences (Patent Document 2). These CPPs can be combined with pharmacoactive ingredients to create pharmacoactive peptides that can be transported into cells (for example, Patent Document 3, Non-Patent Document 1).

[0003] On the other hand, compounds having a polyfluoro structure are known to be stable and low-toxicity in vivo, and to be easily taken up into cells and escaped from endosomes (Non-Patent Literature 2). Furthermore, peptides containing amino acid residues with fluorine atoms introduced into the side chain are CPPs and are useful as DDS (drug delivery system) carriers that deliver active pharmaceutical ingredients to target cells (Patent Literature 4).

[0004] U.S. Patent No. 6,316,003, U.S. Patent No. 6,306,993, International Publication No. 2008 / 089491, International Publication No. 2023 / 048236

[0005] Miyaji et. al., Drug Metabolism and Disposition, 2011, vol.39, p.1946-1953.Zhang et. al., MRS Communications, 2018, vol.8, p.303-313.

[0006] The present invention aims to provide a pharmaceutical composition containing CPP and a medicinal ingredient (AI, Active Ingredient) and having excellent cell membrane permeability.

[0007] When the present inventors used CPP as a DDS carrier for a medicinal ingredient, they found that a CPP-medicinal ingredient complex with extremely excellent cell membrane permeability can be obtained by complexing a helical CPP with the medicinal ingredient, and thus completed the present invention.

[0008] That is, the present invention includes the following aspects. [1] A complex containing a peptide having a helical structure and a medicinal ingredient, wherein the peptide has three or more amino acids peptide-bonded, and at least one side chain of the amino acid residues constituting the peptide has the following general formula (1)

[0009]

[0010] [In the formula, Z １ is a single bond or a divalent, trivalent or tetravalent linking group (excluding an alkylene group); Rf is a C １－３０ alkyl group substituted with at least two fluorine atoms, a C ２－３０ alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms and substituted with at least two fluorine atoms, -SF ５ , -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ Cl (R １０１ , R １０２ , R １０３ , and R １０４ are each independently a hydrogen atom, a fluorine atom or a chlorine atom, but two or more of R １０１ , R １０２ , R １０３ , and R １０４ are fluorine atoms), or a C ８－３０ alkyl group not substituted with a fluorine atom; n3 is 1, 2 or 3, and the black circle means a bond], a pharmaceutical composition having a group represented by the formula. [2] The Rf is a C ６－３０A C atom having an alkyl group, 1 to 5 ether-bonded oxygen atoms between carbon atoms, and substituted with at least 2 fluorine atoms. ６－３０ C that is not substituted with alkyl groups or fluorine atoms ８－３０ The pharmaceutical composition of [1], wherein the Rf is an alkyl group. [3] C, wherein the Rf is substituted with at least two fluorine atoms. １－３０ A C atom having an alkyl group, 1 to 5 ether-bonded oxygen atoms between carbon atoms, and substituted with at least 2 fluorine atoms. ２－３０ Alkyl alkyl group, -SF ５ , or -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ Cl(R) １０１ , R １０２ , R １０３ , and R １０４ Each of these is independently a hydrogen atom, a fluorine atom, or a chlorine atom, but R １０１ , R １０２ , R １０３ , and R １０４ The pharmaceutical composition of [1] or [2], wherein two or more of the amino acids are fluorine atoms. [4] The pharmaceutical composition of any of [1] to [3], wherein the peptide has two or more amino acid residues having the group represented by the general formula (1) in its side chain. [5] The pharmaceutical composition of any of [1] to [4], wherein the peptide is amphiphilic. [6] The pharmaceutical composition of any of [1] to [5], wherein the peptide has a structure in which amino acid residues having the group represented by the general formula (1) are arranged on one side with respect to the central axis of the helical structure, and hydrophilic amino acid residues are arranged on the opposite side. [7] The pharmaceutical composition of [4], wherein the distance between amino acid residues having the group represented by the general formula (1) in its side chain is 3, 6, or 7 amino acid residues. [8] The Rf is the following general formula (f-1) or (f-2)

[0011]

[0012] [wherein, Rf Ｐ This is a fully halogenated C containing at least two or more fluorine atoms. １－１０A fully halogenated carbon atom containing an alkyl group or at least two fluorine atoms and having an ether-bonded oxygen atom between carbon atoms. ２－１０ A pharmaceutical composition according to any of the above [1] to [7], wherein the group is represented as an alkyl group, where n1 is an integer from 0 to 10, n2 is an integer from 0 to 9, and the black circle represents a bond. [9] The Z １ However, alkylene groups, -O-, -S-, -NH-, -N(CH ３ )-,-N(C ２ H ５ )-,-N(C ３ H ７ )-, trivalent nitrogen atom, -C(=O)-, -S(=O) ２ - A cycloalkylene group, a divalent to tetravalent aryl group, a divalent to tetravalent heteroaryl group, or a combination thereof (excluding groups consisting solely of alkylene groups), any of the pharmaceutical compositions described in [1] to [8] above.

[10] The Z １ is -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O) ２ -NH-, -NH-S (=O) ２ -, -S (=O) ２ -NH-S (=O) ２ -, -C(=O)-NH-Ph- (where -Ph- is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group), or any of these groups and C １－６ A pharmaceutical composition of any of the above [1] to [9], which is a combination of alkylene groups.

[11] The Z １ However, the following general formula (2)

[0013]

[0014] [In the formula, Z ２ is a linking group other than a divalent, trivalent, or tetravalent alkylene group; Rh is a hydrogen atom or C １－６A pharmaceutical composition according to any one of [1] to

[10] , wherein the linking group is an alkyl group, and the black circle represents a bond.

[12] A pharmaceutical composition according to any one of [1] to

[11] , wherein the amino acid residue whose side chain is the group represented by the general formula (1) is an amino acid residue to which 1 to 3 of the Rf groups are directly or indirectly linked to the side chain of a natural amino acid.

[13] A pharmaceutical composition according to any one of [1] to

[12] , wherein the pharmacoactive ingredient has a negative charge.

[14] A pharmaceutical composition according to any one of [1] to

[13] , wherein the pharmacoactive ingredient is a nucleic acid, peptide, protein, polysaccharide, or a low molecular weight compound having an acidic group.

[15] A pharmaceutical composition according to any one of [1] to

[14] , wherein the surface tension of the complex is 25 mN / m or more.

[16] The pharmaceutical composition according to any one of [1] to

[15] , wherein the complex is formed by contacting the helical peptide and the pharmacoactive ingredient in a solvent in which both the peptide and the pharmacoactive ingredient are soluble.

[0015] The active ingredient contained in the pharmaceutical composition according to the present invention is a complex of a helical CPP and the active ingredient, and exhibits excellent cell membrane permeability. For this reason, the pharmaceutical composition is extremely useful as a pharmaceutical composition for delivering the active ingredient to target cells.

[0016] This is the CD spectrum of the peptide (ARA) produced in Production Example 1. This is the CD spectrum of the peptide (ARAib) produced in Production Example 2. This is the CD spectrum of the peptide (NRA) produced in Production Example 3. This is the CD spectrum of the peptide (F8-2) produced in Production Example 4. １７ This is the CD spectrum of the peptide (F8-2) produced in Production Example 5. １４ This is the CD spectrum of the peptide (F6-2) produced in Production Example 6. １７ This is the CD spectrum of the peptide (F4-2) produced in Production Example 7. １７ This is the CD spectrum of the peptide (F8-2) produced in Production Example 8. ３４ This is the CD spectrum of the peptide (H12-2) produced in Production Example 9. １７This is the CD spectrum of the peptide (H8-2) produced in Production Example 10. １７ This is the CD spectrum of the peptide (F4-2) produced in Production Example 11. １７ This is the CD spectrum of Aib. The peptide (F6-2) produced in Production Example 12. １７ This is the CD spectrum of Aib). This is the CD spectrum of the peptide (p53_frag) produced in Production Example 14. This is the CD spectrum of the peptide (p53-F_frag) produced in Production Example 15. In Example 1, the peptide (F8-2 １７ ), peptide (F6-2 １７ ), peptide (F8-2 ３４ ), and peptide (F6-2 ３４ This figure shows the results of measuring the surface tension (mN / m) of a peptide-nucleic acid complex containing ) over time. In Example 1, the peptide (F4-2 １７ ), peptide (F6-2 １７ ), or peptide (F8-2 １７ This is a FAM fluorescence image of HeLa cells into which a peptide-nucleic acid complex formed by complexing ) with a FAM-labeled DNA complex, or PEI has been incorporated. In Example 1, the peptide (F4-2 １７ ), peptide (F6-2 １７ ), or peptide (F8-2 １７ This is a GFP fluorescence image of A549 cells into which a peptide-nucleic acid complex formed by complexing ) with GFP-coding pDNA, or PEI has been incorporated. In Example 1, the peptide (F4-2 １７ ), peptide (F6-2 ３４ ), peptide (F8-2 ３４ ), peptide (F6-2 １７ ), or peptide (F8-2 １７ This figure shows the results of FACS analysis of cells that incorporated a peptide-nucleic acid complex formed by combining the peptide (F6-2) with a FAM-labeled DNA complex. In Example 2, the peptide (F6-2) was used. １７), and FAM fluorescence images and transmitted light images of HeLa cells incorporating a peptide-peptide complex formed by complexing a FAM-labeled peptide (FAM-p53-TAD). In Example 2, the peptide (F6-2 １７ ), the peptide (AF ６４７ -F8-2 １７ ), and FAM fluorescence images, AF ６４７ fluorescence images, and Hoechst 33342 images of HeLa cells incorporating a peptide-nucleic acid complex formed by complexing with FAM-DNA. In Example 2, a FAM fluorescence image of HeLa cells incorporating a peptide-nucleic acid complex formed by complexing the peptide (F8-2 １７ ) with FAM-DNA. FIG. 22(A) is a FAM fluorescence image, a Hoechst 33342 stained image, a Lysotracker red stained image, and a Merge image of HeLa cells incorporating a peptide-nucleic acid complex formed by complexing the peptide (F8-2 １７ ) with FAM-DNA in Example 2. FIG. 22(B) is an enlarged view of the region surrounded by the square in the Merge image of FIG. 22(A).

[0017] In the present invention and the specification of the present application, "C ｐ１－ｐ２ " (where p1 and p2 are positive integers satisfying p1 < p2) means a group having 1 or more and p2 or less carbon atoms.

[0018] In the present invention and the specification of the present application, "C １－３０ alkyl group" is an alkyl group having 1 to 30 carbon atoms, which may be linear or branched. "C ２－３０ alkyl group" is an alkyl group having 2 to 30 carbon atoms, which may be linear or branched. C １－３０Examples of the alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, and the like.

[0019] In the present invention and this specification, "C １－１０ alkyl group" is an alkyl group having 1 to 10 carbon atoms, which may be linear or branched. "C ２－１０ alkyl group" is an alkyl group having 2 to 10 carbon atoms, which may be linear or branched. C １－１０ Examples of the C

[0020] alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, and the like. １－６ alkyl group" is an alkyl group having 1 to 6 carbon atoms, which may be linear or branched. C １－６ Examples of the alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, and the like.

[0021] In the present invention and this specification, "C ６－１４ aryl group" is an aromatic hydrocarbon group having 6 to 14 carbon atoms, and C ６－１２ aryl group is particularly preferred. C ６－１４Examples of aryl groups include phenyl, naphthyl, anthryl, and 9-fluorenyl groups, with the phenyl group being particularly preferred.

[0022] In the present invention and this specification, "C which may be substituted" ６－１４ The "aryl group" is an unsubstituted C ６－１４ Aryl group, or C ６－１４ An aryl group is a group in which one or more hydrogen atoms, preferably one to three, bonded to the carbon atom of the aryl group are substituted with other functional groups. When there are two or more substituents, the substituents may be of the same type or different types. The substituents may be a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), or C １－６ alkyl group, C １－６ Alkoxy group and methylenedioxy group (-O-CH ２ Examples include -O-). "C may be substituted." ６－１４ Examples of "aryl groups" include phenyl group, naphthyl group, anthryl group, 4-nitrophenyl group, 4-methoxyphenyl group, 2,4-dimethoxyphenyl group, 3,4-dimethoxyphenyl group, 4-methylphenyl group, 2,6-dimethylphenyl group, 3-chlorophenyl group, and 1,3-benzodioxol-5-yl group.

[0023] In the present invention and this specification, "C ６－１４ Aryl-C １－６ "Alkyl alkyl group" is C １－６ One hydrogen atom bonded to the carbon atom of the alkyl group is C ６－１４ It is a group substituted with an aryl group. C ６－１４ Aryl-C １－６ C in alkyl groups ６－１４ Examples of aryl groups include phenyl, naphthyl, anthryl, and 9-fluorenyl groups, with phenyl or 9-fluorenyl groups being particularly preferred. ６－１４ Aryl-C １－６ C in alkyl groups １－６ As an alkyl group, C １－４ Alkyl groups are preferred. C ６－１４ Aryl-C １－６Examples of alkyl groups include benzyl group, diphenylmethyl group, triphenylmethyl group, 2-phenylethyl group, 9-anthrylmethyl group, and 9-fluorenylmethyl group.

[0024] In the present invention and this specification, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. "Halogen atom other than a fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. As the "halogen atom other than a fluorine atom," a chlorine atom or a bromine atom is preferred, and a chlorine atom is particularly preferred.

[0025] In the present invention and this specification, "C １－６ An "alkoxy group" is a carbon atom with 1 to 6 carbon atoms. １－６ This refers to a group in which an oxygen atom is bonded to the end of an alkyl group. １－６ The alkoxy group may be linear or branched. １－６ Examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy groups.

[0026] In the present invention and this specification, "ether-bonded oxygen atom" refers to an oxygen atom that links carbon atoms together, and does not include oxygen atoms that are linked in series. An alkyl group with Nc carbon atoms (where Nc is an integer of 2 or more) may have a maximum of Nc-1 ether-bonded oxygen atoms.

[0027] Furthermore, in the following, "compound n" refers to the compound represented by formula (n).

[0028] The pharmaceutical composition according to this embodiment contains a complex of a helical peptide and a pharmacoactive ingredient. The peptide has three or more amino acids linked by peptide bonds, and at least one side chain of the amino acid residues constituting the peptide has a group represented by the following general formula (1). Because the peptide has the group represented by general formula (1), it is permeable to cell membranes. That is, the peptide having the group represented by general formula (1) is CPP, and the pharmaceutical composition according to this embodiment contains a complex of helical CPP and a pharmacoactive ingredient (hereinafter sometimes referred to as the "CPP / AI complex") as the active ingredient.

[0029] <Peptide> In this embodiment, the three-dimensional structure of the peptide constituting the CPP / AI complex may be helical, and may be a right-handed helical structure or a left-handed helical structure. It may also be an α-helix structure, a 3-10 helix structure or a π-helix structure. The peptide constituting the CPP / AI complex may form only one helix or two or more helices. The number of amino acid residues constituting one turn of the helical structure is not particularly limited. If there are two or more helices in the peptide, the number of amino acid residues per turn of each helix may be the same or different from each other.

[0030]

[0031] [In the formula, Z １ is a single bond or a divalent, trivalent, or tetravalent linking group (excluding alkylene groups); Rf is a C substituted with at least two fluorine atoms. １－３０ A C atom having an alkyl group, 1 to 5 ether-bonded oxygen atoms between carbon atoms, and substituted with at least 2 fluorine atoms. ２－３０ Alkyl alkyl group, -SF ５ , -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ Cl(R) １０１ , R １０２ , R １０３ , and R １０４Each of these is independently a hydrogen atom, a fluorine atom, or a chlorine atom, but R １０１ , R １０２ , R １０３ , and R １０４ (Two or more of these atoms are fluorine atoms), or C atoms that are not substituted with fluorine atoms. ８－３０ It is an alkyl group; n3 is 1, 2, or 3, and the black circle represents a bond.

[0032] Whether a peptide constituting the CPP / AI complex adopts a helical structure can be determined from its circular dichroism (CD) spectrum. When a peptide adopts a helical structure, there are negative maxima around 208-209 and 222 nm in its CD spectrum, and a positive maxima around 191-193 nm. The term "around" is used because the peak top values ​​can fluctuate by a few nm due to the effects of blueshift and redshift. When a peptide adopts an α-helix structure, the ratio of the peak tops of the negative maxima around 222 nm and around 208 nm ([peak top value of the negative maxima around 222 nm] / [peak top value of the negative maxima around 208 nm]) (hereinafter referred to as the "222 / 208 nm ratio") is close to 1. When a peptide adopts a 3-10 helix structure, the 222 / 208 nm ratio is approximately 0.3 to 0.7. The CD spectrum of a peptide can be measured using a CD spectrometer, which is commonly used for peptide measurement.

[0033] In general formula (1), the black circle represents a binding bond. The group represented by general formula (1) is bonded at the black circle portion to a group or α-carbon atom that constitutes the side chain of the amino acid residue constituting the peptide. Hereafter, an amino acid having the group represented by general formula (1) in its side chain may be called a "CP group amino acid," and a peptide in which at least one side chain of the amino acid residues constituting the peptide has the group represented by general formula (1) may be called a "CP group peptide."

[0034] In general formula (1), Rf is a carbon atom substituted with at least two fluorine atoms. １－３０ Alkyl alkyl group, -SF ５ , -SF ４ -CR １０１ R１０２ -CR １０３ R １０４ C that is not substituted with Cl or fluorine atoms ８－３０ It is an alkyl group.

[0035] C in which Rf is substituted with at least two fluorine atoms １－３０ If it is an alkyl group, then C １－３０ The alkyl group may be a linear group or a branched group. In the case of a branched group, it is preferable that the hydrogen atoms bonded to the carbon atoms constituting the alkylene group portion are substituted with fluorine atoms, or that the group is fully fluorinated. １－３０ As for alkyl groups, C is substituted with two fluorine atoms due to its ease of forming a helical structure and its safety. ４－３０ Alkyl groups are preferred, and C is substituted with two fluorine atoms. ６－３０ Alkyl groups are more preferred, and C is more preferably substituted with two fluorine atoms. ６－２０ Alkyl groups are more preferably C substituted with two fluorine atoms. ６－１５ Alkyl groups are even more preferred, and C is substituted with two fluorine atoms. ６－１２ Alkyl alkyl groups are particularly preferred.

[0036] C in which Rf is substituted with at least two fluorine atoms １－３０ If it is an alkyl group, then C １－３０ The alkyl group may have 1 to 5 ether-bonded oxygen atoms between its carbon atoms. That is, Rf has 1 to 5 ether-bonded oxygen atoms between its carbon atoms and is substituted with at least 2 fluorine atoms. ２－３０ It may be an alkyl group. ２－３０ The alkyl group may be a linear group or a branched group. In the case of a branched group, it is preferable that the hydrogen atoms bonded to the carbon atoms constituting the alkylene group portion are substituted with fluorine atoms, or that the group is fully fluorinated.

[0037] Rf may have one or more hydrogen atoms bonded to the carbon atom further substituted with halogen atoms other than fluorine atoms. Here, the C of Rf １－３０ As an alkyl group, C １－２０Alkyl alkyl groups are preferred, C １－１０ Alkyl alkyl groups are more preferred, C ２－１０ Alkyl alkyl groups are more preferred, C ４－８ Alkyl alkyl groups are even more preferred, C ６－８ Alkyl alkyl groups are particularly preferred. １－３０ Alkyl group C ２－３０ If it is an alkyl group, it may have 1 to 5 ether-bonded oxygen atoms between carbon atoms. In Rf, the number of hydrogen atoms substituted with fluorine atoms is not particularly limited as long as there are 2 or more, for example, 3 or more is preferred, 6 or more is more preferred, and 7 or more is even more preferred.

[0038] Examples of Rf include trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, perfluorodecyl group, difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, Examples include 1,1,2,2,3,3-hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group, 1,1,2,2,3,3-hexafluorohexyl group, 1,1,2,2,3,3-hexafluorooctyl group, 1,1,2,2,3,3-hexafluorodecyl group, 1,1,2,2,3,3-hexafluorooctadecyl group, and 1,1,2,2,3,3-hexafluorohexacosyl group.

[0039] As Rf, a group represented by the following general formula (f-1) or (f-2) is preferred. Here, Rf Ｐ This is a fully halogenated C containing at least two or more fluorine atoms. １－１０ Represents an alkyl group. Rf Ｐ C １－１０ This group consists of an alkyl group in which all hydrogen atoms are replaced by halogen atoms, and at least two of these halogen atoms are fluorine atoms. (Rf) Ｐ If the carbon number is 2 or more, that is, completely halogenated C ２－１０In the case of alkyl groups, there may be 1 to 5 ether-bonded oxygen atoms between the carbon atoms. In general formula (f-2), two Rf Ｐ These groups may be of the same kind or of different kinds.

[0040] In the following general formulas (f-1) or (f-2), n1 is an integer from 0 to 10, and n2 is an integer from 0 to 9. When both n1 and n2 are 0, both represent a simple combination. That is, when n1 is 0, the base represented by general formula (f-1) is Rf Ｐ - and when n2 is 0, the base represented by the general formula (f-2) is (Rf Ｐ ) ２ It is -CH-.

[0041]

[0042] If Rf is a group represented by the general formula (f-1), then Rf is Rf Ｐ The group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 4. Ｐ However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 2. Ｐ However, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, where n1 is an integer from 0 to 2, is more preferable.

[0043] If Rf is a group represented by the general formula (f-2), then Rf is Rf ＰThe group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 4. Ｐ However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 2. Ｐ The group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is an integer from 0 to 2 (however, n2 is 0 or 1, and Rf Ｐ Groups that are trifluoromethyl groups are even more preferred.

[0044] Rf is -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ If the group is represented by Cl, then R １０１ , R １０２ , R １０３ , and R １０４ Each of these is independently a hydrogen atom, a fluorine atom, or a chlorine atom. However, R １０１ , R １０２ , R １０３ , and R １０４ Two or more of them are fluorine atoms. -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ Specifically, the group represented by Cl is -SF ４ -CF ２ -CF ２ Cl, -SF ４ -CF ２ -CFCl ２ , -SF ４ -CF ２-CHF-Cl, -SF ４ -CF ２ - CCl ３ , -SF ４ -CF ２ - CHCl ２ , -SF ４ -CF ２ -CH ２ Cl, -SF ４ -CFCl-CFCl ２ , -SF ４ -CFCl-CHF-Cl, -SF ４ -CHF-CHF-Cl is one example.

[0045] C where Rf is not substituted with a fluorine atom ８－３０ If it is an alkyl group, then C ８－３０ The alkyl group may be a linear group or a branched group. It may also have 1 to 5 ether-bonded oxygen atoms between carbon atoms. C that is not substituted with a fluorine atom of Rf. ８－３０ As for alkyl groups, C is chosen due to its ease of forming a helical structure and its safety. ８－２０ Alkyl alkyl groups are preferred, C ８－１５ Alkyl groups are more preferred, and octyl, nonyl, decyl, undecyl, or dodecyl groups are even more preferred.

[0046] In general formula (1), Z １ The linking group is a single bond or a 2, 3, or 4-valent linking group (excluding alkylene groups), and n3 is 1, 2, or 3. １ The group is not particularly limited to any divalent to tetravalent group other than an alkylene group. For example, Z １ This consists of an alkylene group, an oxygen atom (-O-), a sulfur atom (-S-), -NH-, and -N(CH ３ )-,-N(C ２ H ５ )-,-N(C ３ H ７ )-, trivalent nitrogen atom, -C(=O)-, -S(=O) ２- Examples include groups obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, groups obtained by removing 2 to 4 hydrogen atoms from an aromatic ring, groups obtained by removing 2 to 4 hydrogen atoms from a heterocycle, or combinations thereof. The groups listed above can be used as aryl groups and heteroaryl groups. However, groups consisting only of alkylene groups or groups where the linking portion with Rf is an alkylene group are excluded.

[0047] Z １ However, alkylene group, oxygen atom (-O-), sulfur atom (-S-), -NH-, -N(CH ３ )-,-N(C ２ H ５ )-,-N(C ３ H ７ )-, -C(=O)-, -S(=O) ２ - If the group consists of a cycloalkane with two hydrogen atoms removed, an aromatic ring with two hydrogen atoms removed, a heterocycle with two hydrogen atoms removed, or a combination thereof, then Z １ Z is a divalent linking group, and the group represented by general formula (1) is a group having one Rf group. １ If the group has a trivalent nitrogen atom, the group represented by general formula (1) can be made into a group having two Rf groups by bonding two Rf groups directly to the nitrogen atom or via other divalent linking groups.

[0048] Z １ The linking group may be a linking group having a ring group (a group from which a hydrogen atom has been removed from a ring), or it may be a linking group that does not have a ring group. １ If the ring group has a ring group, the group represented by general formula (1) can be a group having two or three Rf groups. Examples of such ring groups include cycloalkanes, aromatic rings, or groups obtained by removing two to four hydrogen atoms from a heterocycle. As for heterocycles, rings in which one to three carbon atoms of an aromatic ring are replaced with one or more atoms selected from the group consisting of nitrogen atoms, oxygen atoms, and sulfur atoms are preferred. Furthermore, the ring group may be a group obtained by removing hydrogen atoms from a monocycle, or a group obtained by removing hydrogen atoms from a fused ring. Z in general formula (1) １The ring group contained is preferably a group obtained by removing 2 to 4 hydrogen atoms from cyclohexane, benzene, imidazole, or indole. For example, Z １ However, if the linking group has a ring group obtained by removing 2 to 4 hydrogen atoms from benzene, the ring group is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group, with a 1,4-phenylene group or a 1,3,5-substituted phenyl group being preferred.

[0049] Specifically, Z in general formula (1) １ -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O) ２ -NH-, -NH-S (=O) ２ -, -S (=O) ２ -NH-S (=O) ２ Examples include -, -C(=O)-NH-Ph- (where -Ph- is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group). Also, any of these groups and C １－６ It is also preferable that the linking group is a combination of alkylene groups. Z in general formula (1) １ Preferably, the bonding portion with Rf is an oxygen atom.

[0050] Z in general formula (1) １ Preferably, the linking group is represented by the following general formula (2).

[0051]

[0052] In general formula (2), Z ２ This is a single bond, or a 2, 3, or 4 valent linking group (excluding alkylene groups). ２ The group is not particularly limited to any divalent to tetravalent group other than an alkylene group. For example, Z ２ This consists of an alkylene group, an oxygen atom (-O-), a sulfur atom (-S-), -NH-, and -N(CH ３ )-,-N(C ２ H ５ )-,-N(C３ H ７ )-, trivalent nitrogen atom, -C(=O)-, -S(=O) ２ - Examples include groups obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, groups obtained by removing 2 to 4 hydrogen atoms from an aromatic ring, groups obtained by removing 2 to 4 hydrogen atoms from a heterocycle, or combinations thereof. The groups listed above can be used as aryl groups and heteroaryl groups. However, groups consisting only of alkylene groups or groups where the linking portion with Rf is an alkylene group are excluded.

[0053] Z in general formula (2) ２ Specifically, Z １ Similar to those listed above can be used. Z in general formula (2) ２ -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O) ２ -NH-, -NH-S (=O) ２ -, -S (=O) ２ -NH-S (=O) ２ It is preferable that the group is -,-C(=O)-NH-Ph- (where -Ph- is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group).

[0054] In general formula (2), Rh is a hydrogen atom or C １－６ It is an alkyl group. Rh is C １－６ In the case of alkyl groups, Rh is C １－３ Alkyl groups are preferred, and methyl or ethyl groups are more preferred.

[0055] Examples of a base represented by general formula (1) include Z １ Examples include groups in which is a group represented by general formula (2) and Rf is a group represented by general formula (f-1) or (f-2). In particular, Z in general formula (2) ２ is -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O) ２-NH-, -NH-S (=O) ２ -, -S (=O) ２ -NH-S (=O) ２ -, -C(=O)-NH-Ph- (where -Ph- is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group), and Rh is a hydrogen atom or C １－６ The group is preferably an alkyl group in which Rf is represented by the general formula (f-1) or (f-2).

[0056] In this embodiment, the peptide constituting the CPP / AI complex (CP-containing peptide) is preferably a peptide in which the CP-containing amino acid residue is an amino acid residue in which 1 to 3 Rf groups are directly or indirectly linked to the side chain of a natural amino acid. Specifically, the peptide constituting the CPP / AI complex is one in which one or two hydrogen atoms of the amino group of the side chain of an arginine residue, asparagine residue, glutamine residue, or lysine residue are linked to -Rf or -Z. ３ -(Rf)n4(Z ３ is a linking group other than a 2, 3, or 4 valent alkylene group. However, groups in which the linking portion with Rf is an alkylene group are excluded. Also, n4 is 2, 3, or 4.) Amino acid residues substituted with; the hydrogen atom of the imino group of the side chain of the arginine residue is -Rf or -Z ３ Amino acid residues substituted with -(Rf)n4; hydrogen atoms of the carboxyl group in the side chain of an aspartic acid residue or glutamic acid residue are replaced with -Rf or -Z. ３ Amino acid residues substituted with -(Rf)n4; hydrogen atoms of the thiol group in the side chain of a cysteine ​​residue are replaced with -Rf or -Z. ３ Amino acid residues substituted with -(Rf)n4; the methyl group of the methylthio group in the side chain of a methionine residue is replaced with -Rf or -Z. ３ Amino acid residues substituted with -(Rf)n4; hydrogen atoms of the hydroxyl group in the side chain of serine or threonine residues are replaced with -Rf or -Z. ３ Amino acid residues substituted with -(Rf)n4; 1 to 3 hydrogen atoms of the benzene ring in the side chain of a tyrosine or phenylalanine residue are replaced with -Rf or -Z. ３ -(Rf)n4 substituted amino acid residues; 1 to 3 hydrogen atoms of the imidazole ring in the side chain of a histidine residue are replaced with -Rf or -Z３ Amino acid residues substituted with -(Rf)n4; 1 to 3 hydrogen atoms of the indole ring in the side chain of a tryptophan residue are replaced with -Rf or -Z ３ A peptide having at least one amino acid residue substituted with -(Rf)n4 is preferred.

[0057] Z ３ The group is not particularly limited to any divalent to tetravalent group other than an alkylene group. For example, Z ３ This consists of an alkylene group, an oxygen atom (-O-), a sulfur atom (-S-), -NH-, and -N(CH ３ )-,-N(C ２ H ５ )-,-N(C ３ H ７ )-, trivalent nitrogen atom, -C(=O)-, -S(=O) ２ - Examples include groups obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, groups obtained by removing 2 to 4 hydrogen atoms from an aromatic ring, groups obtained by removing 2 to 4 hydrogen atoms from a heterocycle, or combinations thereof. As aryl groups and heteroaryl groups, the groups listed above can be used. However, groups consisting only of alkylene groups or groups where the linking portion with Rf is an alkylene group are excluded. Specifically, Z ３ As for Z １ Ya Z ２ A similar linking group can be used.

[0058] The hydrogen atoms in the amino group, imino group, carboxyl group, hydroxyl group, thiol group, benzene ring, imidazole ring, indole ring, etc. of the side chains of natural amino acids are -Rf or -Z. ３ Substitution of -(Rf)n4 can be carried out by common synthetic reactions such as esterification reactions.

[0059] The CP group peptide used in this embodiment has a side chain of -Z １Examples include peptides that contain at least one amino acid residue that is -(Rf)n3 and have a helical structure. Of the amino acid residues constituting the peptide, at least one may be a CP-containing amino acid residue, but it is preferable that two or more are CP-containing amino acid residues, and all amino acid residues may be CP-containing amino acid residues. One peptide molecule has a side chain of -Z １ If there are two or more amino acid residues that are -(Rf)n3, then these multiple -Z １ -(Rf)n3 may be of the same type or different types. Also, the CP-containing amino acid residues in the peptide may be at the N-terminus, the C-terminus, or anywhere other than the terminal. The CP-containing peptide used in this embodiment is a helical structure with multiple -Z １ It is preferable that -(Rf)n3 is oriented in approximately the same direction with respect to the axis of the helical structure.

[0060] The CP group peptide used in this embodiment may be any peptide consisting of three or more amino acids, preferably a peptide consisting of 3 to 40 amino acids, and more preferably a peptide consisting of 3 to 20 amino acids.

[0061] The amino acid sequence of the CP-containing peptide used in this embodiment is not particularly limited. For example, the amino acid sequence of the CP-containing peptide is preferably one that contains a relatively large number of amino acid residues that have high affinity for the active ingredient. When the active ingredient is an anionic substance such as nucleic acid, the amino acid sequence of the CP-containing peptide is preferably one that contains a large number of cationic amino acid residues such as arginine, lysine, and histidine, and is more preferably one that contains a large amount of arginine because it is easier to form a helical structure.

[0062] The N-terminus of the CP-containing peptide used in this embodiment may be protected with an amino group protecting group. The N-terminus protecting group is not particularly limited as long as it is an amino group protecting group; for example, amino group protecting groups used in peptide synthesis can be used. Examples of amino group protecting groups include carbamate protecting groups such as tert-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyloxycarbonyl (Cbz) group, allyloxycarbonyl (Alloc) group, and 2,2,2-trichloroethoxycarbonyl (Troc) group. Preferably, the tert-butoxycarbonyl (Boc) group or the 9-fluorenylmethyloxycarbonyl (Fmoc) group can be deprotected under mild conditions.

[0063] The C-terminus of the CP-containing peptide used in this embodiment may be protected with a protecting group. The protecting group for the C-terminus is not particularly limited as long as it is a carboxyl group protecting group, and for example, a carboxyl group protecting group used in peptide synthesis can be used. Specifically, the carboxyl group protecting group is a protecting group selected from the group represented by the following general formula (p-1), the 2-(9,10-dioxo)antrylmethyl group, the benzyloxymethyl group, and the phenacyl group. In the general formula (p-1), R ３ C may be substituted. ６－１４ It is an aryl group, R ４ and R ５ Each of these is independently a hydrogen atom or a substituted C ６－１４ This is an aryl group. The black circles represent bonding bonds.

[0064]

[0065] Examples of protecting groups for carboxyl groups include benzyl group, diphenylmethyl group, triphenylmethyl group, 4-nitrobenzyl group, 4-methoxybenzyl group, 2,4-dimethoxybenzyl group, 3,4-dimethoxybenzyl group, 4-methylbenzyl group, 2,6-dimethylbenzyl group, 3-chlorobenzyl group, 9-anthrylmethyl group, piperonyl group, 2-(9,10-dioxo)anthrylmethyl group, benzyloxymethyl group, and phenacyl group. In terms of being able to deprotect under mild conditions, the protecting group for the C-terminal carboxyl group is preferably benzyl group or triphenylmethyl group, and more preferably benzyl group.

[0066] Among the CP group peptides used in this embodiment, the side chain is -Z １ The amino acid residue that is not -(Rf)n3 is not particularly limited and may be an α-amino acid residue, a β-amino acid residue, a γ-amino acid residue, or a δ-amino acid residue. It may also be an L-amino acid residue or a D-amino acid residue. The side chain contained in the CP group peptide used in this embodiment is -Z １ The amino acid residues that are not -(Rf)n3 are preferably amino acids that make up proteins, their D-forms, and amino acid residues of modified amino acids in which the side chains of these are modified.

[0067] Examples of amino acids that make up proteins include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, and histidine. Modified amino acids, which are amino acids that make up proteins, include, for example, amino acids in which the hydrogen atoms of the amino group in the side chain of lysine, arginine, and histidine are replaced with the above-mentioned amino group protecting groups or Pbf(N-ω-(2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl) group; amino acids in which the hydrogen atoms of the carboxyl group in the side chain of aspartic acid and glutamic acid are replaced with the above-mentioned carboxyl group protecting groups or the alkyl group of the tert-butyl group; and amino acids in which the hydrogen atoms of the thiol group of cysteine ​​are replaced with a benzyl group.

[0068] In the case of a CP-containing peptide used in this embodiment, if it has two or more CP-containing amino acid residues, the positional relationship between the CP-containing amino acid residues is not particularly limited as long as the peptide has a helical structure. In the case of a CP-containing peptide used in this embodiment, it is preferable that the spacing between CP-containing amino acid residues is 3, 6, or 7 amino acid residues, that is, the number of amino acid residues in the subregion sandwiched between two CP-containing amino acid residues is 3, 6, or 7, and more preferably 3 or 6, from the viewpoint of easily adopting a helical structure. If there are three or more CP-containing amino acid residues, the spacing between the CP-containing amino acid residues may be different from each other, but it is preferable that they are all the same, that is, that the three or more CP-containing amino acid residues are arranged at equal intervals. In this embodiment, the CP-containing peptide is preferably arranged such that three or more CP-containing amino acid residues are spaced equally apart at intervals of 3, 6, or 7 amino acid residues, and more preferably at intervals of 3 or 6 amino acid residues, as this facilitates the adoption of a 3-10 helix structure, an α-helix structure, or a π-helix structure.

[0069] In this embodiment, the CP-containing peptide is preferably amphiphilic. By complexing a helical, amphiphilic CPP with a pharmacoactive ingredient, a CPP-pharmacoactive ingredient complex with superior cell membrane permeability can be obtained. In particular, the CP-containing peptide used in this embodiment has a structure in which CP-containing amino acid residues are aligned on one side of the central axis of the helical structure of the CP-containing peptide, and hydrophilic amino acid residues are aligned on the opposite side.

[0070] In this embodiment, the CP-containing peptide is preferably one in which two or more CP-containing amino acid residues are arranged at equal intervals of 3, 6, or 7 amino acid residues, and the amino acid residue following the CP-containing amino acid residue is a hydrophilic amino acid residue, in order to easily adopt an amphiphilic helical structure. More preferably, the peptide is preferably one in which two or more CP-containing amino acid residues are arranged at equal intervals of 3 or 6 amino acid residues, and the amino acid residue following the CP-containing amino acid residue is a hydrophilic amino acid residue. Even more preferably, the peptide is preferably one in which two or more CP-containing amino acid residues are arranged at equal intervals of 3 or 6 amino acid residues, and the amino acid residue following the CP-containing amino acid residue is an arginine residue or a lysine residue. Even more preferably, the peptide is preferably one in which two or more CP-containing amino acid residues are arranged at equal intervals of 3 or 6 amino acid residues, and the amino acid residue following the CP-containing amino acid residue is an arginine residue. In this embodiment, the CP-containing peptide is particularly preferably one in which the CP-containing amino acid residues are located at the 1st, 4th, and 7th positions, or at the 1st and 7th positions, and the arginine or lysine residues are located at the 2nd, 5th, and 8th positions, or at the 2nd and 8th positions; more preferably one in which the CP-containing amino acid residues are located at the 1st and 7th positions, and the arginine or lysine residues are located at the 2nd and 8th positions; and even more preferably one in which the CP-containing amino acid residues are located at the 1st and 7th positions, and the arginine residues are located at the 2nd and 8th positions.

[0071] The CP-containing peptide used in this embodiment can be synthesized by general peptide synthesis methods, except that at least a CP-containing amino acid is used as the starting amino acid. For example, it can be synthesized by solid-phase peptide synthesis. The CP-containing peptide can be easily synthesized using an automated peptide synthesizer with CP-containing amino acid residues as starting materials.

[0072] Peptides can be produced by sequentially condensing amino acids with protected amino groups with amino acids whose C-terminus is bonded to a solid phase, and then removing the peptide from the solid phase. It is preferable to use amino acid raw materials in which the amino group is protected with a Boc group or an Fmoc group. It is also preferable to use amino acid raw materials in which the side chain functional group is protected with a protecting group. Examples of protecting groups for side chain functional groups include the Boc group, triphenylmethyl group, benzyl group, and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) group.

[0073] Examples of condensing agents that form peptide bonds include N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (WSC), benzotriazole-1-yloxytrisdimethylaminophosphonium hexafluorophosphate (BOP), benzotriazole-1-yloxytrispirolidinophosphonium hexafluorophosphate (pyBOP), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, and 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU). Furthermore, N-hydroxybenzotriazole (HOBt), (hydroxyimino)cyanoethyl acetate (oxyma), and the above-mentioned condensing agent can also be mixed in a preferred ratio and used.

[0074] Methods for activating the carboxyl terminus may be used to form peptide bonds, and examples of activators include N-hydroxysuccinimide, p-nitrophenyl esters, and pentafluorophenyl esters. Examples of bases used when forming peptide bonds include triethylamine and diisopropylethylamine (DIPEA). Examples of solvents used in the peptide bond formation reaction include chloroform, dichloromethane (DCM), dichloroethane (DCE), acetonitrile (MeCN), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0075] The Boc and Fmoc groups, which are protecting groups for the amino-terminal amino groups of peptides or amino acids, can be removed by trifluoroacetic acid (TFA) or piperidine, respectively. Protecting groups for the side-chain functional groups of amino acid residues of peptides can be removed by, for example, TFA, hydrogen fluoride (HF), or trifluoromethanesulfonic acid.

[0076] Furthermore, in peptide solid-phase synthesis, TFA can be used, for example, to remove peptides with protecting groups attached to the side-chain functional groups of peptides or amino acid residues from the peptide solid-phase synthesis resin. The removal of peptides from the peptide solid-phase resin and the removal of protecting groups from the side-chain functional groups of amino acid residues can be carried out simultaneously within the same reaction system, or they can be carried out independently. As peptide solid-phase synthesis resins for peptide solid-phase synthesis, commercially available resins such as 4-hydroxymethyl-3-methoxyphenoxybutyrate-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, and oxime resins can be used.

[0077] The target peptide or its intermediate can be isolated and purified by various methods, such as ion chromatography, gel filtration chromatography, reverse-phase chromatography, normal-phase chromatography, recrystallization, extraction, and fractional crystallization. Furthermore, the peptides thus obtained can be converted to their respective salts by conventional methods.

[0078] The protecting groups of the amino or carboxyl groups of the manufactured CP-containing peptides can be deprotected as needed. Deprotection can be carried out by conventional methods depending on the type of protecting group.

[0079] <Therapeutic Ingredients> The therapeutic ingredients used in this embodiment are not particularly limited as long as they are functional ingredients that exhibit some kind of physiological activity when taken up into target cells in vivo or in vivo. Examples of such therapeutic ingredients include oligonucleotides, nucleic acids, peptides, proteins, oligosaccharides, polysaccharides, and low molecular weight compounds (compounds with a molecular weight of 500 or less). As the therapeutic ingredients used in this embodiment, substances having a negative charge are preferred because they are more likely to form a stable complex with the CP-containing peptide. As the therapeutic ingredients used in this embodiment, nucleic acids, peptides, proteins, polysaccharides, or low molecular weight compounds having an acidic group are more preferred, and nucleic acids or low molecular weight compounds having an acidic group are even more preferred. Examples of acidic groups include hydroxyl groups, carboxyl groups, and sulfo groups.

[0080] The nucleic acid used as the active ingredient may be DNA, RNA, or a chimeric nucleic acid of DNA and RNA. Examples of DNA include genomic DNA, cDNA, plasmid DNA, and antisense oligonucleotides (ASOs). Examples of RNA include mRNA, siRNA, miRNA, and antisense RNA. The nucleic acid contained in the LNP of the present invention may be single-stranded or double-stranded. It may also be linear or circular. For example, by complexing various nucleic acids used in gene therapy with the CP-containing peptide as the active ingredient, a therapeutic agent with good delivery efficiency to target cells can be obtained.

[0081] The nucleic acid used as the active ingredient may be a nucleic acid composed of natural nucleic acid bases, an artificial nucleic acid, or a nucleic acid containing both natural and artificial nucleic acid bases. Examples of artificial nucleic acids include peptide nucleic acid (PNA), LNA (Locked Nucleic Acid), and alkynyl nucleic acid. For example, the nucleic acid used as the active ingredient in this embodiment may be unmodified, or it may be modified or altered in any part of the nucleic acid by a known method for purposes such as nucleic acid stabilization.

[0082] <CPP / AI Complex> A CPP / AI complex can be produced by mixing the CP-containing peptide and the pharmaceutically active ingredient in a suitable solvent and bringing them into contact in the solvent to complex them. The solvent is not particularly limited as long as it is a solvent in which both the CP-containing peptide and the pharmaceutically active ingredient can be dissolved, and various solvents used for dissolving peptides and nucleic acids, such as phosphate buffer, Tris buffer, and HEPES buffer, can be used as appropriate.

[0083] The CPP / AI complex contained in the pharmaceutical composition of this embodiment preferably has a surface tension of 25 mN / m or more, and more preferably 25 mN / m to 30 mN / m, from the viewpoint of higher stability of the complex. The surface tension of the CPP / AI complex can be measured using pendant drop densitometry. For example, a measurement solution obtained by dissolving the CPP / AI complex to be measured in phosphate-buffered saline (PBS) is extruded from a capillary tube of a pendant drop densitometry device, and the pendant drop (droplet volume 8 μL) created at the tip of the needle is monitored with a camera for a measurement time of 1200 seconds at room temperature. The surface tension of the CPP / AI complex can be determined by fitting the shape of the pendant drop with the Young-Laplace equation (equation (1) below) and converting it into a surface tension value.

[0084]

[0085] The CPP / AI complex contained in the pharmaceutical composition of this embodiment may be one type or two or more types.

[0086] The pharmaceutical composition of this embodiment may consist solely of the CPP / AI complex, or it may contain other components. The amount of the CPP / AI complex in the total composition of the pharmaceutical composition of this embodiment is not particularly limited and can be adjusted as appropriate considering the route of administration, dosage form, etc. The amount of the CPP / AI complex in the total composition of the pharmaceutical composition of this embodiment can be, for example, 0.1 to 99.9% by mass.

[0087] The pharmaceutical composition of this embodiment may be a mixture of a CPP / AI complex and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier generally refers to an inert and non-toxic solid or liquid carrier that does not react with the active ingredient, such as a bulking agent, diluent, or encapsulating material. Various organic or inorganic carrier substances commonly used as pharmaceutical materials can be used as pharmaceutically acceptable carriers, for example, as excipients, lubricants, binders, or disintegrants in solid formulations; or as solvents, solubilizers, suspending agents, isotonic agents, buffers, or analgesics in liquid formulations. Pharmaceutical additives such as preservatives, antioxidants, colorants, and sweeteners may also be used as needed.

[0088] The pharmaceutical composition of this embodiment may be administered orally or parenterally. Parenteral administration may include, but is not limited to, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, vaginal, and intranasal administration. The number of administrations may be a single dose or multiple doses.

[0089] The dosage forms of the pharmaceutical composition of this embodiment include, for example, oral preparations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, and orally disintegrating tablets), capsules (including soft capsules and microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, and film preparations (e.g., orally disintegrating films); and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, and drip infusions), topical preparations (e.g., transdermal preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), and eye drops. These can each be safely administered orally or parenterally (e.g., topically, rectally, or intravenously). These preparations may also be controlled-release preparations such as immediate-release preparations or sustained-release preparations (e.g., sustained-release microcapsules).

[0090] The target population to which the pharmaceutical composition of this embodiment can be applied is not particularly limited. Examples of target populations to which the pharmaceutical composition of this embodiment can be applied include, but are not limited to, mammals (humans, chimpanzees, dogs, cats, horses, cattle, sheep, goats, rats, mice, rabbits, pigs, etc.). In one preferred embodiment, the target population may be humans.

[0091] The pharmaceutical composition of this embodiment and the CPP / AI complex contained therein can be used to introduce pharmacoactive ingredients into cells outside the body. For example, the CPP / AI complex can be introduced into cultured cells by culturing them in a culture medium containing the CPP / AI complex.

[0092] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0093] <Peptide Synthesis> Peptides were synthesized by Fmoc-based peptide synthesis. 53 mg of linkamide resin (0.48 mmol / g) was weighed into a plastic syringe with a filter plate, and the outlet was connected to a "PetiSyzer" (registered trademark) (Hipep Laboratories). 2.0 mL of DMF was added to the resin, and after gentle stirring for 30 minutes, the solvent was removed by vacuum filtration. 2.0 mL of 20% piperidine / DMF (v / v) was added, and after gentle stirring for 5 minutes, the solvent was removed by vacuum filtration. 2.0 mL of DMF was added, and after stirring for 30 seconds, the solvent was removed by vacuum filtration (three times). 0.1 mmol of Fmoc-AA-OH (4 equivalents), Oxyma ((hydroxyimino)cyanoethyl acetate (CAS RN: 3849-21-6)) (4 equivalents), and COMU (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (CAS RN: 1075198-30-9)) (4 equivalents) were dissolved in 2.0 mL of DMF. The resulting solution was added to the resin and stirred gently for 30 seconds. 100 μmol of DIEA was added. The mixture was stirred gently for 60 minutes. After the final amino acid residue was bonded, the synthesized peptide was cleaved and recovered in a solution of 95% TFA, 2.5% TIPS (triisopropylsilyl chloride), and 2.5% water. The crude mixture was then purified by HPLC (ACN-water, 0.1% TFA).

[0094] <CD Spectrum> The CD spectrum of the peptide was measured using a spectrometer ("JASCO J820 Spectrometer," manufactured by JASCO).

[0095] <Zeta Potential> The zeta potential of the peptide was measured in a 5 μM solution using a zeta potential measuring device ("Zetasizer Pro Red," manufactured by Marvern) equipped with a 10 mW laser with an operating wavelength of 633 nm.

[0096] <Cells and Culture Medium> HeLa cells were cultured in DMEM (Nacalai Tesque), and A549 cells were cultured in RPMI1640 (Nacalai Tesque) supplemented with 10% FBS (v / v) heat-inactivated fetal bovine serum (FBS) and a mixed antibiotic / antifungal agent solution (Nacalai Tesque), with 5% CO2. ２ The cells were cultured at 37°C in a humidified incubator containing [specific ingredients / materials]. A549 cells and HeLa cells were provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Bioresource Project.

[0097] <Microscopic Observation> A microscope ("Leica TCS SP8", manufactured by Leica) was used to observe the cells. Fluorescence detection of FAM and GFP was performed at λex = 488 nm and λobs = 500–550 nm.

[0098] <FACS Analysis> For FACS analysis of cells after the introduction of the complex or DNA, a FACS instrument ("Guava® easyCyte", manufactured by Merk) was used.

[0099] [Production Example 1] A peptide (ARA) consisting of the amino acid sequence (ARAARAARA) represented by SEQ ID NO: 1 was synthesized, and its CD spectrum was examined.

[0100]

[0101] MALDI TOF-MS [M+H] + calcd for C 36 H70N 19 O9: 912.56, found: 912.14

[0102] [Production Example 2] A peptide (ARAib) was synthesized in which the hydrogen atoms bonded to the α-carbons of the 3rd, 6th, and 9th alanine atoms in the amino acid sequence (ARAARAARA) represented by Sequence ID No. 1 were replaced with methyl groups, and the CD spectrum was examined.

[0103]

[0104] MALDI TOF-MS [M+H] + calcd for C 39 H 76 N 19O9: 954.6068, found: 955.21

[0105] [Production Example 3] A peptide (NRA) consisting of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 was synthesized, and its CD spectrum was examined.

[0106]

[0107] MALDI TOF-MS [M+H] + calcd for C 38 H 73 N 21 O 11 : 998.57 found: 998.96

[0108] [Manufacturing Example 4] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C) ８ F １７ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ８ F １７ Peptides (F8-2) substituted with amino acid residues that are substituted with a group. １７ We synthesized the compound and examined its CD spectrum.

[0109]

[0110] MALDI TOF-MS [M+H] + calcd for C 66 H 78 F 34 N 21 O 11 : 1986.5641, found: 1987.17

[0111] [Manufacturing Example 5] The first asparagine residue and the fourth alanine residue of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C ８ F １７ Peptide (F8-2) substituted with a residue １４ We synthesized the compound and examined its CD spectrum.

[0112]

[0113] MALDI TOF-MS [M+H] + calcd for C 66 H 78 F 34 N 21 O 11 : 1986.56, found: 1987.35

[0114] [Manufacturing Example 6] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C) ６ F １３ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ６ F １３ Peptides (F6-2) substituted with amino acid residues that are substituted with a group. １７ We synthesized the compound and examined its CD spectrum.

[0115]

[0116] MALDI TOF-MS [M+H] + calcd for C 62 H 78 F 26 N 21 O 11 : 1786.5769 found: 1787.42

[0117] [Manufacturing Example 7] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C) ４ F ９ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ４ F ９ Peptides (F4-2) substituted with amino acid residues that are substituted with a group. １７ We synthesized the compound and examined its CD spectrum.

[0118]

[0119] MALDI TOF-MS [M+H]+ calcd for C 58 H 78 F 18 N 21 O 11 : 1586.59, found: 1587.10

[0120] [Manufacturing Example 8] The third and fourth asparagine residues of the amino acid sequence (ARNNRAARA) represented by Sequence ID No. 3 are Asn(C) ８ F １７ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ８ F １７ Peptides (F8-2) substituted with amino acid residues that are substituted with a group. ３４ We synthesized the compound and examined its CD spectrum.

[0121]

[0122] MALDI TOF-MS [M+H] + calcd for C 66 H 78 F 34 N 21 O 11 : 1986.5641, found: 1987.29

[0123] [Production Example 9] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C １２ H ２５ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C １２ H ２５ Peptides (H12-2) substituted with amino acid residues that are substituted with a group. １７ We synthesized the compound and examined its CD spectrum.

[0124]

[0125] MALDI TOF-MS [M+H] + calcd for C 74 H 128N 21 O 11 : 1487.01, found: 1487.14

[0126] [Production Example 10] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C ８ H １７ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ８ H １７ Peptides (H8-2) substituted with amino acid residues that are substituted with a group. １７ We synthesized the compound and examined its CD spectrum.

[0127]

[0128] MALDI TOF-MS [M+H] + calcd for C 66 H 112 N 21 O 11 : 1374.88, found: 1375.47

[0129] [Production Example 11] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C ４ F ９ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ４ F ９ A peptide (F4-2) in which an amino acid residue is substituted with a group that １７ Aib was synthesized and its CD spectrum was examined.

[0130]

[0131] MALDI TOF-MS [M+H] + calcd for C 61 H 84 F 18 N 21 O 11: 1628.64, found: 1629.16

[0132] [Production Example 12] The first and seventh asparagine residues of the amino acid sequence (NRAARANRA) represented by Sequence ID No. 2 are Asn(C) ６ F １３ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ６ F １３ A peptide (F6-2) in which an amino acid residue is substituted with a group that １７ Aib was synthesized and its CD spectrum was examined.

[0133]

[0134] MALDI TOF-MS [M+H] + calcd for C 65 H 84 F 26 N 21 O 11 : 1828.62, found: 1829.61

[0135] [Manufacturing Example 13] The fourth asparagine residue of the amino acid sequence (ARANRAARA) represented by Sequence ID No. 4 is Asn(C ８ F １７ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ８ F １７ A peptide (F8-1) in which an amino acid residue is substituted with a group that ４ Aib was synthesized and its CD spectrum was examined.

[0136]

[0137] MALDI TOF-MS [M+H] + calcd for C 54 H 80 F17 N 20 O 10 : 1491.61, found:1491.89 CD specter not measured

[0138] [Production Example 14] A fragment peptide of p53 (ETFSDLWKLLLPEN: SEQ ID NO: 5) (p53_frag) was synthesized and its CD spectrum was examined.

[0139]

[0140] MALDI TOF-MS [M+H] + calcd for C 74 H 110 F 17 NaO 22 : 1611.79, found: 1612.26

[0141] [Production Example 15] The fourth serine residue and the eleventh proline residue of the p53 fragment peptide (ETFSDLWKLLLPEN: SEQ ID NO 5) (p53_frag) are Asn(C ８ F １７ ) residue (the hydrogen atom of the amino group in the side chain of the asparagine residue is Z in general formula (1) １ Rf is a phenylene group, and Rf is -C ８ F １７ A peptide (p53-F_frag) was synthesized in which an amino acid residue was substituted with a group (p53-F_frag), and its CD spectrum was examined.

[0142]

[0143] MALDI TOF-MS [M+H] + calcd for C 102 H 117 F 34 N 19 NaO 23 : 2644.79, found: 2646.12

[0144] Figures 1-14 show the CD spectra of the peptides synthesized in manufacturing examples 1-12, 14, and 15. As these spectra show, the peptide (F8-2 １７ ), peptide (F8-2 １４), peptide (F6-2 １７ ), peptide (H12-2 １７ ), peptide (H8-2 １７ Aib), peptide (F6-2 １７ The CD spectra of Aib and the peptide (p53-F_frag) showed negative maxima around 208-209 and 222 nm, a positive maxima around 191-193 nm, and a 222 / 208 nm ratio of approximately 0.3-0.7, confirming that these peptides adopt a 3-10 helix structure. The other peptides, on the other hand, adopted a random structure.

[0145] Peptides (NRA) and (p53-frag) have random structures, whereas peptides (F8-2) are formed by introducing a group represented by general formula (1) into them. １７ The peptide (p53-F_frag) and the peptide (p53-F_frag) adopted a helical structure. These results show that a helical structure can be induced in a randomly structured peptide by introducing an appropriate number of groups represented by general formula (1) in an appropriate arrangement.

[0146] [Example 1] The peptide synthesized in Production Example 1 was complexed with FAM-labeled DNA, and its cell membrane permeability was evaluated. As the FAM-labeled DNA, a 19-base DNA (5'-TTTTTCAGTTGAACCATATA-[FAM]-3', SEQ ID NO: 6) with FAM labeled at the 3' end was used (Eurofins Genomics).

[0147] <Peptide-Nucleic Acid Complex> First, a peptide-nucleic acid complex was prepared by mixing a 0.50 μM peptide with a 45 nM DNA complex using PBS as a solvent to complex the two.

[0148] <Measurement of Surface Tension of Peptide-Nucleic Acid Complexes> The surface tension of peptide-nucleic acid complexes was measured using a pendant drop densitometer (Kyowa Interface Science Co., Ltd.) at room temperature for a measurement time of 1200 seconds. Peptide-nucleic acid complex solution (CHCl) ３ A drop (8 μL, solvent: PBS) was used for measurement. A solution (CHCl2) that does not contain peptide-nucleic acid complexes was also used. ３A drop (8 μL, solvent: PBS) was used as a control.

[0149] Peptide (F8-2 １７ ), peptide (F6-2 １７ ), peptide (F8-2 ３４ ), and peptide (F6-2 ３４ Figure 15 shows the results of measuring the surface tension of the peptide-nucleic acid complexes obtained using these materials by pendant drop densitometry. As shown in Figure 15, a tendency for the surface tension to decrease over time was observed for all complexes. The surface tension was lower for the peptide (F8-2) which had a random structure. ３４ ) and peptide (F6-2 ３４ The peptide (F8-2) which had a helical structure was approximately 31 mN / m at the start of the measurement, but after 1100 seconds it was less than 25 mN / m, a decrease of nearly 20%. In contrast, the peptide (F8-2) which had a helical structure １７ ) and peptide (F6-2 １７ The surface tension of the peptide was approximately 32 mN / m at the start of the measurement and remained above 28 mN / m even after 1100 seconds, indicating a small decrease in surface tension. These results confirm that the complex with the helical peptide was more stable than the complex with the randomly structured peptide.

[0150] <Intracellular uptake of peptide-nucleic acid complexes - 1> HeLa cells (2.0 x 10⁻⁶) ４ Cells (per well) were seeded in an 8-well glass-bottom chamber and stored in DMEM (10% FBS) at 37°C and 5% CO2. ２ The cells were incubated for 24 hours. The cell samples were washed three times (500 μL) with D-PBS and then mixed with FAM-labeled DNA and peptide (peptide (F4-2 １７ ), peptide (F6-2 １７ ), or peptide (F8-2 １７ Serum-free DMEM (500 μL) containing a complex with (5 μM peptide, 0.45 μM DNA), or serum-free DMEM (500 μL) containing FAM-labeled DNA and PEI (polyethyleneimine, 25 kDa, 1 mg / mL) (1 μg), at 37°C and 5% CO2. ２The cells were incubated in [a specific solution]. After 6 hours of incubation, the cells were washed three times with D-PBS (500 μL), and then DMEM (500 μL) was added to each well. The cells were observed under CLSM 6 hours after the start of culture. The cells were then cultured for a further 18 hours, and the cells were also observed under a microscope 24 hours after the start of culture.

[0151] Figure 16 shows FAM fluorescence images of cells at 6 hours (top panel) and 24 hours (bottom panel) after the start of culture. The peptide (F6-2) that had a helical structure was observed. １７ ) and peptide (F8-2 １７ Cells administered with the complex showed a higher proportion of cells incorporating the complex, confirming that its uptake efficiency into cells was higher than that of commonly used PEI.

[0152] <Intracellular uptake of peptide-nucleic acid complexes - 2> A549 cells (2.0 x 10) ４ Cells (per well) were seeded in an 8-well glass-bottom chamber and stored in DMEM (10% FBS) at 37°C and 5% CO2. ２ The cells were incubated for 24 hours. The cell samples were washed three times (500 μL) with D-PBS, and EGFP-encoded pDNA (1 μg) and peptide (peptide (F4-2) were added. １７ ), peptide (F6-2 １７ ), or peptide (F8-2 １７ Add serum-free DMEM (500 μL) containing a complex with (5 μM peptide, 0.45 μM DNA) or serum-free DMEM (500 μL) containing PEI (polyethyleneimine, 25 kDa, 1 mg / mL) (1 μg), and store at 37°C and 5% CO2. ２ The cells were incubated in [a specific solution]. After 24 hours of incubation, the cells were washed three times with D-PBS (500 μL), and then DMEM (500 μL) was added to each well. The cells were observed under a microscope 24 hours after the start of culture.

[0153] Figure 17 shows the GFP fluorescence image of cells 24 hours after the start of culture. The peptide (F6-2) that had a helical structure was observed. １７ ) and peptide (F8-2 １７Cells treated with the complex of ) showed EGFP expression, but peptides (F4-2) had a random structure. １７ In cells administered with the complex of ), EGFP expression was not observed. Also, peptide (F6-2 １７ Rather than a complex of ) peptides (F8-2 １７ The complex of ) showed a higher number of cells expressing EGFP, and this correlated with the chain length (number of carbon atoms) of the Rf group, with longer chain lengths indicating superior nucleic acid uptake efficiency (transfection efficiency).

[0154] <Intracellular uptake of peptide-nucleic acid complexes - 3> HeLa cells (2.0 x 10) ４ Cells (per well) were seeded in an 8-well glass-bottom chamber and stored in DMEM (10% FBS) at 37°C and 5% CO2. ２ The cells were incubated for 24 hours. The cell samples were washed three times (500 μL) with D-PBS and then mixed with FAM-labeled DNA and peptide (peptide (F4-2 １７ ), peptide (F6-2 ３４ ), peptide (F8-2 ３４ ), peptide (F6-2 １７ ), or peptide (F8-2 １７ Add serum-free DMEM (500 μL) containing a complex of (5 μM peptide, 0.45 μM DNA) and heat at 37°C and 5% CO2. ２ The cells were incubated in [a specific solution]. After 4 hours of incubation, the cells were washed three times with D-PBS (500 μL) and subjected to FACS analysis (n=3). Cells treated with only FAM-labeled DNA were used as a control.

[0155]

[0156] Table 1 and Figure 18 show the results of FACS analysis of cells that incorporated each peptide-nucleic acid complex. Cells that incorporated only FAM-labeled DNA were used as a control. As shown in Figure 18, the peptide that took on a helical structure (F6-2) １７ ) and peptide (F8-2 １７Cells administered with the complex showed a high FAM luminance value (RFI), and it was confirmed that the uptake efficiency into cells was higher than that of nucleic acids complexed with randomly structured peptides.

[0157] [Example 2] Peptide (F6-2) synthesized in Production Example 6 １７ ) or the peptide synthesized in Production Example 4 (F8-2 １７ The cell membrane permeability was evaluated by complexing the active ingredient with a fluorescently labeled active ingredient. The fluorescently labeled active ingredients included a peptide (FAM-p53-TAD) (M.W. 66kDa) in which the lysine residue of the p53-TAD peptide (a peptide with TAD added to the C-terminus of SEQ ID NO: 5) was labeled with FAM, and the p53-TAD peptide with AlexaFluor at its N-terminus. ４８８ Labeled peptide (AF ４８８ We used either a protein in which lysine residues of BSA (bovine serum albumin) were labeled with FAM (FAM-BSA), or DNA labeled with FAM at its 3' end (SEQ ID NO: 6) (FAM-DNA, manufactured by Eurofins Genomics).

[0158] <Intracellular uptake of peptide-peptide complexes - 1> First, using serum-free DMEM as a solvent, a 5 μM solution of the peptide from Production Example 6 (F6-2) was prepared. １７ A peptide-peptide complex was produced by mixing ) with 1 μg of FAM-p53-TAD to complex the two. HeLa cells (2.0 × 10⁻⁶) ４ Cells (per well) were seeded in an 8-well glass-bottom chamber and stored in DMEM (10% FBS) at 37°C and 5% CO2. ２ The cells were incubated for 24 hours. The cell samples were washed three times with D-PBS (500 μL), and serum-free DMEM (500 μL) containing the prepared peptide-peptide complex was added. Incubation was performed at 37°C and 5% CO2. ２ The cells were incubated in [a specific solution]. After 2 hours of incubation, the cells were washed three times with D-PBS (500 μL) and then observed under a fluorescence microscope (λex = 488 nm, λem = 500–550 nm). Cells treated with FAM-p53-TAD alone were used as a control.

[0159] Figure 19 shows FAM fluorescence images of cells 2 hours after the addition of the peptide-peptide complex. The scale bar represents 25 μm. The peptide (F6-2) that had a helical structure was shown. １７ Cells administered with the complex of ) showed fluorescence of FAM. These results suggest that even when the active ingredient is a peptide, the peptide (F6-2) takes on a helical structure. １７ It was confirmed that it is taken up into cells by complexing with ).

[0160] <Intracellular uptake of peptide-peptide complexes and peptide-nucleic acid complexes - 3> First, using serum-free DMEM as a solvent, 5 μM of the peptide (F8-2) from Production Example 4 is used. １７ ) and 0.5 μM peptide (AF ６４７ -F8-2 １７ ) (Peptide (F8-2 １７ The N-terminus of ) is AlexaFlour ６４７ A peptide-nucleic acid complex was produced by mixing a peptide labeled with 500 nM FAM-DNA with HeLa cells (2.0 × 10⁻¹⁶). ４ Cells (per well) were seeded in an 8-well glass-bottom chamber and stored in DMEM (10% FBS) at 37°C and 5% CO2. ２ The cells were incubated for 24 hours. The cell samples were washed three times with D-PBS (500 μL), and serum-free DMEM (500 μL) containing the prepared peptide-nucleic acid complex was added. The cells were incubated at 37°C and 5% CO2. ２ The cells were incubated in [a specific solution]. After 4 hours of incubation, the cells were washed three times with D-PBS (500 μL) and then stained with the nuclear stain Hoechst 33342. The stained cells were then examined under a fluorescence microscope (λex = 488 nm, λem = 500–550 nm (FAM), 650–700 nm (AF)). ６４７ Observed using )).

[0161] Figure 20 shows FAM fluorescence images of cells 4 hours after the addition of the peptide-nucleic acid complex. ６４７ Fluorescence images and Hoechst 33342 stained images are shown. The scale bar represents 25 μm. The peptide (F8-2) had a helical structure. １７In cells administered with a complex of ), FAM and AF ６４７ Both fluorescence was confirmed. From these results, the fluorescently labeled peptide (AF ６４７ -F8-2 １７ Even when complexed with both ) and fluorescently labeled nucleic acid (FAM-DNA), the peptide (F8-2) １７ It was confirmed that this can promote uptake into cells.

[0162] <Intracellular uptake of peptide-protein complexes - 4> First, using serum-free DMEM as a solvent, prepare a 5 μM peptide (F8-2) from Production Example 4. １７ A peptide-protein complex was produced by mixing ) with 0.1, 1, 10, or 100 μg / mL of FAM-BSA to complex the two. HeLa cells (2.0 × 10) ４ Cells (per well) were seeded in an 8-well glass-bottom chamber and stored in DMEM (10% FBS) at 37°C and 5% CO2. ２ The cells were incubated for 24 hours. The cell samples were washed three times with D-PBS (500 μL), and serum-free RPMI (500 μL) containing the prepared peptide-protein complex was added. The cells were incubated at 37°C and 5% CO2. ２ The cells were incubated in [a specific solution]. After 2 hours of incubation, the cells were washed three times (500 μL) with D-PBS and then stained with the nuclear stain Hoechst 33342 or the lysosomal stain Lysotracker red. The stained cells were then examined under a fluorescence microscope (λex = 488 nm, λem = 500–550 nm (FAM), 650–700 nm (AF)). ６４７ Observations were made using 580-630 (Lysotracker red).

[0163] Figure 21 shows FAM fluorescence images of cells 2 hours after the addition of the peptide-peptide complex. The scale bar represents 25 μm. The peptide (F8-2) that had a helical structure. １７In cells administered with a complex containing FAM-BSA, the fluorescence of FAM was confirmed to increase in a concentration-dependent manner with FAM-BSA. Furthermore, FAM fluorescence images, Hoechst 33342 staining images, Lysotracker red staining images, and Merge images of cells that incorporated a complex containing 100 μg / mL of FAM-BSA are shown in Figure 22(A). Figure 22(B) is a magnified view of the area enclosed by a rectangle in the Merge image of Figure 22(A). As shown in Figure 22(B), FAM-BSA colocalizes with Lysotracker red (arrow), and peptide (F8-2) is present. １７ This suggests that the complex is taken up into cells via lysosomes.

[0164] The pharmaceutical composition of this embodiment exhibits excellent cell membrane permeability because the active ingredient is complexed with CPP, which has a helical structure. For this reason, it is expected to be used in pharmaceutical fields, for example, as a carrier for introducing the active ingredient into target cells.

Claims

1. A pharmaceutical composition containing a complex of a peptide having a helical structure and a medicinal ingredient, wherein the peptide has three or more amino acids peptide-bonded, and at least one side chain of the amino acid residues constituting the peptide has a group represented by the following general formula (1): [In the formula, Z １ is a single bond or a divalent, trivalent or tetravalent linking group (excluding an alkylene group); Rf is a C １－３０ alkyl group substituted with at least two fluorine atoms, a C ２－３０ alkyl group having 1 to 5 ether-bonded oxygen atoms between carbon atoms and substituted with at least two fluorine atoms, -SF ５ , -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ Cl (R １０１ , R １０２ , R １０３ , and R １０４ are each independently a hydrogen atom, a fluorine atom or a chlorine atom, but two or more of R １０１ , R １０２ , R １０３ , and R １０４ are fluorine atoms), or a C ８－３０ alkyl group not substituted with a fluorine atom; n3 is 1, 2 or 3, and the black circle means a bond].

2. The Rf is a carbon atom substituted with at least two fluorine atoms. ６－３０ A C atom having an alkyl group, 1 to 5 ether-bonded oxygen atoms between carbon atoms, and substituted with at least 2 fluorine atoms. ６－３０ C that is not substituted with alkyl groups or fluorine atoms ８－３０ The pharmaceutical composition according to claim 1, wherein the alkyl group is an alkyl group.

3. The Rf is a carbon atom substituted with at least two fluorine atoms. １－３０ A C atom having an alkyl group, 1 to 5 ether-bonded oxygen atoms between carbon atoms, and substituted with at least 2 fluorine atoms. ２－３０ Alkyl alkyl group, -SF ５ , or -SF ４ -CR １０１ R １０２ -CR １０３ R １０４ Cl(R) １０１ , R １０２ , R １０３ , and R １０４ Each of these is independently a hydrogen atom, a fluorine atom, or a chlorine atom, but R １０１ , R １０２ , R １０３ , and R １０４ The pharmaceutical composition according to claim 1, wherein two or more of the atoms are fluorine atoms.

4. The pharmaceutical composition according to claim 1, wherein the peptide has two or more amino acid residues having a group represented by the general formula (1) in its side chain.

5. The pharmaceutical composition according to claim 1, wherein the peptide is amphiphilic.

6. The pharmaceutical composition according to claim 5, wherein the peptide has a structure in which amino acid residues having the group represented by the general formula (1) are arranged on one side with respect to the central axis of the helical structure, and hydrophilic amino acid residues are arranged on the opposite side.

7. The pharmaceutical composition according to claim 4, wherein the peptide has a side chain with a group represented by the general formula (1) and the spacing between amino acid residues is 3, 6, or 7 amino acid residues.

8. The above Rf is given by the following general formula (f-1) or (f-2) [wherein, Rf Ｐ This is a fully halogenated C containing at least two or more fluorine atoms. １－１０ A fully halogenated carbon atom containing an alkyl group or at least two fluorine atoms and having an ether-bonded oxygen atom between carbon atoms. ２－１０ The pharmaceutical composition according to claim 1, wherein the group is represented as [where n1 is an integer from 0 to 10, n2 is an integer from 0 to 9, and the black circle represents a bond].

9. Said Z １ However, alkylene groups, -O-, -S-, -NH-, -N(CH ３ )-,-N(C ２ H ５ )-,-N(C ３ H ７ )-, trivalent nitrogen atom, -C(=O)-, -S(=O) ２ - A cycloalkylene group, a divalent to tetravalent aryl group, a divalent to tetravalent heteroaryl group, or a combination thereof (excluding groups consisting solely of alkylene groups), as described in claim 1.

10. Said Z １ is -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O) ２ -NH-, -NH-S (=O) ２ -, -S (=O) ２ -NH-S (=O) ２ -, -C(=O)-NH-Ph- (where -Ph- is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5-phenylene group, or a 1,3,5-substituted phenyl group), or any of these groups and C １－６ The pharmaceutical composition according to claim 1, which is a combination of alkylene groups.

11. Said Z １ However, the following general formula (2) [In the formula, Z ２ is a linking group other than a divalent, trivalent, or tetravalent alkylene group; Rh is a hydrogen atom or C １－６ The pharmaceutical composition according to claim 1, wherein the linking group is an alkyl group and represented by the black circle [where the black circle represents a bond].

12. The pharmaceutical composition according to claim 1, wherein the amino acid residue whose side chain is the group represented by the general formula (1) is an amino acid residue to which one to three of the Rf groups are directly or indirectly linked to the side chain of a natural amino acid.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmacoactive ingredient has a negative charge.

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmacoactive ingredient is a nucleic acid, peptide, protein, polysaccharide, or a low molecular weight compound having an acidic group.

15. The pharmaceutical composition according to any one of claims 1 to 12, wherein the surface tension of the composite is 25 mN / m or more.

16. The pharmaceutical composition according to any one of claims 1 to 12, wherein the complex is formed by contacting the helical peptide and the pharmacoactive ingredient in a solvent in which both the peptide and the pharmacoactive ingredient are soluble.

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