KR20200061573A - New peptide and pharmaceutical composition containing the same - Google Patents

Abstract

Provided are a pharmaceutical composition for treating or preventing amyloid diseases, and peptides constituting the same. The pharmaceutical composition for treating amyloid diseases is a peptide in which two or more functional peptide fragments are linked to each link core with their free ends. The functional peptide fragments consist of up to 17 amino acids, and comprise peptide which is a functional peptide fragment containing at least 10 or more of the 17 amino acids of natural Aβ1 to 17 or a pharmaceutically acceptable salt thereof.

Description

A novel peptide and a pharmaceutical composition comprising the same TECHNICAL FIELD

The present disclosure relates to a novel peptide, and more particularly, to a peptide that decomposes or inhibits aggregation of amyloid beta and a pharmaceutical composition comprising the same.

Human amyloid beta (hereinafter Aβ) acts in the brain and causes amyloid disease. Aβ is a peptide composed of 39 to 43 amino acid residues derived from amyloid protein precursor (APP). Amyloid Pre Protein (APP) is sequentially decomposed into Aβ40 or Aβ42 monomers by β- and γ-secretases. Thereafter, Aβ40 or Aβ42 monomers are integrated to form Aβ oligomers, and these oligomers are integrated to form Aβ fibrils, and these fibrils are integrated to form Aβ plaques. Aβ is known to show strong neurotoxicity to the brain from the state of being an oligomer and kill brain cells.

However, the majority of therapeutic agents for the treatment of amyloid disease developed to date employ antibodies against Aβ fragments or specific epitopes in Aβ that cause an immune response to patients, or employ a method of applying chemical drugs. However, in the case of a method using an Aβ fragment or an antibody that causes an immune response, cerebral blood vessel barrier permeability is low, and clinical development is difficult due to side effects such as Amyloid-Related Imaging Abnormalities (ARIAs) caused by immunogenicity. In addition, in the case of chemical drugs, so far, there are no radical treatments for amyloid disease.

The present disclosure seeks to provide peptides that can bind to water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates and disaggregate water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates.

The present disclosure is intended to provide a peptide capable of inhibiting aggregation of Aβ monomers, water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates by binding to Aβ monomers, water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates.

The present disclosure is intended to provide a composition capable of treating amyloid diseases, including peptides capable of binding to and degrading water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates or pharmaceutically acceptable salts thereof.

The present disclosure is intended to provide a composition capable of preventing amyloid disease, including a peptide capable of binding to water-soluble Aβ aggregates and/or insoluble Aβ aggregates and inhibiting their accumulation, or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition for the treatment of amyloid disease according to the embodiments is a peptide in which two or more functional peptide fragments are each linked with a free end to the link core, wherein the functional peptide fragment consists of up to 17 amino acids and is natural Aβ1. Peptide which is a functional peptide fragment containing at least 10 amino acids of 17 amino acids of ~17, or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition for the prevention of amyloid disease according to the embodiments is a peptide in which two or more functional peptide fragments are each linked with a free end to the link core, wherein the functional peptide fragment is composed of up to 17 amino acids and is natural Aβ1 Peptide which is a peptide fragment containing at least 10 or more of 17 amino acids of ~17, or a pharmaceutically acceptable salt thereof.

The peptide according to the embodiments is a peptide in which two or more functional peptide fragments are coupled to the link core with their free ends, wherein the functional peptide fragments are composed of up to 17 amino acids, and at least among the 17 amino acids of natural Aβ1 to 17 It is a peptide fragment containing 10 or more amino acids.

The peptides according to the examples can be used to degrade aggregation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates.

The peptides according to the embodiments can be used to inhibit aggregation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates.

Other details of aspects of the present invention are included in the detailed description below.

The peptides according to the embodiments can bind to water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates to effectively degrade or inhibit aggregation.

The peptides according to the examples contain amino acids similar to the natural Aβ40 or Aβ42, so the specificity for Aβ is strong, the mechanism of action is clear, it is easy to pass through the cerebrovascular barrier (BBB), chemicals such as surfactants, or It has excellent stability due to low or no structural denaturation rate due to light stimulation such as ultraviolet rays, and has a very low possibility of unexpected side effects such as conventional chemical drugs.

The pharmaceutical composition according to the embodiments comprises Aβ-induced cytotoxicity, including a peptide capable of binding to water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates to degrade or inhibit aggregation, or a pharmaceutically acceptable salt thereof. It can alleviate and suppress the death of neurons, thereby preventing or treating amyloid disease.

1 to 6 are schematic diagrams for explaining the structure of a peptide that disaggregates or inhibits aggregation of water-soluble Aβ aggregates and/or insoluble Aβ aggregates according to embodiments.
7 to 9 are graphs showing the results of measuring the Aβ aggregation degradation effect of the peptides prepared by the comparative example and the peptides according to the experimental examples.
10 to 12 are graphs showing the results of measuring the Aβ aggregation inhibitory effect of the peptides prepared by the comparative examples and the peptides according to the experimental examples.
13 is an experimental result showing the result of P101 (0117) directly binding to Aβ.
14A to 14D are experimental results showing the results of administration of P101 (0117) degrading Aβ plaques.
15A to 15D are graphs showing the results of P101(0117) inhibiting Aβ-induced neuronal cell death.
16A to 16E are graphs showing the results of P101(0117) treatment improving spatial work memory and cognitive memory damage of the Aβ-administered mouse model.

Hereinafter, exemplary embodiments will be described in detail so that those skilled in the art can easily implement them. The embodiments may be implemented in various different forms, and are not limited to the specific embodiments described herein.

new Peptide (New Peptide)

As illustrated in FIGS. 1 to 6, the novel peptide is structured to have an effective structure for disaggregating or inhibiting aggregation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates.

Water-soluble Aβ aggregates and/or insoluble Aβ aggregates include Aβ oligomers, Aβ protofibrils, Aβ fibrils, Aβ plaques, and the like.

The novel peptide is a peptide in which two or more functional peptide fragments (FPFs) are linked to the link core (LC) with their free ends. That is, a plurality of functional peptide fragments (FPF) based on the link core (LC) can flow freely with their respective free ends, and thus can directly bind to water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates, compared to peptide fragments composed of monomers. Water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates can be very effectively degraded or effectively inhibited aggregation.

The link core (LC) and the functional peptide fragment (FPF) can be bound either directly or indirectly. Functional peptide fragments may be directly coupled to the link core (LC) as illustrated in FIGS. 1 and 2, and functional peptide fragments (FPF) and the link core (LC) as illustrated in FIGS. 3 to 6. ) May be indirectly bound through a peptide arm (PA).

The link core (LC) may be composed of an amino acid having two or more amino groups or an amino acid having two or more carboxyl groups in order to directly or indirectly bind two or more functional peptide fragments (FPF). The amino acid having two or more amino groups may be Lys(K) or Arg(R). In particular, it may be Lys(K). The amino acid having two or more carboxyl groups may be Asp(D) or Glu(E).

Peptide arms (PA) are functional peptide fragments (FPF) that dissolve or dissolve water-soluble Aβ aggregates and/or Aβ aggregates more smoothly, or do not interfere with these functions, so as not to interfere with these net charges (net charge) It may be a peptide of 0 and / or a flexible peptide. For example, Z constituting the peptide arm (PA) may be glycine (Gly, G) or serine (Ser, S). Furthermore, the peptide arm (PA) may be a peptide comprising 4 to 16 amino acids comprising a GGGS sequence as a repeating unit. The peptide arm (PA) may be a peptide having a GGGSGGGS sequence when considering the degree of flexibility. Of course, if the functionality of the functional peptide fragment (FPF) is sufficiently secured without the peptide arm (PA), the peptide arm (PA) may be omitted.

Functional peptide fragments (FPF) consist of up to 17 amino acids. 1 to 6 illustrate the same sequence as SEQ ID NO: 1 of natural Aβ1 to 17 (SEQ ID NO: 1), but a functional fragment may be any sequence that contains at least 10 amino acids of 17 amino acids of natural Aβ1 to 17. As illustrated in FIGS. 1 to 6, at least 10 or more amino acids may constitute a contiguous sequence.

At least 10 or more amino acids may constitute a non-contiguous sequence.

When at least 10 or more amino acids constitute a contiguous sequence, the functional peptide fragment (FPF) may be a peptide having any one of the sequences of SEQ ID NO: 1-36.

When at least 12 amino acids constitute a non-contiguous sequence, it may be a peptide having any one of the sequences of SEQ ID NO: 37 to 9310.

Sequences constituting non-contiguous sequences of at least 10 to 11 amino acids are not described in Table 1 below, but two or more consecutive amino acids between Xaa or two or more consecutive amino acids at the N or C terminus. Combinations that exist may be suitable.

Furthermore, when the position of Xaa is 1,3,7,11 from the N-terminal, an extremely acidic amino acid may be suitable.

SEQ ID
natural
Aβ1-17
similar
Amino acid number SEQUENCE
NO One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 One 17 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 2 16 Xaa Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 3 16 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Xaa 4 15 Xaa Xaa Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 5 15 Xaa Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Xaa 6 15 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Xaa Xaa 7 14 Xaa Xaa Xaa Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 8 14 Xaa Xaa Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Xaa 9 14 Xaa Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Xaa Xaa 10 14 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Xaa Xaa Xaa

In SEQ ID NO: 1 to 9310 of Table 1, Xaa represents any amino acid selected from 20 amino acids or the amino acid at the corresponding position is deleted. Furthermore, when the position of Xaa is 1, 3, 7, 11 from the N terminal, a polar acidic amino acid may be suitable. As an extremely acidic amino acid, Asp, Glu, etc. may be suitable. In the case of positions 5, 6, 13, 14 and 16 from the N-terminal, a polar basic amino acid may be suitable. As the basic amino acid, Asn, Gln, His, Lys, Arg, etc. may be used. Hydrophobic amino acids may be suitable for positions 2, 12, and 17 from the N terminus. Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and the like can be used as hydrophobic amino acids.

As illustrated in FIGS. 1 to 4, when the new peptide includes two functional peptide fragments (FPF), the new peptide may be a peptide dimer represented by the following general formula (1).

[Formula 1]

FPF-PAx-LC-PAx-FPF

In the above formula, FPF is a peptide having a sequence in which at least 10 amino acid sequences are identical to natural Aβ1 to 17,

The PA is a peptide having 4 to 16 amino acids,

X is 0 or 1,

The LC is an amino acid having at least two amino groups.

When the new peptide is chemically synthesized, the link core (LC) comprises a C terminus, and the free end of a functional peptide fragment (FPF) constitutes an N terminus, respectively.

When the new peptide is biosynthesized, the link core (LC) is located in the middle of the peptide, one of the free ends of the functional peptide fragment (FPF) is the N end, and the other may be the C end.

As illustrated in FIG. 5, when the new peptide includes three functional peptide fragments (FPF), the new peptide may be a peptide trimer represented by the following Formula 2.

[Formula 2]

In the above formula, FPF is a peptide having a sequence in which at least 10 amino acid sequences are identical to natural Aβ1 to 17,

The PA is a peptide having 4 to 16 amino acids,

X is 0 or 1,

The LC is an amino acid having at least two amino groups.

As illustrated in FIG. 6, when the new peptide includes four functional peptide fragments (FPF), the new peptide may be a peptide tetramer represented by Chemical Formula 3.

[Formula 3]

In the above formula, FPF is a peptide having a sequence in which at least 10 amino acid sequences are identical to natural Aβ1 to 17,

The PA is a peptide having 4 to 16 amino acids,

X is 0 or 1,

The LC is an amino acid having at least two amino groups.

Functional peptide fragments (FPF) represented by SEQ ID NO: 1 to 41038 described peptides constituting dimers, trimers, and tetramers, but functional peptide fragments (FPF) are water-soluble Aβ aggregates and/or insoluble even in monomeric state Aβ aggregates can be disaggregated or inhibit aggregation.

The amino acids constituting the peptide are L-form, D-form, and DL-form, and the amino acids constituting the peptide according to the embodiments may include all of them. Peptides include variants, and may include those in which a part of the peptide structure is mutated without changing the main activity by natural or artificial variation.

Peptide Produce

Peptides according to the examples can be synthesized by various methods well known in the art. Specifically, it can be produced in vitro through chemical synthesis, or by genetic recombination and protein expression systems, or by biosynthesis such as cell-free protein synthesis.

In the case of using chemical synthesis, the peptide can be prepared by the method described by Schroder and Lubke, ``The Peptides'' Vol. 1, Acadmeic Press, New York (1965), and the like, by a method such as solution phase synthesis or solid phase synthesis. Can be produced. Examples of methods for forming peptide bonds are acyl azide method acyl halide method, acyl imidazole method, carbodiimide method, phosphonium method, anhydride method, mixed anhydride method, oxidation-reduction method, and Woodward reagent K. And how to do it. Before carrying out the condensation reaction, a carboxyl group, an amino group, etc., which are not involved in the reaction, can be protected, and a carboxyl group, etc., involved in the condensation reaction can be activated by a method known in the art. Examples of the group protecting the carboxyl group include ester-forming groups such as methyl, thi-butyl, aryl, pentafluorophenyl, benzyl, para-methoxybenzyl, or methoxyethoxymethyl. Examples of groups protecting amino groups are tritylcarbonyl, aryloxycarbonyl, cyclohexyloxycarbonyl, trichloroethyloxycarbonyl, benzyloxycarbonyl, thi-butoxycarbonyl, and/or 9-flu Orenyl methyloxycarbonyl and the like. Examples of active forms of carboxyl groups include mixed anhydrides, azides, acylchlorides or active esters [alcohols (eg pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydr Esters with hydroxy-5-norbornene-2,3-dicarboxylimide, N-hydroxysuccinimide, N-hydrooxyphthalamide, or 1-hydroxybenzotriazole); and the like. . Solvents that can be used in the condensation reaction to form peptide bonds are benzene, toluene, hexane, acetone, nitromethane, cyclohexane, ether, chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethylsulfoxide, It may be a single solvent such as pyridine, dioxane, tetrahydrofuran, water, methanol, or ethanol, or a mixed solvent thereof. The reaction temperature may be in the range of about -70°C to 100°C generally used for the reaction, and more preferably in the range of -30°C to 30°C. The reaction for removing the peptide protecting group varies depending on the type of protecting group, but can be removed using an acid compound, a base compound, or a transition metal, etc., which can leave the protecting group without affecting the peptide bond. The protecting group can be removed by acid treatment, for example, with hydrogen chloride, hydrogen bromide, hydrogen fluoride, acetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, trimethylchlorosilane, or mixtures thereof. When carrying out the reaction for removing the protecting group by acid treatment, anisole, phenol, and thioanisol may be added as an auxiliary agent to accelerate the reaction. The protecting group can be removed using a base treatment, for example, ammonia, diethylamine, hydrazine, morpholine, en-methylpyrrolidine, piperidine, sodium carbonate, or mixtures of these bases. The protecting group can be removed using a transition metal treatment, for example, zinc, mercury, palladium/hydrogen, and the like. After completion of the reaction, the peptide may be purified by conventional peptide purification methods, for example, extraction, layer separation, solid precipitation, recrystallization or column chromatography.

Pharmaceutical formulation, formulation, composition

The peptide according to the embodiments may exist in a base form or a pharmaceutically acceptable salt form. Pharmaceutically acceptable salts refer to salts obtained by combinations of the compounds with organic or inorganic acids. Examples of salts include hydrochloride, sulfate, phosphate, acetate, citrate, tartrate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, or paratoluenesulfonate.

The peptide according to the embodiments may further include a targeting sequence, a tag, a labeled residue, a half-life, or an amino acid sequence designed for a specific purpose to increase the stability of the peptide. Peptides according to embodiments may include sugar chain compounds, lipid compounds, nucleic acid compounds, other types of peptides or proteins, and the like. Peptides according to embodiments may be coupled to coupling partners such as effectors, drugs, prodrugs, toxins, peptides, delivery molecules, and the like. The peptide according to the embodiments may be mounted on various formulations such as nanoparticles or liposomes, and may have various forms connected to the inner and outer surfaces of the formulation.

The pharmaceutical composition according to the embodiments may be formulated with a pharmaceutically acceptable and physiologically acceptable adjuvant with an active pharmaceutical active ingredient consisting of a peptide or a pharmaceutically acceptable salt thereof. As an auxiliary agent, excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants or flavoring agents may be used. In addition, it may be further formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers.

Pharmaceutical composition formulations can be made in a variety of formulations, such as injections, suppositories, powders, and transdermal patch preparations, with pharmaceutically acceptable and physiologically acceptable additives.

Pharmaceutically compatible and physiologically acceptable additives can be applied according to a number of factors well known to those skilled in the art, such as the specific bioactive substance employed, its concentration, stability and intended bioavailability; Diseases and disorders or conditions to be treated; The subject to be treated, age, size and general condition; The route used to administer the composition, including, but not limited to, factors such as ocular, topical, transdermal, and muscle should be considered. In general, additives available for administration of physiologically active substances other than the oral administration route include D5W (5% glucose in water), dextrose and an aqueous solution containing physiological salt within 5% of the volume, and topical injection in lesions In this case, various injectable hydrogels can be used to enhance the therapeutic effect and increase the duration. In addition, pharmaceutically available additives may include additional ingredients that can enhance the stability of the active ingredients such as preservatives and antioxidants. The peptides or pharmaceutically acceptable salts, inhibitors, or compositions thereof of the present invention can be formulated by any suitable method in the art, for example, disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA (latest edition), etc. With reference to the method, it can be preferably formulated according to each disease or component.

The peptide according to the embodiments or a pharmaceutically acceptable salt thereof may be stored as a physiological saline solution, and may be lyophilized in an ampoule after adding a pharmaceutical additive or excipient, etc. It can be dissolved in saline solution and used as an injection.

Use as an agent for preventing, improving or treating amyloid disease

The peptides according to the embodiments can decompose water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates very effectively or effectively inhibit aggregation. Accordingly, a composition comprising the peptide according to the embodiments or a pharmaceutically acceptable salt thereof as a pharmaceutical component can prevent or treat amyloid disease caused by aggregation of Aβ. Prevention refers to any action that suppresses disease or delays the onset of disease by administration of the composition. Treatment refers to any act that ameliorates (improves) or beneficially alters a disease by administering a composition.

Amyloid diseases include Alzheimer's disease, cerebral amyloid vasculopathy, Down's syndrome, amyloid stroke, systemic amyloid disease, Dutch type amyloidosis, Niemann-Pick disease, etc. Any disease caused by (aggregation) can be targeted.

In particular, it was confirmed that the peptides according to the examples caused degradation and aggregation inhibition of Aβ in vitro (see FIGS. 7 to 12 ), thereby reducing toxicity induced by Aβ (FIGS. 15A to 15A ). 15d), it was confirmed that intravenous administration of peptides according to the examples reduces Aβ plaques in the brain of the APP/PS1 transgenic mouse model (see FIGS. 14A-14D ). In addition, when the peptides according to the embodiments were administered to the AD mouse model induced through Aβ administration, it was confirmed that Aβ induced cytotoxicity can be reduced by controlling apoptotic signaling (FIGS. 15A to 15A ). 15d), it was confirmed that the effect of improving the cognitive decline (see FIGS. 16a to 16e). These results indicate that the peptide according to the examples may be an active pharmaceutical ingredient of a pharmaceutical composition for treating Alzheimer's disease (AD) disease.

Thus, the present disclosure can treat amyloid diseases by administering the peptides according to the embodiments or pharmaceutically acceptable salts thereof to mammals including humans in need of administration. Mammals can also be administered to livestock such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs or cats, where amyloid disease can be inhibited or reduced by administering the peptides according to the embodiments or pharmaceutically acceptable salts thereof. Can be used. The pharmaceutically effective amount of the peptide or its pharmaceutically acceptable salt is not particularly limited, but may be appropriately changed depending on the severity of intestinal disease symptoms, the patient's age, weight, health status, sex, route of administration, and treatment period.

The peptide according to the embodiments or a pharmaceutically acceptable salt thereof is mainly parenterally, for example, topical injection, intravenous or subcutaneous injection, intraventricular or intrathecal administration, transdermal administration, or nasal administration or intrarectal administration. To be administered. The dosage may be based on an adult (60 kg in weight), but it is natural that it may vary depending on weight, physical condition, etc.

Other uses

The peptides or salts thereof according to the embodiments can be used to diagnose degenerative neurological diseases. In the case of a diagnostic method, the step of reacting a peptide or a salt thereof, or a composition comprising the same, with a sample according to the embodiments, and comparing the beta amyloid concentration in the sample before and after reaction with the peptide or a salt thereof, or a composition comprising the same Through the steps, it is possible to diagnose the presence of degenerative neurological diseases.

For example, it is possible to diagnose degenerative neurological diseases by comparing concentrations before and after reacting a sample to be analyzed with a peptide or a salt thereof according to embodiments, or a composition containing them. Specifically, (a) measuring the first concentration of Aβ before administering the peptide according to the embodiments or a salt thereof, or a composition comprising them to a sample (blood or plasma, etc.), and (b) according to the embodiments. After administering a peptide or a salt thereof, or a composition containing them to a sample, the second concentration of Aβ is measured, and (c) the measured first concentration and the second concentration are compared, and if the second concentration is high, degenerative neurological disease Can be diagnosed. Samples that can be used at this time may include all forms of bio-fluids, such as blood, plasma, and serum, as well as cerebrospinal fluid, saliva, urine, tears, and runny nose.

Such diagnostic compositions and diagnostic methods are applicable not only to in vitro diagnostics, but also to in vivo diagnostics. For example, it is possible to diagnose by comparing the concentration of Aβ before and after administering the peptide according to the embodiments or a salt thereof, or a composition comprising them to the body. The concentration of Aβ can be analyzed by imaging methods. The diagnostic method is not particularly limited, and may include all various methods known in the art. For example, the method described in Korean Patent Publication No. 2014-0128265, Korean Patent Publication No. 2014-0128266, or the like can be used.

Peptides according to the embodiments or salts thereof may be used as food compositions, such as health functional food for the prevention of cognitive impairment. When applied as a food composition, it may contain various flavoring agents, natural carbohydrates, and the like as additional ingredients, like a conventional food composition.

Hereinafter, preferred experimental examples are provided to help understanding of the present invention, but the following experimental examples are only illustrative of the present invention and the scope of the present invention is not limited to the following examples.

Peptide Produce

Manufacturing example 1: P101(0117) (NH ₂ - SEQ ID NO:1 - GGGSGGGS - εK ( COOH )- GGGSGGGS - SEQ ID NO:1(parallel) -NH ₂ ) Produce

Lysine (Fmoc-Lys(Fmoc)-OH) (1.1 mmol) in which the amine moiety was protected with a protecting group was dissolved in a mixture of 2 ml of DMF:DCM (Dimethylformamide:dichloromethane=1:1 volume ratio), and then 156 μl of DIC ( N,N'-diisopropylcarbodiimide) was added and then activated in an ultrasonic bath for 2 minutes. Thereafter, 0.051 g of DMAP (4-dimethylaminopyridine) was dissolved in the shaken mixture, and the mixture was shaken for 1 hour in a resin swollen from the cartridge (TentaGel R PHB 0.125 mmol, substrate) for 1 hour to perform anhydrous coupling reaction to react with the resin. Lysine was bound. The resin after the reaction was completed was washed for 20 seconds each in the order of DMF, DCM and DMF.

In order to remove Fmoc of lysine bound to the resin, it was removed by shaking twice with 15 ml of 20% piperidine (1:4 piperidine/DMF, v/v) for 15 minutes twice.

On the other hand, using Fmoc-protected amino acids, the peptide arms (2.2 mmol) and the functional peptide fragments (2.2 mmol) of SEQ ID 1 (Aβ 1-17) having the H2N-GGGSGGGS-COOH amino acid sequence are in sequence. 5 ml of 0.2 M amino acid, 1 ml of 1.0 M oxyma, and 2 ml of 0.5 M DIC were added in order to react, followed by reaction with the lysine with the protecting group removed for 50 minutes.

During each reaction, the substrate-bound peptide-based resin was washed with DMF, DCM and DMF for 20 seconds, respectively, and the Fmoc-protecting group was treated with 20% piperidine (1:4 piperidine/DMF, v/v) ) It was removed by shaking twice with 6 ml for 15 minutes twice.

After all of the above processes, the Fmoc-protecting group is removed and the completed substrate-coupled protein dimer is washed with DCM and stored under reduced pressure with TFA/anisole/TIS (95:2.5:2.5, v/v/v). After shaking for 4 hours, the resin and the protein dimer were separated, and TFA was completely removed using a vacuum concentrator. Thereafter, the protein dimer was treated with 30 ml of diethyl ether and centrifuged at 3000 rpm for 15 minutes. After removing the supernatant of diethyl ether, 30 ml of diethyl ether was added again, centrifuged for 15 minutes at 3000 rpm, and the precipitated white protein dimer was dissolved in 25% acetonitrile aqueous solution and lyophilized to parallelize the white solid powder. A three-dimensional protein dimer was obtained. After lyophilization, it was purified using HPLC.

Manufacturing example  2: P101(0317) (NH ₂ - SEQ ID NO:4 - GGGSGGGS - εK ( COOH )- GGGSGGGS - SEQ ID NO:4 (parallel)-NH ₂ ) Produce

P101 was performed in the same manner as in Production Example 1, except that it was prepared to be a functional peptide fragment sequence of SEQ ID NO:4 (Aβ03 to 17) instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1 to 17). 0317) was prepared. In SEQ ID NO:4 used in Preparation Example 2, the amino acid at position 1 and 2 was deleted (X=0).

Manufacturing example 3: P101(0517) (NH ₂ - SEQ ID NO:11 - GGGSGGGS - εK ( COOH )- GGGSGGGS - SEQ ID NO:11 (parallel)-NH ₂ ) Produce

P101 was performed in the same manner as in Preparation Example 1, except that it was prepared to be a functional peptide fragment sequence of SEQ ID NO:11 (Aβ05-17) instead of the functional peptide fragment of SEQ ID NO:1 (Aβ1-17). 0517). For SEQ ID NO:11 used in Preparation Example 3, the amino acid at position 1, 2, 3, 4 was deleted (X=0).

Manufacturing example 4: P101(0717) (NH ₂ - SEQ  ID NO:22 - GGGSGGGS - εK ( COOH )- GGGSGGGS - SEQ ID NO:22  (parallel)-NH ₂ ) Produce

P101 was performed in the same manner as in Preparation Example 1, except that it was prepared to be a functional peptide fragment sequence of SEQ ID NO:15 (Aβ01 to 13) instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1 to 17). 0717). For SEQ ID NO:11 used in Preparation Example 4, the amino acid at position 1, 2, 3, 4, 5, 6 was deleted (X=0).

Comparative manufacturing example 1: C101(0119) (NH ₂ - Aβ1 ~19- GGGSGGGS - εK ( COOH )- Aβ1 ~19(parallel)-NH ₂ ) Produce

C101 (0119) was prepared in the same manner as in Preparation Example 1, except that a peptide having a sequence of natural Aβ1 to 19 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1 to 17). .

Comparative manufacturing example  2: C101(0121) (NH ₂ - Aβ1 ~21- GGGSGGGS - εK ( COOH )- Aβ1 ~21(parallel)-NH ₂ ) Produce

C101 (0121) was prepared in the same manner as in Production Example 1, except that a peptide having a sequence of natural Aβ1 to 21 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1 to 17). .

Comparative manufacturing example  3: C101(0123) (NH ₂ - Aβ1 ~23- GGGSGGGS - εK ( COOH )- Aβ1 ~23(parallel)-NH ₂ ) Produce

C101 (0121) was prepared in the same manner as in Production Example 1, except that a peptide having a sequence of natural Aβ1 to 21 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1 to 17). .

Comparative manufacturing example  4: C101(0125) (NH ₂ - Aβ1 ~25- GGGSGGGS - εK ( COOH )- Aβ1 ~25(parallel)-NH ₂ ) Produce

C101 (0125) was prepared in the same manner as in Preparation Example 1, except that a peptide having a sequence of natural Aβ1 to 25 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1 to 17). .

Comparative manufacturing example 5: C101(0917) (NH ₂ - Aβ9 ~17- GGGSGGGS - εK ( COOH )- Aβ9 ~17(parallel)-NH ₂ ) Produce

C101 (0917) was prepared in the same manner as in Preparation Example 1, except that a peptide having a sequence of natural Aβ9-17 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1-17). .

Comparative manufacturing example 6: C101(1117) (NH ₂ - Aβ11 ~17- GGGSGGGS - εK ( COOH )- Aβ11 ~17(parallel)-NH ₂ ) Produce

C101 (1117) was prepared in the same manner as in Preparation Example 1, except that a peptide having a sequence of natural Aβ11-17 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1-21). .

Comparative manufacturing example  7: C101(1317) (NH ₂ - Aβ13 ~17- GGGSGGGS - εK ( COOH )- Aβ13 ~17(parallel)-NH ₂ ) Produce

C101 (1317) was prepared in the same manner as in Preparation Example 1, except that a peptide having a sequence of natural Aβ13-17 was linked instead of a functional peptide fragment of SEQ ID NO:1 (Aβ1-21). .

Amyloid beta ( Aβ ) Coagulation decomposition and/or aggregation inhibition effect measurement

Sample preparation

Peptides consisting of Aβ1 to 17 monomer fragments (MAβ(0117)), peptide dimers prepared according to Preparation Examples 1 to 4, and Comparative Preparation Examples 1 to 7, were respectively dissolved using 1% DMSO at a concentration of 2.5M. Then, the materials were diluted with 1% DMSO solution and used.

In the amyloid beta (Aβ) solution, Aβ1-42 monomers were dissolved in DMSO to a concentration of 10 mM. Then, it was diluted to a concentration of 100 uM using DW and stored on ice.

On the other hand, Thioflavin-T was dissolved in 50 mM glycine buffer (pH 8.5) to a concentration of 5 mM. Thereafter, the mixture was diluted to 5 uM using 50 mM glycine buffer (pH8.5) and stored in the dark with the light blocked.

Amyloid beta ( Aβ ) Coagulation decomposition effect measurement

The previously prepared amyloid beta (Aβ) solution was respectively placed in a 1.5 mL microcentrifuge tube to be 50 uM, and reacted at 37° C. for 72 hours. Peptide consisting of Aβ1~17 monomer fragments for each of the 1.5mL micro-centrifuge tubes (MAβ(0117)), peptide dimers prepared according to Preparation Examples 1 to 4 and Comparative Preparation Examples 1 to 7, respectively, have different concentrations of 50uM and 250uM. Each was treated with and reacted at 37° C. for 48 hours.

After the reaction was completed, the reaction solution was placed in each well of 25 uL in a 96-well fluorescence assay plate, and 75 uL of the previously prepared Thioflavin-T solution was added to each well. After reacting at room temperature for 5 minutes in the dark, fluorescence values were measured at 450 nm excitation and 485 nm emission using a multi-mode microplate reader.

As a result of the measurement, only the amyloid beta (Aβ) was treated, and the fluorescence value of the group aggregated for 72 hours was converted into a% value and displayed as 100, and the obtained fluorescence value was converted to a relative value and shown in FIGS.

It can be seen from the results of FIG. 7 that the P101(0117) peptide having two functional peptide fragments compared to the monomer (MAβ(0117)) has a cohesive decomposition effect of almost 3-4 times or more.

From the results of FIG. 8, it can be seen that a functional peptide fragment consisting of up to 17 amino acids can maximize the effect of agglutination.

It can be seen from the results of FIG. 9 that when the functional peptide fragment has the same amino acid sequence of natural Aβ1 to 17 and at least 10 amino acid sequences, it is possible to maintain a cohesive effect.

Amyloid beta ( Aβ ) Measurement of the effect of inhibiting aggregation

The previously prepared amyloid beta (Aβ) solution was placed in a 1.5 mL microcentrifuge tube to be 50 uM, and then a peptide consisting of Aβ1 to 17 monomer fragments per 1.5 mL microcentrifuge tube (MAβ(0117)), Preparation Examples 1 to 4, Peptide dimers prepared according to Comparative Preparation Examples 1 to 7 were respectively treated at different concentrations of 50 uM and 250 uM, and then reacted at 37° C. for 72 hours.

After the reaction was completed, the reaction solution was added to each well of 25 uL in a 96-well fluorescence assay plate, and 75 uL of the previously prepared Thioflavin-T solution was added to each well. After reacting in the dark for 5 minutes at room temperature, fluorescence values were measured at 450 nm excitation and 485 nm emission using a multi-mode microplate reader.

As a result of measurement, only the amyloid beta (Aβ) was treated, and the fluorescence value of the group aggregated for 72 hours was converted into a% value, expressed as 100, and the obtained fluorescence value was converted to a relative value, and shown in FIGS.

From the results of FIG. 10, it can be seen that the P101 (0117) peptide having two functional peptide fragments compared to the monomer (MAβ (0117)) has an aggregation inhibition effect of almost 3-4 times or more.

It can be seen from the results of FIG. 11 that a functional peptide fragment consisting of up to 17 amino acids can maximize aggregation inhibitory effect.

From the results of FIG. 12, it can be seen that the functional peptide fragment can maintain the aggregation inhibitory effect when the amino acid sequence of natural Aβ1 to 17 and at least 10 amino acid sequences are the same.

P101(0117) and With Aβ oligomer Direct interaction confirmation

To understand how P101(0117) reduces amyloid plaques and neuronal cell death in 2xTg-AD mice, the present inventors have determined that between P101(0117) and Aβ42 aggregates by in vitro (in vitro) physical and biological assays and structural screening. The presence of direct interactions was investigated. We evaluated binding kinetics using surface plasmon resonance analysis and affinity using the Biacore T200 system. The results are shown in FIG. 13.

Surface plasmon resonance analysis was performed using a Biacore T200 instrument and Series S carboxymethylated (CM5) dextran matrix sensor chip (GE Healthcare). HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P20) was used as a running buffer at 25°C. First, the present inventors dissolved Aβ42 peptide in DMSO (20 mg/ml, w/v) and diluted 100-fold in distilled water to make 200 μg/ml. The prepared Aβ42 peptide was cultured at 37°C for 7 days. Finally, the cultured Aβ42 peptide (200 μg/ml) was diluted with 10 mM sodium acetate solution (pH 4.0) to prepare 40 μg/ml of Aβ42 aggregate. Then, the agglomerates were immobilized covalently on the chip surface by amine bonding chemistry. The activated carboxymethyl group remaining on the surface was blocked by administration of 1 M ethanolamine (pH 8.0). The immobilization value of the Aβ42 aggregate was 6000 RU and the theoretical Rmax of the Aβ42 aggregate was 7017 RU. P101 (0117) was prepared with PBS-T running buffer (10 mM phosphate, 135 mM NaCl, 27 mM KCl and 0.005% surfactant P20) containing 1% DMSO as a serially diluted sample. For this binding and kinetics analysis, P101 (0117) was administered at a flow rate of 30 μl/min for 120 seconds.

As shown in FIG. 13, after fixing the Aβ42 aggregate as a ligand on the CM5 sensor chip through amine coupling, P101 proteins of different concentrations (62.5 to 5000 nM) were flowed through the channel. Although it is difficult to calculate the exact binding constant due to Aβ aggregated in various forms, the result of substantial curve fitting efficacy (0.0843 RU2 of calculated χ) shows that P101(0117) is directly related to Aβ42 aggregate. Binding and it was found to be a dose-dependent interaction.

2xTg -Check the effect of brain amyloid plaque reduction in AD mice

Aβ40 or Aβ42 monomers accumulate to form Aβ oligomers, and these oligomers accumulate to form Aβ fibrils, and these fibrils accumulate to form Aβ plaques and thus accumulated Aβ plaques Since it is known to cause memory deficiency in AD patients, it was investigated whether brain amyloid plaque reduction occurs when the peptide (P101(0117)) according to the embodiment is administered.

(1) 2xTg-AD mouse model preparation

Doubly mutated transgenic mice (2xTg-AD, phylogenetic name; B6.Cg-Tg (APPswe, PS1dE9) 85Dbo/J) and wild type (C57BL/6J) mice mutated with two genes were Jackson Laboratory (Bar Harbor, Maine, USA). These 2xTg-AD mice were maintained as double hemizygotes across the wild type mice. The genotypes of all APP and PS1 genes were confirmed by PCR analysis using tail DNA according to the standard PCR conditions of the Jackson Laboratory. The mice were housed in 4-6 animals per plastic cage in the animal breeding room, maintained at 21±1°C, alternating the 12-hour contrast cycle, and freely ingested food and water. At the beginning of the experiment, 27 mice were evaluated at 4 months of age with 2xTg-AD (n = 13, male) and wild type (n = 14, male). All animal experiments were conducted in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978).

(2) P101(0117) treatment on mouse model

Intravenous administration model: 100 μmol of unagglomerated P101 (0117) was administered intravenously (i.v.) twice a week for 4 weeks into the tail vein of a 12-month-old 2xTg-AD mouse. The control group received the same amount of vehicle (2.5% DMSO in PBS). The administration was performed with a 1 mL Kovacs-syringe.

Intracerebroventricular (ICV) administration model: Synthetic Aβ42 and P101 (0117) were dissolved in sterile saline 250 and 25 pmole (5 μL 50 μM, 5 μL 5 μM), respectively. Mice were anesthetized with 2% avertin (20 μg/g, IP) prior to ICV administration. Lysed Aβ42 and P101 (0117) were administered to the ICV region of the mouse brain according to the previously reported protocol. Specifically, we administered Aβ42 to the ICV region at 1.0 mm posterior of the bregma, 1.8 mm lateral to the sagittal, 3.6 mm abdomen, and 2.4 mm deep using a Hamilton syringe with a 26-gauge stainless-steel needle.

(3) Brain and plasma preparation

Mice were anesthetized with 2% avertin (20 mg/g, i.p.). Blood was collected from the vena cava and transferred to an EDTA tube using a protease inhibitor cocktail (Roche Diagnostics, cat# 11836170001) and the tube was gently shaken. Plasma was obtained from the EDTA-treated blood and immediately frozen with dry ice and stored in a freezer at -80°C. After collecting the blood, the mice were perfused with 0.9% NaCl and the brain excised. The hemibrains were fixed overnight at 4° C. in 4% paraformaldehyde (pH 7.4). For cryopreservation, the brain was soaked in 30% sucrose for 48 hours. The other hemibrains were dissected into the cortical and hippocampal regions and homogenized on ice for 30 min in lysis buffer (10 mM Tris-HCl, 5 mM EDTA in 320 mM sucrose, pH 7.4) containing 1X protease inhibitor cocktail. The sample was centrifuged at 4500°C for 13500 rpm for 30 minutes. The BCA protein analysis kit (ThermoFisher, cat #23225) was used to measure lysate concentration according to the manufacturer's instructions. The absorbance was read at 560 nm.

(4) Immunohistochemical staining and ThS staining

Brains were cut to a thickness of 35 μm using a cryostat (Leica CM1800) and stained with GFAP (Milipore AB5541). Alexa Fluor 568-conjugated secondary antibody (Life technologies) was used for fluorescence detection. ThS staining was performed in ethanol (50%) at a concentration of 500 μM for 7 minutes. Then, the sections were rinsed sequentially in 100, 90 and 70% ethanol for 10 seconds and placed in PBS. Images were obtained using a Leica DM2500 fluorescence microscope.

(5) Results

We used the APP/PS1 (2xTg-AD) mouse model. The model over-produced human Aβ and showed positive amyloid plaques in the cortex and hippocampus of 6-month-old mice. The inventors administered P101 (0117) 8 times intravenously in 10 month old 2×Tg-AD mice for 4 weeks (FIG. 14A ). Mice were sacrificed after anesthesia with 2% avertin (20 μg/g, i.p.) 3 days after the last dose. The inventors took blood from the inferior vena cava with a 1 mL syringe. The collected blood was transferred directly to an EDTA tube and plasma was separated. The obtained brain was fixed with 4% paraformaldehyde, and a 30% sucrose solution was used for cryopreservation.

To stain the frozen brain with 30 μm for staining with thioflavin S (ThS), make tissue sections, stain β sheet rich Aβ plaques, and glial fiber acidic protein Glial cells were stained using (glial fibrillary acidic protein; GFAP) antibody (FIG. 13B). As shown in FIG. 14B, when compared with a control mouse (a mouse administered with vehicle (10% DMSO in PBS)), the number of plaques and the total plaque area were significantly reduced in 2xTg-mouse administered with P101 (0117). (Fig. 14c and 14d). It has been reported that the total or relative concentrations of Aβ40 and Aβ42 are related to the development of familial and sporadic Alzheimer's disease.

Overall, these results indicate that administration of P101 (0117) degrades Aβ42 plaques present in the 2xTg-AD model mouse brain and induces it into the Aβ42 peptide.

P101(0117) By Aβ Confirm the effect of improving induced cytotoxicity

The loss of nerve cells induced by amyloid protein is a key feature of AD. Cell viability analysis (MTT analysis) was performed using two cells (BV2 and HT22 cells) to investigate whether P101 (0117) affects neuronal cell death. In addition, in order to discover the signaling mechanism of P101 (0117) in cytotoxicity, the present inventors analyzed released apoptotic signaling molecules released from cells by Western blot analysis using the above cultures.

(1) Cell culture (BV2 cells, HT22 cells)

BV2 and HT22 cells were obtained from the Korea Cell Line Bank (Seoul National University, Korea). Cells were cultured in Dulbecco's 65 modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 units/ml penicillin, 50 μg/ml streptomycin. Major astrocytes were prepared from newborn C57BL/6 mice (P0-3) as previously described. The cerebral cortex and hippocampus were incised and ground to a single cell suspension by grinding and plated with 0.1 mg/ml poly-D-lysine (PDL) pre-coated 10 cm dish. These cells were treated with 25 mM glucose, 10% (vol/vol) heat-activated equine serum, 10% (v/v) heat-inactivated fetal bovine serum, 2 mM glutamine and 1000 units/ml penicillin-streptomycin. Maintained in supplemented DMEM medium. On the third day of culture, the cells were washed with PBS and the medium was replaced. The next day, cells were transferred to a new medium at a density suitable for the experiment. All cell cultures were maintained at 37°C in a 5% CO2 incubator.

(2) Cell viability analysis (MTT analysis) method

The cultured BV2 and HT22 cells were 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazorium bromide (3-(4,5-dimethyl-2-thiazolyl)-2, Cell viability was analyzed using 5-diphenyltetrazolium bromide (MTT) analysis.

The experiment was performed with DMEM supplemented with starvation medium, 2% FBS.

BV2, HT22 cells (5×10 ³ cells/well) and major astrocytes (2×104 cells/well) were cultured in 96-well plates. After adding MTT reagent (15 μM) to each well, Aβ40, Aβ42, and P101 (0117) were treated. After incubation for 4 hours, 100 μl of the solubilization solution was added and left at room temperature for 16 hours. The formazan product was measured using an EnSpire® Multimode Plate Reader at a wavelength of 570 nm. In addition, neuronal cell death was visualized using the Live/Dead®Viability/Cytotoxicity kit (Molecular Probes) according to the manufacturer's instructions. The results are as shown in Figs. 15A to 15D.

(3) Western blot Assay method

Samples of the prepared cells were separated by SDS-PAGE for Western blot analysis and transferred to PVDF membrane. The membrane was treated at 4° C. overnight using a primary antibody, and detected using a secondary antibody conjugated with Horseradish Peroxidase (HRP). Deployment of the blot was performed with a Clarity Western ECL substrate (Bio-Rad) according to the manufacturer's instructions.

(4) Results

The MTT assay was followed by treatment with Aβ40 (FIG. 15A) or Aβ42 (FIG. 15B) alone, and cell proliferation in combination with hippocampal nerve cell line HT22, microglial cell line BV2, and P101 (0117) in major astrocytes. Was performed to quantify. 15A and 15B, Aβ40 induced neuronal cell death was significantly reduced after treatment with P101 (0117) in all three cell lines.

The results were further confirmed by a survival experiment, LIVE/DEAD®Viability/Cytotoxicity Assay for visually detecting cell death. Cells treated with nothing showed minimal degree of endogenous cell death, whereas cells treated with Aβ40 showed almost 50% cell death. Neuronal cell death was confirmed to be significantly recovered when the cells were treated with P101 (0117) with Aβ40 (FIG. 15C). Therefore, it was confirmed that P101 (0117) can block neuronal cell death induced by Aβ40 and Aβ42 in vitro (in vitro).

15D, different proteins were identified in the data (Bcl-2 protein soldier Bax is a pro-apoptotic protein; Cleaved caspase-3 is a cell). Basic in apoptosis; poly(ADP-ribose) polymerase-1 (Poly) which induces cell death in response to DNA damage and other genotoxic stress factors after proteolysis by caspase (ADP-ribose) polymerase-1, PARP1); cytochrome-C is released during cell death from mitochondria to the cytoplasm; and neuroglia fibers expressed in astrocytes to protect neurons from tissue degeneration and limit inflammation Western blot analysis of BV2 cell extracts treated with glial fibrillary acidic protein (GFAP) Aβ40 and P101 (0117) compared to Aβ40 alone treatment with Bax, cleaved caspase-3, and cleavage Decreased levels of cleaved PARP-1 (cleaved PARP-1) The analysis of major astrocytes showed that the combination treatment of Aβ40 and P101 (0117) was Bax, cleaved caspase-3, cleaved PARP-1, and It showed a decrease in the level of cytochrome-c The combination of Aβ40 with P101 (0117) showed an increase in GFAP.

It was confirmed that P101(0117) treated cells reduced Aβ induced apoptosis proteins and decreased apoptotic cell death.

Confirmation of the effect of suppressing the spatial working memory impairment of P101(0117)

We have previously confirmed that P101 (0117) binds directly to Aβ42 aggregates and lowers Aβ induced cytotoxicity in neuronal cell lines and major astrocytes.

To determine whether P101(0117) can improve Aβ induced memory impairment in vivo (in vivo), the present inventors conducted P101(0117) and Aβ42 before conducting behavioral tests using the AD mouse model administered with Aβ42. The appropriate ratio between was confirmed. The behavior test will be described later.

In addition, mice were sacrificed and brains dissected after behavioral testing. The present inventors confirmed the level change of the cell death signaling factor in the hippocampus region by Western blot, and the results are shown in FIG. 16g, and the brain dissection method and Western blot analysis are the same as those shown in the experimental examples described above.

(1) Behavior test method

The Y-maze test was performed 4 days after ICV administration to the mouse model. The Y-maze device is a plastic black maze with three passage-shaped arms labeled A, B and C. Each arm was 40 cm long, 12 cm high, 10 cm high at the top, and 3 cm wide at the bottom. The arms converged to the middle, forming a 4 cm equilateral triangle on the longest axis. The mouse was placed at the tip of one arm and allowed to move freely through the maze for 12 minutes. The series of arm movements were recorded manually. It was considered entry when all four limbs of the mouse were within one of the arms. When one mouse entered three different arms in succession, alternation counted. The impulsive alternation behavior was calculated based on the following equation.

Before each test, the labyrinth interior was rinsed with 70% ethanol solution to remove scent cues.

In the habituation phase to perform a new subject recognition test, mice were able to explore a new subject recognition test box for 10 minutes. In the familiarization phase, mice were placed in a box and interacted with two identical objects, a black plastic pyramid-shaped object 5 cm from the wall, for 10 minutes. In the test phase, one of the subjects was replaced with a new subject, a ceramic sheep-shaped doll, and mice were tested for 10 minutes. All sessions were recorded with EthoVision software, and the time taken to interact with each subject was manually analyzed.

(2) Results

We prepared one vehicle and four Aβ42 aggregate samples to induce AD-like behavior by intraventricular (ICV) administration in 8-week-old C57Bl/6 mice (male, n = 7-11 per group) ( Figure 16a). Among them, in order to confirm the inhibitory and therapeutic effects of Aβ42 aggregate toxicity of P101 (0117), two substances were pre-mixed and incubated (Group D) and immediately before ICV administration (Group E). The inventors conducted a Y-maze test and a new subject recognition test on the 4th day after ICV administration (FIG. 16B).

The Y-maze is a test that measures spatial working memory. In the Y-maze, the subject mouse can move freely, and the sequence of entry is recorded and analyzed. As shown in Fig. 16c, as a result of administration of Group D and Group E, the alternating ratio increased to an almost the same level as Group A, which is spatial cognitive function in both pre-aggregation or post-aggregation administration of P101(0117). It is said to improve to the normal level. It was confirmed that the control of the experiment was well performed by observing the difference in the overall entries was insignificant.

As shown in Fig. 16D, the NOR test was performed to evaluate cognitive recognition memory. All mouse groups explored two subjects on the affinity trail for almost the same amount of time. When a new object was presented, the Group B mice were unable to distinguish between the new and familiar objects. However, Group D and Group E with P101 (0117) co-administered to Aβ42 aggregates showed a significant increase in the time spent exploring new subjects compared to Group B.

As shown in Fig. 16E, in the group D and Group E showing improved memory impairment in the behavioral test, a decrease in protein levels of Bax and truncated caspase-3 was observed. No change in cytochrome-c expression was observed. Thus, as a result, it is suggested that P101 (0117) restores cognitive impairment in the AD mouse model induced by Aβ42 administration.

Stability experiment (KR1198047)

Stability evaluation was performed in human serum to evaluate the resistance of the radiolabeled peptide to proteolytic degradation and to predict its stability in vivo. 10 μCi of the radiolabeled peptide was added to human serum and cultured at 37° C. for 72 hours.

Partial sample samples were obtained at appropriate time intervals to measure the stability of the radiolabeled peptide. Through RP-HPLC analysis, it was confirmed that only desired radiolabeled peptides were observed. In addition, it can be seen that a high stability of 98% or more was maintained for a period of 72 hours or more. The results of the stability experiment in human serum indicate that the prepared peptide has high stability against hydrolysis and metabolic resistance. Although the experimental examples have been described above, the scope of rights is not limited thereby. The embodied form can be implemented in various ways within the scope of the detailed description of the invention and the accompanying drawings, and it is natural that this also belongs to the scope of rights.

Claims (26)

A peptide in which two or more functional peptide fragments are linked to the link core with respective free ends,
The functional peptide fragment is composed of up to 17 amino acids and is a functional peptide fragment comprising at least 10 or more amino acids among 17 amino acids of natural Aβ1 to 17, or a pharmaceutically acceptable salt thereof. Therapeutic pharmaceutical composition. A peptide in which two or more functional peptide fragments are linked to the link core with respective free ends,
The functional peptide fragment is composed of up to 17 amino acids, and a peptide that is a peptide fragment comprising at least 10 or more amino acids among 17 amino acids of natural Aβ1 to 17, or prevention of amyloid disease, including a pharmaceutically acceptable salt thereof Dragon pharmaceutical composition. The method according to claim 1 or 2,
The amyloid disease is Alzheimer's disease, cerebral amyloid vasculopathy, Down's syndrome, amyloid stroke, systemic amyloid disease, Dutch type amyloidosis, or Neiman-Pick disease. The method according to claim 1 or 2,
The amyloid disease is Alzheimer's disease pharmaceutical composition. According to claim 1,
The peptide or pharmaceutically acceptable salt thereof is a pharmaceutical composition having a water-soluble Aβ aggregate and / or water-insoluble Aβ aggregate degradation activity. According to claim 1,
The peptide or a pharmaceutically acceptable salt thereof is a pharmaceutical composition having an activity of inhibiting the accumulation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates. The method according to claim 1 or 2,
The link core is a pharmaceutical composition that is an amino acid having at least two or more amino groups or an amino acid having two or more carboxyl groups. The method according to claim 1 or 2,
The link core and the functional fragment is a pharmaceutical composition indirectly coupled by a peptide arm. The method of claim 8,
The peptide arm is a pharmaceutical composition that is a flexible peptide without net charge. The method of claim 9,
The peptide arm is a pharmaceutical composition comprising a repeating unit of GGGS. The method according to claim 1 or 2,
Wherein the link core is C-terminal, and each free end is N-terminal. The method according to claim 1 or 2,
The link core is located in the middle of the peptide, at least one of the free end of the N-terminal, at least one of the C-terminal pharmaceutical composition. The method according to claim 1 or 2,
The pharmaceutical composition comprising at least 10 or more amino acids contiguous sequence. The method of claim 1
The pharmaceutical composition of the at least 10 or more amino acids comprises a non-contiguous sequence. A peptide in which two or more functional peptide fragments are linked to each link core with their free ends,
The functional peptide fragment is a peptide that is composed of up to 17 amino acids and is a peptide fragment comprising at least 10 or more amino acids among 17 amino acids of natural Aβ1 to 17. The method of claim 15,
The peptide is a peptide having an activity to decompose by binding to water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates. The method of claim 15,
The peptide is a peptide having an activity of inhibiting the accumulation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates. The method of claim 15,
The link core and the functional fragment are peptides indirectly linked by a peptide arm. The method of claim 15,
The peptide arm is a flexible peptide without net charge. The method of claim 19,
The peptide arm is a peptide comprising a repeat unit of GGGS. The method of claim 15,
The link core is a C-terminal, and each free end is an N-terminal peptide. The method of claim 15,
Wherein the link core is located in the middle of the peptide, at least one of the free end of the N-terminal, at least the other C-terminal peptide. The method of claim 15
The at least 10 amino acids are peptides constituting the adjacent sequence. The method of claim 15
The peptide comprising at least 10 or more amino acids is a non-contiguous sequence. A method for degrading aggregation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates using the peptides according to any one of claims 15, 16, and 18 to 24,
A method in which two or more functional peptide fragments of the peptide are degraded by binding to water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates, respectively. A method for inhibiting aggregation of water-soluble Aβ aggregates and/or water-insoluble Aβ aggregates using the peptides according to any one of claims 15 and 17 to 24,
A method in which two or more functional peptide fragments of the peptide are each combined with the water-soluble Aβ aggregate and/or the water-insoluble Aβ aggregate to inhibit aggregation.

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