Sugar chain-modified neuropeptide derivative comprising neuropeptide sequence and sugar chain, pharmaceutical composition, nasal / nasal drop preparation, and use of sugar chain-modified neuropeptide derivative

Abstract

The present invention provides a sugar chain-modified neuropeptide derivative having a neuropeptide sequence, a membrane permeation enhancing sequence, an endosome escape enhancing sequence, and a sugar chain.

Description

Technical Field

[0001] The present invention relates to a glycan-modified neuropeptide derivative containing a neuropeptide sequence and a glycan, a pharmaceutical composition, a nasal and nasal drop preparation, and uses of the glycan-modified neuropeptide derivative. Background Art

[0002] It is known that central nervous system diseases such as Alzheimer's disease, vascular dementia, and amyotrophic lateral sclerosis are areas with low treatment satisfaction and few effective therapeutic drugs, that is, areas with high medical needs, and the development of new therapeutic drugs is expected. On the other hand, for example, in the treatment of depression, low molecular weight drugs are used and certain therapeutic effects can be obtained. However, about 30 to 40% of patients show treatment resistance to existing antidepressants, so the development of therapeutic drugs with new pharmacological mechanisms is expected.

[0003] In view of the above background, in recent years, neuropeptides and the like having a mechanism of action different from that of low molecular weight drugs have attracted attention as candidates for their therapeutic drugs. For example, it is known that glucagon-like peptide-1 (GLP-1), a peptide composed of 37 amino acid residues derived from proglucagon, and glucagon-like peptide-2 (GLP-2) composed of 33 amino acid residues both play a role of binding to a G protein-coupled receptor (GPCR) and transmitting signals. As the pharmacological action of GLP-1 (active forms include 7-37 and 7-36 amide) in the brain, reports related to the improvement effect on learning disorders have been made (for example, Non-Patent Documents 1 to 2). In addition, as the pharmacological action of GLP-2 in the brain, antidepressant effects, blood pressure lowering effects, and learning disorder improvement effects that are also effective in treatment-resistant depression model animals have been reported (for example, Non-Patent Documents 3 to 9). In addition, it has been reported that neuromedin U (NmU) containing 23 amino acid residues also binds to GPCR in the brain and shows an improvement effect on learning disorders (for example, Non-Patent Document 10). In addition, research and development have been carried out on peptides with central effects, such as enkephalin (5 amino acid residues), pasireotide (5 amino acid residues), octreotide (7 amino acid residues), lanreotide (7 amino acid residues), oxytocin (9 amino acid residues), somatostatin-14 (14 amino acid residues), dynorphin (17 amino acid residues), somatostatin-28 (28 amino acid residues), ghrelin (28 amino acid residues), orexin B (28 amino acid residues), galanin (30 amino acid residues), β-endorphin (31 amino acid residues), orexin A (33 amino acid residues), neuropeptide Y (36 amino acid residues), insulin (51 amino acid residues), galanin-like peptide (60 amino acid residues), insulin-like growth factor-1 (70 amino acid residues), nerve growth factor (118 amino acid residues), leptin (166 amino acid residues), etc.

[0004] Against the background of high medical needs for central nervous system diseases, the main factors can be cited as follows: due to the tight intercellular junctions represented by the blood-brain barrier (BBB), the transfer of drugs from the blood to the brain tissue is extremely restricted, and it is difficult to deliver drugs to the target site. For example, macromolecules over 500 Da cannot pass through the BBB 100%, and more than 98% of molecules less than 500 Da cannot pass through the BBB (Non-Patent Document 11). Therefore, when conducting pharmacodynamic and pharmacological tests on drugs for central nervous system diseases, intracerebroventricular administration, which directly administers drugs into the brain, can be used. However, the invasiveness of intracerebroventricular administration is very high, and it is not practical for clinical application. Therefore, considering clinical application, non-invasive nasal administration, that is, intranasal administration to the nasal cavity anatomically close to the brain, has attracted attention. In fact, it has been reported that in animal experiments, most peptides are transferred to the center via the olfactory bulb or cerebrospinal fluid through intranasal administration (for example, Non-Patent Document 12). However, the practical application of peptides capable of exhibiting central effects has not been achieved by intranasal administration of peptides. One of the main reasons is that a drug delivery system (DDS) that takes into account the characteristics of the nasal mucosa structure has not been developed.

[0005] The nasal mucosa is covered by olfactory epithelium and respiratory epithelium. In the human nasal mucosa, the olfactory epithelium accounts for about 3%, and the respiratory epithelium accounts for about 97% (Non-Patent Document 13). Therefore, in the human body, in order to efficiently transfer peptides to the central nervous system through intranasal administration, it is effective to transfer peptides from the respiratory epithelium rather than the olfactory epithelium.

[0006] The following three pathways are considered to be the main pathways for the transfer of intranasally administered drugs to the central nervous system. (1) Pathways for transfer from the nasal mucosa to the blood and then through the BBB to the central nervous system. (2) Pathways for transfer from the olfactory epithelium to the olfactory bulb or for diffusion from the intercellular spaces of the olfactory epithelium into the cerebrospinal fluid and then to the central nervous system. (3) Pathways for transfer from the respiratory epithelium via the trigeminal nerve to the central nervous system. In considering the clinical application of drugs, for the central transfer of peptides by nasal administration, the pathway of (3) that exploits the above characteristics of the human nasal mucosa structure is the most suitable option. However, it has been reported that there are a very large number of capillaries in the layer of the respiratory epithelium, i.e., the lamina propria, and the vascular permeability is high, so peptides are systemically absorbed through the intercellular spaces of the respiratory epithelium. For example, a nasal drop of calcitonin is clinically used as a therapeutic agent for osteoporosis, and this preparation is designed to be systemically absorbed from the nasal mucosa by nasal administration of calcitonin (Non-Patent Document 14). Therefore, to efficiently transfer peptides to the central nervous system, it is important to inhibit the permeation of peptides through the intercellular spaces. From this perspective, a nose-to-brain pathway system has been proposed, in which a neuropeptide derivative obtained by adding a cell membrane penetration enhancing sequence and an endosome escape enhancing sequence to a neuropeptide is administered nasally so that it reaches the action sites, i.e., the hippocampus and the hypothalamus, and thus exhibits an effect on the central nervous system (Patent Document 1). Prior Art Documents Patent Documents

[0007] Patent Document 1: International Publication No. WO 2016 / 035820 Non-Patent Documents

[0008] Non-Patent Document 1: Neuroscience Research 64(2009)67 - 74 Non-Patent Document 2: Journal of Neuroscience Research 92(2014)446 - 454 Non-Patent Document 3: Behavioural Brain Research 204(2009)235 - 240 Non-Patent Document 4: Neuroscience 212(2012)140 - 148 Non-Patent Document 5: Life Sciences 93(2013)889 - 896 Non-Patent Document 6: Neuroscience Letters 550(2013)104 - 108 Non-Patent Document 7: Behavioural Brain Research 243(2013)153-157 Non-Patent Document 8: Neuropeptides 49(2015)7-14 Non-Patent Document 9: Neuroscience 294(2015)156-165 Non-Patent Document 10: Neuroscience Research 61(2008)113-119 Non-Patent Document 11: NeuroRx 2(2005)3-14 Non-Patent Document 12: European Journal of Pharmaceutical Sciences40(2010)385-403 Non-Patent Document 13: Toxicologic Pathology 19(1991)321-336 Non-Patent Document 14: European Journal of Pharmaceutics and Biopharmaceutics88(2014)8-27 Non-Patent Document 15: International Journal of Pharmaceutics 515(2016)37-45 Non-Patent Document 16: Nature 422(2003)37-44 Non-Patent Document 17: Neuroscientist 20(2014)71-81 Summary of the Invention

[0009] The neuropeptide derivatives obtained by adding a cell-penetration enhancing sequence and an endosome escape enhancing sequence as described in Patent Document 1 are shown to be transferred to the central nervous system by nasal administration and exhibit central effects. However, the neuropeptide derivatives described in Patent Document 1 have high lipophilicity and are mostly poorly soluble in aqueous solvents. For this reason, it is considered that a sequence containing highly hydrophobic amino acid residues such as phenylalanine is used to promote endosome escape, and thus the water solubility of the neuropeptide derivatives is low. Therefore, in the examples of Patent Document 1, 16% dimethyl sulfoxide (DMSO) was used to dissolve the neuropeptide derivatives for testing. However, it has been reported that the organic solvent DMSO has cytotoxicity and irritation to the eyes and skin, and its toxicity during clinical application is a concern. Therefore, when formulating this derivative into a clinical preparation, it is necessary to improve its solubility in aqueous solvents. Generally, additives such as surfactants and inclusion compounds are used to improve the solubility of poorly soluble drugs in aqueous solvents. However, it has been reported that cytotoxicity occurs when these additives are used (Non-Patent Document 15). In addition, there is room for improvement in the retention in the central nervous system, the persistence of the drug effect, and the effect of reducing the pharmaceutically effective amount of the neuropeptide derivatives described in Patent Document 1. That is, the subject of the present invention is to provide a neuropeptide derivative, a pharmaceutical composition, a nasal / drop nasal preparation, and the use of a glycan-modified neuropeptide derivative, which are excellent in retention in the central nervous system, persistence of drug effect, and effect of reducing the pharmaceutically effective amount. [Means for Solving the Problem]

[0010] Specific means for achieving the above subject include the following embodiments. <1> A glycan-modified neuropeptide derivative having a neuropeptide sequence, a central transfer enhancing sequence, and a glycan-modifying molecule containing a glycan, wherein the central transfer enhancing sequence contains a membrane penetration enhancing sequence and an endosome escape enhancing sequence. <2> The glycan-modified neuropeptide derivative according to <1>, wherein the glycan-modified neuropeptide derivative further has a spacer sequence disposed between the neuropeptide sequence and the central transfer enhancing sequence, and the glycan-modifying molecule is bonded to the spacer sequence. <3> The glycan-modified neuropeptide derivative according to <2>, wherein the spacer sequence contains a lysine residue, and the glycan-modifying molecule is bonded to the lysine residue. <4> The glycan-modified neuropeptide derivative according to any one of <1> to <3>, wherein the glycan-modifying molecule further contains a linking group. <5> The glycan-modified neuropeptide derivative according to <4>, wherein the linking group contains an alkylene group having 3 to 15 carbon atoms. <6>The glycopeptide-modified neuropeptide derivative according to any one of <1> to <5>, wherein the glycopeptide-modifying molecule is bonded to the C-terminal side or the N-terminal side of the neuropeptide sequence. <7>The glycopeptide-modified neuropeptide derivative according to any one of <1> to <6>, wherein the number of monosaccharide residues in each glycopeptide is 5 to 20. <8>The glycopeptide-modified neuropeptide derivative according to any one of <1> to <7>, wherein the number of amino acid residues in the neuropeptide sequence is 200 or less. <9>The glycopeptide-modified neuropeptide derivative according to any one of <1> to <8>, wherein the membrane penetration enhancing sequence is cationic. <10>The glycopeptide-modified neuropeptide derivative according to any one of <1> to <9>, wherein more than half of the total number of amino acid residues in the membrane penetration enhancing sequence are basic amino acid residues. <11>The glycopeptide-modified neuropeptide derivative according to any one of <1> to <10>, wherein the endosome escape enhancing sequence is an amino acid sequence selected from FFLIPKG, LILIG, FFG, FFFFFG, and FFFFFFFG. <12>A pharmaceutical composition comprising the glycopeptide-modified neuropeptide derivative according to any one of <1> to <11> as an active ingredient. <13>The pharmaceutical composition according to <12>, which is used for the treatment of mental and neurological diseases or neurodegenerative diseases. <14>The pharmaceutical composition according to <12>, which is used for the treatment of depression or dementia. <15>A nasal drop preparation comprising the glycopeptide-modified neuropeptide derivative according to any one of <1> to <11> as an active ingredient. <16>The nasal drop preparation according to <15>, which is used for the treatment of mental and neurological diseases or neurodegenerative diseases. <17>The nasal drop preparation according to <15>, which is used for the treatment of depression or dementia. <18>The use of the glycopeptide-modified neuropeptide derivative according to any one of <1> to <11> in nasal drop administration. Advantages of the Invention

[0011] According to the present invention, it is possible to provide a neuropeptide derivative, a pharmaceutical composition, a nasal drop preparation, and the use of a glycopeptide-modified neuropeptide derivative, which have excellent retention in the central nervous system, persistence of drug efficacy, and effect of reducing the pharmaceutically effective amount. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1It is a graph showing the solubility of various GLP-2 derivatives in Example 1. Figure 2 It is a graph showing the antidepressant-like effects after nasal administration of various GLP-2 derivatives in Example 2. Figure 3 It is a graph showing the influence of PBS on the antidepressant-like effects of the glycan-modified GLP-2 derivative (C-terminal 11 sugars) in Example 3. Figure 4 It is a graph showing the brain distribution of the PAS-CPP-GLP-2 derivative (sugar-free) and the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) after nasal administration using an optical imaging device in Example 4. Figure 5 It is a graph showing the results of quantifying the brain transfer amounts of PAS-CPP-GLP-2 (sugar-free) and the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) using ELISA in Example 5. Figure 6A It is a graph showing the brain distribution of the PAS-CPP-GLP-2 derivative (sugar-free) and the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) using immunostaining 5 minutes after the start of nasal administration in Example 6. Figure 6B It is a graph showing the brain distribution of the PAS-CPP-GLP-2 derivative (sugar-free) and the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) using immunostaining 20 minutes after the start of nasal administration in Example 6. Figure 7A It is a graph qualitatively and quantitatively showing the brain distribution 5 minutes after the start of nasal administration in Example 7. Figure 7B It is a graph qualitatively and quantitatively showing the brain distribution 20 minutes after the start of nasal administration in Example 7. Figure 7C It is a graph qualitatively and quantitatively showing the brain distribution 60 minutes after the start of nasal administration in Example 7. Figure 8 It is a graph showing the localization state of the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) in the trigeminal nerve 5 minutes after the start of nasal administration in Example 8. Figure 9 It is a graph obtained by confirming the metastasis of the glycan-modified GLP-2 derivative administered nasally to the trigeminothalamic tract in Example 9. Figure 10 It is a graph showing the antidepressant-like effects after nasal administration of the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) and the PAS-CPP-GLP-2 derivative (sugar-free) in Example 10. Figure 11 This is a graph showing the antidepressant-like effects after intranasal administration of PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) and PAS-CPP-GLP-2 derivative without sugar chain modification (sugar-free) in Example 11. Figure 12 This is a graph showing the intervention of macropinocytosis, which is the uptake mechanism of PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) in ingested neuronal cells NeuroA2 in Example 12. Figure 13A This is a graph showing the enhanced effect of the improvement in learning and memory after intranasal administration of sugar chain-modified PAS-CPP-GLP-1 derivative (C-terminal 11 sugars) and PAS-CPP-GLP-1 derivative (sugar-free) in Example 13. Figure 13B This is a graph showing the improvement effect on learning and memory when PAS-CPP-GLP-1 derivative (sugar-free) is administered intranasally or into the lateral cerebral ventricle. Figure 14A This is a graph showing the results obtained by studying the brain metastasis of PAS-CPP-GLP-1 derivative (sugar-free) and sugar chain-modified PAS-CPP-GLP-1 derivative (N-terminal 11 sugars) using an in vivo imager in Example 14. Figure 14B This is a graph showing the quantitative results of the brain metastasis amounts of PAS-CPP-GLP-1 derivative (sugar-free) and sugar chain-modified PAS-CPP-GLP-1 derivative (N-terminal 11 sugars) in Example 14. Figure 14C This is a graph showing the improvement effect on learning and memory after intranasal administration of PAS-CPP-GLP-1 derivative (sugar-free) and sugar chain-modified PAS-CPP-GLP-1 derivative (N-terminal 11 sugars) in Example 14. Figure 14D This is a graph showing the improvement effect on learning and memory 60 minutes after intranasal administration of PAS-CPP-GLP-1 derivative (sugar-free) and sugar chain-modified PAS-CPP-GLP-1 derivative (N-terminal 11 sugars) in Example 14. Figure 14E This is a graph showing the improvement effect on learning impairment 5 minutes after intranasal administration of PAS-CPP-GLP-1 derivative (sugar-free) and sugar chain-modified PAS-CPP-GLP-1 derivative (N-terminal 11 sugars) in Example 14. Figure 14F This is a graph showing the antidepressant-like effects after intranasal administration of PAS-CPP-GLP-2 derivative (sugar-free) in Example 14. Figure 14GThis is a graph showing the antidepressant-like effect after intranasal administration of a glycan-modified PAS-CPP-GLP-2 derivative (N-terminal 11 sugars) in Example 14. Figure 15A This is a graph showing the uptake of various glycan-modified PAS-CPP-GLP-2 derivatives in nerve cells in Example 15. Figure 15B This is a graph showing the antidepressant-like effect of various glycan-modified PAS-CPP-GLP-2 derivatives in Example 15. Figure 15C This is a graph showing the neuroactivity of various glycan-modified PAS-CPP-GLP-2 derivatives at the site of action in Example 15. Figure 15D This is a graph showing the quantitative results of the brain transfer amount of various glycan-modified PAS-CPP-GLP-2 derivatives in Example 15. Figure 16 This is a graph showing the usefulness of PAS-CPP of the PAS-CPP-GLP-2 derivative. Figure 17 This is a graph showing the effect of a glycan-modified PAS-CPP-GLP-1 derivative on inhibiting β-amyloid-induced neurodegeneration in Example 16. Figure 18 This is a graph showing the effect of a glycan-modified PAS-CPP-GLP-1 derivative on inhibiting β-amyloid-induced neurodegeneration in Example 16. Figure 19 This is a graph showing the effect of a glycan-modified PAS-CPP-GLP-1 derivative on inhibiting β-amyloid-induced neurodegeneration in Example 16. Detailed Description of the Invention

[0013] Hereinafter, embodiments 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, a numerical range represented by "~" means a range in which the values described before and after "~" are respectively used as the minimum value and the maximum value. 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. Amino acid residues contained in the amino acid sequence are sometimes represented by single-letter notations well-known in the art (for example, a glycine residue is represented as "G"). In this specification, "treatment" refers to the action or effect of eliminating or reducing symptoms, and in addition, it also refers to the action or effect of inhibiting the aggravation of the symptoms. "Antidepressant action" or "antidepressant effect" means, in addition to the action or effect of eliminating or alleviating the symptoms of depression, the action or effect of inhibiting the aggravation of the symptoms. "Learning disorder improvement action" or "learning disorder improvement effect" means, in addition to the action or effect of eliminating or alleviating the symptoms of learning disorder, the action or effect of inhibiting the aggravation of the symptoms.

[0014] <Glycan-modified neuropeptide derivative> The glycan-modified neuropeptide derivative of the present invention has a neuropeptide sequence, a central transfer enhancing sequence, and a glycan-modified molecule containing a glycan. The central transfer enhancing sequence contains a membrane penetration enhancing sequence (Cell penetrating peptide, hereinafter also referred to as CPP) and an endosome escape enhancing sequence (Penetration accelerating sequence, hereinafter also referred to as PAS).

[0015] In order to solve the problem of the above-mentioned neuropeptide derivative (i.e., poor solubility in aqueous solvents) without using cytotoxic additives such as surfactants and inclusion compounds, the present inventor created a substance obtained by adding a glycan to a neuropeptide derivative (hereinafter, also referred to as a glycan-modified neuropeptide derivative) in order to improve the water solubility of the neuropeptide derivative. Generally, the water solubility and membrane permeability of neuropeptide derivatives are in a trade-off relationship. Although the addition of a glycan can improve the water solubility of neuropeptide derivatives, there is a concern that the membrane permeability of the cell membrane will decrease, the dosage required to exhibit the drug effect will increase, or the drug effect will not be exhibited. However, it was surprisingly found that when a neuropeptide derivative added with a glycan was administered intranasally, compared with a neuropeptide derivative without the addition of a glycan, it showed a high degree of efficient central metastasis and retention in the central nervous system that was difficult to conceive based on previous insights. In addition, it was found that the persistence of the drug effect was improved and the pharmaceutically effective amount was also reduced. Furthermore, it was found that not only water-insoluble neuropeptide derivatives but also water-soluble neuropeptide derivatives showed the same effect by glycan modification.

[0016] As the transfer pathway of the glycan-modified neuropeptide of the present invention to the central nervous system after intranasal administration, the following pathways can be mainly cited: It is transferred to the principal sensory trigeminal nucleus (Pr5) of the pons in the brainstem via the trigeminal nerve and trigeminal ganglion in the nasal cavity, and then transferred to the central nervous system such as the hippocampus and hypothalamus. As a transfer pathway from the main sensory nucleus of the trigeminal nerve to the central nervous system, the trigeminothalamic tract can be cited. The trigeminothalamic tract is sometimes also called the trigeminal-thalamic tract. In the present invention, the trigeminothalamic tract is a concept that also includes the trigeminal-thalamic tract.

[0017] The sugar chain-modified neuropeptide derivative of the present invention has a membrane penetration-enhancing sequence and an endosome escape-enhancing sequence. As shown in the reference examples described later, derivatives obtained by adding both a membrane penetration-enhancing sequence and an endosome escape-enhancing sequence to the neuropeptide sequence exhibit antidepressant effects as central actions. In contrast, derivatives obtained by adding only a membrane penetration-enhancing sequence or only an endosome escape-enhancing sequence to the neuropeptide sequence do not exhibit antidepressant effects. That is, the sugar chain-modified neuropeptide derivative of the present invention exhibits excellent central actions by having both a membrane penetration-enhancing sequence and an endosome escape-enhancing sequence.

[0018] The use of the sugar chain-modified neuropeptide derivative is not particularly limited as long as it utilizes the pharmacological effect exhibited by the sugar chain-modified neuropeptide derivative acting on the central nervous system. Examples of such pharmacological effects include antidepressant effects, learning disorder improvement effects, anti-anxiety effects, feeding inhibition effects, cognitive disorder improvement effects, blood pressure lowering effects, analgesic effects, sleep effects, anti-epileptic effects, and the like. Therefore, the sugar chain-modified neuropeptide derivative of the present invention can be suitably used for the treatment of neuropsychiatric diseases and neurodegenerative diseases such as antidepressants, learning disorder improvers, anti-anxiety agents, appetite suppressants, cognitive disorder improvers, blood pressure lowering agents, analgesics, hypnotics, and anti-epileptic agents.

[0019] The number of amino acid residues contained in the sugar chain-modified neuropeptide derivative is not particularly limited. As shown in the examples described later, it was confirmed that the sugar chain-modified neuropeptide is taken into nerve cells by macropinocytosis. Macropinocytosis is a mechanism of intracellular uptake caused by the remodeling of the actin cytoskeleton and the formation of a wavy structure of the flowing plasma membrane, and the size of the endosomal vesicles produced is larger than 1 μm. Therefore, even if the molecular weight of the sugar chain-modified neuropeptide derivative is large, it can be expected to be taken into cells (Non-Patent Document 16).

[0020] For example, the total number of amino acid residues of the neuropeptide derivative of the sugar chain-modified neuropeptide derivative is determined by the total number of the neuropeptide sequence, the membrane penetration-enhancing sequence, the endosome escape-enhancing sequence, and the spacer sequence. For example, the total number of amino acid residues of the neuropeptide derivative can be 250 or less, can be 200 or less, and can be 150 or less. The number of residues of amino acid residues contained in the sugar chain-modified neuropeptide derivative can be, for example, 10 or more, can be 20 or more, and can be 30 or more.

[0021] Each of the amino acid residues constituting the sugar chain-modified neuropeptide derivative may 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 sugar chain-modified neuropeptide derivative is not particularly limited and may be any of extraction from organisms or natural substances, genetic engineering methods, organic synthetic chemical methods, etc.

[0022] (Neuropeptide sequence) The neuropeptide sequence in the sugar chain-modified neuropeptide derivative is not particularly limited as long as it is a sequence derived from a peptide that acts on the central nervous system and exhibits a pharmacological effect. In the sugar chain-modified neuropeptide derivative, the method for adding a membrane penetration enhancing sequence, an endosome escape enhancing sequence, and a sugar chain to the neuropeptide sequence is not particularly limited and can be carried out by a known method.

[0023] The number of amino acid residues contained in the neuropeptide sequence included in the sugar chain-modified neuropeptide is not particularly limited as long as the sugar chain neuropeptide of the present invention is taken into cells by macropinocytosis, considering the characteristics of macropinocytosis. The total number of amino acid residues contained in the neuropeptide sequence may be 5 to 200, may be 5 to 170, may be 9 to 120, may be 9 to 70, may be 9 to 60. The number of amino acid residues contained in the neuropeptide sequence may be 5 or more, may be 10 or more, may be 15 or more. The number of amino acid residues contained in the neuropeptide sequence may be 200 or less, may be 170 or less, may be 120 or less, may be 70 or less, may be 60 or less, may be 51 or less.

[0024] In one embodiment, the neuropeptide sequence is an amino acid sequence derived from a neuropeptide having central activity. As neuropeptides, specifically, GLP-1 (23 amino acid residues), GLP-2 (37 amino acid residues), enkephalin (5 amino acid residues), pasireotide (5 amino acid residues), oxytocin enkephalin (5 amino acid residues), octreotide (7 amino acid residues), lanreotide (7 amino acid residues), oxytocin (9 amino acid residues), somatostatin-14 (14 amino acid residues), dynorphin (17 amino acid residues), somatostatin-28 (28 amino acid residues), ghrelin (28 amino acid residues), orexin B (28 amino acid residues), galanin (30 amino acid residues), β-endorphin (31 amino acid residues), orexin A (33 amino acid residues), neuropeptide Y (36 amino acid residues), insulin (51 amino acid residues), galanin-like peptide (60 amino acid residues), insulin-like growth factor-1 (70 amino acid residues), nerve growth factor (118 amino acid residues), leptin (166 amino acid residues), dynorphin (17 amino acid residues), ghrelin (28 amino acid residues), orexin B (28 amino acid residues), galanin (30 amino acid residues), β-endorphin (31 amino acid residues), orexin A (33 amino acid residues), neuropeptide Y (36 amino acid residues), insulin (51 amino acid residues), galanin-like peptide (60 amino acid residues), insulin-like growth factor-1 (70 amino acid residues), nerve growth factor (118 amino acid residues), leptin (166 amino acid residues), etc. can be mentioned.

[0025] In one embodiment of the neuropeptide derivative, the neuropeptide sequence is an amino acid sequence derived from a peptide of the following (a1) to (a2) or (b).

[0026] (a1) A peptide containing the amino acid sequence represented by HADGSFSDEMNTILDNLAARDFINWLIQTKITD (GLP-2, SEQ ID NO: 1) (a2) A peptide containing the amino acid sequence represented by HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (GLP-1: active form 7-36 amide, SEQ ID NO: 2) (b) A peptide having a central activity and containing an amino acid sequence in which one or more amino acid residues in the amino acid sequence (a1) or (a2) are deleted, substituted, or added.

[0027] Among the above-mentioned peptides, GLP-2 exhibits antidepressant and blood pressure-lowering effects, and GLP-1 exhibits an effect of improving learning disorders. Therefore, the usefulness of these peptides as neuropeptide sequences is great. It should be noted that the "amino acid sequence derived from a peptide" refers to the part corresponding to the amino acid sequence of a peptide when the amino acid sequence of a certain peptide is bonded to another amino acid sequence to form a peptide.

[0028] In the case where the neuropeptide sequence is derived from "a peptide (b) having an amino acid sequence in which one or more amino acid residues in (a1) or (a2) are deleted, substituted, or added and having a central action", the number of amino acid residues deleted, substituted, or added is not particularly limited as long as the neuropeptide sequence can achieve the effects of the present invention. For example, it is 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

[0029] (membrane penetration enhancing sequence) The membrane penetration enhancing sequence contained in the central transfer enhancing sequence is an amino acid sequence derived from a peptide having the action of crossing cell membranes, that is, a membrane-permeable peptide.

[0030] Examples of membrane-permeable peptides that constitute a 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, SEQ ID NO: 3), TAT (GRKKRRQRRR, 10 amino acid residues, SEQ ID NO: 4), mini penetratin (RRMKWKK, 7 amino acid residues, SEQ ID NO: 5), R9FC (RRRRRRRRRFFC, 12 amino acid residues, SEQ ID NO: 6), AIP6 (RLRWR, 5 amino acid residues, SEQ ID NO: 7), DPV3 (RKKRRRESRKKRRRES, 16 amino acid residues, SEQ ID NO: 8), DPV6 (GRPRESGKKRKRKRLKP, 17 amino acid residues, SEQ ID NO: 9), Pep-1 (KETWWETWWTEWSQPKKKRKV, 21 amino acid residues, SEQ ID NO: 10), MPG (GLAFLGFLGAAGSTMGAWSQPKKKRKV, 27 amino acid residues, SEQ ID NO: 11), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL, 27 amino acid residues, SEQ ID NO: 12), MAP (KLALKALKALKAALKLA, 17 amino acid residues, SEQ ID NO: 13), W / R (RRWWRRWRR, 9 amino acid residues, SEQ ID NO: 14), CADY (GLWRALWRLLRSLWRLLWRA, 20 amino acid residues, SEQ ID NO: 15), EB-1 (LIRLWSHLIHIWFQNRRLKWKK, 22 amino acid residues, SEQ ID NO: 16), HRSV (RRIPNRRPRR, 10 amino acid residues, SEQ ID NO: 17), PTD-5 (RRQRRRTSKLMKR, 13 amino acid residues, SEQ ID NO: 18), TAT47-57 (YGRKKRRQRRR, 11 amino acid residues, SEQ ID NO: 19), TP2 (PLIYLRLLRGQF, 12 amino acid residues, SEQ ID NO: 20), TP10 (AGYLLGKINLHALAALAKKIL, 21 amino acid residues, SEQ ID NO: 21), a cationic sequence bonded to heparan, a cationic sequence bonded to RNA, a cationic sequence bonded to DNA, and the like.

[0031] As a membrane-permeable peptide constituting the membrane permeation enhancing sequence, as a whole peptide, it is preferably a positively charged membrane-permeable peptide or a cationic membrane-permeable peptide. For example, it is preferably 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). As such a membrane-permeable peptide, oligomeric arginine (Rn, n is the number of arginine residues, that is, 6 - 12), 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. can be mentioned. 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 the glycan-modified neuropeptide derivative can be more efficiently introduced into cells.

[0032] The number of amino acid residues of the membrane permeation enhancing sequence 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, further more preferably an oligomeric arginine containing 7 to 9 arginine residues, and further more preferably an oligomeric arginine containing 8 arginine residues.

[0033] (Endosome escape enhancing sequence) It is considered that the endosome escape enhancing sequence contained in the central transfer enhancing sequence shortens the residence time of the neuropeptide 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 glycan-modified neuropeptide derivative to the central nervous system can be achieved in a shorter time.

[0034] The structure of the endosome escape enhancing sequence is not particularly limited. 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 mentioned.

[0035] There are no particular limitations on the positions of the membrane penetration enhancing sequence and the endosome escape enhancing sequence in the glycan-modified neuropeptide derivative. For example, the membrane penetration enhancing sequence can be located on the side closer to the neuropeptide sequence, and the endosome escape enhancing sequence can be located on the side closer to the neuropeptide sequence. From the viewpoint of more efficiently achieving the effects of the present invention, the membrane penetration enhancing sequence is more preferably located on the side closer to the neuropeptide sequence, and the membrane penetration enhancing sequence is further preferably located on the side closer to the neuropeptide sequence, and an endosome escape enhancing sequence is present on the N-terminal side or the C-terminal side of the membrane penetration enhancing sequence.

[0036] (Glycan-modified molecule) There are no particular limitations on the types of glycans contained in the glycan-modified molecule. Examples of glycans include: 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. Among them, N-linked glycans are preferred, and complex type glycans are more preferred.

[0037] There are no particular limitations on the structure of the glycan, which can be a double-stranded structure or other structures (such as a branched structure). Examples of monosaccharides constituting the glycan include 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. The monosaccharides constituting the glycan can be D-isomers or L-isomers. The monosaccharides constituting the glycan can be α-anomers or β-anomers.

[0038] The number of glycan-modified molecules possessed by the glycan-modified neuropeptide 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 glycan-modified neuropeptide 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".

[0039] The sugar chain-modified molecule can be composed only of a sugar chain or can be composed of a sugar chain and a part other than the sugar chain. That is, the sugar chain can be directly bonded to an amino acid residue constituting the sugar chain-modified neuropeptide derivative, or can be indirectly bonded to an amino acid residue constituting the sugar chain-modified neuropeptide derivative. In the present specification, both the state in which the sugar chain is directly bonded to an amino acid residue constituting the sugar chain-modified neuropeptide derivative and the state in which the sugar chain is indirectly bonded to an amino acid residue constituting the sugar chain-modified neuropeptide derivative are included in the state of "the sugar chain-modified molecule is bonded to an amino acid residue constituting the sugar chain-modified neuropeptide derivative". As the state in which the sugar chain is indirectly bonded to an amino acid residue constituting the sugar chain-modified neuropeptide derivative, a state of bonding via an amino acid residue such as a cysteine residue or an asparagine residue, a hydrocarbon group, etc. can be cited.

[0040] The sugar chain-modified molecule is preferably bonded to a position where the functions of the membrane permeability enhancing sequence and the endosomal escape enhancing sequence can be well maintained, and is preferably bonded to the neuropeptide sequence. When the sugar chain-modified molecule is bonded to the neuropeptide sequence, the bonding position of the sugar chain-modified molecule is not particularly limited as long as it is a position where the stability and activity of the neuropeptide sequence are not reduced. That is, it can be the N-terminus of the neuropeptide sequence, the C-terminus, or a position other than the termini. As long as the physiological activity of the neuropeptide sequence is not affected, a part of the neuropeptide sequence can be replaced with an amino acid such as a cysteine residue or an asparagine residue that is easily bonded to the sugar chain-modified molecule. Or, an amino acid residue can be introduced at the terminus of the neuropeptide sequence to bond the sugar chain-modified molecule to the amino acid residue.

[0041] In order not to affect the functions of the membrane permeability enhancing sequence and the endosomal escape enhancing sequence, the bonding position of the sugar chain-modified molecule in the neuropeptide sequence is preferably a position far from the membrane permeability enhancing sequence and the endosomal escape enhancing sequence. The position where the sugar chain-modified molecule is bonded to the neuropeptide is not particularly limited as long as it does not affect the functions of the membrane permeability enhancing sequence and the endosomal escape enhancing sequence and does not affect the physiological activity of the neuropeptide. For example, when the membrane permeability enhancing sequence and the endosomal escape enhancing sequence are added to the N-terminal side of the neuropeptide sequence, the sugar chain-modified molecule is preferably bonded to the C-terminal side of the neuropeptide sequence. When the membrane permeability enhancing sequence and the endosomal escape enhancing sequence are added to the C-terminal side of the neuropeptide sequence, the sugar chain-modified molecule is preferably bonded to the N-terminal side of the neuropeptide sequence.

[0042] From the viewpoint of adjusting the distance between the sugar chain-modified molecule and the central transfer enhancing sequence, the sugar chain-modified molecule further includes a linking group.

[0043] The type of the linking group included in the sugar chain-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 sugar chain-modified molecule and the central transport enhancing sequence, the number of carbon atoms of the alkylene group is preferably 3 to 15, more preferably 4 to 12, and still more preferably 5 to 10.

[0044] The number of monosaccharide residues in each sugar chain contained in the sugar chain-modified molecule is not particularly limited. For example, it can be in the range of 5 to 20, and can be in the range of 5 to 15. The research results of the present inventors have shown that when a sugar chain-modified molecule containing a sugar chain with a specific number or more of monosaccharide residues is added to a neuropeptide derivative, the solubility of the neuropeptide derivative in an aqueous solvent is improved. Specifically, the number of monosaccharide residues in each sugar chain is preferably 5 or more, and more preferably 10 or more.

[0045] (Spacer sequence) The sugar chain-modified neuropeptide derivative may also have a spacer sequence disposed between the neuropeptide sequence and the central transport enhancing sequence. By having a spacer sequence between the neuropeptide sequence and the central transport enhancing sequence, an effect of preventing a decrease or impairment of the neuropeptide sequence activity can be expected. When the sugar chain-modified neuropeptide derivative has a spacer sequence, the spacer sequence can be disposed between the neuropeptide sequence and the membrane penetration enhancing sequence, or can be disposed between the neuropeptide sequence and the endosomal escape enhancing sequence.

[0046] Generally, the membrane penetration enhancing sequence contains basic amino acids. Therefore, when the neuropeptide sequence contains acidic amino acid residues, for example, by the presence of 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 neuropeptide sequence, thereby preventing a decrease or impairment of the neuropeptide sequence activity.

[0047] From the viewpoint of ensuring the distance between the sugar chain-modified molecule and the central transport enhancing sequence, the spacer sequence preferably contains a lysine residue (K). Lysine is an amino acid having 4-aminobutyl in the side chain. By bonding the sugar chain-modified molecule to the terminal amino group of 4-aminobutyl, an alkylene group having 4 carbon atoms can exist between the sugar chain-modified molecule and the spacer sequence. Therefore, the distance between the sugar chain-modified molecule and the central transport enhancing sequence can be appropriately ensured.

[0048] As the spacer sequence containing a lysine residue, there can be mentioned 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 with a lysine residue.

[0049] For the sugar chain-modified neuropeptide derivatives, in addition to the addition of the above-mentioned sugar chains, various modifications can be carried out according to the use. For example, amino modifications (such as biotinylation, myristoylation, palmitoylation, acetylation, maleimidation, etc.), carboxyl modifications (such as amidation, esterification, etc.), thiol modifications (such as farnesylation, geranylation, methylation, palmitoylation, etc.), hydroxyl modifications (such as phosphorylation, sulfation, octanoylation, palmitoylation, palmitoleoylation, etc.), various fluorescent labels (such as 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 one of the neuropeptide sequence, membrane penetration enhancing sequence, endosome escape enhancing sequence, and spacer sequence of the sugar chain-modified neuropeptide derivative.

[0050] The combination of the neuropeptide sequence, membrane penetration enhancing sequence, and endosome escape enhancing sequence constituting the sugar chain-modified neuropeptide derivative is not particularly limited and can be selected according to the use. In one embodiment, the sugar chain-modified neuropeptide derivative may sequentially have an endosome escape enhancing sequence, a membrane penetration enhancing sequence, an optionally configured spacer sequence, and a neuropeptide sequence starting from the N-terminal side. In one embodiment, the sugar chain-modified neuropeptide derivative may sequentially have an endosome escape enhancing sequence, a membrane penetration enhancing sequence, an optionally configured spacer sequence, and a neuropeptide sequence starting from the C-terminal side. In the above constitution, the endosome escape enhancing sequence can be selected from FFLIPKG, LILIG, FFG, FFFFFG, or FFFFFFFG. In the above constitution, the membrane penetration enhancing sequence can be selected from oligomeric arginine (for example, Rn, n = 6 to 12). In the above constitution, the spacer sequence can be selected from sequences containing glycine residues (for example, Gn, n = 2 to 6), sequences containing glycine residues and cysteine residues (for example, GCG), and sequences containing glycine residues and lysine residues (for example, GKG).

[0051] <Pharmaceutical composition> The pharmaceutical composition of the present invention contains the above sugar chain-modified neuropeptide derivative as an active ingredient. By containing the sugar chain-modified neuropeptide derivative as an active ingredient, the pharmaceutical composition of the present invention has excellent transferability to the central nervous system upon nasal administration and can efficiently exhibit pharmacological effects. Therefore, for example, it is useful for the treatment of diseases that require daily administration of pharmaceutical preparations at home. Therefore, a preferred dosage form of the pharmaceutical composition can be a nasal / nasal drop preparation.

[0052] There is no particular limitation on the mental or neurological disorder or neurodegenerative disease to be treated with the pharmaceutical composition, as long as the therapeutic effect is exhibited by acting on the central nervous system with the neuropeptide sequence of the sugar chain-modified neuropeptide derivative. Examples of the mental or neurological disorder or neurodegenerative disease to be treated include depression, learning disorder, anxiety, eating disorder, cognitive disorder, hypertension, sleep disorder, epilepsy, Alzheimer's disease, vascular cognitive impairment, amyotrophic lateral sclerosis, etc.

[0053] Specific examples of the pharmaceutical composition include antidepressants, learning disorder improvers, anxiolytics, appetite suppressants, cognitive disorder improvers, antihypertensives, analgesics, hypnotics, antiepileptics, etc. The type of the neuropeptide sequence of the sugar chain-modified neuropeptide derivative can be selected according to the disease to be treated. For example, a pharmaceutical composition containing a sugar chain-modified neuropeptide derivative having a neuropeptide sequence derived from GLP-2 is useful as an antidepressant. In addition, GLP-2 exhibits an antihypertensive effect, and thus, it is considered that the efficacy is particularly high when administered to patients with depression complicated by hypertension caused by strong stress. A pharmaceutical composition containing a sugar chain-modified neuropeptide derivative having a neuropeptide sequence derived from GLP-1 is useful as a learning disorder improver and can be expected to be used as a therapeutic agent for cognitive impairment.

[0054] The details and preferred modes of the sugar chain-modified neuropeptide derivative contained in the pharmaceutical composition are as described above. From the viewpoint of the delivery property to the brain, the use method of the pharmaceutical composition is preferably intranasal or intranasal drip administration. The pharmaceutical composition may contain components other than the sugar chain-modified neuropeptide derivative. Specific examples of the components that can be contained in addition to the pharmaceutical composition include the medium used in the preparation of the pharmaceutical composition and formulation additives. Examples of the formulation additives include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizing agents, stabilizers, isotonic agents, suspending agents, emulsifiers, solvents, thickeners, mucolytic agents, wetting agents, preservatives, etc. The dosage of the pharmaceutical composition can be selected according to the type of the disease, the symptoms, body weight, age, etc. of the patient, the administration method, etc. The pharmaceutical composition of the present invention is particularly preferably an intranasal or intranasal drip preparation. That is, one embodiment of the present invention is the use of the present invention in intranasal or intranasal drip administration.

[0055] <Intranasal or intranasal drip preparation> The intranasal drop preparation of the present invention contains the above-mentioned neuropeptide derivative as an active ingredient. By containing the sugar chain-modified neuropeptide derivative as an active ingredient, the intranasal drop preparation of the present invention has excellent brain transferability 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.

[0056] The intranasal drop preparation may contain components other than the sugar chain-modified neuropeptide derivative. As components other than the sugar chain-modified neuropeptide derivative, the above substances can be cited as media and formulation additives used in the preparation of pharmaceutical compositions.

[0057] An embodiment of the present invention includes the use of the above-mentioned neuropeptide derivative or a pharmaceutical composition containing the same as an active ingredient in intranasal drop administration. The details and preferred modes of the sugar chain-modified neuropeptide derivative and the pharmaceutical composition in this use are as shown above.

[0058] <Method for treating mental or neurodegenerative diseases> An embodiment of the present invention includes a method for treating mental or neurodegenerative diseases, which comprises administering the above sugar chain-modified neuropeptide derivative or pharmaceutical composition to a patient. The details and preferred modes of the sugar chain-modified neuropeptide derivative and the pharmaceutical composition in this method are as shown above. Specific examples of the mental or neurodegenerative diseases to be treated by the above method include depression, learning disorder, anxiety, eating disorder, cognitive disorder, hypertension, sleep disorder, epilepsy, Alzheimer's disease, vascular cognitive impairment, amyotrophic lateral sclerosis, etc. The method of administering the sugar chain-modified neuropeptide derivative or pharmaceutical composition to a patient is not particularly limited, and intranasal administration is preferred. Examples

[0059] Examples are given below to further specifically illustrate the present invention. The materials, amounts used, ratios, treatment sequences, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention is not restrictively interpreted as the specific examples shown below.

[0060] <Preparation of GLP-2 derivative with sugar chain modification at C-terminus> As a glycan-modified GLP-2 derivative, a glycan-modified GLP-2 derivative, namely PAS-CPP-GLP-2 (C-terminal 11 sugars), is prepared by a conventional method, which is configured in sequence from the N-terminal side with an endosome escape enhancing sequence (PAS: FFLIPKG), a membrane penetration enhancing sequence (CPP: RRRRRRRR), a spacer sequence (GG), and an amino acid sequence derived from GLP-2 as a neuropeptide sequence. As the neuropeptide sequence, a sequence of a molecule in which a glycan containing 11 monosaccharide residues is added via a cysteine residue at the C-terminus of GLP-2 is used. The structure of the prepared glycan-modified GLP-2 derivative is shown below. The fluorescent label of PAS-CPP-GLP-2 (C-terminal 11 sugars) used in some embodiments is prepared by adding a fluorescent label (FITC or ICG) to the endosome escape enhancing sequence.

[0061] [Chemical Formula 1]

[0062] As the neuropeptide sequence, a sequence of a molecule in which a glycan containing 5 monosaccharide residues is added via a cysteine residue at the C-terminus of GLP-2 is used. Except for this, it is the same as the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars), and a glycan-modified GLP-2 derivative, namely PAS-CPP-GLP-2 derivative (C-terminal 5 sugars), is prepared. The structure of the prepared glycan-modified GLP-2 derivative is shown below.

[0063] [Chemical Formula 2]

[0064] As the neuropeptide sequence, a sequence of a molecule without a glycan added via a cysteine residue at the C-terminus of GLP-2 is used. Except for this, it is the same as the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars), and an unglycosylated GLP-2 derivative, namely PAS-CPP-GLP-2 derivative (sugar-free), is prepared. The structure of the prepared GLP-2 derivative is shown below. The fluorescent label of PAS-CPP-GLP-2 derivative (sugar-free) used in some embodiments is prepared by adding a fluorescent label (FITC or ICG) to the endosome escape enhancing sequence.

[0065] [Chemical Formula 3]

[0066] <Example 1: Evaluation of the solubility of GLP-2 derivatives in aqueous solvents> The Milli-Q aqueous solutions of the prepared PAS-CPP-GLP-2 (sugar-free), PAS-CPP-GLP-2 (5 sugars at the C-terminus), and PAS-CPP-GLP-2 (11 sugars at the C-terminus) were dispensed into microtubes to be 5 nmol / tube, 30 nmol / tube, 60 nmol / tube, 120 nmol / tube, and 200 nmol / tube, and freeze-dried. 200 μL of PBS (Dulbecco’s Phosphate Buffered Saline; Sigma-Aldrich, the same below) was added to the freeze-dried samples, and after sonication, they were allowed to stand overnight. Then, the concentration was measured. Specifically, the sample suspension was centrifuged, and the OD (280 nm) of the supernatant obtained using a spectrophotometer (NanoDrop TM 2000c spectrometer; Thermo Fisher Scientific K.K.) was measured to calculate the peptide concentration.

[0067] As Figure 1 shown, the PAS-CPP-GLP-2 derivative (sugar-free) was completely insoluble in PBS. The solubility of the sugar chain-modified GLP-2 derivatives (5 sugars or 11 sugars at the C-terminus) in PBS was improved. In particular, the solubility of the GLP-2 derivative (11 sugars at the C-terminus) increased linearly.

[0068] <Example 2: Evaluation of the Effect of Sugar Chain Modification of GLP-2 Derivatives on Pharmacological Activity> To evaluate the effect of sugar chain modification on the antidepressant-like effect of GLP-2 derivatives (11 sugars at the C-terminus, 5 sugars at the C-terminus), the forced swimming test (FST) of mice was performed.

[0069] (Preparation and Administration of the Administration Solution) The following method was used to evaluate whether the pharmacological activity could be maintained by sugar chain modification. After completely dissolving the PAS-CPP-GLP-2 derivative (sugar-free) and PAS-CPP-GLP-2 (5 sugars or 11 sugars) in DMSO, PBS was added to make the final concentration of DMSO 16% by mass, and the administration solutions were prepared respectively. The concentration of each GLP-2 derivative was set to 0.6 nmol / 4 μL. Use an integrated all-in-one small animal anesthetic (MK-AT210D, Muromachi Kikai Co., Ltd., the same hereinafter). After anesthetizing the mice with isoflurane, administer the liquid medicine into the nasal cavity. Specifically, bring the tip of the needle of the anesthetic close to the nasal cavity of the mouse in a horizontal manner, and perform nasal administration of a total of 4 μL (0.6 nmol / mouse), 2 μL for each single nostril, in such a way that the droplets are inhaled through spontaneous breathing. In the control (Vehicle) group, 4 μL of a PBS solution containing 16% by mass of DMSO (hereinafter, also referred to as 16% DMSO) was administered nasally. Twenty minutes before the test session of the forced swimming test (FST) conducted by the following method, nasal administration was carried out.

[0070] (Forced swimming test) In a transparent plastic cylindrical tank with a diameter of 18 cm and a height of 50 cm, inject water adjusted to a water temperature of 25°C ± 1°C to a height of 7 cm, and place 7-week-old male ddY mice in it and let them swim for 15 minutes. Then take out the mice, wipe their bodies with a towel, and put them back into the cage. This series of processes is used as the training stage. Twenty-four hours after the end of the training stage, a 15-minute test session is carried out by the same method as the training stage. All situations during the test are recorded with a camera. During the test, when the mice are placed in the tank, they struggle desperately to escape from the tank, and when they realize that they cannot escape, they will give up escaping and gradually stop moving. This state is called the immobile state and is defined as a depressive-like state. The time of this immobile state (immobile time) is measured within the first 6 minutes from the start time of the test session. The presence or absence of an antidepressant-like effect is judged by the length of the immobile time. In the examples described in this specification, the forced swimming test was carried out by the above method.

[0071] As Figure 2 shown, regardless of the presence or absence of sugar chain modification, the immobile time of the administration group of the GLP-2 derivative was significantly shortened compared with the control group, showing an antidepressant-like effect. The above results suggest that the sugar chain (11 sugars) bonded to the C-terminus of GLP-2 does not affect the efficacy of the PAS-CPP-GLP-2 derivative.

[0072] <Example 3: Evaluation of the effect of PBS on the efficacy of the sugar chain-modified GLP-2 derivative> To evaluate the effect of PBS on the antidepressant-like effect of the sugar chain-modified GLP-2 derivative (C-terminal 11 sugars), a forced swimming test (FST) of mice was carried out.

[0073] (Preparation of the administration liquid and nasal administration) The PAS-CPP-GLP-2 derivative (sugar-free) and PAS-CPP-GLP-2 (11 sugars at the C-terminus) were each mixed with PBS (the sugar-free one was in a suspension state, and the 11-sugar one was in a completely dissolved state), and dosing solutions were prepared respectively. The concentration of each GLP-2 derivative was set at 0.6 nmol / 4 μL. Using an integrated small animal anesthetic device, the mice were anesthetized with isoflurane, and then the dosing solution was administered into the nasal cavity. Transnasal administration was performed at 2 μL per single nostril, totaling 4 μL (0.6 nmol / mouse). The control (Vehicle) group was only administered an equal amount of 16% DMSO transnasally. Transnasal administration was performed 20 minutes before the test phase of the forced swimming test (FST) conducted by the following method.

[0074] As Figure 3 shown, the administration group of the PAS-CPP-GLP-2 derivative (sugar-free) did not show a significant antidepressant-like effect compared with the control group. The reason is considered to be that the PAS-CPP-GLP-2 derivative is insoluble in PBS. On the other hand, the administration group of the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) showed a significant antidepressant-like effect compared with the control group. The reason is considered to be that the PAS-CPP-GLP-2 derivative is dissolved in PBS by sugar chain modification. From the above results, it can be known that the sugar chain-modified PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) can exhibit a central effect even when using an aqueous solvent such as PBS. Accordingly, it can be known that there is no problem of reduced membrane permeability and reduced drug efficacy of the derivative caused by the increased water solubility accompanied by sugar chain modification, which was initially worried about.

[0075] <Example 4: Evaluation of the effect of sugar chain modification on the central metastasis of GLP-2 derivatives> To evaluate the effect of sugar chain modification on the central metastasis of GLP-2 derivatives, a light imaging device was used to study the metastasis to the central nervous system.

[0076] (Preparation of dosing solution) After completely dissolving the dosing solutions of the PAS-CPP-GLP-2 derivative (sugar-free) fluorescently labeled with ICG and the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) labeled with ICG in DMSO, PBS was added so that the final concentration of DMSO became 16% to prepare. The PAS-CPP-GLP-2 derivative (11 sugars) was completely dissolved in PBS, and DMSO was used to make the conditions equal to those of the PAS-CPP-GLP-2 derivative (sugar-free). The concentrations of PAS-CPP-GLP-2 derivative (sugar-free) and PAS-CPP-GLP-2 derivative (11 sugars) were adjusted to be 3.0 nmol / 4 μL, respectively.

[0077] (Evaluation of the central transfer pathway using a light imaging device) Using an integrated small animal anesthetic, 7-week-old male ddY mice were anesthetized with isoflurane, and then a dosing solution prepared by intranasal administration of 4 μL in total (2 μL per single nostril) into the nasal cavity, or 16% DMSO as a control, was administered. To observe the transfer to the brain over time, the brain was removed 5 minutes, 10 minutes, 20 minutes, 60 minutes, and 90 minutes after intranasal administration, and immersed and fixed overnight with 4% paraformaldehyde (4% PFA) solution. Then, using a brain matrix (RBM-2000S, ASI), sagittal plane sections with a thickness of 2 mm on both the left and right sides starting from the center of the brain were prepared. The sections were placed on a petri dish, and measurements were performed using a light imaging device (Clairvivo OPT plus, Shimadzu Corporation). The measurement conditions were set as follows: excitation wavelength: 785 nm, fluorescence wavelength: 849 nm, exposure time: 6 seconds.

[0078] The results are shown in Figure 4 . Surprisingly, 5 minutes after intranasal administration, fluorescence was observed only in the case of administering PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) (lower panel), from the posterior half of the brain to the vicinity of the hippocampus and hypothalamus. 10 minutes after intranasal administration, fluorescence was observed in both the case of administering PAS-CPP-GLP-2 (sugar-free) (middle panel) and the case of administering PAS-CPP-GLP-2 derivative (C-terminal 11 sugars). In the case of administering PAS-CPP-GLP-2 (sugar-free), fluorescence was observed near the hippocampus, and in the case of administering PAS-CPP-GLP-2 derivative (C-terminal 11 sugars), fluorescence was intensively observed near the observed site at the 5-minute time point, and its intensity increased. 20 minutes after intranasal administration, fluorescence was also observed in both PAS-CPP-GLP-2 (sugar-free) and PAS-CPP-GLP-2 derivative (C-terminal 11 sugars). It was found that for both derivatives, stronger fluorescence was observed near the hippocampus compared to 10 minutes after intranasal administration, and in particular, more fluorescence was present in PAS-CPP-GLP-2 derivative (C-terminal 11 sugars). 60 minutes after intranasal administration, no fluorescence was observed in PAS-CPP-GLP-2 (sugar-free), whereas relatively strong fluorescence was observed in PAS-CPP-GLP-2 derivative (C-terminal 11 sugars). No fluorescence was observed in the nasal administration group of PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) even 90 minutes after nasal administration, indicating its disappearance from the brain. In addition, it was found that PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) remained in the brain for a longer time compared to PAS-CPP-GLP-2 (sugar-free).

[0079] <Example 5: Effect of sugar chain modification on the amount of GLP-2 derivative transferred to the brain> In Example 4, it was qualitatively shown that most of the drug was transferred to the brain in the case of PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) compared to PAS-CPP-GLP-2 (sugar-free). Therefore, in Example 5, the amounts of GLP-2 transferred to the brain of PAS-CPP-GLP-2 (sugar-free) and PAS-CPP-GLP-2 derivative (11 sugars) were quantified by ELISA to quantitatively study the effect of sugar chain modification on the amount of GLP-2 derivative transferred to the brain.

[0080] (Quantification of the amount of GLP-2 transferred to the brain using ELISA) 16% DMSO or various GLP-2 derivatives (6.0 nmol / mouse) were administered nasally to mice. The brains were removed 20 minutes after administration and homogenized using a BioMasher II (Nippi, Tokyo, Japan). After centrifugation at 1000×g (4°C) for 15 minutes, the supernatant was recovered to prepare a sample. The amount of GLP-2 derivative present in the sample was quantified using a GLP-2 ELISA kit. First, each well of the assay plate was filled with a washing solution (350 μL), and the washing operation was repeated 3 times by pipetting. Then, a labeled antigen solution (40 μL), a sample (25 μL), and a specific antibody solution (50 μL) were sequentially added to each well and mixed. The assay plate was sealed with a sealing cap and left standing at 4°C for 18 hours. Then, the washing operation was performed 3 times, an SA-HRP solution (100 μL) was added, and the mixture was shaken at room temperature for 1 hour (100 rpm). Just before the reaction ended, a chromogenic reagent solution was prepared by dissolving 1 OPD tablet in 25 mL of a substrate solution (0.1 M citrate buffer containing 0.03% hydrogen peroxide). Then, the washing operation was repeated 5 times, the chromogenic reagent solution (100 μL) was added, and the mixture was shielded from light and left standing at room temperature for 1 hour. Finally, an enzyme reaction termination solution (100 μL) was added, and the absorbance at 490 nm was measured using an ARVO (PerkinElmer Japan Co., Ltd., Kanagawa, Japan) to calculate the concentration of the GLP-2 derivative.

[0081] As a result, in the group administered with the GLP-2 derivative without sugar chain modification, the result was below the detection limit. On the other hand, in the group administered with the sugar chain-modified GLP-2 derivative, 10.598 ± 0.998 pmol / g( Figure 5 ) was detected. Therefore, it was found that sugar chain modification increased the amount of GLP-2 derivative transferred into the brain.

[0082] <Example 6: Evaluation of Brain Distribution of Sugar Chain-Modified GLP-2 Derivative> To evaluate the brain distribution of the sugar chain-modified GLP-2 derivative (C-terminal 11 sugars) administered via nasal administration, frozen brain sections were prepared, immunostained, and observed. The brain sections were prepared from the following tissues: the hippocampus (HIP) and the hypothalamus (DMH), which are considered to be the sites of action of GLP-2, and the olfactory bulb (OB) containing the olfactory nerve, which has a high possibility of being a transfer pathway for GLP-2, and the tissue near the pons and the main sensory nucleus of the trigeminal nerve (Pr5).

[0083] (Preparation of Frozen Brain Sections) Using an integrated small animal anesthetic apparatus, 7-week-old male ddY mice were anesthetized with isoflurane, and then a 16% DMSO solution (3.0 nmol / 4 μL) of PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) or 16% DMSO as a control was administered via nasal administration at 2 μL per single nostril for a total of 4 μL. The brains were removed 5 minutes and 20 minutes after the start of administration. In the brains 5 minutes after the start of nasal administration, perfusion fixation was not performed, and the brains were infiltrated and fixed overnight at 4°C with a 4% PFA solution. The brains were removed 20 minutes after the start of nasal administration according to the following method. The mice were fixed in the dorsal position under isoflurane anesthesia, and the chest was incised. PBS was then perfused through the left ventricle followed by 4% PFA to fix the tissues throughout the body. After removing the brains, they were stored in a 4% PFA solution. On the day after all brain samples were removed, they were replaced with 20% sucrose overnight (4°C), and further replaced and placed in 30% sucrose overnight (4°C). Then, 30-μm-thick frozen sections were prepared using a cryostat (CM3050S; Leica Microsystems).

[0084] (Immunohistochemical Staining) Place the frozen brain sections on a glass slide and draw a circle around the sections using an immunohistochemistry pen (liquid blocker). Add blocking buffer within the circle and block for 30 minutes at room temperature. Then, add a primary antibody solution containing GLP-2 polyclonal antibody diluted 200-fold with blocking buffer and incubate for 1 hour at room temperature. After washing 3 times with 1×PBS, add a secondary antibody solution containing Alexa Fluor (registered trademark) 568 goat anti-mouse IgG H&L diluted 500-fold with 1% BSA / PBS solution and incubate for 1 hour at room temperature. After washing 3 times with 1×PBS, mount with ProLong (registered trademark) Diamond Antifade Mountant. After confirming the solidification of the mounting medium, perform fluorescence observation and image acquisition using a confocal laser microscope (TCS SP8; Leica, WetZlar, Germany) and software (Leica Application Suite X Software; Leica).

[0085] As Figure 6A shown, 5 minutes after the start of nasal administration, in the case of administering PAS-CPP-GLP-2 derivative (C-terminal 11 sugar), strong fluorescence was observed near Pr5, but almost no fluorescence was observed in other regions. As a control, in the case of administering 16% DMSO, almost no fluorescence was observed in any region. As Figure 6B shown, 20 minutes after the start of nasal administration, in the case of administering PAS-CPP-GLP-2 derivative (C-terminal 11 sugar), fluorescence was confirmed in the HIP, and fluorescence was also clearly confirmed in the DMH. In addition, although fluorescence was also confirmed in Pr5 and OB, the fluorescence in OB was weak. HIP and DMH are regions considered to be the sites of action of GLP-2. As a control, in the case of administering 16% DMSO, almost no fluorescence was observed in any region. The above results demonstrate that: Figure 2 and Figure 3 the time to onset of efficacy in the efficacy test shown in is consistent, and the glycan-modified GLP-2 derivative reaches the site of action within 20 minutes after nasal administration and exhibits efficacy. Furthermore, it is suggested that the glycan-modified GLP-2 derivative is mainly delivered from the principal sensory trigeminal nucleus (Pr5) of the pons in the brainstem to the sites of action in the hippocampus and hypothalamus via the trigeminal nerve of the respiratory epithelium and exhibits efficacy. The above results overturned the theory of the common sense of neuroscience that axoplasmic transport is very slow (50 - 400 mm / day in the fast case, 0.2 - 8 mm / day in the slow case: Non-Patent Document 17).

[0086] <Example 7: Influence of Glycan Modification on the Brain Distribution of GLP-2 Derivatives> Figure 6A and Figure 6B (Example 6) The results suggest that the glycan-modified GLP-2 derivative is mainly delivered from the principal sensory trigeminal nucleus (Pr5) of the pons in the brainstem to the action sites in the hippocampus and hypothalamus via the trigeminal nerve of the respiratory epithelium and exhibits pharmacological effects, but the influence of glycan modification on the brain distribution of GLP-2 derivatives has not been elucidated. Therefore, in Example 7, in order to clarify the influence of glycan modification on the brain distribution of GLP-2 derivatives, qualitative and quantitative studies were conducted.

[0087] (Preparation of Frozen Sections) Mice were administered 16% DMSO or various GLP-2 derivatives (3.0 nmol / mouse) via nasal cavity. The brains were removed 5 minutes, 10 minutes, 20 minutes, and 60 minutes after the start of administration. For the brains 5 minutes and 10 minutes after the start of administration, perfusion fixation was not performed, and they were infiltrated and fixed overnight at 4°C in a 4% PFA solution. The brains were removed 20 minutes and 60 minutes after the start of administration according to the following method. The mice were fixed in the dorsal position under isoflurane anesthesia, and the chest was incised. PBS was then perfused through the left ventricle followed by 4% PFA to fix the tissues throughout the body. After removing the brains, they were stored in a 4% PFA solution. On the day after all brain samples were removed, they were replaced with 20% sucrose overnight (4°C) and further replaced with 30% sucrose and replaced overnight (4°C). Then, 30-μm-thick frozen sections were prepared using a cryostat (CM3050S; Leica Microsystems). Nissl staining images were compared with a brain atlas (Paxinos and Franklin, 2003) to prepare sections containing the olfactory bulb (OB) including the olfactory nerve, the pons · principal sensory trigeminal nucleus (Pr5), the hippocampus (HIP), and the dorsomedial hypothalamic nucleus (DMH).

[0088] (Immunostaining Method and Fluorescence Observation in Evaluation of Brain Distribution) The frozen sections were placed on glass slides, and a circle was drawn around the sections using an immunohistochemistry pen (liquid blocker). 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 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 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. Then, after washing 3 times with 1×PBS, ProLong TMDiamond Antifade Mountant was used for mounting. After confirming the curing of the mounting medium, fluorescence observation was carried out using a confocal laser microscope (TCSSP8; Leica, WetZlar, Germany) and software (Leica Application Suite X Software; Leica, WetZlar, Germany). In addition, the fluorescence intensity of the obtained images was calculated using the same software, and the average fluorescence intensity of each group was quantified.

[0089] PAS-CPP-GLP-2 (sugar-free) or PAS-CPP-GLP-2 (11 sugars at the C-terminus) was administered to mice via the nose. The brain was removed 5 minutes, 20 minutes, or 60 minutes after the start of administration, and the drug distribution in the olfactory bulb (OB) containing the olfactory nerve, which is highly likely to be a transfer pathway, the principal sensory trigeminal nucleus of the pons (Pr5), the hippocampus (HIP), and the hypothalamus (DMH), which are considered to be the sites of action of GLP-2, was observed. As a result, 5 minutes after the start of administration, significant fluorescence was observed in the OB and Pr5 containing the olfactory nerve in either the case of administering PAS-CPP-GLP-2 (sugar-free) or PAS-CPP-GLP-2 (11 sugars at the C-terminus). Stronger fluorescence was observed in Pr5 than in OB ( Figure 7A )

[0090] 20 minutes after the start of administration, although significant fluorescence was observed in the OB when PAS-CPP-GLP-2 (sugar-free) was administered, in either the case of administering PAS-CPP-GLP-2 (sugar-free) or PAS-CPP-GLP-2 (11 sugars at the C-terminus), the fluorescence observed in the OB and Pr5 was weaker than that 5 minutes after the start of administration. On the other hand, in either the case of administering PAS-CPP-GLP-2 (sugar-free) or PAS-CPP-GLP-2 (11 sugars at the C-terminus), strong fluorescence was observed in the HIP and DMH, which are the sites of action of GLP-2 ( Figure 7B ). The situation 20 minutes after the start of administration was consistent with the onset time of the efficacy of the GLP-2 derivative, and the results demonstrated that both PAS-CPP-GLP-2 (sugar-free) and PAS-CPP-GLP-2 (11 sugars at the C-terminus) reached the site of action and exhibited efficacy.

[0091] Sixty minutes after administration, although significant fluorescence was observed in the OB and Pr5 of PAS-CPP-GLP-2 (C-terminal 11 sugars), the fluorescence was weaker compared to that 5 minutes after administration. In the action sites of GLP-2, namely the HIP and DMH, no fluorescence was observed in PAS-CPP-GLP-2 (sugar-free), whereas significant fluorescence was observed in PAS-CPP-GLP-2 (C-terminal 11 sugars) ( Figure 7C ). As a result, it was suggested that the effect of prolonging the duration of action of the GLP-2 derivative caused by the significant sugar chain modification in Example 10 ( Figure 10 ) was demonstrated.

[0092] <Example 8: Observation of Trigeminal Nerve Sections> Using an integrated small animal anesthetic device, 7-week-old male ddY mice were anesthetized with isoflurane, and then a 16% DMSO solution (concentration 3.0 nmol / 4 μL) of a PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) fluorescently labeled with FITC or 16% DMSO as a control was administered intranasally at 2 μL per single nostril for a total of 4 μL. The trigeminal nerve was excised 5 minutes after administration. The excised trigeminal nerve was infiltrated and fixed in 4% PFA overnight. On the day after excision, it was replaced with 20% sucrose overnight (4 °C), and further replaced with 30% sucrose and replaced overnight (4 °C). Then, 20-μm-thick frozen sections were prepared using a cryostat (CM3050S; Leica Microsystems). Place the frozen section on a glass slide and draw a circle around the section using an immunohistochemistry pen (liquid blocker). Add 10 mM CuSO4 / CH3COONH4 with autologous fluorescence suppression effect into the circle and impregnate for 15 minutes. Wash three times with 1×PBS, add blocking buffer, and block at room temperature for 30 minutes. Then, add the primary antibody solution containing Neuro-Chrom (registered trademark) Pan Neuronal Marker Antibody-Rabbit diluted 1000-fold with the blocking solution and incubate at room temperature for 2 hours. After washing three times with 1×PBS, add the secondary antibody solution containing AlexaFluor (registered trademark) 568 goat anti-mouse IgG H&L and 40 ng / ml DAPI diluted 500-fold with 1% BSA / PBS solution and incubate at room temperature for 1 hour. After washing three times with 1×PBS, mount with ProLong (registered trademark) Diamond Antifade Mountant. After confirming the solidification of the mounting medium, perform fluorescence observation and image acquisition using a confocal laser microscope (TCS SP8; Leica) and software (Leica Application Suite X Software; Leica).

[0093] Figure 8 A is an image of a trigeminal nerve section excised after administration of 16% DMSO, Figure 8 B is an image of a trigeminal nerve section excised after administration of a 16% DMSO solution of PAS-CPP-GLP-2 derivative (C-terminal 11 sugar), Figure 8 C of Figure 8 is an enlarged image of the portion enclosed by the box of B of As Figure 8 shown, after administration of the PAS-CPP-GLP-2 derivative (C-terminal 11 sugar), in the excised trigeminal nerve section, a large amount of green fluorescence representing the PAS-CPP-GLP-2 derivative (C-terminal 11 sugar) was observed, and at the same time, yellow fluorescence (the particularly bright part in the figure) representing the overlapping state of red fluorescence representing nerve fibers and green fluorescence representing the PAS-CPP-GLP-2 derivative (C-terminal 11 sugar) was observed. These results suggest that the nasally administered PAS-CPP-GLP-2 derivative (C-terminal 11 sugar) transfers to the center through the trigeminal nerve containing trigeminal nerve fibers.

[0094] <Example 9: Evaluation of the metastasis of the glycosylated GLP-2 derivative administered nasally to the trigeminothalamic tract> In Example 8, it was shown that the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) was transferred from the trigeminal nerve of the respiratory epithelium to the principal sensory trigeminal nucleus (Pr5) which is its projection target. In Example 9, it was investigated whether the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) was transferred to the trigeminothalamic tract, a neural pathway connecting Pr5 and the ventral posteromedial nucleus of the thalamus (VPM).

[0095] (Observation of Sections of the Trigeminothalamic Tract) Mice were administered 16% DMSO or a glycan-modified GLP-2 derivative (3.0 nmol / mouse) via the nose. The brain was removed 15 minutes after administration and infiltrated and fixed overnight in 4% PFA. Starting from the day after removal, it was replaced with 30% sucrose and left overnight at 4 °C. Then, cryosections of the trigeminothalamic tract with a thickness of 30 μm were prepared using a cryostat (CM3050S; Leica Microsystems, WetZlar, Germany). 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) was added and incubated overnight at 4 °C. After washing 3 times with 1×PBS, a primary antibody solution containing a GLP-2 polyclonal antibody (1:200) diluted with 1% BSA / PBS solution was added and incubated at room temperature for 2 hours. After washing 3 times with 1×PBS, a secondary antibody solution containing Alexa Fluor 568 goat anti-mouse IgG H&L diluted 1000-fold with 1% BSA / PBS solution and 40 ng / ml DAPI was added and incubated at room temperature for 1 hour. After washing 3 times with 1×PBS, fluorescence observation of the trigeminothalamic tract was performed using a confocal laser microscope (TCS SP8; Leica, WetZlar, Germany) and software (Leica Application Suite X Software; Leica, WetZlar, Germany).

[0096] Observation was performed on the trigeminothalamic tract, a neural pathway connecting the principal sensory trigeminal nucleus (Pr5), which is the projection target of the trigeminal nerve, and the ventral posteromedial nucleus of the thalamus (VPM). As a result, in the case of the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars), a large amount of fluorescence of the drug was observed in the nerve bundles constituting the trigeminothalamic tract ( Figure 9 of C), and in addition, yellow fluorescence indicating a state where the green fluorescence representing nerve fibers overlapped with the red fluorescence representing the drug was observed ( Figure 9 of D). According toFigure 8 and Figure 9 The results shown strongly suggest that the glycan-modified GLP-2 derivative administered via the nose transfers from the respiratory epithelium to the principal sensory trigeminal nucleus (Pr5) via the trigeminal nerve, and further transfers to the action site, i.e., the thalamus, via the trigeminothalamic tract.

[0097] <Example 10: Evaluation of the Persistence of the Pharmacological Effect of the Glycan-Modified GLP-2 Derivative> In Example 5, results were obtained that were persistent compared to localization to the action sites, i.e., the hippocampus and hypothalamus. Along with these results, it was considered that the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) might have a persistent central effect, i.e., an antidepressant-like effect, and thus an evaluation of the persistence of the antidepressant-like effect was conducted.

[0098] Using an integrated small animal anesthetic apparatus, 7-week-old male ddY mice were anesthetized with isoflurane, and in the control (vehicle) group, 16% DMSO was administered at 2 μL per single nostril for a total of 4 μL, and in the GLP-2 administration group, a 16% DMSO solution (0.6 nmol / 4 μL) of the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) or the PAS-CPP-GLP-2 derivative (sugar-free) was administered at 2 μL per single nostril for a total of 4 μL, and the forced swim test was performed. Nasal administration was performed 20 minutes before the start of the test phase of the forced swim test. For the other control groups and the GLP-2 administration group, nasal administration was performed in the same manner as above 60 minutes before the start of the test phase of the forced swim test (FST).

[0099] As Figure 10 shown, when the forced swim test was performed 20 minutes after nasal administration (left graph), either the PAS-CPP-GLP-2 derivative (sugar-free) or the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) showed a significantly shorter immobility time compared to the control (vehicle) group and exhibited an antidepressant-like effect. When the forced swim test was performed 60 minutes after nasal administration (right graph), a significant difference was confirmed only in the PAS-CPP-GLP-2 derivative (11 sugars) compared to the control (vehicle) group, and an antidepressant-like effect was exhibited. Taking the above results and the results of Example 5 into comprehensive consideration, it can be seen that the glycan-modified GLP-2 derivative exists longer in the action sites, i.e., the hippocampus and hypothalamus, compared to the GLP-2 derivative without glycan modification, and can sustain the antidepressant-like effect.

[0100] <Example 11: Evaluation of the Pharmacological Effect Enhancement Effect of the Glycan-Modified GLP-2 Derivative> In Example 6, it was observed that the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) tended to localize more at the action sites, namely the hippocampus and hypothalamus, compared to the PAS-CPP-GLP-2 derivative (sugar-free). Therefore, it was considered that the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) might exhibit antidepressant-like effects with a smaller dosage compared to the PAS-CPP-GLP-2 derivative (sugar-free). Thus, it was investigated whether the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) could exhibit antidepressant-like effects with a smaller dosage compared to the PAS-CPP-GLP-2 derivative (sugar-free).

[0101] Using an integrated small animal anesthetic apparatus, 7-week-old male ddY mice were anesthetized with isoflurane. In the control (Vehicle) group, 16% DMSO was administered at 2 μL per single nostril, for a total of 4 μL. In the GLP-2 administration group, a 16% DMSO solution (0.6 nmol / 4 μL) of the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) or the PAS-CPP-GLP-2 derivative (sugar-free) was administered at 2 μL per single nostril, for a total of 4 μL. In the GLP-2 administration group (1 / 2), a 16% DMSO solution (0.3 nmol / 4 μL) of the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) or the PAS-CPP-GLP-2 derivative (sugar-free) was administered at 2 μL per single nostril, for a total of 4 μL, and the forced swimming test was performed. Intranasal administration was carried out 20 minutes before the start of the test phase of the forced swimming test.

[0102] As Figure 11 shown, when the PAS-CPP-GLP-2 derivative (sugar-free) was administered, although antidepressant-like effects were exhibited at a dosage of 0.6 nmol / mouse, significant antidepressant-like effects were not exhibited at a dosage of 0.3 nmol / mouse. On the other hand, when the PAS-CPP-GLP-2 derivative (11 sugars) was administered, antidepressant-like effects equivalent to those at 0.6 nmol / mouse were exhibited even at a dosage of 0.3 nmol / mouse. The above results suggest that the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) exhibits antidepressant-like effects with a smaller dosage compared to the PAS-CPP-GLP-2 derivative (sugar-free), and sugar chain modification has the effect of enhancing the efficacy of neuropeptide derivatives.

[0103] <Example 12: Evaluation of the Uptake Pathway of Sugar Chain-Modified GLP-2 Derivatives into Nerve Cells> Studying the uptake pathway of glycan-modified GLP-2 derivatives into nerve cells is important in exploring neuropeptide derivatives that can be applied to clinical use. Therefore, the uptake pathway of glycan-modified GLP-2 derivatives into NeuroA2, a nerve cell, was studied. Specifically, to confirm whether the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) induces macropinocytosis and is taken up by NeuroA2, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), which specifically inhibits the uptake of macropinocytosis, was used for the study.

[0104] (Preparation of the exposure solution for cells) To fully block the uptake pathway of macropinocytosis, 30 minutes before exposing the GLP-2 derivative to the cells, a culture medium containing EIPA was added to NeuroA2 cells (pretreatment). The culture medium was prepared as follows: EIPA was dissolved using DMSO and diluted with 10% DMEM to a final concentration of 1%. After the pretreatment, EIPA dissolved in DMSO was added to the FITC-PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus), and the solution prepared by diluting with the culture medium so that the final concentrations were 0.045 μg / μL (derivative) and 100 μM (EIPA), respectively, was added to NeuroA2 cells. For use in exposing the control group of cells, a DMSO solution of the PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) (0.045 μg / μL in terms of the derivative) was prepared.

[0105] (Study of the intracellular uptake pathway) Neuro2A cells were seeded at 2×10 5 cells / well in a 12-well plate and incubated for 24 hours to confirm that the cells were completely confluent. Then, 500 μL / well of the solution containing EIPA (pretreatment) was added to the cells in the EIPA addition group, and they were left standing in the incubator for 20 minutes. Then, 500 μL / well of the EIPA solution containing the FITC-PAS-CPP-GLP-2 derivative (11 sugars at the C-terminus) prepared was added, and they were left standing in the incubator. To the cells in the control group, a solution without EIPA was added, and the same operation was performed. Thirty minutes after exposure, cells were washed once with 1×PBS at 500 μL / well, and the cells were recovered into a tube by trypsin treatment. After centrifugation at 1000 rpm for 5 minutes, FACS buffer was added at a volume of 1000 μL / tube to prepare a cell suspension, and the cells were centrifuged again at 1000 rpm for 5 minutes. After adding FACS buffer again at a volume of 1000 μL / tube and resuspending the cells, the cell suspension was filtered through a nylon mesh filter and allowed to stand in an ice bath before measurement. Measurement was performed using a fully automated analytical flow cytometer system BD FACSCalibur (registered trademark) (Becton, Dickinson and Company) with the fluorescence intensity of FITC as the measurement target, and analysis was performed using FlowJo (FlowJo Software).

[0106] Macropinocytosis is a mechanism of intracellular uptake caused by the remodeling of the actin cytoskeleton and the formation of a fluctuating structure of the fluid plasma membrane. The size of the endosomal vesicles generated is larger than 1 μm, and efficient uptake can be expected (Non-Patent Document 16). Therefore, the intracellular uptake amount was measured using EIPA treatment that can specifically inhibit macropinocytosis, and whether macropinocytosis-based uptake occurs was investigated. As a result, as Figure 12 shown, in Neuro2A cells, the intracellular uptake amount of PAS-CPP-GLP-2 derivative (C-terminal 11 sugars) in NeuroA2 cells was significantly reduced in the presence of EIPA. From these results, it was found that the glycan-modified neuropeptide derivative was taken into cells by macropinocytosis.

[0107] <Example 13: Evaluation of the effect of reducing the pharmaceutically effective amount of glycan-modified GLP-1 derivative> GLP-2 derivatives are peptides that are poorly soluble in water, and it has been confirmed that glycan modification not only improves solubility but also enhances the pharmacological effect. However, not all of the neuropeptide derivatives to which PAS-CPP is added as the object of the present invention are poorly soluble in water. For example, PAS-CPP-GLP-1 has high solubility in water, so PAS-CPP-GLP-1 was dissolved in PBS for experiments in the evaluation of the pharmacological effect. Therefore, it is important in demonstrating the usefulness of glycan modification that not only poorly soluble PAS-CPP-GLP-2 but also PAS-CPP-GLP-1 with high solubility in water shows enhanced pharmacological effect through glycan modification. Therefore, it was investigated whether the glycan-modified GLP-1 derivative shows an excellent pharmacological effect compared to the unmodified GLP-1 derivative.

[0108] (Preparation of GLP-1 derivative) In the PAS-CPP-GLP-2 derivative (C-terminal 11 sugars), the GLP-2 as the neuropeptide sequence was changed to GLP-1, and otherwise, the PAS-CPP-GLP-1 derivative (C-terminal 11 sugars) was prepared in the same manner. The structures of the prepared glycopeptide-modified GLP-1 derivatives are shown below.

[0109] [Chemical formula 4]

[0110] In the PAS-CPP-GLP-2 derivative (sugar-free), the GLP-2 as the neuropeptide sequence was changed to GLP-1, and otherwise, the PAS-CPP-GLP-1 derivative (sugar-free) was prepared in the same manner. The structures of the prepared glycopeptide-modified GLP-1 derivatives are shown below.

[0111] [Chemical formula 5]

[0112] (Preparation of LPS-induced dementia model mice) Lipopolysaccharide (SIGMA-Aldrich) was dissolved in 0.01 M PBS at a concentration of 10 μg / 5 μL to prepare an LPS administration solution. After anesthetizing 7-week-old male ddY mice with isoflurane, the LPS administration solution was administered into the lateral cerebral ventricle of the LPS administration group at a dose of 10 μg / mouse. Specifically, using a 50 μL syringe (Hamilton(R) GASTIGHT(R) syringe, 1700) and a 28G×3 mm intracerebral needle (Natsume Seisakusho Co., Ltd.), considering the pressure difference, 5 μL of the LPS administration solution was administered over 15 seconds. The control group was administered 5 μL of 0.01 M PBS.

[0113] (Preparation and administration of the administration solution) The PAS-CPP-GLP-1 derivative (sugar-free) is soluble in PBS, so the PAS-CPP-GLP-1 derivative (sugar-free) and the PAS-CPP-GLP-1 derivative (C-terminal 11 sugars) were separately dissolved in PBS to prepare an administration solution (0.2 nmol / 4 μL). After anesthetizing the mice with isoflurane, the administration solution was administered intranasally at 2 μL per single nostril for a total of 4 μL (0.2 nmol / mouse). In the control group and the LPS group, 4 μL of PBS was administered at 2 μL per single nostril. The administration was performed 20 minutes before the following Y-maze test.

[0114] (Y-maze test) As an experimental apparatus, a Y-shaped maze with 120° arms made of black acrylic plates was used. The dimensions of the arms were 10 cm at the upper part of the cross-section, 3 cm at the bottom, 12 cm in height, and 40 cm in length. Mice were placed at the end of the Y-shaped maze, and the arms that the mice moved into were recorded successively within 8 minutes. The sum of the number of times the mice entered each arm was taken as the "total arm entries", and among them, the value obtained by dividing the "number of times of continuously entering 3 different arms" by the value obtained by subtracting 2 from the total arm entries, and multiplying this value by 100 was calculated as the spontaneous alternation rate (Percent alternation). This spontaneous alternation rate (Alternation) was used as an index of learning and memory behavior.

[0115] As Figure 13A shown, the group administered with PAS-CPP-GLP-1 derivative (sugar-free) did not show a significant learning and memory improvement effect compared with the LPS group administered only with PBS. In contrast, the group administered with PAS-CPP-GLP-1 derivative (11 sugars at the C-terminus) showed a significant learning and memory improvement effect compared with the LPS group administered only with PBS. It should be noted that, as Figure 13B shown, when the dosage of PAS-CPP-GLP-1 derivative (sugar-free) was increased, a tendency to show a significant learning and memory improvement effect was observed.

[0116] The dosage of PAS-CPP-GLP-1 derivative (sugar-free) was changed to the Figure 12 shown amount (nmol / mouse), and the Y-maze test was carried out in a state where the administration methods were intranasal administration and intracerebroventricular administration, respectively. As Figure 13B shown, in the case of intracerebroventricular administration (i.c.v.) of PAS-CPP-GLP-1 derivative (sugar-free), a spontaneous alternation rate (Alternation) of more than 60% was shown at 0.9 nmol / mouse. In contrast, in the case of intranasal administration (i.n.), a spontaneous alternation rate (Alternation) of more than 60% was shown at 0.45 nmol / mouse. Therefore, it was suggested that the intranasal administration of PAS-CPP-GLP-1 derivative (sugar-free) was transferred to the action site more efficiently compared with intracerebroventricular administration.

[0117] In addition, taking into comprehensive consideration Figure 13A and Figure 13BFrom the results shown, it can be said that even in the case of nasal administration, the PAS-CPP-GLP-1 derivative (11 sugars) shows an alternation rate equal to or higher than that of the PAS-CPP-GLP-1 derivative (sugar-free) administered into the lateral ventricle of the brain at a dosage of about one-fourth or less (0.2 nmol / mouse). This is a surprising result that cannot be conceived based on the common general knowledge in the art, and it shows the usefulness of the sugar chain-modified neuropeptide derivative of the present invention.

[0118] In addition, the PAS-CPP-GLP-1 derivative (sugar-free) is different from the PAS-CPP-GLP-2 derivative (sugar-free) in that it has high water solubility and sufficient solubility. Therefore, when evaluating the drug efficacy, the drug solution does not need to be dissolved in DMSO, but can be dissolved in PBS. That is, from this example, it can be seen that even for a peptide derivative showing high water solubility, the drug efficacy can be enhanced by sugar chain modification (which means a reduction in the pharmaceutically effective amount). The above situation shows that the method of sugar chain modification is effective regardless of the solubility of the peptide derivative having the functional sequence (PAS-CPP) in water, and it improves the generality of the application of sugar chain modification in peptides having the functional sequence (PAS-CPP).

[0119] <Example 14: Study on Neuropeptide Derivatives with Sugar Chain-Modified N-Terminus> (Preparation of Sugar Chain-Modified GLP-1 Derivative (11 Sugars at the N-Terminus)) A PAS-CPP-GLP-1 derivative (sugar-free) was obtained, which has, from the N-terminal side, an endosomal escape enhancement sequence (PAS: FFLIPKG), a membrane penetration enhancement sequence (CPP: RRRRRRRR), a spacer sequence containing a cysteine residue (GCG), and an amino acid sequence derived from GLP-1 as a neuropeptide sequence. The structure of the obtained GLP-1 derivative is shown below.

[0120] [Chemical Formula 6]

[0121] Next, a sugar chain (11 sugars) was bonded to the cysteine residue as the spacer sequence to obtain a PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) with the N-terminus of GLP-1 modified by a sugar chain. The structure of the obtained sugar chain-modified GLP-1 derivative is shown below.

[0122] [Chemical Formula 7]

[0123] (Preparation of Drug Solution) Completely dissolve the administration solutions of the ICG-fluorescently labeled PAS-CPP-GLP-1 derivative (sugar-free) and the ICG-fluorescently labeled PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) in DMSO. Then, prepare by adding PBS such that the final concentration of DMSO becomes 16%. The PAS-CPP-GLP-1 derivative (sugar-free) completely dissolves in PBS, but the ICG-PAS-CPP-GLP-1 fluorescently labeled with ICG does not dissolve in PBS. On the other hand, the ICG-PAS-CPP-GLP-1 derivative with a sugar chain modification at the N-terminus (11 sugars at the N-terminus) completely dissolves in PBS. Therefore, to make the conditions the same, DMSO is used as the solvent. Adjust such that the concentrations of the PAS-CPP-GLP-1 derivative (sugar-free) and the PAS-CPP-GLP-2 derivative (11 sugars at the N-terminus) become 3.0 nmol / 4 μL, respectively.

[0124] (Evaluation of the central transfer pathway using an optical imaging device) Anesthetize 7-week-old male ddY mice with isoflurane using an integrated small animal anesthetic device. Then, intranasally administer to the mice the prepared administration solution at 2 μL per single nostril, totaling 4 μL, or 16% DMSO as a control. To observe the time-dependent transfer into the brain, remove the brains 20 minutes after intranasal administration and immerse and fix them overnight in a 4% paraformaldehyde (4% PFA) solution. Then, using a brain matrix (RBM-2000S, ASI), prepare sagittal plane sections 2 mm thick on both the left and right starting from the center of the brain. Place the sections on a petri dish and perform measurements using an optical imaging device (Clairvivo OPT plus, Shimadzu Corporation). The measurement conditions are set as follows: excitation wavelength: 785 nm, fluorescence wavelength: 849 nm, exposure time: 6 seconds. The results are shown in Figure 14A .

[0125] As Figure 14A shown, it is suggested that the sample administered with the PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) has a greater amount of transfer to the center compared to the sample administered with the PAS-CPP-GLP-1 derivative (sugar-free), and the transferability to the center of the PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) is improved by sugar chain modification.

[0126] (Quantification of the amount of brain transfer using ELISA) Mice were administered 16% DMSO or various GLP-1 derivatives (3.6 nmol / mouse) via nasal route. Brains were removed 5 minutes and 20 minutes after the administration, and homogenized using BioMasher II (Nippi, Tokyo, Japan). After centrifugation at 1000×g for 15 minutes, the supernatant was recovered to prepare samples. The amount of GLP-1 derivatives present in the samples was quantified using a GLP-1 ELISA kit. First, each well of the assay plate was filled with the washing solution (350 μL), and the washing operation was repeated 3 times by pipetting. Next, the labeled antigen solution (40 μL), the sample (25 μL), and the specific antibody solution (50 μL) were added to each well in sequence and mixed. The assay plate was sealed with a sealing cap and left standing at 4°C for 18 hours. Then, the washing operation was performed 3 times, the SA-HRP solution (100 μL) was added, and it was soaked at room temperature for 1.5 hours (60 rpm). Just before the reaction ended, a chromogenic reagent solution was prepared by dissolving 1 OPD tablet in 25 mL of the substrate solution (0.1 M citrate buffer containing 0.03% hydrogen peroxide). Then, the washing operation was repeated 5 times, the chromogenic reagent solution (100 μL) was added to the sample and shielded from light, and it was left standing at room temperature for 1 hour. Finally, the enzyme reaction termination solution (100 μL) was added, and the absorbance at 490 nm was measured using ARVO (PerkinElmer Japan Co., Ltd., Kanagawa, Japan), and the concentration of the GLP-1 derivative was calculated. The results are shown in Figure 14B .

[0127] As Figure 14B shown, it was suggested that the sample administered with the PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) had a higher concentration compared to the sample administered with the PAS-CPP-GLP-1 derivative (sugar-free), and the metastasis of the PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) to the central nervous system was enhanced by sugar chain modification.

[0128] (Evaluation of the effect of reducing the pharmaceutically effective amount of the GLP-1 derivative (11 sugars at the N-terminus) modified with a sugar chain at the N-terminus) (Preparation of LPS-induced dementia model mice) Lipopolysaccharide (SIGMA-Aldrich) was dissolved in 0.01 M PBS at a concentration of 10 μg / 5 μL to prepare the LPS administration solution. After anesthetizing 7-week-old male ddY mice with isoflurane, an LPS administration solution was administered into the lateral cerebral ventricle of the LPS administration group at a dose of 10 μg / mouse. Specifically, using a 50 μL syringe (Hamilton(R) GASTIGHT(R) syringe, 1700) and a 28G×3 mm intracerebral needle (Natsume Seisakusho Co., Ltd.), 5 μL of the LPS administration solution was administered over 15 seconds considering the pressure difference. The control group was administered 5 μL of PBS

[0129] (Preparation and administration of the administration solution) PAS-CPP-GLP-1 derivative (sugar-free) is soluble in PBS, so various administration solutions were prepared by dissolving PAS-CPP-GLP-1 derivative (sugar-free) and PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) in PBS respectively After anesthetizing the mice with isoflurane, various administration solutions were administered into the nasal cavity at 2 μL per single nostril, totaling 4 μL. In the control group and the LPS group, 4 μL of PBS was administered at 2 μL per single nostril. Administration was performed 20 minutes before the Y-maze test. The Y-maze test was performed in the same manner as in Example 13. The results are shown in Figure 14C .

[0130] As Figure 14C shown, the sample administered with PAS-CPP-GLP-1 derivative (sugar-free) showed efficacy at 0.45 nmol / mouse. In contrast, the sample administered with PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) showed efficacy at 0.1 nmol / mouse. Based on the above results, it is suggested that the pharmaceutically effective amount of PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) is reduced by sugar chain modification

[0131] (Evaluation of the efficacy persistence of GLP-1 derivative with sugar chain modification at the N-terminus) The test was performed 60 minutes after the start of administration. Otherwise, the Y-maze test was performed in the same manner as in Additional Example 3. The results are shown in Figure 14D .

[0132] As Figure 14D shown, the sample administered with PAS-CPP-GLP-1 derivative (sugar-free) did not show efficacy 60 minutes after the start of administration. In contrast, the sample administered with PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) showed efficacy 60 minutes after the start of administration. Based on the above results, it is suggested that the efficacy persistence of PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) is improved by sugar chain modification

[0133] (Evaluation of the rapid onset of action of GLP-1 derivative with sugar chain modification at the N-terminus) The experiment was carried out 5 minutes after administration. Except for this, the Y-maze test was carried out in the same manner as in Additional Example 3. The results are shown in Figure 14E .

[0134] As Figure 14E shown, the sample administered with the PAS-CPP-GLP-1 derivative (sugar-free) did not show a pharmacological effect 5 minutes after administration. In contrast, the sample administered with the PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) showed a pharmacological effect 5 minutes after administration. Based on the above results, it is suggested that the rapid onset of the pharmacological effect of the PAS-CPP-GLP-1 derivative (11 sugars at the N-terminus) is improved by sugar chain modification.

[0135] (Evaluation of the effect of reducing the pharmaceutically effective amount of a GLP-2 derivative (11 sugars at the N-terminus) with a sugar chain-modified N-terminus) GLP-1 was changed to GLP-2, and PAS-CPP-GLP-2 derivatives (sugar-free) and PAS-CPP-GLP-2 derivatives (11 sugars at the N-terminus) having the following structures were prepared.

[0136] [Chemical Formula 8]

[0137] [Chemical Formula 9]

[0138] After completely dissolving the PAS-CPP-GLP-2 derivative (sugar-free or 11 sugars at the N-terminus) in DMSO, 16% DMSO was added to prepare a dosing solution. Using an integrated small animal anesthetic, mice were anesthetized with isoflurane, and the dosing solution was administered intranasally. Specifically, the tip of the needle of the anesthetic was brought close to the nasal cavity of the mouse in a horizontal manner, and 4 μL in total, 2 μL for each single nostril, was administered intranasally by the method of inhaling droplets through spontaneous breathing. In the control (Vehicle) group, 4 μL of 16% DMSO was administered intranasally. Intranasal administration was carried out 20 minutes before the test phase of the forced swimming test (FST). The results of the forced swimming test are shown in Figure 14F and Figure 14G .

[0139] As Figure 14F and Figure 14G shown, the sample administered with the PAS-CPP-GLP-2 derivative (sugar-free) showed a pharmacological effect at 0.6 mmol. In contrast, the sample administered with the PAS-CPP-GLP-2 derivative (11 sugars at the N-terminus) showed a pharmacological effect at 0.3 nmol. Based on the above results, it is suggested that the pharmaceutically effective amount of the PAS-CPP-GLP-2 derivative (11 sugars at the N-terminus) is reduced by sugar chain modification.

[0140] <Example 15: Study of GLP-2 derivative (N-terminal 11 sugars, C6 linker: C6) with sugar chain modification via a linker group> (Production of sugar chain-modified GLP-2 derivative) An endosome escape enhancement sequence (PAS: FFLIPKG), a membrane penetration enhancement sequence (CPP: RRRRRRRR), a spacer sequence containing a cysteine residue (GCG), and an amino acid sequence derived from GLP-1 as a neuropeptide sequence were sequentially arranged from the N-terminal side to produce a PAS-CPP-GLP-2 derivative (N-terminal 11 sugars) in a state where a sugar chain (11 sugars) was bonded to a cysteine residue as a spacer sequence. The structure of the produced GLP-2 derivative is shown below.

[0141] [Chemical Formula 10]

[0142] The cysteine residue as a spacer sequence in the PAS-CPP-GLP-2 derivative (N-terminal 11 sugars) was changed to a lysine residue (K) to produce a PAS-CPP-GLP-2 derivative (N-terminal 11 sugars, C6 linker: C6) in a state where a sugar chain-modified molecule containing a sugar chain (11 sugars) and an alkylene group with 6 carbon atoms as a linker group was bonded to the lysine residue in the spacer sequence. The structure of the produced GLP-2 derivative is shown below.

[0143] [Chemical Formula 11]

[0144] (Evaluation of intracellular uptake into nerve cells) Neuro2A cells were seeded at 2×10 5 cells / well in a 12-well plate and incubated for 24 hours. After confirming that the cells were completely confluent, solutions containing various prepared derivatives were added at 500 μL / well, respectively, and left standing in an incubator. Thirty minutes after the start of exposure, the cells were washed once with 500 μL / well of 1×PBS, and the cells were recovered into a tube by trypsin treatment. After centrifugation at 1000 rpm for 5 minutes, 1000 μL / tube of FACS buffer was added to prepare a cell suspension, and centrifugation was performed again at 1000 rpm for 5 minutes. After adding 1000 μL / tube of FACS buffer again and suspending it, filtration was performed using a nylon mesh filter, and it was left standing in an ice bath before measurement. The measurement was performed using a fully automated analytical flow cytometer system BD FACSCalibur TM (Becton, Dickinson and Company), and the analysis was performed using FlowJo (FlowJo Software). The results are shown inFigure 15A In the figure, "N11" represents no linking group, and "C6" represents a linking group having 6 carbon atoms.

[0145] Such as Figure 15A As shown, the glycan-modified GLP-2 derivative (N-terminal 11-glycan, C6 linking group: C6) in which the glycan is bonded to the peptide derivative via a linking group has an increased intracellular uptake compared to the glycan-modified GLP-2 derivative (N-terminal 11-glycan, no linking group: N11) in which the glycan is bonded to the peptide derivative without a linking group.

[0146] (Evaluation of the effect of reducing the pharmaceutically effective amount) Each derivative was completely dissolved in PBS to prepare a dosing solution. Using an integrated small animal anesthetic apparatus, mice were anesthetized with isoflurane, and then the dosing solution was administered intranasally. Specifically, the tip of the needle of the anesthetic apparatus was brought close to the nasal cavity of the mouse in a horizontal manner, and intranasal administration of 4 μL in total, 2 μL per single nostril, was performed by inhalation of droplets through spontaneous breathing. In the control (vehicle) group, 4 μL of PBS was administered intranasally, and the forced swimming test (FST) was performed. Intranasal administration was performed 20 minutes before the test phase. The results of the forced swimming test are shown in Figure 15B .

[0147] Such as Figure 15B As shown, the glycan-modified GLP-2 derivative (N-terminal 11-glycan, C6 linking group: C6) in which the glycan is bonded to the peptide derivative via a linking group has a shorter immobility time compared to the glycan-modified GLP-2 derivative (N-terminal 11-glycan, no linking group: N11) in which the glycan is bonded to the peptide derivative without a linking group, and an antidepressant-like effect was confirmed.

[0148] (Evaluation of the neuronal activity at the site of action of the antidepressant-like effect (DMH: dorsomedial hypothalamic nucleus)) Twenty minutes after intranasal administration of PBS or various glycan-modified GLP-2 derivatives (0.15 nmol / mouse) to mice, the brains were removed, infiltrated and fixed overnight in 4% PFA. On the day after removal, they were replaced and placed in 30% sucrose and left overnight (4 °C). Then, cryosections of the hippocampus and hypothalamus with a thickness of 40 μm were prepared using a cryostat (CM3050S; Leica Microsystems, WetZlar, Germany). The sections were placed on glass slides, and BLOXALL was added TMBlocking solution was used to block for 10 minutes at room temperature. After washing three times with 1×PBS, Normal Horse Serum with 0.3% Triton X-100 was added and permeabilized at room temperature for 20 minutes. The primary antibody solution containing Anti-c-Fos antibody (1:10,000) diluted with 2.5% BSA / PBS solution was added and incubated overnight at 4°C. After washing three times with 1×PBS, the secondary antibody solution containing biotinylated horse anti-mouse / rabbit IgG secondary antibody was added and incubated for 2 hours at room temperature. After washing three times with 1×PBS, VECTSTAIN Elite ABC Reagent was added and incubated for 1.5 hours at room temperature. After washing three times with 1×PBS, ImmPACT TM DAB EqV solution was added and incubated for 10 minutes at room temperature. After washing with purified water, mounting was performed, and the expression of c-Fos in HIP and DMH was observed using a fluorescence microscope (BIO-REVO BZ-9000; Keyence, Osaka, Japan). In addition, c-Fos positive cells in the obtained images were counted, the average value of each group was calculated and quantified. The results are shown in Figure 15C .

[0149] As Figure 15C shown, in the glycan-modified GLP-2 derivative (N-terminal 11 glycans, C6 linker: C6) in which a glycan is bonded to a peptide derivative via a linker group, a significant increase in c-Fos expression was confirmed in the site of action of the antidepressant-like effect, which is a result reflecting the antidepressant-like effect confirmed in the forced swimming test.

[0150] (Effect of glycan binding position on central transfer amount) Mice were administered intranasally (6.0 nmol / mouse) with the PAS-CPP-GLP-2 derivatives prepared in Example 1, namely, the PAS-CPP-GLP-2 derivative (C-terminal 11-sugar, no linker, C11), the PAS-CPP-GLP-2 derivative (N-terminal 11-sugar, no linker, N11), and the PAS-CPP-GLP-2 derivative (N-terminal 11-sugar, with C6 linker, C6) in which the sugar chain was modified via an alkylene group having 6 carbon atoms. The brain was removed 20 minutes after the administration and homogenized using a BioMasher (registered trademark) II (Nippi, Tokyo, Japan). After centrifugation at 1000×g (4°C) for 15 minutes, the supernatant was recovered to prepare a sample. A GLP-2 ELISA kit was used to quantify the GLP-2 derivatives present in the sample. First, each well of the assay plate was filled with 350 μL of the washing solution, and the washing operation was repeated 3 times by pipetting. Then, 40 μL of the labeled antigen solution, 25 μL of the sample, and 50 μL of the specific antibody solution were sequentially added to each well and mixed. The assay plate was sealed with a sealing cap and allowed to stand at 4°C for 18 hours. Then, the washing operation was performed 3 times, 100 μL of the SA-HRP solution was added, and the mixture was shaken at room temperature for 1 hour (100 rpm). Just before the reaction ended, a chromogenic reagent solution was prepared by dissolving 1 OPD tablet in 25 mL of the substrate solution (0.1 M citrate buffer containing 0.03% hydrogen peroxide). Then, the washing operation was repeated 5 times, 100 μL of the chromogenic reagent solution was added, and the mixture was shielded from light and allowed to stand at room temperature for 1 hour. Finally, 100 μL of the enzyme reaction termination solution was added, and the absorbance at 490 nm was measured using an ARVO (PerkinElmer Japan Co., Ltd., Kanagawa, Japan) to calculate the concentration of the GLP-2 derivative. The results are shown in Figure 15D . In the figure, C11 represents the PAS-CPP-GLP-2 derivative (C-terminal 11-sugar, no linker), N11 represents the PAS-CPP-GLP-2 derivative (N-terminal 11-sugar, no linker), and C6 represents the PAS-CPP-GLP-2 derivative (N-terminal 11-sugar, with C6 linker).

[0151] As Figure 15D shown, the amount of transfer to the central nervous system of C6 in which the sugar chain is bonded to the peptide derivative via a linker is increased compared to C11 and N11 in which the sugar chain is bonded to the peptide derivative without a linker.

[0152] <Evaluation of the Effects of Adding a Membrane Permeation Enhancing Sequence and an Endosomal Escape Enhancing Sequence> Prepared by conventional methods respectively: GLP-2 derivatives (CPP-GLP-2) configured successively with a membrane penetration enhancing sequence (CPP: RRRRRRRR), a spacer sequence (GG), and an amino acid sequence derived from GLP-2 as a neuropeptide sequence; GLP-2 derivatives (PAS-GLP-2) configured successively with an endosome escape enhancing sequence (PAS: FFLIPKG), a spacer sequence (GG), and an amino acid sequence derived from GLP-2 as a neuropeptide sequence; GLP-2 derivatives (PAS-CPP-GLP-2) configured successively with an endosome escape enhancing sequence (PAS: FFLIPKG), a membrane penetration enhancing sequence (CPP: RRRRRRRR), a spacer sequence (GG), and an amino acid sequence derived from GLP-2 as a neuropeptide sequence. To exclude the influence of sugar chain modification, this reference example was carried out in a state where the GLP-2 derivative was not sugar chain-modified.

[0153] After completely dissolving the various prepared GLP-2 derivatives or GLP-2 in DMSO, PBS was added so that the final concentration of DMSO became 16% by mass, and the dosing solutions were respectively prepared. The concentration of the GLP-2 derivative or GLP-2 was set to 0.6 nmol / 4 μL. Using an integrated small animal anesthetic, 7-week-old male ddY mice were anesthetized with isoflurane, and then intranasal administration of 2 μL per single nostril for a total of 4 μL (0.6 nmol / mouse) was performed. In the control (vehicle) group, 4 μL of 16% DMSO was intranasally administered. Intranasal administration was carried out 20 minutes before the test stage of the forced swimming test.

[0154] As Figure 16 shown, in the group administered with PAS-CPP-GLP-2, the immobility time was significantly shortened compared with the control group, showing an antidepressant-like effect. In contrast, in the groups administered with PAS-GLP-2, CPP-GLP-2 or GLP-2, no significant difference in the immobility time was found, and no antidepressant-like effect was shown.

[0155] <Example 16: Study on Sugar Chain-Modified GLP-1 Derivatives Using a β-Amyloid-Induced Neurodegenerative Model> (Preparation of β-Amyloid-Induced Neurodegenerative Model Mice) Prepared with physiological saline such that the concentration of amyloid-β protein fragment (Aβ1-42) becomes 1 mg / mL, incubated at 37 °C for 4 days to prepare an Aβ1-42 solution. After anesthetizing mice with isoflurane, the Aβ1-42 solution (3 μL) was administered into the lateral ventricle. The administration was performed using a 50 μL syringe (Hamilton(R) GASTIGHT(R) syringe, 1700) and a 28G×3 mm intracerebral needle (Natsume Seisakusho Co., Ltd.), taking into account the pressure difference and taking 15 seconds.

[0156] The prepared neurodegenerative model mice were divided into a vehicle group and a GLP-1 derivative administration group. The vehicle group was administered PBS via nasal administration, and the GLP-1 derivative administration group was administered a PBS solution of the N-terminal glycan PAS-CPP-GLP-1 (11 sugars) used in Example 14 (the administration amount was 0.1 nmol / mouse or 1.0 nmol / mouse) via nasal administration. Nasal administration was performed once a day for 7 days. After anesthetizing mice with isoflurane using an integrated small animal anesthetic device, nasal administration was performed using a micropipette with a volume of 2 - 20 μL. The tip of the micropipette needle was brought close to the nasal cavity of the mouse in a horizontal manner, and nasal administration of a total of 4 μL (2 μL per nostril) was performed by inhaling the droplets through spontaneous breathing.

[0157] (Preparation of frozen brain sections) The brains of each group were removed and immersed in a 4% PFA solution, infiltrated and fixed overnight at 4 °C. Then, they were replaced and placed in 30% sucrose and left overnight (4 °C) for replacement. Then, frozen sections (thickness 30 μm) of the hippocampal CA1 region, hippocampal dentate gyrus, and cerebral cortex were prepared using a cryostat (CM3050S; Leica Microsystems, WetZlar, Germany), respectively. For comparison, frozen sections were also prepared from mice administered PBS via nasal administration without intracerebroventricular administration of Aβ1-42.

[0158] (Immunohistochemical staining of neurodegenerative cells) Place the prepared frozen sections on the sealing cover, let them dry, and then immerse them in a basicalcohol solution for 5 minutes, 70% ethanol solution for 2 minutes, and PBS for 2 minutes in sequence. The basic alcohol is obtained by mixing 20 mL of 5% sodium hydroxide aqueous solution and 80 mL of absolute ethanol. After drying the immersed sections to a certain extent, immerse them in 0.04% potassium permanganate solution for 3 minutes and PBS for 2 minutes in sequence. Then, immerse them in 0.0001% Fluoro-Jade B staining solution (FJB) and 2.5 μg / mL DAPI for 10 minutes, and wash them 3 times with purified water. The 0.0001% FJB solution is prepared with 0.1% acetic acid solution. Let the sections dry, and then immerse them in xylene for 1 minute. After drying the sections completely again, use Fluoromount TM Seal the section sealing cover on the glass slide, and observe the fluorescence of the hippocampal CA1 region, hippocampal dentate gyrus, and cerebral cortex using a confocal laser microscope. Then, use Image J to measure the fluorescence intensity of the green fluorescence of neurodegenerative cells.

[0159] The measurement results of neurodegeneration in the hippocampal CA1 region are shown in Figure 17 The measurement results of neurodegeneration in the hippocampal dentate gyrus are shown in Figure 18 The measurement results of neurodegeneration in the cerebral cortex are shown in Figure 19 . As Figure 17 , Figure 18 and Figure 19 show, it can be seen that in the model mice prepared with Aβ1-42, in the group administered with the GLP-1 derivative, neurodegeneration was significantly inhibited in any of the hippocampal CA1 region, hippocampal dentate gyrus, and cerebral cortex.

[0160] The disclosure of Japanese Patent Application No. 2022-180380 is incorporated herein by reference in its entirety. For all the documents, patent applications, and technical standards described in this specification, the practice 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 glycan-modified neuropeptide derivative, characterized in that, It has a neuropeptide sequence, a central transfer enhancing sequence, and a sugar chain-modified molecule containing a sugar chain, and the central transfer enhancing sequence contains a membrane penetration enhancing sequence and an endosomal escape enhancing sequence.

2. The glycan-modified neuropeptide derivative according to claim 1, wherein, The sugar chain-modified neuropeptide derivative also has a spacer sequence disposed between the neuropeptide sequence and the central transfer enhancing sequence.

3. The glycan-modified neuropeptide derivative according to claim 2, wherein, The spacer sequence contains a lysine residue, and the sugar chain-modified molecule is bonded to the lysine residue.

4. The glycan-modified neuropeptide derivative according to claim 1, wherein, The sugar chain-modified molecule also contains a linking group.

5. The glycan-modified neuropeptide derivative according to claim 4, wherein, The linking group contains an alkylene group having 3 to 15 carbon atoms.

6. The glycan-modified neuropeptide derivative according to claim 1, wherein, The sugar chain-modified molecule is bonded to the C-terminal side or the N-terminal side of the neuropeptide sequence.

7. The glycan-modified neuropeptide derivative according to claim 1, wherein, The number of monosaccharide residues in each sugar chain is 5 to 20.

8. The glycan-modified neuropeptide derivative according to claim 1, wherein, The number of amino acid residues in the neuropeptide sequence is 200 or less.

9. The glycan-modified neuropeptide derivative according to claim 1, wherein, The membrane penetration enhancing sequence is cationic.

10. The glycan-modified neuropeptide 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.

11. The glycan-modified neuropeptide derivative according to claim 1, wherein, The endosomal escape enhancing sequence is an amino acid sequence selected from FFLIPKG, LILIG, FFG, FFFFFG, and FFFFFFFG.

12. A pharmaceutical composition, characterized in that, Comprising the sugar chain-modified neuropeptide derivative according to any one of claims 1 to 11 as an active ingredient.

13. The pharmaceutical composition according to claim 12, wherein, The pharmaceutical composition is used for the treatment of mental and neurological diseases or neurodegenerative diseases.

14. The pharmaceutical composition according to claim 12, wherein, The pharmaceutical composition is used for the treatment of depression or dementia.

15. A nasal drop preparation, characterized in that, Comprising the sugar chain-modified neuropeptide derivative according to any one of claims 1 to 11 as an active ingredient.

16. The nasal drop preparation according to claim 15, wherein, The nasal drop preparation is used for the treatment of mental and neurological diseases or neurodegenerative diseases.

17. The nasal drop preparation according to claim 15, wherein, The nasal drop preparation is used for the treatment of depression or dementia.

18. Use of the glycan-modified neuropeptide derivative according to any one of claims 1 to 11 in nasal drop administration.

Images