Peptide and association or composition containing said peptide

Abstract

The present invention provides a peptide that can be utilized in active targeting DDS. Provided according to an embodiment of the present invention are: a peptide that has a hydrophobic region and a hydrophilic region lower in hydrophobicity than the hydrophobic region, located on at least one end side of the hydrophobic region, that has a first target binding site capable of binding to a first target at one end and a second target binding site capable of binding to a second target at the other end; a derivative of the peptide; or a salt of the peptide or derivative of the peptide.

Description

[Technical field]

[0001] The present invention relates to a peptide and an association or composition comprising said peptide. [Background technology]

[0002] Peptides, which are composed of several to several tens of amino acid residues, have the advantages of being easy to produce and have low quality control, being less susceptible to antigenicity, and being easily absorbed into tissues, compared to proteins such as antibodies. On the other hand, peptides have properties such as biological instability and aggregability, making them difficult to directly apply to pharmaceuticals.

[0003] In addition, various drug delivery systems (DDS) have been developed. For example, Patent Document 1 discloses that spherical nanoparticles formed by self-association of amphiphilic peptide molecules that include a hydrophilic portion containing a positively charged amino acid residue and a hydrophobic portion covalently bonded to the hydrophilic portion are used as a carrier formulation for hydrophobic drugs. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2017-525676 Summary of the Invention [Problem to be solved by the invention]

[0005] In recent years, active targeting DDS incorporating targeting molecules (ligand molecules) has been actively researched and developed, and the targeting properties of peptides, which have properties different from those of antibodies, have also attracted attention.

[0006] In view of the above, a primary object of the present invention is to provide a peptide that can be used in an active targeting-type DDS. [Means for solving the problem]

[0007] According to one aspect of the present invention, there are provided peptides as set forth in [1] to [8] below, peptide aggregates as set forth in [9] to

[11] , and peptide compositions as set forth in

[12] to

[13] below. [1] A peptide or a derivative thereof, or a salt thereof, which has a hydrophobic region and a hydrophilic region located at at least one end of the hydrophobic region and having a lower hydrophobicity than the hydrophobic region, and which has a first target binding site at one end capable of binding to a first target, and a second target binding site at the other end capable of binding to a second target. [2] The peptide or derivative thereof according to [1], or a salt thereof, wherein the first target and the second target are different from each other. [3] The peptide or derivative thereof, or a salt thereof, according to [1] or [2], wherein the number of constituent amino acid residues is 10 to 40. [4] A peptide or derivative thereof, or a salt thereof, according to any one of [1] to [3], which has the hydrophilic region on both ends of the hydrophobic region. [5] The peptide or derivative thereof according to [4], or a salt thereof, wherein the hydrophobic region comprises 2 to 15 amino acid residues, of which 60% or more are selected from nonpolar amino acid residues and tyrosine residues. [6] The peptide or derivative thereof according to [4] or [5], or a salt thereof, wherein the number of amino acid residues constituting the two hydrophilic regions is independently 3 to 24. [7] The peptide or derivative thereof, or a salt thereof, according to any one of [1] to [3], which has the hydrophilic region only on one end side of the hydrophobic region. [8] The peptide or derivative thereof according to any one of [1] to [7], or a salt thereof, wherein at least one of the first target binding site and the second target binding site has physiological activity. [9] A peptide association comprising the peptide or its derivative or a salt thereof according to any one of [1] to [8].

[10] The peptide association of [9], further comprising a drug.

[11] The peptide complex according to [9] or

[10] , which satisfies at least one of the following (i) and (ii): (i) the binding affinity of the peptide or derivative thereof, or a salt thereof, for the first target is equal to or greater than the binding affinity of a non-self-associating peptide comprising the first target binding site for the first target; (ii) the binding affinity of the peptide or a derivative thereof, or a salt thereof, for the second target is equal to or greater than the binding affinity of the non-self-associating peptide comprising the second target binding site for the second target.

[12] A peptide composition comprising an aqueous medium and the peptide association according to [9] or

[11] , wherein the peptide association is formed by association mediated by intermolecular interactions comprising hydrophobic interactions between the hydrophobic regions of the peptides.

[13] The peptide composition according to

[12] , wherein the aggregate further contains a drug. Effect of the Invention

[0008] The peptide according to the embodiment of the present invention has a configuration in which a hydrophobic region with relatively high hydrophobicity and a hydrophilic region with low hydrophobicity are arranged at both ends, and a target binding site having affinity for a target is arranged. A peptide having such a configuration can form an aggregate in an aqueous medium in which the end of the hydrophilic region is arranged on the outside by associating with the hydrophobic region through hydrophobic interaction between the hydrophobic regions. The aggregate can be used for an active targeting type DDS by further containing a drug or using a target binding site having physiological activity. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1(a) and (b) are schematic diagrams illustrating the structures of a peptide according to a first embodiment of the present invention and a peptide association formed by the peptide, respectively. [Diagram 2] FIG. 1 shows the results of a Helical Wheel analysis of an example of a hydrophobic region. [Diagram 3]FIG. 2 is a schematic diagram illustrating the relationship between the target binding site and the hydrophobic or hydrophilic region in the peptide according to the first embodiment of the present invention. [Figure 4] 1(a) and 1(b) are schematic diagrams illustrating the structures of a peptide according to a second embodiment of the present invention and a peptide association formed by the peptide, respectively. [Diagram 5] 1 is a graph showing the relationship between peptide concentration and fluorescence intensity derived from pyrene. [Figure 6] 1 is a graph showing the relationship between peptide concentration and ANS-derived fluorescence intensity. [Figure 7] 1 is a graph showing the results of evaluating the blood retention of a peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments. In addition, each embodiment can be appropriately combined unless it is inappropriate in the context. Furthermore, in this specification, "~" representing a numerical range includes the upper and lower numerical limits.

[0011] As used herein, natural amino acids include alanine (A), leucine (L), arginine (R), lysine (K), asparagine (N), methionine (M), aspartic acid (D), phenylalanine (F), cysteine ​​(C), proline (P), glutamine (Q), serine (S), glutamic acid (E), threonine (T), glycine (G), tryptophan (W), histidine (H), tyrosine (Y), isoleucine (I), and valine (V). In addition, unnatural amino acids include 2,3-diaminopropionic acid (DAP), 2,4-diaminobutanoic acid (DAB), ornithine (Orn), isoserine (Ise), and the like. Of these, A, L, M, F, I, V, P, W, and G are classified as nonpolar (hydrophobic) amino acids, while acidic amino acids such as D and E, basic amino acids such as R, K, H, DAP, DAB, Orn, and polar uncharged amino acids such as N, Q, S, T, C, Y, and Ise are classified as polar (hydrophilic) amino acids.

[0012] A. Peptides The peptide according to the embodiment of the present invention has a hydrophobic region and a hydrophilic region located at at least one end of the hydrophobic region and having a lower hydrophobicity than the hydrophobic region, a first target binding site capable of binding to a first target at one end, and a second target binding site capable of binding to a second target at the other end. A peptide having such a configuration (hereinafter, sometimes referred to as a "self-associating peptide") can form an aggregate by associating with the hydrophobic regions in an aqueous medium through hydrophobic interactions. If necessary, a drug may be bound to the peptide. The drug may be grafted, for example, to a side chain of the peptide.

[0013] The number of amino acid residues constituting the self-associating peptide is, for example, 10 to 40, preferably 12 to 38, more preferably 15 to 31, and even more preferably 18 to 24. The number of amino acid residues constituting the self-associating peptide means the number of amino acid residues constituting the main chain of the self-associating peptide, and the same applies to the number of amino acid residues constituting each region or site described below.

[0014] The self-associating peptides may be composed entirely of natural amino acid residues, may be composed entirely of non-natural amino acid residues, or may contain both natural and non-natural amino acid residues.

[0015] The self-associating peptide may be in the form of a derivative or a salt, so long as the effects of the present invention are obtained. In this specification, unless otherwise inappropriate in the context, a peptide may be a derivative of the peptide, or a salt of the peptide or its derivative.

[0016] Examples of peptide derivatives include those in which functional groups such as the N-terminal amino group, the C-terminal carboxyl group, the carboxyl group of the side chain, the amino group, the guanidino group, the hydroxyl group, and the thiol group of the peptide are substituted with various substituents. The substituents are not particularly limited, and examples thereof include alkyl groups, acyl groups, hydroxyl groups, amino groups, alkylamino groups, nitro groups, amide groups, sulfonyl groups, phosphate groups, halogens, and various protective groups. These substituents may be further substituted with halogens such as fluorine. The substitution may also be the introduction of labels such as fluorescent labels and biotin labels.

[0017] The salt of the peptide is preferably a pharmacologically acceptable salt. Examples of pharmacologically acceptable salts include acid addition salts and base addition salts. Examples of acid addition salts include inorganic acid salts and organic acid salts. Examples of inorganic acid salts include hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc. Examples of organic acid salts include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc. Examples of base addition salts include inorganic base salts and organic base salts. Examples of inorganic base salts include sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, etc. Examples of organic base salts include triethylammonium salt, triethanolammonium salt, pyridinium salt, diisopropylammonium salt, etc.

[0018] A-1. First embodiment Fig. 1(a) is a schematic diagram illustrating the structure of a self-associating peptide according to a first embodiment of the present invention. Self-associating peptide 10a has a hydrophobic region 12, a first hydrophilic region 14 located at one end of the hydrophobic region 12, and a second hydrophilic region 16 located at the other end. As shown in Fig. 1(b), self-associating peptide 10a associates in an aqueous medium through hydrophobic interaction between the hydrophobic regions 12 to form a peptide association 100a. In self-associating peptide 10a, either the end on the first hydrophilic region 14 side or the end on the second hydrophilic region 16 side may be the N-terminus.

[0019] The hydrophobic region 12 typically has nonpolar amino acid residues or tyrosine residues at both ends and is composed of, for example, 2 to 15, preferably 3 to 13, more preferably 4 to 12, and further preferably 6 to 10 amino acid residues.

[0020] In one embodiment, for example, 60% or more, preferably 60% to 100%, more preferably 70% to 90% of the amino acid residues constituting the hydrophobic region are selected from nonpolar amino acid residues and tyrosine residues. Although tyrosine is classified as a polar amino acid, it has very low solubility, and therefore, when included in the hydrophobic region, it can contribute to the self-association of the peptide.

[0021] In one embodiment, the total number of nonpolar amino acid residues and tyrosine residues contained in the hydrophobic region is, for example, 2 or more, preferably 3 to 13, and more preferably 4 to 10.

[0022] The nonpolar amino acid residues contained in the hydrophobic region are, for example, selected from L, A, W, F, V, I, and G, preferably selected from L, A, W, F, and G, and more preferably selected from L, A, W, and F. These nonpolar amino acid residues may be advantageous in terms of self-association of the peptide. The nonpolar amino acid residues contained in the hydrophobic region may be of only one type, or may be of two or more types.

[0023] The hydrophobic region may contain polar amino acid residues. From the viewpoint of peptide synthesis, when the hydrophobic region contains only one type of nonpolar amino acid residue, it is preferable to intersperse polar amino acid residues. The number of polar amino acid residues that can be contained in the hydrophobic region may be, for example, 1 to 6, or for example, 2 to 4. The ratio of the number of polar amino acid residues to the total number of amino acid residues constituting the hydrophobic region may be, for example, 7% to 40%, or for example, 13% to 30%. The polar amino acid residues contained in the hydrophobic region may be only one type, or may be two or more types.

[0024] By using polar amino acid residues as the constituent amino acid residues of the hydrophobic region, the hydrophobicity of the hydrophobic region can be flexibly adjusted within a wide range.

[0025] In one embodiment, the hydrophobic region contains charged amino acid residues, preferably acidic amino acid residues. The number of charged amino acid residues in the hydrophobic region is, for example, 1 to 6, or for example, 2 to 4. Two or more charged amino acid residues may be arranged contiguously or non-contiguously. For example, the charged amino acid residues may be arranged at equal intervals in the hydrophobic region. In one embodiment, the charged amino acid residues may be arranged via non-polar amino acid residues so that they are gathered on one side of a line passing through the central axis of the α-helix in the helical wheel projection, preferably adjacent to each other in the helical wheel projection, as illustrated in FIG. 2. By arranging the charged amino acid residues in this way, the aggregation of the peptide can be controlled. For example, a peptide in which polar amino acid residues such as charged amino acid residues are arranged so as to be gathered on one side of the α-helix structure can take a so-called amphipathic helix structure, and the degree of aggregation can be controlled by utilizing the electrostatic repulsion caused by the structure. The helical wheel projection can be obtained using the Gene Inspector (R) The sequence can be obtained using amino acid sequence analysis software such as those listed above.

[0026] The hydrophobic region may have a hydropathic index of, for example, -1.6 or more, preferably -0.9 to 4.5, and more preferably 0.4 to 2.8. In the present specification, the "hydropathic index of the hydrophobic region" means the average value of the hydropathic indexes of all amino acids constituting the hydrophobic region. The following values ​​are used as the hydropathic indexes of natural amino acids. [Table 1]

[0027] The first hydrophilic region 14 and the second hydrophilic region 16 each typically have a polar amino acid residue (excluding tyrosine residue) at the end of the hydrophobic region 12 and have a lower hydrophobicity than the hydrophobic region 12. The difference in hydrophobicity between the hydrophobic region and the first or second hydrophilic region is not limited as long as the self-associating peptide can associate with the hydrophobic regions in an aqueous medium through hydrophobic interaction to form an aggregate. In the aggregate, the hydrophobic region of the self-associating peptide is preferably located inside, and the first and second hydrophilic regions are located outside. The difference between the hydrophobic index of the hydrophobic region and the hydropathic index of the first or second hydrophilic region (Hydropathy Index of the hydrophobic region - Hydropathy Index of the hydrophilic region) is, for example, 0 or more, preferably 0.4 to 6.4, more preferably 1 to 6. In this specification, the "Hydropathy Index of a hydrophilic region" means the average value of the hydropathic indexes of all amino acids constituting the hydrophilic region.

[0028] The number of amino acid residues constituting the first hydrophilic region or the second hydrophilic region is, independently, for example, 3 to 24, preferably 3 to 18, more preferably 3 to 15, and further preferably 5 to 15.

[0029] The ratio of the number of amino acid residues constituting the first hydrophilic region to the number of amino acid residues constituting the hydrophobic region is not limited as long as the effects of the present invention can be obtained, and is, for example, 20% to 400%, preferably 30% to 300%. The ratio of the number of amino acid residues constituting the second hydrophilic region to the number of amino acid residues constituting the hydrophobic region is not limited as long as the effects of the present invention can be obtained, and is, for example, 20% to 400%, preferably 30% to 300%.

[0030] In one embodiment, for example, 20% or more, 30% or more, or 40% or more, preferably 50% to 100% of the amino acid residues constituting the first hydrophilic region or the second hydrophilic region are polar amino acid residues excluding tyrosine residues. The difference between the ratio of the number of polar amino acid residues excluding tyrosine residues to the number of amino acid residues constituting the hydrophilic region and the ratio of the number of polar amino acid residues excluding tyrosine residues to the number of amino acid residues constituting the hydrophobic region is, for example, 10% or more, preferably 20% or more, and can be, for example, 30% or more, 40% or more, or 50% or more.

[0031] Self-associating peptides containing charged amino acid residues, i.e., acidic amino acid residues and / or basic amino acid residues, in the hydrophilic region can form aggregates whose particle size can be changed by adjusting the pH of the aqueous medium. In particular, self-associating peptides containing one or more, for example, two or more or three or more histidine residues in the hydrophilic region can form aggregates whose particle size can be changed in the range from neutral to slightly acidic.

[0032] The self-associating peptide has a first target-binding site at one end capable of binding to a first target, and a second target-binding site at the other end capable of binding to a second target. A self-associating peptide having such a configuration can form an aggregate in which the first target-binding site and the second target-binding site are exposed to the outside, and an aggregate capable of binding to the target of each target-binding site can be obtained.

[0033] The first target-binding site and the second target-binding site are composed of amino acid sequences that can selectively bind to the first target and the second target to form a biological binding pair with the target, respectively. Such amino acid sequences may be known amino acid sequences, or may be obtained by screening an amino acid sequence that can selectively bind to a target of interest using an mRNA display method, a cDNA display method, a Phage Display method, or the like.

[0034] The first target and the second target can be appropriately selected according to the purpose. The first target and the second target are typically substances derived from living organisms, viruses, etc., and examples thereof include substances that are specifically present in living cells such as cancer cells, bacteria, fungi, and viruses, or substances produced therefrom.

[0035] The first target and the second target may be different from each other or may be the same. When the first target and the second target are different substances, a peptide having directivity to two targets is obtained, and as a result, a DDS having directivity to two targets can be realized. Whether the first target and the second target are the same substance or different substances, when the peptide forms a self-association, the affinity (Avidity) for the target per peptide can be improved compared to a single peptide that binds to the first target or the second target. When the first target and the second target are the same substance, the first target binding site and the second target binding site can bind to the same site or different sites of the substance. When the first target and the second target are the same substance, the amino acid sequences of the first target binding site and the second target binding site may be the same or different from each other.

[0036] In one embodiment, at least one of the first target binding site and the second target binding site has physiological activity. According to such an embodiment, the peptide itself has physiological activity due to the end region having physiological activity and can exert a medicinal effect, so it is not necessarily required to make the peptide or peptide association carry a drug. The physiological activity of the target binding site can be appropriately selected depending on the application of the peptide, etc. The physiological activity can be, for example, a hormone action, a neurotransmission action, an antitumor action, an antibacterial action, an action to regulate an enzyme activity, etc.

[0037] The first target-binding site and the second target-binding site are located at both ends of the self-associating peptide, respectively, and as long as they can bind to the first target and the second target, the number of constituent amino acid residues is not particularly limited and can be, for example, 3 to 24, or for example, 5 to 15. In the self-associating peptide exemplified in Figure 1(a), the first hydrophilic region 14 coincides with the first target-binding site, and the second hydrophilic region 16 coincides with the second target-binding site, but it is not necessary for the hydrophilic regions and the target-binding sites to coincide.

[0038] For example, the self-associating peptide illustrated in FIG. 3 has, from the N-terminus to the C-terminus, a first hydrophilic region composed of p amino acid residues, a hydrophobic region composed of q amino acid residues, and a second hydrophilic region composed of r amino acid residues, in that order; the region from the N-terminus amino acid residue (A1) to the second amino acid residue (B2) from the N-terminus of the hydrophobic region is the first target binding site, and the region from the second amino acid residue (C2) from the N-terminus of the second hydrophilic region to the C-terminus amino acid residue (C3). r ) is the second target binding site.

[0039] In contrast to the illustrated example, the second target binding site may extend beyond the second hydrophilic region and include part of the hydrophobic region, or the first target binding site may be part of the first hydrophilic region.

[0040] From the viewpoint of forming an assembly in which both target-binding sites are positioned on the outside, it is preferable that, for example, 3 to 15, and preferably 6 to 10, amino acid residues are interposed between the C-terminal amino acid residue of the first target-binding site and the N-terminal amino acid residue of the second target-binding site.

[0041] A-2. Second embodiment Fig. 4(a) is a schematic diagram illustrating the structure of a self-associating peptide according to a second embodiment of the present invention. Self-associating peptide 10b has a hydrophobic region 12 and a first hydrophilic region 14 located at one end of the hydrophobic region 12. As shown in Fig. 4(b), self-associating peptide 10b associates in an aqueous medium through hydrophobic interaction between the hydrophobic regions 12 to form a peptide association 100b. In self-associating peptide 10b, either the end on the hydrophobic region 12 side or the end on the first hydrophilic region 14 side may be the N-terminus.

[0042] The hydrophobic region 12 typically has a nonpolar amino acid residue or a polar amino acid residue selected from tyrosine, serine, and threonine residues at the end of the first hydrophilic region 14. The hydrophobic region preferably contains at least one selected from proline, tryptophan, glycine, tyrosine, serine, and threonine residues, more preferably at least one selected from tryptophan, glycine, or tyrosine residues. If the particle structure of the peptide association is excessively stable, the second target binding site located at the end of the hydrophobic region 12 may not function sufficiently. However, since the above amino acid residues show a negative Hydropathy Index and have a relatively low degree of hydrophobicity, such a problem can be avoided by including these amino acid residues in the hydrophobic region. The hydrophobic region 12 is composed of, for example, 3 to 37, preferably 4 to 15, more preferably 5 to 13, and even more preferably 6 to 12 amino acid residues.

[0043] In one embodiment, for example, 50% or more of the amino acid residues constituting the hydrophobic region are nonpolar amino acid residues or polar amino acid residues selected from tyrosine residues, serine residues, and threonine residues, and preferably 50% or more (e.g., 50% to 80%) are selected from proline residues, tryptophan residues, glycine residues, tyrosine residues, serine residues, and threonine residues, and more preferably tryptophan residues, glycine residues, and tyrosine residues.

[0044] In one embodiment, the total number of nonpolar amino acid residues and polar amino acid residues selected from tyrosine residues, serine residues, and threonine residues contained in the hydrophobic region is, for example, 3 or more, preferably 4 to 12, and more preferably 6 to 10.

[0045] The nonpolar amino acid residues contained in the hydrophobic region may vary depending on the amino acid sequence of the target binding site, but in addition to the above, it is preferable that the hydrophobic region contains nonpolar amino acid residues similar to those described in the first embodiment.

[0046] The hydrophobic region may further contain polar amino acid residues other than tyrosine residues, serine residues, and threonine residues. The number of polar amino acid residues (excluding tyrosine residues, serine residues, and threonine residues) that can be contained in the hydrophobic region may be, for example, 1 to 6, or for example, 2 to 4. The ratio of the number of polar amino acid residues (excluding tyrosine residues, serine residues, and threonine residues) to the total number of amino acid residues constituting the hydrophobic region may be, for example, 7% to 50%, or for example, 13% to 30%. The polar amino acid residues (excluding tyrosine residues, serine residues, and threonine residues) contained in the hydrophobic region may be only one type, or may be two or more types. The number of charged amino acid residues in the hydrophobic region may be, for example, 0 to 6, or for example, 2 to 4.

[0047] The first hydrophilic region 14 typically has a polar amino acid residue (excluding tyrosine, serine, and threonine residues) at the end of the hydrophobic region 12, and has a lower hydrophobicity than the hydrophobic region 12. The difference in hydrophobicity between the hydrophobic region and the first hydrophilic region is not limited as long as the self-associating peptide can form an aggregate by association in an aqueous medium through hydrophobic interactions between the hydrophobic regions. In the aggregate, typically, the hydrophobic region of the self-associating peptide is disposed inside, and the first hydrophilic region is disposed outside. When intermolecular hydrogen bonds (crosslinks) can be formed in an aqueous solution, the aqueous peptide solution can exhibit gel-like properties. The hydrophobic index of the hydrophobic region is as described for the first embodiment. The difference between the hydropathic index of the hydrophobic region and the hydropathic index of the first hydrophilic region (hydropathic index of hydrophobic region - hydropathic index of hydrophilic region) may be, for example, -0.3 or more, preferably 0 to 6, and more preferably 0.4 to 5.

[0048] The first hydrophilic region comprises, for example, 3-24, and preferably 5-15, amino acid residues.

[0049] The ratio of the number of amino acid residues constituting the first hydrophilic region to the number of amino acid residues constituting the hydrophobic region is not limited as long as the effects of the present invention are obtained, and is, for example, 25% to 400%, preferably 50% to 200%.

[0050] In one embodiment, for example, 20% or more, 30% or more, or 40% or more, preferably 50% to 100% of the amino acid residues constituting the first hydrophilic region are polar amino acid residues. For example, 20% or more, 30% or more, 40% or more, or 50% or more of the amino acid residues constituting the first hydrophilic region may be polar amino acid residues excluding tyrosine residues, serine residues, and threonine residues. In addition, the difference between the ratio of the number of polar amino acid residues excluding tyrosine residues, serine residues, and threonine residues to the number of amino acid residues constituting the first hydrophilic region and the ratio of the number of polar amino acid residues excluding tyrosine residues, serine residues, and threonine residues to the number of amino acid residues constituting the hydrophobic region is, for example, 10% or more, preferably 20% or more, and may be, for example, 30% or more, 40% or more, or 50% or more.

[0051] The self-associating peptide 10b in the illustrated example has a first target-binding site capable of binding to a first target at the end on the first hydrophilic region 14 side, and a second target-binding site capable of binding to a second target at the end on the hydrophobic region 12 side. A self-associating peptide having such a configuration can form an aggregate in which the first target-binding site is exposed to the outside and can bind to the first target in the aggregate state, and when the aggregate structure collapses, the second target-binding site appears from the inside of the aggregate to the outside and can bind to the second target. The first target-binding site and the second target-binding site are as described in relation to the first embodiment.

[0052] A-3. Method for producing self-associating peptides Methods for producing self-associating peptides include chemical synthesis methods such as solid-phase synthesis, stepwise elongation, and liquid-phase synthesis, fermentation, and enzymatic methods. Among these, solid-phase synthesis is preferred. Examples of solid-phase synthesis include Fmoc synthesis and Boc synthesis.

[0053] B. Peptide aggregates According to another aspect of the present invention, there is provided a peptide complex comprising the peptide described in section A or a derivative thereof, or a salt of either of them. A peptide complex can be formed by association of two or more molecules of self-associating peptides through intermolecular interactions. The intermolecular interactions for forming the complex typically include hydrophobic interactions between hydrophobic regions of the peptides, and may further include other intermolecular interactions such as hydrogen bonds, electrostatic interactions, van der Waals interactions, etc. between peptide molecules.

[0054] The peptide association is preferably a core-shell type association as shown in Figures 1(b) and 4(b). In a core-shell type peptide association, self-associating peptides are associated such that the hydrophobic region is located inside (core) and the hydrophilic region is located outside (shell). Furthermore, self-associated peptides may associate with each other to form larger associations (e.g., secondary associations).

[0055] The average particle size of the peptide association may be any appropriate value depending on the purpose. The average particle size of the peptide association when administered intravenously is, for example, less than 300 nm, preferably 100 nm or less, more preferably 5 nm to 50 nm. The average particle size of the peptide association when used via other administration routes may be, for example, 3000 nm or less, preferably 1000 nm or less. The particle size of the peptide association can be adjusted, for example, by the hydrophobicity of the hydrophobic region and / or hydrophilic region, the pH of the aqueous medium, and the like. Specifically, when the hydrophobicity of the hydrophobic region and / or hydrophilic region is high, the particle size of the peptide association tends to be small. In addition, by shifting the ionization equilibrium of the charged amino acid residue side chains from a dissociated state to a non-dissociated state, the hydrophobicity of the hydrophobic region and / or hydrophilic region increases, and as a result, the particle size of the peptide association tends to be small. In particular, when the histidine content is high, the particle size of the peptide association can be adjusted in the vicinity of physiological conditions, that is, neutral to weakly acidic.

[0056] In one embodiment, the self-associating peptide forming the peptide complex as a whole exhibits the same or higher binding affinity to the first target as the non-self-associating peptide comprising the first target-binding site, and preferably exhibits a higher binding affinity. Specifically, the dissociation constant (K D ) is the dissociation constant (K) of the non-self-associating peptide containing the first target binding site to the first target. D ) or less, and preferably less than that. The dissociation constant (K D ) is, for example, 10 -6 M or less, preferably 10 -7 It can be M or less.

[0057] In one embodiment, the self-associating peptide forming the peptide complex as a whole exhibits the same or higher binding affinity to the second target as the non-self-associating peptide comprising the second target-binding site, and preferably exhibits a higher binding affinity. Specifically, the dissociation constant (K D ) is the dissociation constant (K D ) or less, and preferably less than that. Note that the dissociation constant (K D ) is, for example, 10 -6 M or less, preferably 10 -7 The dissociation constant (K D ) refers to the binding affinity of the peptide complex for a first target or a second target in terms of the peptide, and can be obtained by calculating the dissociation constant using the molar concentration of the peptide used to form the complex instead of the molar concentration of the peptide complex.

[0058] The peptide complex may further contain a drug. Since the peptide complex can exhibit targeting due to the target-binding site, a peptide complex containing a drug can deliver the drug to a desired target. The drug may be contained in the peptide complex by being supported (e.g., encapsulated) or by being bound to a self-associating peptide (e.g., bound to a peptide side chain via a linker as necessary).

[0059] As the drug, any appropriate drug can be used depending on the purpose. Examples include antitumor agents, signal transduction inhibitors, metabolic antagonists, analgesics, anti-inflammatory agents, antibacterial agents, contrast agents, and the like. Among them, hydrophobic drugs are preferred. Hydrophobic drugs can be suitably carried inside the peptide association. Examples of hydrophobic antitumor agents include paclitaxel, topotecan, camptothecin, cisplatin, daunorubicin hydrochloride, methotrexate, mitomycin C, docetaxel, vincrestine sulfate, and derivatives thereof.

[0060] A peptide association can be obtained, for example, by dissolving a self-associating peptide in an organic solvent, and if necessary, air-drying the resulting solution, for example, by drying to a film under a nitrogen stream atmosphere, and if necessary, further drying under reduced pressure to remove the organic solvent, and then adding and mixing an aqueous medium to cause the self-associating peptide to self-associate.

[0061] Examples of organic solvents that dissolve the self-associating peptides include water-immiscible organic solvents such as dichloromethane, chloroform, diethyl ether, dibutyl ether, ethyl acetate, and butyl acetate; water-miscible organic solvents such as methanol, ethanol, propyl alcohol, isopropyl alcohol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, acetonitrile, acetone, and tetrahydrofuran; and mixed solvents of these.

[0062] Examples of the aqueous medium include water and buffer solutions. Examples of the buffer solution include phosphate buffer solution, phosphate buffered saline, citrate buffer solution, Tris buffer solution, TAPS buffer solution, MES buffer solution, HEPES buffer solution, etc. The pH of the aqueous medium is, for example, 3 to 8, preferably 5 to 7.4.

[0063] Alternatively, peptide complexes may be produced by stirring a mixture containing the self-associating peptide and, where necessary, a drug. The mixture is preferably stirred by applying energy such as ultrasonic waves.

[0064] C. Peptide Compositions According to another aspect of the present invention, there is provided a peptide composition comprising an aqueous medium and the peptide complex described in Section B. As described in Section B, the peptide complex is formed by association via intermolecular interactions (typically, hydrophobic interactions between hydrophobic regions of self-associating peptides). The peptide composition may further comprise a drug. The drug may be supported (e.g., encapsulated) in the peptide complex, or may be bound to the self-associating peptide (e.g., bound to a peptide side chain via a linker as necessary). The peptide complex, drug, and aqueous medium are as described in Section B.

[0065] In one embodiment, the peptide composition is a pharmaceutical composition containing a physiologically active self-associating peptide and / or a drug as an active ingredient, and in the pharmaceutical composition, the peptide association can function as a DDS for efficiently delivering the active ingredient to a target. In such an embodiment, the peptide composition is prepared into any appropriate dosage form depending on the administration mode. For example, the peptide composition can be a parenteral preparation such as an injection such as a subcutaneous injection, an intravenous injection, an intramuscular injection, an intraperitoneal injection, or a drip infusion, an eye drop, a nasal drop, or an external preparation (e.g., an eye ointment, an oral ointment, etc.). Alternatively, the peptide composition can be an oral preparation in a form that prevents decomposition in gastric acid, such as an enteric coating.

[0066] The peptide composition may further contain any appropriate additive depending on the application, etc. Examples of the additive include an isotonicity agent, a buffering agent, a preservative, a thickening agent, a stabilizer, a pH adjusting agent, etc. EXAMPLES

[0067] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0068] [Experimental Example 1: Synthesis of peptides] The peptides were synthesized using the peptide synthesis service provided by Toray Research Center, Inc. The amino acid sequences of the obtained peptides are shown in Table 2.

[0069] In Table 2, the peptides of the Examples are divided into three regions by two hyphens. The N-terminal region and C-terminal region of the peptides of Examples 1-1 to 1-5 are an HA binding site capable of binding to hydroxyapatite (HA) and an Rgp binding site capable of binding to Arg-gingipain (Rgp), respectively. The N-terminal region and C-terminal region of the peptides of Examples 2-1 and 2-2 are an amino acid sequence derived from a CD73 binding peptide capable of binding to CD73 and an amino acid sequence derived from an FcRn binding peptide capable of binding to fetal Fc receptor (FcRn), respectively. The underlined regions indicate hydrophobic regions. On the other hand, the peptides of the Reference Examples are amphipathic peptides, and the N-terminal region separated by one hyphen is a hydrophilic region (also an Rgp binding site) and the C-terminal region is a hydrophobic region. The amino acid sequences constituting each target binding site are sequences described in previously published papers (e.g., P. Steinbauer, et al. Single-Molecule Force Spectroscopy Reveals Adhesion-by-Demand in Statherin at the Protein-Hydroxyapatite Interface Langmuir 2020, 36, 13292-13300.) or sequences determined by the cDNA display method. [Table 2]

[0070] [Experimental Example 2: Formation of peptide aggregates] 5 mg of the peptides of Examples 1-1 to 1-5 and Reference Examples 1-1 to 1-8 were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (40 mM). 4 μL of the stock solution was dropped into a 10 mM HEPES, 150 mM NaCl buffer solution (pH 7) being stirred with a micromixer (Taitec, E-36) to dilute the solution 100-fold. The diluted solution was vortexed and then allowed to stand overnight at room temperature. Next, 200 μL of the diluted solution was used as a measurement sample and the particle size distribution was measured by dynamic light scattering at 25° C. using a Litesizer 500 (Anton Paar). The main peak of the scattering intensity was determined as the average particle size of the aggregate, and the results are shown in Table 3. However, when the polydispersity index exceeded 20%, the main peak was determined with reference to the volume-based particle size distribution.

[0071] [Table 3]

[0072] As shown in Table 3, all of the peptides of Examples 1-1 to 1-5 formed aggregates with an average particle size of 500 nm or less in HEPES buffer. It was also confirmed that the amphipathic peptides of Reference Examples 1-1 to 1-8 formed aggregates and that the particle size could change depending on the amino acid sequence of the hydrophobic region. Although an average particle size of 5 nm was measured for Example 1-5 and Reference Example 1-5, this particle size was larger than the size of one peptide molecule, so it is believed that an aggregate containing two or more peptide molecules was formed.

[0073] [Experimental Example 3: Evaluation of pH dependence of particle size of peptide aggregates] 5 mg of the peptides of Examples 2-1 to 2-2 were dissolved in DMSO to prepare stock solutions (50 mg / mL). 4 μL of the stock solution was dropped into 10 mM MES, 150 mM NaCl buffer (pH 6.0) being stirred with a micromixer (Taitec, E-36) to obtain a 100-fold dilution to obtain a dilution solution A. Dilution solution A was vortexed and then allowed to stand overnight at room temperature. 100 μL of the dilution solution A was used to dilute with 300 μL of the same MES buffer or 10 mM HEPES, 150 mM NaCl buffer (pH 7.4), and the solution was stirred for 1 hour to obtain a dilution solution B. 200 μL of each of dilution solutions A and B after standing overnight were used as measurement samples, and the particle size distribution was measured at 25 ° C. using Litesizer 500 (Anton Paar). The results of the average particle size obtained according to Experimental Example 2 are shown in Table 4.

[0074] [Table 4]

[0075] As shown in Table 4, the average particle size of the peptide aggregates of Examples 2-1 and 2-2 is smaller in a buffer solution of pH 7 than in a buffer solution of pH 6, indicating that the average particle size of these peptide aggregates is pH-dependent. The pH dependence of the average particle size is presumably due to changes in the ionization state of charged amino acid residues, particularly histidine side chains, depending on the pH of the aqueous medium, resulting in changes in the hydrophobicity of the hydrophilic region.

[0076] [Experimental Example 4: Evaluation of binding affinity to the second target (Rgp)] The binding affinity of the peptide of Example 1-2 and the Rgp-binding peptide A (RRKRR: SEQ ID NO: 16) constituting the Rgp-binding site of the peptide to Rgp was measured by the SPR method using "Biacore-T100" (GE Healthcare). The measurement was performed according to a conventional method. Specifically, for the measurement of the peptide of Example 1-2, streptavidin (Extravidin SIGMA E2511) was bound to a chip (CM5 sensor chip) by amine coupling, and then a 20-fold molar ratio of NHS-biotinylation reagent (Thermo Fisher Scientific, EZ-Link (R) Rgp was immobilized on a CM5 sensor chip by direct amine coupling to measure Rgp-binding peptide A, which is biotinylated with NHS-SS-Biotin. Next, the peptide stock solution (DMSO) was diluted with running buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween, 0.2% DMSO) to prepare final peptide concentrations of 25, 100, 400, and 1600 nM. Using these four concentrations of peptide solutions, sensorgrams were obtained showing the mass change due to the binding of the peptide to the Rgp immobilized on the chip. The dissociation constant K D The results are shown in Table 5.

[0077] [Table 5]

[0078] As shown in Table 5, the self-associating peptide of Example 1-2 exhibited higher binding affinity to Rgp (second target) than Rgp-binding peptide A (non-self-associating peptide containing a second target binding site). This is believed to be because the peptide of Example 1-2 formed an aggregate in the peptide solution, which enabled it to bind to the target at multiple points, resulting in an avidity effect.

[0079] [Experimental Example 5: Evaluation of binding affinity to the first target (HA)] The peptides of Examples 1-2 and 1-5 and the HA-binding peptide A (DSpSEEKW: SEQ ID NO: 17) constituting the HA-binding site of the peptides were attached to hydroxyapatite beads (1 μm BcMag (R) The binding affinity of the peptide to Hydroxyapatite Magnetic Particles (Bioclone, model number FO-101) was evaluated. Specifically, the beads were weighed and suspended in 100 mM sodium phosphate buffer (pH 7.4) / 20% ethanol to a concentration of 30 mg / mL. The beads were irradiated with ultrasound and uniformly dispersed, and 267 μL of the bead suspension was taken in a 1.5 mL tube. The beads were fixed on a magnetic stand (Takara Bio, Magnetic Stand (6 tubes), product code 5328), the supernatant was removed, and 100 μL of peptide solution was added. The peptide solution was prepared by diluting a 100 mM stock solution (DMSO) to 40 μM with 10 mM HEPES (pH 7.4) / 1% DMSO, and then diluting it 4-fold to 5-fold in sequence. After stirring for 4 hours at 4°C, the tube was placed on a magnetic stand to fix the beads, and the supernatant was collected. The peptide concentration in the supernatant was measured using 50 μL of the supernatant with a plate reader (Thermo Fisher Scientific, VARIO-SCAN) based on the fluorescence intensity derived from the tryptophan contained in the sequence (excitation wavelength: 280 nm, emission wavelength: 355 nm). The peptide concentration in the supernatant was subtracted from the total peptide concentration to determine the amount of peptide bound to the beads, and the dissociation constant K of the peptide with respect to the hydroxyapatite beads was calculated by curve fitting the resulting adsorption isotherm to a theoretical formula (Microsoft Excel). D The results are shown in Table 6.

[0080] [Table 6]

[0081] As shown in Table 6, the self-associating peptide of Example 1-2 showed almost the same binding affinity to HA (first target) as HA-binding peptide A (non-self-associating peptide containing a first target binding site). From the results of Experimental Examples 4 and 5, it was found that the self-associating peptide of Example 1-2 can bind to both the first target and the second target with almost the same or higher affinity as the target-binding peptide alone. On the other hand, the self-associating peptide of Example 1-5 showed a higher binding affinity to HA than HA-binding peptide A. These indicate that the second hydrophilic region (corresponding to the second target binding site in these peptides) affects the secondary structure, aggregate formation mode and particle size of the self-associating peptide as a whole, and therefore may also affect the affinity of the first target binding site for the first target. The difference between the results of Examples 1-2 and 1-5 is thought to be due to the difference in the ease of occurrence of the Avidity effect, which allows binding to the target at multiple points.

[0082] [Experimental Example 6: Formation of pyrene-inclusion complexes] The fluorescent probe method has been widely used as a technique for measuring the critical micelle concentration (CMC) of surfactant solutions. When hydrophobic fluorescent molecules are added to an aqueous surfactant solution and excited, weak fluorescence is emitted from the fluorescent molecules dispersed in the solution when the surfactant concentration is low. On the other hand, when the surfactant concentration exceeds the CMC, the hydrophobic fluorescent molecules are incorporated into the inside of the micelles surrounded by the hydrophobic groups of the surfactant (hydrophobic environment), and emit strong fluorescence. In this experimental example, pyrene was used as the fluorescent probe, and a peptide was used instead of a surfactant, and the change in fluorescence properties with changes in peptide concentration was investigated. 1) Pyrene (FUJIFILM Wako Pure Chemical) was weighed, 80% ethanol was added, and the mixture was dissolved in a vortex mixer to prepare a 5 mM stock solution. The stock solution was diluted 500-fold with 80% ethanol to adjust the concentration to 10 μM. Meanwhile, the peptides were dissolved in DMSO stock solution in PBS (Thermo Fisher Scientific, BupH (R)Phosphate Buffered Saline Packs) to prepare a 2-fold dilution series (2, 1, 0.5, 0.25, 0.125, 0.0625 mM) at 10-fold concentrations of the final concentration. 2) 142.4 μL of PBS, 16 μL of peptide solution (final concentration: 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 mM), and 1.6 μL of 10 μM pyrene solution (final concentration 0.1 μM) were added to a 1.5 mL tube and mixed with a vortex mixer. As a control, 158.4 μL of PBS and 1.6 μL of 10 μM pyrene solution were added and mixed with a vortex mixer. Then, the tube was left to stand at room temperature for 24 hours in the dark. 3) 150 μL of the reaction solution was transferred to each well of a plate (Non-Treated Black Polystyrene 96-well plate (Corning 3694)). 150 μL of PBS was added to each well as a control. (R) Fluorescence spectra were scanned using a multi-detection mode microplate reader (excitation wavelength 333 nm, emission wavelength 360-420 nm). Peak fluorescence intensity (384 nm) was plotted against peptide concentration (logarithmic value). The results are shown in Figure 5.

[0083] In the peptide of Example 1-1, the fluorescence intensity clearly increased with increasing peptide concentration. The increase in fluorescence intensity means that a peptide association was formed that provides a hydrophobic environment capable of encapsulating pyrene. In addition, in the plot of FIG. 5, the concentration at the intersection (inflection point) of the two obtained straight lines was taken as the critical aggregation concentration (CAC), and the CAC value of the peptide of Example 1-1 was 50 μM. The peptides of Examples 1-2 and 1-5 had almost the same fluorescence intensity up to 0.1 mM. Although a tendency for the fluorescence intensity to increase was observed for all peptides at 0.2 mM, it was determined that it would be difficult to determine the CAC value.

[0084] [Experimental Example 7: Formation of ANS inclusion complex] 1) ANS (8-anilino-1-naphthalenesulfonic acid) (Tokyo Chemical Industry Co., Ltd.) was weighed and dissolved in 80% ethanol to prepare a 5 mM stock solution. The stock solution was diluted 500 times with 80% ethanol to adjust the concentration to 10 μM. Meanwhile, a 2-fold dilution series of peptide solutions (2, 1, 0.5, 0.25, 0.125, 0.0625 mM) was prepared in the same manner as in Experimental Example 6. 2) 142.4 μL of PBS, 16 μL of peptide solution (final concentration: 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 mM), and 1.6 μL of 10 μM ANS solution (final concentration 0.1 μM) were added to a 1.5 mL tube and mixed with a vortex mixer. As a control, 158.4 μL of PBS and 1.6 μL of 10 μM ANS solution were added and mixed. Then, the tube was left to stand at room temperature for 1 hour in the dark. 3) 150 μL of the reaction solution was transferred to each well of a plate (Non-Treated Black Polystyrene 96-well plate (Corning 3694)). 150 μL of PBS was added to each well as a control. (R) Fluorescence spectra were scanned using a multi-detection mode microplate reader (excitation wavelength 370 nm, emission wavelength 450-600 nm). Fluorescence intensity at 490 nm was plotted against peptide concentration (logarithmic value). The results are shown in Figure 6.

[0085] As shown in FIG. 6, the fluorescence intensity increased with increasing peptide concentration for all peptides. ANS is widely used as a probe molecule, like pyrene, because its fluorescence intensity increases significantly when placed in a hydrophobic environment compared to pure water. The increase in fluorescence intensity means that a peptide complex that provides a hydrophobic environment capable of encapsulating ANS was formed. However, considering the results for pyrene shown in FIG. 5 and that pyrene is more hydrophobic than ANS, it can be considered that the hydrophobic environment that the peptide complexes of Examples 1-2 and 1-5 can present at, for example, 0.1 mM is not hydrophobic enough to encapsulate pyrene, although it can encapsulate ANS. In addition, when the concentration at the intersection (inflection point) of the two obtained lines in the plot diagram of FIG. 6 was taken as CAC, the CAC value for all peptides was 50 μM.

[0086] In Experiments 6 and 7, the formation of peptide aggregates was confirmed not only by particle size measurement using dynamic light scattering but also by the fluorescent probe method. It was also found that the ability to form aggregates and the hydrophobic environment that can be presented can vary depending on the peptide sequence.

[0087] [Experimental Example 8: Evaluation of blood dynamics] The three types of fluorescein (FAM)-labeled peptides shown in Table 7 were purchased from Eurofins Genomics, Inc. 1 mg of each peptide was dissolved in 4 mL of PBS to prepare an administration solution (0.25 mg / mL). With regard to each peptide, "GYSEWRKWEL" is a peptide sequence confirmed to have affinity for IL-6R, and "HWRGWV" is a peptide sequence reported to have affinity for the Fc region of immunoglobulin (Journal of Chromatography A, 1218, (2011), 8344-8352, etc.), and each can function as a target binding site. The peptide of Example 3-1 has a hydrophobic region (LLLLLLLGG) underlined in Table 7, and a first hydrophilic region (GYSEWRKWE) and a second hydrophilic region (HWRGWV) located on the N-terminal and C-terminal sides thereof, both of which have a lower hydrophobicity than the hydrophobic region. The peptides of Example 3-1 and Comparative Example 2 have been confirmed to form aggregates in water.

[0088] [Table 7]

[0089] After an acclimation period of 7 days or more was allowed after receiving and inspecting the mice, 18 Crl:CD1 (ICR) mice (male) (Jackson Laboratory Japan, Inc.) were divided into 6 groups of 3 mice each as shown in Table 8 and used for the experiment. The administration solution was administered into the tail vein of non-fasting mice using a syringe equipped with an injection needle (1 mg / 4 mL / kg). The administration volume for each individual was calculated from the body weight measured on the day of administration. After administration, approximately 0.1 mL of blood was collected from the jugular vein under no anesthesia using a heparinized syringe equipped with an injection needle at the designated collection time points shown in Table 8. At the final collection time point, the entire amount of blood was collected from the posterior vena cava under isoflurane anesthesia. The collected blood was promptly transferred to a polypropylene tube and cooled on ice. The entire amount of collected blood was centrifuged (approximately 10,000×g, 3 minutes, 4°C), and the plasma sample was promptly separated into a sample storage container and shielded from light. The plasma sample was frozen and stored until measurement (temperature control: approximately -80°C).

[0090] [Table 8]

[0091] The frozen and thawed plasma sample (10 μL) was diluted 10-fold with PBS (90 μL) and analyzed using a plate reader (TECAN, SPARK (R) The fluorescence intensity was measured (excitation wavelength 495 nm, emission wavelength 520 nm) using a multi-detection mode microplate reader. The plasma concentration of the peptide was calculated from the calibration curves prepared in each of Comparative Example 1, Comparative Example 2, and Example 3-1. The obtained plasma concentration was logarithmically plotted against the time after administration, and four time points including the final blood sampling time point were exponentially approximated (Microsoft Excel), and the elimination half-life was calculated from the slope of the line. The results are shown in Figure 7 and Table 9. In Figure 7, the symbols represent the average value, the bars represent the standard deviation, and when there is no standard deviation, it is within the symbol (n=3).

[0092] [Table 9]

[0093] As is clear from FIG. 7 and Table 9, the peptide of Example 3-1 has a significantly longer half-life after intravenous administration than the peptides of Comparative Examples 1 and 2. In addition, when Comparative Example 1 and Comparative Example 2 are compared, Comparative Example 2 showed a longer half-life. This effect of improving blood retention is thought to be due to the increase in the apparent molecular weight of the peptide due to the formation of an aggregate. Furthermore, Example 3-1 is thought to show a significantly longer half-life than Comparative Example 2 due to the influence of the peptide sequence of HWRGWV functioning to interact with the Fc region of immunoglobulin present in the blood, etc. [Industrial Applicability]

[0094] The peptide, peptide association, and peptide composition according to embodiments of the present invention can be suitably used in, for example, a DDS system, a pharmaceutical composition, and the like. [Explanation of symbols]

[0095] 10 Self-Associating Peptides 12 Hydrophobic region 14 First hydrophilic region 16 Second hydrophilic region 100 Peptide complex

Claims

1. A peptide association comprising a self-associating peptide or derivative thereof, or a pharmacologically acceptable salt thereof, comprising a hydrophobic region, a first hydrophilic region located on the N-terminal side of the hydrophobic region and having a lower hydrophobicity than the hydrophobic region, and a second hydrophilic region located on the C-terminal side of the hydrophobic region and having a first target-binding site at the N-terminal end that selectively binds to a first target and a second target-binding site at the C-terminal end that selectively binds to a second target, wherein the hydrophobic region is located inside and the first hydrophilic region and the second hydrophilic region are located on the outside, the number of amino acid residues constituting the hydrophobic region is 6 to 12, of which 70% or more are selected from nonpolar amino acid residues and tyrosine residues; the hydrophobic region has nonpolar amino acid residues or tyrosine residues at both ends; the first hydrophilic region and the second hydrophilic region each independently have 5 to 15 amino acid residues; the first hydrophilic region and the second hydrophilic region each have a polar amino acid residue other than a tyrosine residue at a terminal on the hydrophobic region side; 50% or more of the amino acid residues constituting the first hydrophilic region are selected from polar amino acid residues excluding tyrosine residues; 50% or more of the amino acid residues constituting the second hydrophilic region are selected from polar amino acid residues excluding tyrosine residues; the non-polar amino acids are selected from alanine, leucine, methionine, phenylalanine, isoleucine, valine, proline, tryptophan, and glycine; the polar amino acid is selected from aspartic acid, glutamic acid, arginine, lysine, histidine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, asparagine, glutamine, serine, threonine, cysteine, tyrosine, and isoserine; The derivative is a peptide association in which the N-terminal amino group, the C-terminal carboxyl group, and one or more groups selected from a side chain carboxyl group, an amino group, a guanidino group, a hydroxyl group, and a thiol group of the self-associating peptide are substituted with a substituent selected from an alkyl group, an acyl group, a hydroxyl group, an amino group, an alkylamino group, a nitro group, an amide group, a phosphate group, and a halogen.

2. The peptide association of claim 1, wherein the hydrophobic region comprises at least one amino acid residue selected from leucine, glutamic acid, alanine, methionine, and lysine.

3. The peptide complex of claim 1, wherein the first target and the second target are different from each other.

4. The peptide complex of claim 1, wherein the self-associating peptide comprises 15 to 40 amino acid residues.

5. The peptide complex of claim 1, wherein at least one of the first target-binding site and the second target-binding site has biological activity.

6. The peptide complex of claim 1, further comprising a drug.

7. The peptide complex of claim 1 , which satisfies at least one of the following (i) and (ii): (i) the binding affinity of the peptide or derivative thereof, or a salt thereof, for the first target is equal to or greater than the binding affinity of a non-self-associating peptide comprising the first target binding site for the first target; (ii) the binding affinity of the peptide or derivative thereof, or a salt thereof, for the second target is equal to or greater than the binding affinity of a non-self-associating peptide comprising the second target binding site for the second target.

8. A method for producing a peptide complex comprising the steps of: A peptide composition, wherein the peptide association is formed by association via intermolecular interactions comprising hydrophobic interactions between the hydrophobic regions of the peptides.

9. The peptide composition of claim 8 , wherein the association further comprises a drug.

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