Physiologically active peptide derivative for treating ocular diseases, pharmaceutical composition, nonasal / nonasal

Abstract

The present invention provides a physiologically active peptide derivative for use in the treatment of ocular diseases having a physiologically active peptide sequence and a functional sequence comprising a membrane permeation enhancing sequence and an endosome escape enhancing sequence.

Description

Technical Field

[0001] The present invention relates to a bioactive peptide derivative for treating ophthalmic diseases, a pharmaceutical composition, a nasal / nasal drop preparation, and the use of the bioactive peptide derivative. Background Art

[0002] Intravitreal injection is a method for treating ophthalmic diseases in which a pharmaceutical preparation is injected into the eyeball using a syringe (for example, refer to Patent Document 1). For example, in order to treat ophthalmic diseases such as age-related macular degeneration, a treatment is performed in which an anti-VEGF substance is injected into the eyeball, and the anti-VEGF substance has an effect of reducing abnormal blood vessels (choroidal neovascularization) generated from the choroid located under the retina toward the retina. Prior Art Documents Patent Documents

[0003] Patent Document 1 Japanese Unexamined Patent Application Publication No. 2022-81491 Summary of the Invention

[0004] Although intravitreal injection can effectively administer a pharmaceutical preparation into the eyeball, the physical and mental burdens on the patient and the risk of infectious diseases are great. Therefore, it is desired to develop a method for administering a pharmaceutical preparation by a less invasive method. In addition, in recent years, peptides showing bioactive effects have been used for the treatment of various diseases. Therefore, the use of peptides as therapeutic agents for ophthalmic diseases is likely to increase in the future.

[0005] In view of the above problems, an object of the present invention is to provide a novel bioactive peptide derivative for treating ophthalmic diseases, a pharmaceutical composition, a nasal / nasal drop preparation, and the use of the bioactive peptide derivative.

[0006] Specific means for achieving the above problems include the following embodiments. <1> A bioactive peptide derivative for treating ophthalmic diseases, which has a bioactive peptide sequence and a functional sequence, and the functional sequence includes a membrane penetration enhancing sequence and an endosome escape enhancing sequence. <2> The bioactive peptide derivative according to <1>, wherein the bioactive peptide derivative further has a sugar chain-modified molecule containing a sugar chain. <3> The bioactive peptide derivative according to <2>, wherein the sugar chain-modified molecule is bonded to the C-terminal side or the N-terminal side of the bioactive peptide sequence. <4> The bioactive peptide derivative according to <2>, wherein the number of monosaccharide residues of each sugar chain is 5 to 20. <5> The bioactive peptide derivative according to any one of <1> to <4>, wherein the number of amino acid residues of the bioactive peptide sequence is 200 or less. <6> The bioactive peptide derivative according to any one of <1> to <5>, wherein the membrane penetration enhancing sequence is cationic. <7> The bioactive peptide derivative according to any one of <1> to <6>, wherein more than half of the total number of amino acid residues of the membrane penetration enhancing sequence are basic amino acid residues. <8> The bioactive peptide derivative according to any one of <1> to <7>, wherein the endosome escape enhancing sequence is an amino acid sequence selected from FFLIPKG, LILIG, FFG, FFFFFG, and FFFFFFFG. <9> The bioactive peptide derivative according to any one of <1> to <8>, wherein the bioactive peptide derivative reaches the optic nerve and retina via the lateral geniculate body. <10> A pharmaceutical composition comprising the bioactive peptide derivative according to any one of <1> to <9> as an active ingredient. <11> A nasal drop preparation comprising the bioactive peptide derivative according to any one of <1> to <9> as an active ingredient. <12> Use of a bioactive peptide derivative in the treatment of eye diseases, characterized in that the bioactive peptide derivative has a bioactive peptide sequence and a functional sequence, and the functional sequence comprises a membrane penetration enhancing sequence and an endosome escape enhancing sequence. Advantages of the Invention

[0007] According to the present invention, it is possible to provide a novel bioactive peptide derivative, a pharmaceutical composition, a nasal drop preparation, and the use of a bioactive peptide derivative for treating eye diseases. Brief Description of the Drawings

[0008] Figure 1 It is a diagram showing the fluorescence observation results of the mouse eyeball and optic nerve in Example 1. Figure 2 It is a diagram showing the fluorescence observation results of the mouse eyeball and optic nerve in Example 2. Figure 3 It is a diagram showing the fluorescence observation results of the mouse optic nerve in Example 3. Figure 4 It is a diagram showing the fluorescence observation results of the mouse optic nerve in Example 4. Figure 5 It is a diagram showing the fluorescence observation results of the mouse retina in Example 5. Figure 6 It is a diagram showing the fluorescence observation results of the mouse lateral geniculate body in Example 6. Figure 7 It is a diagram showing the fluorescence observation results of the mouse eyeball in Example 7. Figure 8 This is a figure showing the fluorescence observation results of the optic nerve of the mouse in Example 8. Figure 9 This is a figure showing the fluorescence observation results of the retina of the mouse in Example 8. Detailed implementation manners

[0009] Hereinafter, the implementation manners of the present invention will be described. These descriptions and examples are illustrative descriptions and examples of the present invention and do not limit the scope of the present invention. In this specification, the numerical range represented by "~" means: the range including the values described before and after "~" as the minimum value and the maximum value respectively. The description of the amino acid sequence is as follows: the left side is the N-terminal side and the right side is the C-terminal side. The amino acid residues contained in the amino acid sequence are sometimes represented by the single-letter notations well-known in the art (for example, representing the glycine residue as "G"). In this specification, "treatment" means the action or effect of disappearing or reducing symptoms, and in addition, it also means the action or effect of inhibiting the deterioration of the symptoms.

[0010] <Physiologically active peptide derivative> The physiologically active peptide derivative of the present invention has a physiologically active peptide sequence and a functional sequence, and the functional sequence includes a cell penetrating peptide (hereinafter also referred to as CPP) and a penetration accelerating sequence (hereinafter also referred to as PAS). The physiologically active peptide derivative of the present invention is used for the treatment of eye diseases.

[0011] The present inventors observed that: if a peptide bonded with a functional sequence is administered to a mouse via the nose, the peptide transfers to the eyeball or optic nerve of the mouse, and the functional sequence includes a cell penetrating peptide and a penetration accelerating sequence. That is, the physiologically active peptide derivative of the present invention can enable a peptide as a therapeutic agent for eye diseases to effectively act on the affected part by a non-invasive method such as nasal administration. Therefore, it is useful as a therapeutic agent for eye diseases containing a peptide as an active ingredient.

[0012] The use of the physiologically active peptide derivative is not particularly limited as long as it is a use of the pharmacological effect manifested by the action of the physiologically active peptide sequence contained in the physiologically active peptide derivative on the eyeball or optic nerve. As the use of the physiologically active peptide derivative, for example, the treatment or improvement of various eye diseases, eye injuries, eye fatigue or symptoms caused by eye fatigue, etc. can be cited.

[0013] As the eye diseases that are the application objects of the present invention, specifically, optic neuritis, retrobulbar neuritis, neuromyelitis optica, age-related macular degeneration, myopic choroidal neovascularization, branch retinal vein occlusion, central retinal vein occlusion, diabetic retinopathy, diabetic macular edema, allergic conjunctivitis, viral conjunctivitis, bacterial conjunctivitis, infective endophthalmitis, blepharochalasis, chalazion, dacryocystitis, canaliculitis, orbital cellulitis, malignant tumor, retinitis pigmentosa, retinal rupture, intraocular lymphoma, thyroid ophthalmopathy, sarcoidosis, dry eye, Vogt-Koyanagi-Harada disease, macular hole, corneal infection, uveitis, glaucoma, optic neuropathy, cataract, Behcet's syndrome, etc. can be cited. However, the application objects of the invention are not limited thereto.

[0014] There is no particular limitation on the total number of amino acid residues of the bioactive peptide derivative. For example, the total number of amino acid residues of the bioactive peptide derivative can be 250 or less, can be 200 or less, can be 150 or less. The number of residues of the amino acid residues contained in the bioactive peptide derivative can be, for example, 10 or more, can be 20 or more, can be 30 or more.

[0015] Each of the amino acid residues constituting the bioactive peptide derivative can be either an L-isomer or a D-isomer as long as the effects of the present invention can be achieved. The method for preparing the bioactive peptide derivative is not particularly limited and can be any one of extraction from organisms or natural substances, genetic engineering methods, organic synthetic chemical methods, etc.

[0016] (Bioactive peptide sequence) The bioactive peptide sequence in the bioactive peptide derivative is not particularly limited as long as it is a sequence derived from a peptide that exhibits a pharmacological effect by acting on the eyeball or optic nerve. The method of bonding the bioactive peptide sequence, the membrane penetration enhancing sequence, and the endosome escape enhancing sequence in the bioactive peptide derivative is not particularly limited and can be carried out by a known method.

[0017] As the peptides that exhibit a pharmacological effect by acting on the eyeball or optic nerve, specifically, GLP-1 (23 amino acid residues), GLP-2 (37 amino acid residues), G-CSF (granulocyte colony-stimulating factor, 174 or 177 amino acid residues), PEG-G-CSF (PEGylated G-CSF, 174 or 177 amino acid residues), erythropoietin (165 amino acid residues), PEGylated erythropoietin (165 amino acid residues), vasoactive intestinal peptide (VIP, 28 amino acid residues), oxytocin (9 amino acid residues), transforming growth factor TGF-β (112 amino acid residues), etc. can be cited.

[0018] The number of amino acid residues contained in the bioactive peptide sequence is not particularly limited. The total number of amino acid residues contained in the bioactive peptide sequence can be from 5 to 200, can be from 5 to 170, can be from 9 to 120, can be from 9 to 70, can be from 9 to 60. The number of amino acid residues contained in the bioactive peptide sequence can be 5 or more, can be 10 or more, can be 15 or more. The number of amino acid residues contained in the bioactive peptide sequence can be 200 or less, can be 170 or less, can be 120 or less, can be 70 or less, can be 60 or less, can be 51 or less.

[0019] (Membrane permeability enhancing sequence) The membrane permeability enhancing sequence is an amino acid sequence derived from a peptide (membrane permeable peptide) having the effect of transferring into cells through cell membranes.

[0020] Examples of the membrane-permeable peptides that constitute the membrane permeability enhancing sequence include: oligomeric arginine (Rn, where n is the number of arginine residues, i.e., 6 to 12), oligomeric lysine (Kn, where n is the number of lysine residues, i.e., 6 to 12), penetratin (RQIKIWFQNRRMKWKK, 16 amino acid residues, sequence number 3), TAT (GRKKRRQRRR, 10 amino acid residues, sequence number 4), mini penetratin (RRMKWKK, 7 amino acid residues, sequence number 5), R9FC (RRRRRRRRRRFFC, 12 amino acid residues, sequence number 6), AIP6 (RLRWR, 5 amino acid residues, sequence number 7), DPV3 (RKKRRRESRKKRRRES, 16 amino acid residues, sequence number 8), DPV6 (GRPRESGKKRKRKRLKP, 17 amino acid residues, sequence number 9), Pep-1 (KETWWETWWTEWSQPKKKRKV, 21 amino acid residues, sequence number 10), MPG (GLAFLGFLGAAGSTMGAWSQPKKKRKV, 27 amino acid residues, sequence number 11), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL, 27 amino acid residues, sequence number 12), MAP (KLALKALKALKAALKLA, 17 amino acid residues, sequence number 13), W / R (RRWWRRWRR, 9 amino acid residues, sequence number 14), CADY (GLWRALWRLLRSLWRLLWRA, 20 amino acid residues, sequence number 15), EB-1 (LIRLWSHLIHIWFQNRRLKWKK, 22 amino acid residues, sequence number 16), HRSV (RRIPNRRPRR, 10 amino acid residues, sequence number 17), PTD-5 (RRQRRRTSKLMKR, 13 amino acid residues, sequence number 18), TAT47-57 (YGRKKRRQRRR, 11 amino acid residues, sequence number 19), TP2 (PLIYLRLLRGQF, 12 amino acid residues, sequence number 20), TP10 (AGYLLGKINLHALAALAKKIL, 21 amino acid residues, sequence number 21), a cationic sequence bonded to heparan, a cationic sequence bonded to RNA, a cationic sequence bonded to DNA, and the like.

[0021] As a membrane-permeable peptide constituting the membrane permeation-enhancing sequence, a positively charged membrane-permeable peptide or a cationic membrane-permeable peptide is preferred. For example, a cationic membrane-permeable peptide containing an amino acid sequence rich in basic amino acid residues such as arginine, lysine, histidine, tryptophan, etc. (for example, amino acid residues with more than half of the total number of amino acid residues being basic) is preferred. Examples of such membrane-permeable peptides include oligomeric arginine, TAT derived from the Tat protein of human immunodeficiency virus type 1 (HIV-1), cell-penetrating peptides, Pep-1, MPG, MAP, CADY, EB-1, Transportan, etc. It is considered that a membrane-permeable peptide containing an amino acid sequence rich in basic amino acid residues induces macropinocytosis, which is a type of endocytosis in the process of cells taking in substances outside the cells. Thus, it is considered that a bioactive peptide derivative can be more effectively introduced into cells.

[0022] There is no particular limitation on the number of amino acid residues of the membrane permeation-enhancing sequence. For example, it is preferably 5 to 27. In addition, more than half of the total number of amino acid residues of the membrane permeation-enhancing sequence is preferably a basic amino acid residue, more preferably a peptide particularly containing an arginine residue among basic amino acid residues, further preferably an oligomeric arginine containing 6 to 12 arginine residues, still further preferably an oligomeric arginine containing 7 to 9 arginine residues, and still further preferably an oligomeric arginine containing 8 arginine residues.

[0023] (Endosome escape-enhancing sequence) It is considered that the endosome escape-enhancing sequence shortens the residence time of the bioactive peptide derivative introduced into cells in endosomes and enables endosome escape in a shorter time. As a result, it is considered that the transfer and distribution of the bioactive peptide derivative to the eyeball or optic nerve can be achieved in a shorter time.

[0024] There is no particular limitation on the structure of the endosome escape-enhancing sequence. For example, sequences promoting endosome escape such as FFLIPKG (SEQ ID NO: 22), LILIG (SEQ ID NO: 23), FFG, FFFFFG (SEQ ID NO: 24), FFFFFFFG (SEQ ID NO: 25), etc. can be cited.

[0025] There is no particular limitation on the positions of the membrane permeation-enhancing sequence and the endosome escape-enhancing sequence in the bioactive peptide derivative. For example, the membrane permeation-enhancing sequence can be located on the side closer to the bioactive peptide sequence, and the endosome escape-enhancing sequence can be located on the side closer to the bioactive peptide sequence. From the viewpoint of more effectively achieving the effects of the present invention, the membrane permeation-enhancing sequence is more preferably located on the side closer to the bioactive peptide sequence, and the membrane permeation-enhancing sequence is further preferably located on the side closer to the bioactive peptide sequence, and an endosome escape-enhancing sequence exists on the N-terminal side or C-terminal side of the membrane permeation-enhancing sequence.

[0026] (Glycan-modified molecule) From the viewpoint of promoting the transfer and distribution of the bioactive peptide derivative to the eyeball or optic nerve, the bioactive peptide derivative preferably has a glycan-modified molecule containing a glycan.

[0027] The type of glycan contained in the glycan-modified molecule is not particularly limited. As the glycan, specifically, N-linked glycans such as high-mannose type, complex type, and hybrid type (a combination of high-mannose type and complex type), O-linked glycans, and proteoglycans such as mucin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate can be mentioned. Among them, N-linked glycans are preferred, and complex type glycans are more preferred.

[0028] The structure of the glycan is not particularly limited and can be a double-stranded structure or other structures (such as a branched structure). As the monosaccharides constituting the glycan, glucose, mannose, galactose, fructose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, fucose, sialic acid, N-acetylneuraminic acid, N-glycolylneuraminic acid, deaminoneuraminic acid, glucuronic acid, iduronic acid, galacturonic acid, xylose, ribose, deoxyribose, etc. can be mentioned. The monosaccharides constituting the glycan can be D-isomers or L-isomers. The monosaccharides constituting the glycan can be α-anomers or β-anomers.

[0029] The number of glycan-modified molecules possessed by the bioactive peptide derivative can be 1 or 2 or more, and the number of glycans possessed by 1 glycan-modified molecule can be 1 or 2 or more. The number of glycans possessed by the bioactive peptide derivative can be 1 or 2 or more. In this specification, the number of glycans is counted according to the base of each glycan. That is, a set of monosaccharide residues connected starting from 1 base is counted as "1 strand".

[0030] The glycan-modified molecule can be composed only of glycans or can be composed of glycans and parts other than glycans. That is, the glycan can be directly bonded to the amino acid residue constituting the bioactive peptide derivative or can be indirectly bonded to the amino acid residue constituting the bioactive peptide derivative. In this specification, both the state where the glycan is directly bonded to the amino acid residue constituting the bioactive peptide derivative and the state where the glycan is indirectly bonded to the amino acid residue constituting the bioactive peptide derivative are included in the state of "the glycan-modified molecule is bonded to the amino acid residue constituting the bioactive peptide derivative". As the state where the glycan is indirectly bonded to the amino acid residue constituting the bioactive peptide derivative, a state of bonding through an amino acid residue such as a cysteine residue or an asparagine residue, a linking group, etc. can be mentioned.

[0031] The glycan-modified molecule is preferably bonded to a position that can well maintain the function of the membrane permeability promoting sequence and the endosome escape enhancing sequence, and is preferably bonded to a bioactive peptide sequence. When the glycan-modified molecule is bonded to a bioactive peptide sequence, the bonding site of the glycan-modified molecule is not particularly limited as long as it is a position where the stability and pharmacological action of the bioactive peptide sequence are not reduced. That is, it can be the N-terminus of the bioactive peptide sequence, the C-terminus, or a position other than the termini. As long as the pharmacological action of the bioactive peptide sequence is not affected, a part of the bioactive peptide sequence can be replaced with an amino acid such as a cysteine residue or an asparagine residue that is easily bonded to the glycan-modified molecule. Alternatively, an amino acid residue can be introduced at the end of the bioactive peptide sequence to bond the glycan-modified molecule to the amino acid residue.

[0032] From the viewpoint of reducing the influence of the glycan on the function of the functional sequence, the bonding position of the glycan-modified molecule in the bioactive peptide sequence is preferably a position that can appropriately ensure the distance from the functional sequence. As a method for ensuring the distance between the glycan-modified molecule and the functional sequence, the following can be cited: when the functional sequence is added to the N-terminal side of the bioactive peptide sequence, bonding the glycan-modified molecule to the C-terminal side of the bioactive peptide sequence; when the functional sequence is added to the C-terminal side of the bioactive peptide sequence, bonding the glycan-modified molecule to the N-terminal side of the bioactive peptide sequence.

[0033] As another method for ensuring the distance between the glycan-modified molecule and the functional sequence, the following can be cited: the glycan-modified molecule further contains a linking group, and the glycan is bonded via the linking group. The type of the linking group contained in the glycan-modified molecule is not particularly limited. For example, an alkylene group, a polyethylene glycol (PEG) group, etc. can be cited. When the linking group is an alkylene group, from the viewpoint of appropriately ensuring the distance between the glycan-modified molecule and the functional sequence, the number of carbon atoms of the alkylene group is preferably 3 to 15, more preferably 4 to 12, and further preferably 5 to 10.

[0034] The number of monosaccharide residues of each glycan contained in the glycan-modified molecule is not particularly limited. For example, it can be in the range of 5 to 20, or can be in the range of 5 to 15. From the viewpoint of improving the solubility of the bioactive peptide derivative in an aqueous solvent, the number of monosaccharide residues of each glycan is preferably 5 or more, more preferably 10 or more.

[0035] (Spacer sequence) The bioactive peptide derivative may also have a spacer sequence disposed between the bioactive peptide sequence and the functional sequence. By having a spacer sequence between the bioactive peptide sequence and the functional sequence, it is possible to expect the effect of preventing the reduction or impairment of the activity of the bioactive peptide sequence. When the bioactive peptide derivative has a spacer sequence, the spacer sequence may be disposed between the bioactive peptide sequence and the membrane penetration enhancing sequence, or may be disposed between the bioactive peptide sequence and the endosome escape enhancing sequence.

[0036] Generally, the membrane penetration enhancing sequence is composed of basic amino acids. Therefore, when the bioactive peptide sequence contains acidic amino acid residues, for example, by having a spacer sequence containing 1 to 10, preferably 2 to 6 neutral amino acid residues such as glycine, the membrane penetration enhancing sequence interacts with the bioactive peptide sequence, thereby preventing the reduction or impairment of the activity of the bioactive peptide sequence.

[0037] The spacer sequence may contain a lysine residue (K). Lysine is an amino acid having a 4-aminobutyl side chain. By bonding a sugar chain-modifying molecule to the terminal amino group of 4-aminobutyl, an alkylene group having 4 carbon atoms can be present between the sugar chain-modifying molecule and the spacer sequence. Therefore, the distance between the sugar chain-modifying molecule and the functional sequence can be appropriately ensured.

[0038] As a spacer sequence containing a lysine residue, a spacer sequence obtained by substituting one or more amino acid residues in a spacer sequence containing 1 to 10, preferably 2 to 6 neutral amino acid residues such as glycine can be mentioned.

[0039] In addition to the above-mentioned addition of sugar chains, the bioactive peptide derivative can be subjected to various modifications according to the use. For example, amino modification (biotinylation, myristoylation, palmitoylation, acetylation, maleimideation, etc.), carboxyl modification (amidation, esterification, etc.), thiol modification (farnesylation, geranylation, methylation, palmitoylation, etc.), hydroxyl modification (phosphorylation, sulfation, octanoylation, palmitoylation, palmitoleoylation, etc.), various fluorescence labeling (FITC, FAM, ICG, Rhodamine, BODIPY, NBD, MCA, etc.), PEGylation, introduction of unnatural amino acids, D-amino acids, etc. can be carried out. The modification can be carried out in any of the bioactive peptide sequence, the membrane penetration enhancing sequence, the endosome escape enhancing sequence, and the spacer sequence of the bioactive peptide derivative.

[0040] The combination of the bioactive peptide sequence, the membrane penetration enhancing sequence, and the endosome escape enhancing sequence constituting the bioactive peptide derivative is not particularly limited and can be selected according to the use. In one embodiment, the bioactive peptide derivative may sequentially have an endosome escape enhancing sequence, a membrane penetration enhancing sequence, a spacer sequence optionally provided, and a bioactive peptide sequence starting from the N-terminal side. In one embodiment, the bioactive peptide derivative may sequentially have an endosome escape enhancing sequence, a membrane penetration enhancing sequence, a spacer sequence optionally provided, and a bioactive peptide sequence starting from the C-terminal side. In the above constitution, the endosome escape enhancing sequence may be selected from FFLIPKG, LILIG, FFG, FFFFFG, or FFFFFFFG. In the above constitution, the membrane penetration enhancing sequence may be selected from oligomeric arginine (e.g., Rn, n = 6 to 12). In the above constitution, the spacer sequence may be selected from a sequence containing a glycine residue (e.g., Gn, n = 2 to 6), a sequence containing a glycine residue and a cysteine residue (e.g., GCG), and a sequence containing a glycine residue and a lysine residue (e.g., GKG).

[0041] <Medicinal composition> The medicinal composition of the present invention contains the above bioactive peptide derivative as an active ingredient. By containing the bioactive peptide derivative as an active ingredient, the medicinal composition of the present invention has excellent transferability to the eyeball or optic nerve upon nasal administration and can effectively exhibit a pharmacological effect. Therefore, for example, it is useful for the treatment of eye diseases that require daily administration of a pharmaceutical preparation at home. Therefore, as a preferred dosage form of the medicinal composition, a nasal drop preparation can be cited.

[0042] The details and preferred modes of the bioactive peptide derivative contained in the medicinal composition are as shown above. From the viewpoint of the delivery property to the eyeball or optic nerve, the use method of the medicinal composition is preferably nasal drop administration. The medicinal composition may contain components other than the bioactive peptide derivative. Specific examples of the components that can be contained in addition to the medicinal composition include the medium used in the preparation of the medicinal composition and formulation additives. As formulation additives, excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizing agents, stabilizers, isotonic agents, suspending agents, emulsifiers, solvents, thickeners, mucolytic agents, wetting agents, preservatives, etc. can be cited. The dosage of the medicinal composition can be selected according to the type of disease, the symptoms, body weight, age, etc. of the patient, and the administration method. The medicinal composition of the present invention is particularly preferably a nasal drop preparation. That is, one embodiment of the present invention is the use of the bioactive peptide derivative of the present invention or the medicinal composition containing the same in nasal drop administration.

[0043] <Nasal drop preparation> The nasal drop preparation of the present invention contains the above-mentioned bioactive peptide derivative as an active ingredient. By containing the bioactive peptide derivative as an active ingredient, the nasal drop preparation of the present invention has excellent transferability to the eyeball or optic nerve and can effectively exhibit pharmacological effects. In addition, since it is a low-invasive administration method, it is suitable for improving the symptoms of diseases that require daily administration at home.

[0044] The nasal drop preparation may contain components other than the bioactive peptide derivative. As components other than the bioactive peptide derivative, the above substances can be cited as media and formulation additives used in the preparation of pharmaceutical compositions.

[0045] An embodiment of the present invention includes the use of a pharmaceutical composition containing the above-mentioned bioactive peptide derivative as an active ingredient in nasal drop administration. The details and preferred forms of the bioactive peptide derivative and the pharmaceutical composition in this use are as shown above.

[0046] <Method for treating eye diseases> An embodiment of the present invention includes a method for treating eye diseases, which comprises administering the above-mentioned bioactive peptide derivative or pharmaceutical composition to a patient. The details and preferred embodiments of the bioactive peptide derivative and the pharmaceutical composition in this method are as shown above. The treatment target of the above method, that is, eye diseases, can be cited as the above-mentioned eye diseases as the application target of the bioactive peptide derivative. The method of administering the bioactive peptide derivative or pharmaceutical composition to a patient is not particularly limited, and nasal administration is preferred. Examples

[0047] Examples are given below to further specifically illustrate the present invention. The materials, amounts used, ratios, processing sequences, etc. shown in the following examples can be appropriately changed as long as they do not depart from the gist of the present invention. Therefore, the scope of the present invention is not restrictively interpreted as the specific examples shown below.

[0048] In the following examples, GLP-2 used in the preparation of the bioactive peptide derivative is a peptide containing the amino acid sequence shown by HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 1), and GLP-1 is a peptide containing the amino acid sequence shown by HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (active form 7-36 amide, SEQ ID NO: 2).

[0049] <Example 1: Transfer of GLP-2 derivative to the eyeball and optic nerve> A GLP-2 derivative (C-terminal 11 sugars) was prepared by a general method, which has an endosome escape enhancing sequence (PAS: FFLIPKG), a membrane penetration enhancing sequence (CPP: RRRRRRRR), a spacer sequence (GG), and a GLP-2-derived amino acid sequence as a bioactive peptide sequence arranged in sequence from the N-terminal side. As the bioactive peptide sequence, a sequence in which a sugar chain containing 11 monosaccharide residues is added via a cysteine residue at the C-terminus of GLP-2 was used. The structure of the prepared GLP-2 derivative is shown below.

[0050] [Chemical Formula 1]

[0051] Male ddY mice (7 weeks old, the same below) were administered intranasally with 16% DMSO (solvent) or a DMSO solution of ICG-labeled GLP-2 derivative (3.0 nmol / mouse or 12.0 nmol / mouse). The eyeballs were enucleated 20 minutes after administration and infiltrated and fixed in 4% PFA solution overnight. Then, the eyeballs were washed with 1×PBS, placed on a petri dish, and measured using a light imaging device (Clairvivo OPT plus; Shimadzu, Kyoto, Japan, the same below). The measurement conditions were set as follows: excitation wavelength: 785 nm, fluorescence wavelength: 849 nm, exposure time: 6 sec. The results are shown in Figure 1 .

[0052] As Figure 1 shown, it was confirmed that the intranasally administered GLP-2 derivative was transferred to the eyeball and optic nerve.

[0053] <Example 2: Transfer of GLP-1 Derivative to Eyeball and Optic Nerve> The bioactive peptide sequence was changed from GLP-2 to GLP-1, and otherwise the same as in Example 1, a GLP-1 derivative (C-terminal 11 sugars) with a sugar chain added to the C-terminal side of GLP-1 was prepared. The structure of the prepared GLP-1 derivative is shown below.

[0054] [Chemical Formula 2]

[0055] Male ddY mice were administered intranasally with 16% DMSO (solvent) or a DMSO solution of ICG-labeled GLP-1 derivative (0.9 nmol / mouse). The eyeballs were enucleated 5 minutes and 20 minutes after administration and infiltrated and fixed in 4% PFA solution overnight. Then, the eyeballs were washed with 1×PBS, placed on a petri dish, and measured using a light imaging device. The measurement conditions were set as follows: excitation wavelength: 785 nm, fluorescence wavelength: 849 nm, exposure time: 6 sec. The results are shown inFigure 2 .

[0056] As Figure 2 shown, it was confirmed that the GLP-1 derivative (C-terminal 11 sugars) administered via the nose was transferred to the eyeball and optic nerve.

[0057] <Example 3: Distribution of GLP-2 Derivative to Optic Nerve> PBS (solvent) or a PBS solution (3.0 nmol / mouse) of the GLP-2 derivative used in Example 1 was administered via the nose to mice. The eyeballs were removed 20 minutes after the start of administration and infiltrated and fixed in 4% PFA overnight. The next day, the fixative was replaced with 30% sucrose and left overnight (4°C). Then, cryosections of the optic nerve with a thickness of 10 μm were prepared using a cryostat (CM3050S; Leica Microsystems, WetZlar, Germany, the same below). The sections were placed on glass slides, blocking buffer was added, and blocking was performed at room temperature for 30 minutes. Then, a primary antibody solution containing Neuro-Chrom TM Pan Neuronal Marker Antibody-Rabbit (1:500) and GLP-2 polyclonal antibody (1:200) diluted with the blocking buffer was added, and incubation was carried out at room temperature for 2 hours. After washing 3 times with 1×PBS, a secondary antibody solution containing Alexa Fluor (registered trademark) 568 goat anti-mouse IgG H&L and 40 ng / ml DAPI diluted 500-fold with 1% BSA / PBS solution was added, and incubation was carried out at room temperature for 1 hour. After washing 3 times with 1×PBS, mounting was performed, and fluorescence observation was carried out using software (Leica Application Suite X Software; Leica, WetZlar, Germany, the same below) with a confocal laser microscope (TCS SP8; Leica, WetZlar, Germany, the same below). The results are shown in Figure 3 .

[0058] As Figure 3 shown, it was confirmed that the GLP-2 derivative (C-terminal 11 sugars, corresponding to punctate fluorescence) administered via the nose was distributed in the optic nerve.

[0059] <Example 4: Distribution of GLP-1 Derivative to Optic Nerve> A PAS-CPP-GLP-1 derivative (sugar-free) was obtained, which has an endosome escape enhancing sequence (PAS: FFLIPKG), a membrane penetration enhancing sequence (CPP: RRRRRRRR), a spacer sequence containing a cysteine residue (GCG), and an amino acid sequence derived from GLP-1 as a neuropeptide sequence, arranged successively from the N-terminal side. The structure of the obtained GLP-1 derivative is shown below.

[0060] [Chemical Formula 3]

[0061] Next, a sugar chain (11-sugar) was bonded to the cysteine residue of the spacer sequence to obtain a PAS-CPP-GLP-1 derivative (N-terminal 11-sugar) in which the N-terminal side of GLP-1 was modified with a sugar chain. The structure of the obtained GLP-1 derivative is shown below.

[0062] [Chemical Formula 4]

[0063] PBS (solvent) or a PBS solution (3.6 nmol / mouse) of the GLP-1 derivative (N-terminal 11-sugar) was administered to mice via nasal cavity. Twenty minutes after the administration, the eyeballs were enucleated. Similar to Example 3, frozen sections of the optic nerve were prepared and fluorescence observation was performed. The results are shown in Figure 4 .

[0064] As Figure 4 shown, it was confirmed that the GLP-1 derivative (N-terminal 11-sugar, equivalent to punctate fluorescence) administered via nasal cavity was distributed in the optic nerve.

[0065] <Example 5: Distribution of GLP-1 Derivative in Retina> PBS (solvent) or a PBS solution (3.6 nmol / mouse) of the GLP-1 derivative (N-terminal 11-sugar) used in Example 4 was administered to mice via nasal cavity. Twenty minutes after the administration, the eyeballs were enucleated. Similar to Example 4, frozen sections of the retina were prepared and fluorescence observation was performed. The results are shown in Figure 5 .

[0066] As Figure 5 shown, it was confirmed that the GLP-1 derivative (N-terminal 11-sugar, equivalent to fluorescence indicated by arrows) administered via nasal cavity was distributed in the retina.

[0067] <Example 6: Distribution of GLP-2 Derivative in Lateral Geniculate Nucleus> The physiologically active peptide sequence was changed from GLP-2 to GLP-1. Except for this, it was the same as in Example 4, and a GLP-2 derivative (N-terminal 11 sugars) with a sugar chain added to the N-terminal side of GLP-2 was prepared. The structure of the prepared GLP-2 derivative is shown below.

[0068] [Chemical formula 5]

[0069] PBS or a PBS solution of the GLP-2 derivative (3.0 nmol / mouse) was administered intranasally to mice. Five minutes after the administration, the brain was removed and infiltrated and fixed overnight at 4°C in a 4% PFA solution. On the day after the removal, the brain samples were replaced with 20% sucrose overnight (4°C), and further replaced with 30% sucrose and replaced overnight (4°C). Then, cryosections of the lateral geniculate body with a thickness of 30 μm were prepared using a cryostat. The prepared sections were placed on glass slides, and a circle was drawn around the sections using an immunohistochemistry pen (liquid blocker). A blocking buffer was added inside the circle and blocked at room temperature for 30 minutes. Then, a primary antibody solution containing a GLP-2 polyclonal antibody diluted 200-fold with the blocking buffer was added and incubated at room temperature for 1 hour. After washing 3 times with 1×PBS, a secondary antibody solution containing Alexa Fluor (registered trademark) 568 goat anti-mouse IgG H&L diluted 500-fold with a 1% BSA / PBS solution was added and incubated at room temperature for 1 hour. After washing 3 times with 1×PBS, it was sealed with ProLong TM Diamond Antifade Mountant. After confirming the curing of the mounting medium, fluorescence observation was performed using a confocal laser microscope and software. The results are shown in Figure 6 .

[0070] As Figure 6 shown, fluorescence corresponding to the intranasally administered GLP-2 derivative was observed in the lateral geniculate body, suggesting that the intranasally administered GLP-2 derivative is transferred to the eyeball and optic nerve via the lateral geniculate body. This is the discovery of a new pathway that has not been reported so far, that is, the intranasally administered peptide is transferred to the eyeball and optic nerve via the lateral geniculate body of the thalamus, and at the same time suggests that the intranasally administered peptide is applicable to the treatment of ocular diseases such as optic neuritis and optic neuropathy.

[0071] <Example 7: Transfer of a GLP-2 derivative with a sugar chain bonded via a linking group to the eyeball> The spacer sequence (GCG) of the GLP-2 derivative (N-terminal 11-sugar) prepared in Example 6 was changed to the spacer sequence (GKG), and a GLP-2 derivative (C6 linker N-terminal 11-sugar) was prepared in which a sugar chain (11-sugar) and an alkylene group having 6 carbon atoms as a linking group were molecularly bonded to the lysine residue (K) in the spacer sequence. The structure of the prepared GLP-2 derivative is shown below.

[0072] [Chemical Formula 6]

[0073] Mice were administered intranasally with 16% DMSO (solvent) or a DMSO solution (3.0 nmol / mouse) of an ICG-labeled GLP-2 derivative (C6 linker N-terminal 11-sugar). The eyeballs were enucleated 5 minutes, 10 minutes, 20 minutes, 60 minutes, or 90 minutes after the start of administration and infiltrated and fixed overnight in a 4% PFA solution. Then, the eyeballs were washed with 1×PBS, placed on a petri dish, and measured using a light imaging device. The measurement conditions were set as follows: excitation wavelength: 785 nm, fluorescence wavelength: 849 nm, exposure time: 6 sec. The results are shown in Figure 7 .

[0074] As Figure 7 shown, it was confirmed that the intranasally administered GLP-2 derivative (C6 linker N-terminal 11-sugar) was transferred to the eyeballs within 5 minutes after the start of administration. In addition, it was confirmed that the (C6 linker N-terminal 11-sugar) transferred to the eyeballs was also present in the eyeballs 90 minutes after the start of administration.

[0075] <Example 8 Distribution of GLP-2 Derivative Bonded with Sugar Chain via Linker Group to Optic Nerve and Retina> Mice were administered intranasally with PBS (solvent) or a PBS solution (3.0 nmol / mouse) of a GLP-2 derivative (C6 linker N-terminal 11-sugar). The eyeballs were enucleated 20 minutes after the start of administration and infiltrated and fixed overnight in 4% PFA. On the day after enucleation, the eyeballs were replaced and placed in 30% sucrose and left overnight (4°C). Then, 10-μm-thick cryosections of the optic nerve and retina were prepared using a cryostat. The sections were placed on glass slides, blocking buffer was added, and blocking was performed at room temperature for 30 minutes. Then, a primary antibody solution containing Neuro-Chrom TM Pan Neuronal MarkerAntibody-Rabbit (1:500) and GLP-2 polyclonal antibody (1:200) diluted with blocking buffer was added, and incubation was performed at room temperature for 2 hours. After washing 3 times with 1×PBS, a solution containing Alexa Fluor TMSecondary antibody solution of 568 goat anti-mouse IgG H&L and 20 μg / ml DAPI was incubated at room temperature for 1 hour. After washing 3 times with 1×PBS, mounting was performed. Then, fluorescence observation was carried out using a confocal laser microscope and software. The results of fluorescence observation of the part corresponding to the optic nerve are shown in Figure 8 and the results of fluorescence observation of the part corresponding to the retina are shown in Figure 9 . As Figure 8 and Figure 9 show, it was confirmed that the GLP-2 derivative administered via the nose (C6-linked group, N-terminal 11 sugars, corresponding to punctate fluorescence) was transferred to the retinal ganglion cell and the inner reticulated layer within 20 minutes from the start of administration.

[0076] The disclosure of Japanese Patent Application No. 2022-180381 is incorporated herein by reference in its entirety. For all documents, patent applications, and technical standards described in this specification, the act of incorporating each document, patent application, and technical standard by reference is equivalent to the case where they are specifically and separately described, and they are incorporated herein by reference.

Claims

1. A physiologically active peptide derivative for treating eye diseases, characterized in that, It has a bioactive peptide sequence and a functional sequence, and the functional sequence includes a membrane penetration enhancing sequence and an endosome escape enhancing sequence.

2. The bioactive peptide derivative according to claim 1, wherein, The bioactive peptide derivative also has a sugar chain-modified molecule containing a sugar chain.

3. The bioactive peptide derivative according to claim 2, wherein The sugar chain-modified molecule is bonded to the C-terminal side or the N-terminal side of the bioactive peptide sequence.

4. The bioactive peptide derivative according to claim 2, wherein, The number of monosaccharide residues in each sugar chain is 5 to 20.

5. The bioactive peptide derivative according to claim 1, wherein, The number of amino acid residues in the bioactive peptide sequence is 200 or less.

6. The bioactive peptide derivative according to claim 1, wherein, The membrane penetration enhancing sequence is cationic.

7. The bioactive peptide derivative according to claim 1, wherein, More than half of the total number of amino acid residues in the membrane penetration enhancing sequence are basic amino acid residues.

8. The bioactive peptide derivative according to claim 1, wherein, The endosome escape enhancing sequence is an amino acid sequence selected from FFLIPKG, LILIG, FFG, FFFFG, and FFFFFFFG.

9. The bioactive peptide derivative according to claim 1, wherein, The bioactive peptide derivative reaches the optic nerve and retina via the lateral geniculate body.

10. A pharmaceutical composition, characterized in that, Comprising the bioactive peptide derivative according to any one of claims 1 to 9 as an active ingredient.

11. A nasal drop preparation, characterized in that, Comprising the bioactive peptide derivative according to any one of claims 1 to 9 as an active ingredient.

12. Use of a physiologically active peptide derivative in the treatment of ocular diseases, characterized in that, The bioactive peptide derivative has a bioactive peptide sequence and a functional sequence, and the functional sequence includes a membrane penetration enhancing sequence and an endosome escape enhancing sequence.

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