JP2008019245A - Intranasal agent for prevention and treatment of alzheimer's disease containing humanin derivative or fused peptide composed of humanin derivative and neurotropic peptide as active component - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a noninvasive and effective agent for the prevention and treatment of Alzheimer's disease by using a humanin derivative such as colivelin and provide a prevention and treatment method. <P>SOLUTION: The invention provides an intranasal agent for the prevention and treatment of Alzheimer's disease including a humanin derivative such as colivelin or a fused peptide composed of the derivative and a neurotropic peptide as an active component, and a method for the prevention and treatment of Alzheimer's disease or injury relating to Alzheimer's disease comprising the transnasal administration of the intranasal agent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention administers a nasal administration agent for preventing or treating Alzheimer's disease or improving Alzheimer's disease-related injury, which contains a humanin derivative or a fusion peptide of the derivative and a neurotropic peptide as an active ingredient, and the therapeutic agent. The present invention relates to methods for preventing and treating Alzheimer's disease.

The development of effective new anti-AD treatments is strongly desired due to the very limited effects on current Alzheimer's disease (AD) treatment. Current clinically used treatments for AD, such as acetylcholinesterase inhibitors such as donepezil, antioxidants such as vitamin E, and NMDA excitotoxicity inhibitors, can slightly slow the progression of AD pathology. (Sano et al., 1997; Reisberg et al., 2003; Lane et al., 2004). Therefore, several promising strategies have been considered to develop AD treatments separate from the development of such drugs.

  Nerve loss, which is thought to be directly related to the main pathological or neurological signs or symptoms of AD (Mattson et al., 2004) It is considered the most promising candidate.

  The present inventor has implemented a functional screening method that does not bias a compound that prevents cell death using a cDNA library prepared from the occipital lobe in the autopsy brain after death of an AD patient, the “Destrap Screening Method” Thus, a cDNA encoding a 24-amino acid peptide MAPRGFSCLLLLTSEIDLPVKRRA named humanin (HN) was identified (Patent Document 1). HN antagonizes various FAD genes, anti-APP antibodies, and neuronal cell death caused by all AD-related injuries such as beta amyloid peptide (Aβ) (Hashimoto et al., 2001a and b; Nishimoto et al., 2005).

Collivelin is a humanin (NH) derivative that has a strong activity (EC50 <100 femtomolar) at the C-terminus of activity-dependent neurotrophic factor (ADNF), a peptide having the amino acid sequence: PAGASRLLLLTGEIDLP ( AGA- (C8R) HNG17) is a fused peptide (Chiba et al., 2005). In vitro, corivelin is associated with various AD-related injuries by activating JAK2 / STAT3 prosurvival axis via NH 2 receptor and Ca2 + / calmodulin-dependent protein kinase IV (CaMKIV) via ADNF receptor. It exhibits neuroprotective action that antagonizes various types of neurotoxicity. Furthermore, colivelin has also been shown to suppress Aβ-induced hippocampal nerve loss in vivo.

Thus, when colivelin is delivered directly into the cerebrospinal fluid by intracerebroventicular (icv) administration, toxic Aβ icv administration and various cholinergic actions such as 3-quinuclinidyl benzylate (3-QNB) Colliverin inhibits AD-related memory loss or memory impairment (amnesia) in vivo because it inhibits the impairment of spatial working memory induced by intraperitoneal administration of systemic inhibitors (Chiba et al., 2005) It is thought to suppress.
Tokurei WO01 / 021787 Polypeptide that suppresses neuronal cell death, Humanin WO03 / 097687 Neuroprotective Polypeptides and Methods of Use WO2005 / 097156 “Neurodegenerative disease therapeutic agent”

Since AD therapeutic agents must be used safely and for a long period of time, in order to clinically apply humanin derivatives such as colivelin, they are less invasive and as small as possible in view of side effects. However, it is desired to develop an effective administration method.

Accordingly, an object of the present invention is to provide a noninvasive and effective drug for preventing and treating Alzheimer's disease and a method for preventing and treating using a humanin derivative such as colivelin.

That is, the present invention relates to the following aspects.
[Aspect 1] A nasal administration agent for prevention and treatment of Alzheimer's disease containing a humanin derivative or a fusion peptide of the derivative and a neurotropic peptide as an active ingredient.
[Aspect 2] A nasal administration agent for protecting / ameliorating Alzheimer's disease-related injury, comprising as an active ingredient a humanin derivative or a fusion peptide of the derivative and a neurotropic peptide.
[Aspect 3] The nasal administration agent according to aspect 2, wherein the Alzheimer's disease-related disorder is memory injury.
[Aspect 4] The nasal administration agent according to aspect 2, wherein the Alzheimer's disease-related disorder is neurodegeneration. [Aspect 5] The agent for intranasal administration according to aspect 2, wherein the Alzheimer's disease-related injury is a decrease in the number of choline acetyltransferase immunoresponsive neurons.
[Aspect 6] The nasal administration agent according to any one of Aspects 1 to 5, wherein the humanin derivative is a peptide having the amino acid sequence: PAGASRLLLLTGEIDLP (AGA- (C8R) HNG17).
[Aspect 7] The nasal administration agent according to Aspect 6, wherein the neurotrophic peptide is vasoactive intestinal peptide (VIP), activity-dependent neurotrophic factor (ADNF) or insulin-like growth factor (IGF-1).
[Aspect 8] The nasal administration agent according to any one of Aspects 1 to 7, wherein the fusion peptide is a peptide consisting of 26 amino acids in which AGA- (C8R) HNG17 is bound to the C-terminus of ADNF.
[Aspect 9] A method for preventing or treating Alzheimer's disease or Alzheimer's disease-related injury, which comprises nasally administering the nasal administration agent according to any one of Aspects 1 to 8.
[Aspect 10] Use of a humanin derivative or a fusion peptide of the derivative and a neurotrophic peptide for the production of a nasal administration agent for preventing or treating Alzheimer's disease.
[Aspect 11] The use according to Aspect 10, wherein the fusion peptide is a peptide consisting of 26 amino acids to which a peptide having the amino acid sequence: PAGASRLLLLTGEIDLP (AGA- (C8R) HNG17) is bound at the C-terminus of ADNF.

The present invention provides a highly effective drug for preventing and treating AD and a method for preventing and treating AD that are non-invasive and exhibit superior effects in a smaller dose compared to conventional intraperitoneal administration, etc. It became possible to carry out treatment safely for a long period of time.

  In the present specification, “humanin derivative” refers to neuronal cell death caused by AD-related injury equal to or higher than that of the above-mentioned polypeptide consisting of 24 amino acids (humanin) disclosed in WO01 / 021787. A polypeptide having an antagonistic action or an inhibitory action and derivatives thereof.

Accordingly, specific examples of humanin derivatives include, for example, the formula (I) described in International Publication WO01 / 021787 pamphlet.
^{Pro-Xn 1 - (Cys /} bXaa) - (Leu / Arg) -Xn 2 -Leu-Thr- (Gly / Ser) -Xn 3 -Pro (I)
(Wherein “Cys / bXaa” is Cys or a basic amino acid, “(Leu / Arg)” is Leu or Arg, “(Gly / Ser)” is Gly or Ser, and Xn ¹ , Xn ² , and Xn ³ each independently represents any amino acid of 10 residues or less)
A polypeptide having the amino acid sequence shown below (hereinafter also simply referred to as “polypeptide”).

  Furthermore, as a more specific example, SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26 to 29, 32, 33, 37 to 40, 46 described in the pamphlet of International Publication No. WO01 / 021787 An amino acid sequence selected from the group consisting of 48, 54, and 60, wherein one or more amino acids comprise an amino acid sequence substituted, deleted, inserted and / or added, and against neuronal cell death caused by AD-related injury And a polypeptide having an antagonistic action or an inhibitory action. A peptide having the amino acid sequence: PAGASRLLLLTGEIDLP (AGA- (C8R) HNG17) is a preferred specific example.

The active ingredient of the present invention includes a fusion peptide of a humanin derivative and a neurotrophic peptide, such as a vasoactive intestinal peptide (VIP), an activity-dependent neurotrophic factor (ADNF), or an insulin-like growth factor (IGF-1). A fusion peptide formed by binding a humanin derivative to the C-terminus of a neurotropic peptide such as

  A humanin derivative and a fusion peptide of the derivative and a neurotropic peptide can be easily synthesized by any method known to those skilled in the art.

  Furthermore, the humanin derivative of the present invention includes a compound having a form in which the functional group of the above polypeptide is modified by a known method by modification, addition, mutation, substitution, or deletion. Such modification of the functional group can be performed using any method known to those skilled in the art, for example, protection of the functional group present in the polypeptide, control of polypeptide stability or tissue migration, or activity of the polypeptide. It can be performed for the purpose of control.

  That is, the polypeptide may be naturally modified by post-translational modification or the like. It may be artificially modified. Modifications include modifications such as peptide backbone, amino acid side chain, amino terminus, or carboxyl terminus. The polypeptide may be branched or cyclic. Modifications include acetylation, acylation, ADP ribosylation, amidation, covalent bonds such as [flavin, nucleotides, nucleotide derivatives, lipids, lipid derivatives, or phosphatidylinositol], cross-linking, cyclization, disulfides Examples include, but are not limited to, bond formation, demethylation, pyroglutamine oxidation, carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, phosphorylation, ubiquitination and the like. Furthermore, the polypeptide may be any salt and ester known to those skilled in the art. The polypeptide of the present invention can be produced by known peptide synthesis techniques, and can also be produced by expressing DNA encoding these polypeptides.

  In the present invention, “having an antagonizing action or an inhibitory action on nerve cell death caused by AD-related injury” refers to antagonizing or inhibiting at least one of the above-mentioned nerve cell deaths related to AD. That is, the above-mentioned humanin-like polypeptides include those having an activity of suppressing at least one of these neuronal cell deaths related to AD. Even if the suppression of cell death is not complete suppression, it may be suppressed significantly. The inhibitory activity of neuronal cell death can be assayed according to the method described in the following examples or other methods (see, for example, International Publication No. WO00 / 14204).

  As already mentioned, previous studies have revealed that neuronal cell death occurs in Alzheimer's disease. For this reason, the agent (nasal administration agent) of the present invention can also be used as an agent for protecting neurodegeneration in Alzheimer's disease. In addition to Alzheimer's disease, it is also possible to prevent diseases caused by neuronal cell death due to cerebral ischemia (T. Kirino, 1982, Brain Res., 239) using the agent of the present invention. 57-69). In addition, Parkinson's disease with dementia (MH Polymeropoulos et al., 1997, Science, 276: 2045-2047), diffuse Lewy body disease (MG Spillantini et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 6469-6473), dementia associated with Down's syndrome and the like are also targets for treatment and prevention. In addition, APLP1, which is a related molecule of APP, is said to be a causative gene of congenital nephrotic syndrome (Lenkkeri, U. et al., 1998, Hum. Genet. 102: 192-196). Renal diseases are also subject to treatment and prevention.

  The drug of the present invention contains a humanin derivative or a fusion peptide of the derivative and a neurotropic peptide as an active ingredient, and can be formulated by a known pharmaceutical method. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, a mixture of an appropriate organic solvent such as isopropanol and physiological saline, vegetable oil, emulsifier, suspending agent, surfactant In addition, it is conceivable to formulate and administer it in appropriate combination with a stabilizer, sustained-release agent and the like. The agents of the present invention can be in any form known to those skilled in the art suitable for nasal administration routes such as nasal drops, inhalants, and sprays.

  The agents of the present invention can be administered nasally (in the nasal cavity) to a patient using any method and means known to those skilled in the art. For example, depending on the tissue transferability of the active ingredient in the drug of the present invention, therapeutic purpose, patient weight, age, symptoms, etc., a drug of about several tens of μl per treatment once to several times a day is appropriate Can be administered over a period of time. By such nasal administration, the blood-brain barrier that restricts the entry of substances from the bloodstream to the central nerve by the action of the dendritic transport process mechanism of olfactory neurons consisting of both extracellular and intracellular transport systems Bypass (Illum et al., 2000; Throne et al., 2004), drugs can reach the brain or central nervous system. In particular, neurotropic peptides such as vasoactive intestinal peptide (VIP), activity-dependent neurotropic factor (ADNF) or insulin-like growth factor (IGF-1) are delivered to the brain or cerebrospinal fluid by nasal administration (Gozes et al., 1996, 2000; Throne et al., 1997; Throne et al., 2004).

A person skilled in the art can appropriately select the amount of the active ingredient in the drug according to the tissue transferability of the active ingredient in the drug of the present invention, the purpose of treatment, the patient's weight, age, symptoms and the like. For example, in the treatment of Alzheimer's disease or the like, when administration is performed for the purpose of protecting neuronal degeneration, the compound may be administered at a concentration that effectively suppresses neurodegeneration around the target cell. preferable. That is, the active ingredient can have a concentration in the range of several hundred pmol to several tens nmol, for example.

  Hereinafter, the present invention will be described in more detail with reference to examples. The technical scope of the present invention is not limited by these descriptions.

Experimental materials and methods
Peptide colibellin (amino acid sequence: SALLRSIPAPAGASRLLLLTGEIDLP), ADNF (SALLRSIPA) and AGA- (C8R) HNG17 (amino acid sequence: PAGASRLLLLTGEIDLP) were synthesized by Asahi Techno Glass Company (Funabashi, Japan).

The policy on the use of animals and humans in neuroscience research by the Society for Animal and Treatment Neurology and guidelines for the management and use of laboratory animals at Keio University School of Medicine were conducted. All experiments were approved by the Animal Experiment Committee at Keio University. Seven-week-old CD-1 (ICR) was purchased from Charles River Japan. As described in the previous literature (Kawasaki et al., 2004; Yamada et al., 2005), this animal is a 12-hour day / night cycle (7:00 AM) in an animal facility free of special pathogens. -7: 00 PM) (temperature: 23 + 1 ° C, humidity: 50 + 5%).

  The AD mouse model was generated by repeated i.c.v. administration of Aβ (Yamada et al., 2005). Briefly, stereotaxy was performed in the left ventricle (front and rear: +0.3 mm, side: 1.0 mm, horizontal direction: 3.0 mm from bregma) of 8-week-old CD-1 mice under 10% Nembutal anesthesia. Cannule (C315GS-4, plastic One Inc., Roanoke, VA) was implanted using a fixed surgical device. 10 days after cannula implantation, model animals were assigned to control group, Aβ administration group, or Aβ administration + colivelin treatment group, 3 μl sterile distilled water (DDW), or 3 μl Aβ25-35 peptide (1 nmol) in DDW at icv A given amount of synthetic colibellin peptide administered every 3 weeks (10 times) and dissolved in 10 μl of solvent (sterile saline containing 5% sefsol and 20% isopropanol) or 10 μl of the same solvent Was administered nasally once a day for 3 weeks (FIG. 4). Behavioral tests were performed 2 days after the last i.c.v. administration or 1 day after the last administration of colivelin.

  A cholinergic drug-induced memory loss model was created as described in the literature (Chiba et al., 2005). Briefly, 8 hours old CD-1 mice were given a predetermined amount of a solvent containing or not containing colivelin (5% sefsol and 20%) 24 hours and 30 minutes before the Y-maze test (YM). 10 μl of isopropanol-containing sterile saline) was administered intranasally (FIGS. 1A and C). 3-QNB dissolved scopolamine (1 mg / kg) dissolved in 0.2 ml sterile distilled water subcutaneously 30 minutes before YM, while dissolved in 0.2 ml sterile distilled water (containing 20% methanol) (0.5 mg / kg) was administered intraperitoneally 15 minutes before YM to induce memory loss (memory impairment). As a control test, colivelin was dissolved in sterile distilled water at a concentration of 0.5 mg / ml, and 1 nmol was intraperitoneally and subcutaneously administered 24 hours before and 30 minutes before the YM test, respectively.

Behavior test
OF and YM were performed as described in the literature (Kawasumi et al., 2004; Yamada et al., 2005; Chiba et al., 2005). OF acclimated the mouse to the experimental environment and left the mouse free in a 100 cm side gray plastic field for 3 minutes. The device for YM is three gray arms (40 cm long, 12 cm high, bottom width 3 cm, and top width 10 cm) connected to each other at an angle of 120 ° C. Each mouse was placed at the end of the arm and allowed to explore the arm freely for 8 minutes. The percentage of voluntary changes (Spontaneous Alternation: SA%), which is an indicator of spatial working memory, is “the previous two choices relative to the total number of choices in the test minus 2 (the first two cannot be evaluated)” The ratio of selecting different arms (successful selection) is defined. For example, if the mouse has entered 10 times (1-2-3-2-3-1-2-3-2-1), 5 times during the selection of 8 times ((10-2) times) As a result, SA% is 62.5%.

Immunohistochemistry Immunohistochemical analysis was performed as described in the literature (Kawasumi et al., 2004; Yamada et al., 2005; Chiba et al., 2005). After behavioral testing, PBS was perfused through the heart and fixed with ethanol containing 5% acetic acid (most experiments) or 4% PFA (for ADNF immunohistochemistry). The brain was embedded in paraffin, and a 10 μm coronal section was prepared on a New-Silane glass slide (Muto Pure Chemicals, Tokyo, Japan). Next, the sample was deparaffinized and washed in methanol and PBS. For immunohistochemical detection, reaction with anti-ChAT antibody (1:50 dilution, Chemicon USA) or anti-ADNF antibody (affinity purification, 1:50 dilution), and ABC method (Vectastain Elite Kit, Vector, CA, USA) Visualized. ChAT immunoreactive neurons in 5 medial septums with 50 μm spacing (approximately 0.6-0.9 mm ahead of bregma) were counted and ChAT immune response in 3 mice (N = 3) per treatment group The average total number of sex neurons was compared. The ADNF immune response was visualized with FITC-conjugated anti-rabbit IgG secondary antibody (Sigma).

Immunoblot analysis Immunoblot analysis was performed as described in the literature (Hashimoto et al., 2001a, b; Chiba et al., 2005). Lysate (50 μg / g) dissolved in a phosphatase inhibitor cocktail (Sigma) containing lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Trito-X100, Complete protease inhibitor) from the brain sample lane) was subjected to SDS-PAGE, and proteins fractionated on the gel were transferred to a PVDF membrane. Membranes are reacted with phosphorylated STAT3 antibody (1: 200) or total STAT3 antibody (1: 1000), then 1: 5000 dilution of horseradish (HRP) labeled anti-rabbit IgG antibody (BioRad Laboratories, Hercules, CA USA) And reacted. Antigen bands were visualized by ECL method (Amsharm Pharmacia Biotech, Uppsala, Sweden).

Statistical analysis All values in the figure showing in vitro tests are mean + SD (standard deviation). Moreover, all the values in the figure which shows an in-vivo test are an average value + SEM (standard error). Statistical analysis was performed by one-way ANOVA followed by Fisher's PLSD, which was significant at p <0.05.

Results: Suppressing anticholinergic drug-induced memory impairment (memory loss) by short-term nasal administration of colivelin
The progression of AD cognitive dysfunction causes the cholinergic system to malfunction in the central nervous system (CNS) (Bartus et al., 1982; Coyle et al., 1983). That is, administration of anticholinergic agents such as scopolamine, 3-quinuclidinyl benzylate (3-QNB) and ethylcholine aziridinium (AF64A) induces memory impairment in rodents and humans (Fisher et al ., 2004; Krejcova et al., 2004).

Using the Y-maze test described above, the effect of nasal administration of colibellin on spatial working memory damage induced by scopolamine was examined (FIG. 1A). In the same test, SA%, an index of spatial walking memory, was 65.6 + 2.0% in control mice, but 46.8 + 2.2% in scopolamine-treated mice, and scopolamine treatment caused spatial working memory impairment. (Mamiya et al. 2001; Tajima et al., 2005). On the other hand, when nasal administration of colibellin was performed, as shown in FIG. 1B, the spatial working memory disturbance induced by scopolamine treatment was alleviated in a dose-dependent manner (200 pmol colivelin administration: 50.9 + 2.3%, 1 nmol colivelin administration: 55.0 + 2.5%, 5 nmol colibellin administration: 59.2 + 3.1%).

  Furthermore, a similar test was performed using 3-QNB as another drug (FIG. 1C). As a result, as shown in FIG. 1D, the spatial working memory disorder was alleviated in a dose-dependent manner by nasal administration of colibellin. For comparison, donepezil (5 mg / kg) was only able to slightly block spatial working memory failure.

  Administration of AGA- (C8R) HNG17 or ADNF alone at the dose tested did not have an effect on 3-QNB-induced spatial working memory impairment (FIGS. 2A, B). Therefore, it is considered that the effect of colivelin, which is a fusion peptide to which these are bound, is not due to the effect of each peptide alone, but due to the remarkable synergistic action of both peptides in vivo due to the fusion peptide.

Results: Enhanced effect of long-term repeated nasal administration of neuroprotective peptides
It has been reported that daily nasal administration of ADNF for more than one week has a neuroprotective effect against memory impairment induced by the anticholinergic drug AF64A (Gozes et al., 2000). Thus, when combined with the above results shown in FIG. 2, this effect appears to be due to increased ADNF concentrations in vivo with long-term repeated administration.

  To verify this, mice were administered AGA- (C8R) HNG17, ADNF and colivelin at 1 nmol per treatment per day for 7 days, and the effect of each peptide was evaluated (FIG. 3). In contrast to short-term administration, administration of AGA- (C8R) HNG17 and colivelin effectively alleviates 3-QNB-induced spatial working memory impairment, although ADNF alone has had little effect. Was shown (FIG. 3B).

Results: Relieving Aβ25-35-induced memory impairment by long-term repeated colivelin nasal administration
It is known that repeated icv administration of Aβ induces memory impairment in rodents (Flood et al., 1991; Delobette et al., 1997; Yamada et al., 1999; Yamaguchi and Kawashima., 2001; Stepanichev et al., 2003b). Therefore, according to the literature (Yamada et al., 2005; Chiba et al., 2005), the therapeutic effect by nasal administration of colibellin on Aβ toxicity in vivo was examined as described above.

As a result, SA% in control mice was 66.1 + 2.1%, whereas in mice treated with Aβ25-35, it was 53.3 + 1.7%, and repeated administration of Aβ can induce damage to spatial working memory. Indicated. On the other hand, SA% was 68.0 + 2.4% when nasal administration (1 nmol) in addition to Aβ25-35 was administered, and SA% was 62.4 when ADNF was administered nasally in addition to Aβ25-35. + 3.0%, indicating that nasal administration of colibellin and ADNF has a significant therapeutic effect on Aβ25-35-induced memory impairment, and that colivelin is more effective than ADNF (Fig. 4B).

Results: Prevention of Aβ25-35-induced decrease in the number of ChAT-positive neurons by colivelin
Aβ icv administration reduces choline acetyltransferase (ChAT) expression levels in the inner septum of mouse neurons, and between the degree of spatial working memory impairment and the number of ChAT immunoreactive neurons in the inner septum Have been reported to have a positive correlation (Tajima et al., 2005; Yamada et al., 2005).

Therefore, the present inventor examined the neuroprotective effect of colivelin in vivo by the immunohistochemical analysis described above. From the results shown in FIG. 5, the number of ChAT immunoreactive neurons in the medial septum (232.3 + 16.0) in mice injected with Aβ25-35 is significantly reduced compared to the control (555.6 + 57.4) In addition, it was observed that the number of ChAT immunoreactive neurons was almost the same as the control (514.0 + 21.1) in the mice treated with colivelin, and the decrease in the number of ChAT immunoreactive neurons by repeated administration of Aβ25-35, It was found that nasal administration of colivelin was significantly inhibited.

Results: Transport to the CNS via the olfactory bulb of colibellin nasal administration In order to study the transport route of nasally administered corivelin to the CNS, in the brain section of a mouse administered 5 times nasally 5 nmol colivelin at 15 minute intervals Coliberin was immunostained as described above. That is, when a mouse brain section containing the olfactory bulb is fixed with 4% paraformaldehyde (PFA) and immunostained with an anti-ADNF antibody that recognizes koliberin, the corbelin immunostaining is performed in the ventral region of the olfactory bulb of the mouse treated with koliberin. Was observed (FIG. 6B, D). On the other hand, no colivelin immunostaining was observed in the control mice (FIGS. 6A and 6C). Therefore, these results strongly suggested that intranasally administered colivelin is transported to the CNS via the olfactory bulb. Consistent with previous findings that nasally administered compounds enter the CNS via both extracellular and intracellular transport pathways, colivelin immunostaining was detected in both the olfactory bulb nerve axon and extracellular matrix. . E and F are negative controls obtained by staining a control mouse and a colibellin-treated mouse with only the secondary antibody, respectively.

Results: Effects of colivelin via the JAK2 / STAT3 axis in vivo It has been found in vitro that the intracellular humanin (HN) signaling pathway is via JAK2 and STAT3 (Hashimoto et al., 2005 ). It is also known that STAT3 is activated by phosphorylation of tyrosine 705 (Tyr705) by JAK kinase. In order to confirm whether the same HN-induced prosurvival pathway is activated in vivo and that corivelin acts, we investigated whether STAT3 is phosphorylated in vivo by nasal administration of corivelin. . As shown in FIG. 1A (short-time administration protocol), AGA- (C8R) HNG17, ADNF or colivelin was administered nasally twice to CD-1 mice. Brain samples were prepared immediately after YM. The immunoblot analysis described above increased phosphorylated STAT3 (Tyr705) in the olfactory bulb of colivelin-treated mice, but did not change the total amount of STARA3. These results suggested that STAT3 was phosphorylated by nasal administration of colibellin (FIG. 7). In contrast, administration of AGA- (C8R) HNG17 did not increase STAT3 phosphorylation levels, and this increase in STAT3 phosphorylation levels is essential for alleviating (improving) memory impairment mediated by colivelin. Was suggested.

  STAT3 is phosphorylated by NH stimulation, and the main kinase involved in this phosphorylation is JAK2. In order to examine the relationship between the administration of colivelin and the phosphorylation of STAT3, according to the experimental protocol shown in FIG. 1A, 24 hours before the Y-maze test (YM), AGAK treatment (5 mg / mg) was performed. kg) and tested the effect of scopolamine-induced memory impairment on suppression by colivelin. As shown in FIG. 8, the subcutaneous injection of AG490 prevented the suppression of SA% reduction induced by subcutaneous administration of scopolamine by colibellin. On the other hand, AG490 had no effect on SA% of control mice or scopolamine treated mice. In contrast, AG43 (no JAK2 inhibitory activity) treatment (5 mg / kg), which is an analog of AG490, has an effect on the SA% of control mice, scopolamine-treated mice, or nasally administered mice. (FIG. 8B). From the above facts, it can be concluded that the suppression of scopolamine-induced memory impairment by colivelin is also via the JAK2 / STAT3 axis in vivo.

Results: Comparison with the administration method of corivelin To investigate which method is superior when the same amount of corivelin is administered intranasally, intraperitoneally and subcutaneously, a scopolamine-induced memory loss model was used. Coliberin was administered intranasally, intraperitoneally and subcutaneously to compare its effects. As a result, as shown in FIG. 9, scopolamine-induced memory impairment was significantly improved by intranasal administration, but not by the other two administration methods. It has been demonstrated that the administration has unexpectedly significant effectiveness compared to the other two administration methods.

  By using a nasal administration agent containing the humanin derivative of the present invention or a fusion peptide of the derivative and a neurotropic peptide as an active ingredient, a noninvasive and highly effective drug for the prevention and treatment of Alzheimer's disease and Prevention and treatment methods have been provided, and it has become possible to safely carry out Alzheimer's disease treatment for a long period of time.

  The contents described in the documents cited in the present specification constitute the disclosure of the present specification as a part of the present specification.

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Experimental protocol (A, C) on the effect of short-term colivelin nasal administration by Y-maze test on anticholinergic drug-induced memory impairment (memory loss) and graphs showing the results (B, D) ). Values are mean + SEM (standard error) (* p <0.05, ** p <0.01). It is the graph which showed the influence which it has on the 3-QNB induced memory impairment effect by single nasal administration of AGA- (C8R) HNG17 or ADNF. The value is an average value + SEM (standard error). It is the experiment protocol (A) regarding the enhancement of the effect by long-term repeated nasal administration of a neuroprotective peptide, and the graph (B) which shows the result. In addition, a value is an average value + SEM (standard error) (* p <0.05, ** p <0.01). It is the experiment protocol (A) regarding the relief | moderation of A (beta) 25-35-induced memory impairment by long-term repeated coryvelin nasal administration, and the graph (B) which shows the result. In addition, a value is an average value + SEM (standard error) (** p <0.01). FIG. 6 shows the inhibition of Aβ25-35-induced decrease in the number of ChAT-positive neurons by colivelin. It is the photograph (A) obtained by immunohistochemically dyeing a brain section with an anti-ChAT antibody, and the graph (B) which shows the result. In addition, a value is an average value + SEM (standard error) (** p <0.01). It is the photograph obtained by the immunohistochemical dyeing | staining which shows the transport to CNS through the olfactory bulb of the coryvelin nasal administration. A, B, E and F are prepared by coronal cutting, while C and D are prepared by sagittal cutting. It is the photograph obtained by the immunoblot analysis which shows that STAT3 was phosphorylated by nasal administration of colivelin. Results (A) of the effects on the inhibition of scopolamine-induced memory impairment by AG490, a JAK2 inhibitor, by colivelin (A), and (B) the results (B) of AG43, which is a similar compound to AG490, as a comparative control experiment. It is a graph to show. In addition, a value is an average value + SEM (standard error) (* p <0.05). It is a result which shows the effect with respect to scopolamine-induced memory loss of colivelin administered intranasally, intraperitoneally and subcutaneously. In addition, a value is an average value + SEM (standard error) (* p <0.05).

Claims (11)

A nasal administration agent for the prevention and treatment of Alzheimer's disease comprising a humanin derivative or a fusion peptide of the derivative and a neurotropic peptide as an active ingredient. A nasal administration agent for protecting and improving Alzheimer's disease-related injury, comprising a humanin derivative or a fusion peptide of the derivative and a neurotropic peptide as an active ingredient. The nasal administration agent according to claim 2, wherein the Alzheimer's disease-related disorder is memory injury. The nasal administration agent according to claim 2, wherein the Alzheimer's disease-related disorder is neurodegeneration. The nasal administration agent according to claim 2, wherein the Alzheimer's disease-related injury is a decrease in the number of choline acetyltransferase immunoresponsive neurons. The nasal administration agent according to any one of claims 1 to 5, wherein the humanin derivative is a peptide having the amino acid sequence: PAGASRLLLLTGEIDLP (AGA- (C8R) HNG17). The nasal administration agent according to claim 6, wherein the neurotropic peptide is vasoactive intestinal peptide (VIP), activity-dependent neurotrophic factor (ADNF) or insulin-like growth factor (IGF-1). The nasal administration agent according to any one of claims 1 to 7, wherein the fusion peptide is a peptide consisting of 26 amino acids in which AGA- (C8R) HNG17 is bound to the C-terminus of ADNF. A method for the prophylaxis or treatment of Alzheimer's disease or Alzheimer's disease-related injury, which comprises nasally administering the nasal administration agent according to any one of claims 1 to 8. Use of a humanin derivative or a fusion peptide of a neurotropic peptide and a humanin derivative for the production of a nasal agent for the prevention / treatment of Alzheimer's disease. The use according to claim 10, wherein the fusion peptide is a peptide consisting of 26 amino acids in which a peptide having the amino acid sequence: PAGASRLLLLTGEIDLP (AGA- (C8R) HNG17) is bound to the C-terminus of ADNF.

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