WO2015023132A1 - Composition for treating and preventing multiple sclerosis - Google Patents

Abstract

The present invention relates to a composition for treating and preventing multiple sclerosis. More specifically, the present invention relates to a composition which contains a peptide derived from telomerase and is effective in the treatment and prevention of multiple sclerosis. Further, the present invention provides a method for treating and preventing multiple sclerosis using the fact that a peptide having a sequence of the sequence ID number according to the present invention, a peptide having a sequence having 80% homology with the above sequence, or a peptide fragment thereof, has excellent effects of treating and preventing multiple sclerosis. More specifically, the present invention provides a method for treating and preventing multiple sclerosis without side effects by administering the peptide at a low dosage.

Description

Compositions for the treatment and prevention of multiple sclerosis

The present invention relates to a composition for treating and preventing multiple sclerosis. More specifically, the present invention relates to a composition for treating and preventing multiple sclerosis as a composition comprising a peptide derived from telomerase.

Multiple sclerosis is a representative disease of autoimmune inflammatory neurodegenerative disease, which is rare in Asians and Africans, but most frequently in Europeans, especially Caucasian women in their 30s. In Caucasian whites, the frequency affects 200 people per 100,000.

Multiple sclerosis involves visual impairment, dysfunction of the limbs and resulting gait disturbances, skin sensation impairment with pain or numbness, defecation and urination disorders, speech and hearing disorders, cognitive impairment, etc. Often it gets worse. This neurological disorder is due to damage to myelin sheaths surrounding the axons of nerve cells due to inflammatory responses in the central nervous system. The lesions are scattered in the white matter areas of the brain and spinal cord. In the lesions, inflammatory reactions involving perivenous inflammatory cell infiltration and bullous myelin sheaths of nerve fibers are observed. In addition, loss and appearance of oligodendrocytes Proliferation of glial cells.

The cause and pathological progress of multiple sclerosis have not yet been fully understood, but it is known to be an autoimmune reaction involving both cellular and humoral immune systems (Engelhardt, Clin. Exp. Neuroimmunol. 1: 79-93, 2010) ).

Multiple sclerosis is an immunological disease involving both cellular and humoral immune systems, as mentioned earlier, and various immunotherapies have been attempted: 1) desensitization of autoreactive T cells using immuno-tolerant antigens, and 2) T cells. Immunization using monoclonal antibodies or interferon-gamma against directional cytokines, B cell surface molecules or cell adhesion molecules, 3) plasma separation and the like have been attempted. These therapies have positive results, but their use is limited due to undesirable side effects (Steinman & Zamvil, Ann. Neurol. 60: 12-21, 2006). Therefore, the development of a safe drug without side effects and toxicity for patients with multiple sclerosis is urgently needed.

[Preceding technical literature]

[Patent Documents]

KR 2013-0078395 A

[Non-Patent Documents]

ENGELHARDT, Britta, 'T cell migration into the central nervous system during health and disease: different molecular keys allow access to different central nervous system compartments', Clinical and Experimental Neuroimmunology, Vol. 1, No. 2, pp.79-93, (2010)

Accordingly, the present inventors have completed the present invention as a result of diligent efforts to develop a composition for treating and preventing multiple sclerosis with excellent effects while minimizing side effects.

The inventors have found that a peptide derived from telomerase can have an excellent effect in treating and preventing compositions for treating and preventing multiple sclerosis and completed the present invention.

An object of the present invention is to provide a composition having an effect on the treatment and prevention of multiple sclerosis treatment and prevention.

According to one aspect of the present invention, there is provided a composition for treating and preventing multiple sclerosis comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof. .

In the composition according to one aspect of the invention, the fragment may be a fragment consisting of three or more amino acids.

The composition according to one aspect of the present invention may be one containing the peptide at a low dose.

The composition according to one aspect of the present invention may include the peptide at a concentration of 10 nmol / kg or less.

The composition according to one aspect of the present invention may be one containing the peptide at a concentration of 5 nmol / kg or less.

The composition according to one aspect of the present invention may be one containing the peptide at a concentration of 1 nmol / kg or less.

The composition according to one aspect of the present invention may be one containing the peptide at a concentration of 0.2 nmol / kg or less.

The composition according to one aspect of the invention may be a pharmaceutical composition.

The composition according to one aspect of the present invention may be a food composition.

According to another aspect of the present invention there is provided a method for treating and preventing multiple sclerosis, wherein the above-mentioned composition is administered to a subject in need thereof.

The method for treating and preventing multiple sclerosis according to one aspect of the present invention may use a composition comprising a low dose of a peptide.

A peptide having a sequence of SEQ ID NO: 1 according to the present invention (hereinafter referred to as peptide "RIA") or a peptide or fragment having a sequence having 80% homology with the sequence is excellent in the treatment and prevention of multiple sclerosis. Has the effect, especially when administered at a low dose has the advantage of showing a good effect.

1 is an experiment for identifying an effective dosage route, in which the peptide RIA is administered in separate doses by intraperitoneal and subcutaneous injection (experimental day 0) at the induction of experimental autoimmune encephalomyelitis (EAE) (200 days). A graph showing the results of nmol / kg intraperitoneal injection (intraperitoneal, IP) and 50 nmol / kg subcutaneous injection (subcutaneous, SC).

FIG. 2 is an experiment to identify an effective dosage route, in which doses of peptide RIA were administered in separate doses by intraperitoneal and subcutaneous injection when EAE symptoms reached peak (day 22) (200 nmol IP, 50 nmol SC) ) Is a graph showing the results.

Figure 3 is a graph showing the results of administration of RIA 50nmol / kg, RIA 25nmol / kg, RIA 10nmol / kg during EAE induction (day 0).

4 is a graph showing the results of administration of peptide RIA after RIA 50nmol / kg, RIA 25nmol / kg, RIA1 0nmol / kg, and RIA 5nmol / kg after symptom onset (day 22).

Figure 5 is a graph showing the results of administration of RIA 25nmol / kg, RIA 5nmol / kg, RIA 1nmol / kg, RIA 0.2nmol / kg at EAE induction (day 0).

Figure 6 is a graph showing the results of administration of RIA 25nmol, RIA 5nmol, RIA 1nmol, RIA 0.2nmol peptide RIA after the onset of symptoms (day 22).

Figure 7 shows drainage lymph nodes after primary antigenic stimulation (myelin oligodendroglial protein, MOG _35-45 ), peptide RIA 1nmol / kg, 0.2nmol / kg and PBS after primary antigenic stimulation It is a graph showing the results of T cell proliferation after in vitro secondary antigenic stimulation to cells isolated from lymph nodes.

The present invention may be variously modified and may have various embodiments. Hereinafter, the present invention will be described in more detail. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Telomere is a genetic material repeatedly present at the end of a chromosome and is known to prevent damage to the chromosome or binding to another chromosome. Each time a cell divides, the telomeres become slightly shorter. After a certain number of cell divisions, the telomeres become very short, and the cells stop dividing and die. On the other hand, elongation of telomeres is known to prolong cell life. For example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening, so that cancer cells can continue to proliferate without dying. The inventors have confirmed that the peptide derived from telomerase is effective in the treatment and prevention of multiple sclerosis and have completed the present invention.

In one aspect of the invention, a peptide of SEQ ID NO: 1, a peptide that is a fragment of SEQ ID NO: 1, or a peptide having a sequence homology of at least 80% with the peptide sequence is used in telomerase, specifically in human ( Homo sapiens ) telomerase. Peptides derived.

Peptides disclosed herein can include peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. In addition, the peptides disclosed herein, peptides or fragments thereof comprising SEQ ID NO: 1 and one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids Or peptides with seven or more amino acids changed.

In one aspect of the invention, amino acid changes belong to a property that allows the physicochemical properties of the peptide to be altered. For example, amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.

As used herein, "amino acid" includes not only the 22 standard amino acids that are naturally incorporated into the peptide, but also D-isomers and modified amino acids. Accordingly, in one aspect of the invention the peptide may be a peptide comprising D-amino acids. Meanwhile, in another aspect of the present invention, the peptide may include a non-standard amino acid or the like which has been post-translational modified. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-elimination deimidization) , Deamidation) and structural changes (eg, formation of disulfide bridges). It also includes changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that occur during the linkage with crosslinkers to form peptide conjugates.

Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source. On the other hand, the peptides disclosed herein may be artificial variants, comprising an amino acid sequence in which one or more amino acids are substituted, deleted and / or inserted compared to peptides that are fragments of SEQ ID NO: 1. Amino acid changes in the wild type polypeptide as well as in artificial variants include conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein. Examples of conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art. The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa. Other examples of conservative substitutions are shown in the following table.

Table 1

Original amino acid	Exemplary residue substitutions	Preferred residue substitution
Ala (A)	val; leu; ile	Val
Arg (R)	lys; gln; asn	Lys
Asn (N)	gln; his; asp, lys; arg	Gln
Asp (D)	glu; asn	Glu
Cys (C)	ser; ala	Ser
Gln (Q)	asn; glu	Asn
Glu (E)	asp; gln	Asp
Gly (G)	ala	Ala
His (H)	asn; gln; lys; arg	Arg
Ile (I)	leu; val; met; ala; phe; norleucine	Leu
Leu (L)	norleucine; ile; val; met; ala; phe	Ile
Lys (K)	arg; gln; asn	Arg
Met (M)	leu; phe; ile	Leu
Phe (F)	leu; val; ile; ala; tyr	Tyr
Pro (P)	ala	Ala
Ser (S)	thr	Thr
Thr (T)	ser	Ser
Trp (W)	tyr; phe	Tyr
Tyr (Y)	trp; phe; thr; ser	Phe
Val (V)	ile; leu; met; phe; ala; norleucine	Leu

Practical modifications in the biological properties of the peptide include (a) their effect on maintaining the structure of the polypeptide backbone, eg, sheet or helical conformation, within the substitution region, (b) the charge of the molecule at the target site. Or their effect in maintaining hydrophobicity, or (c) their effect in maintaining the bulk of the side chains, is carried out by selecting significantly different substitutions. Natural residues are divided into the following groups based on common side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will be made by exchanging a member of one of these classes for another class. Any cysteine residue that is not involved in maintaining the proper conformation of the peptide can generally be substituted with serine to improve the oxidative stability of the molecule and to prevent abnormal crosslinking. Conversely, cysteine bond (s) can be added to the peptide to improve its stability.

Another type of amino acid variant of the peptide is a change in the glycosylation pattern of the antibody. By change is meant the deletion of one or more carbohydrate residues found in the peptide and / or the addition of one or more glycosylation sites that are not present in the peptide.

Glycosylation of peptides is typically either N-linked or O-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to asparagine side chains. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation means attaching one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine You can also use

The addition of glycosylation sites to the peptide is conveniently performed by changing the amino acid sequence to contain one or more of the above mentioned tripeptide sequences (for N-linked glycosylation sites). Such changes may also be made by adding or replacing one or more serine or threonine residues with the sequence of the original antibody (for O-linked glycosylation sites).

In addition, a peptide having a sequence of SEQ ID NO: 1, a peptide which is a fragment of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence according to an aspect of the present invention has low intracellular toxicity and stability in vivo. This has the advantage of being high. SEQ ID NO: 1 in the present invention is a telomerase-derived peptide consisting of 16 amino acids as follows.

SEQ ID NO: 1 EARPALLTSRLRFIPK

In one aspect of the present invention, the composition for treating and preventing multiple sclerosis comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof as an active ingredient. To provide.

In another aspect of the present invention, there is provided a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof, for use in the treatment or prevention of multiple sclerosis. It may be used for.

In another aspect of the present invention, a composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof, a composition for treating or preventing multiple sclerosis It may be used for the preparation of.

In another aspect of the invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1 for use in treating or preventing multiple sclerosis, or for preparing a composition for treating or preventing multiple sclerosis, said amino acid Peptides are peptides or fragments thereof that have at least 80% sequence homology with a sequence.

Multiple sclerosis treatment and prophylactic composition according to one aspect of the present invention comprises a peptide comprising an amino acid sequence of SEQ ID NO: 1 in one aspect, a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof 0.01g / L to 1kg / L, specifically 0.1g / L to 100 / L, more specifically may be included in the content of 1g / L to 10g / L, but if the difference in effect according to the dose can be appropriately adjusted have. When included in the above range or less, it is not only appropriate to exhibit the intended effect of the present invention, but also satisfies both the stability and safety of the composition, it may be appropriate to include in the above range in terms of cost-effectiveness. .

The composition according to one aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.

In one aspect of the present invention, the composition comprises a peptide comprising an amino acid sequence of SEQ ID NO: 1, a therapeutic agent for multiple sclerosis comprising a peptide which is a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof as an active ingredient, and Provided is a prophylactic pharmaceutical composition. The pharmaceutical composition according to one aspect of the present invention may be administered orally, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intradural or subcutaneous.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. Formulations for parenteral administration may be, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

Pharmaceutical compositions according to one aspect of the invention may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavoring or sweetening agents as needed. Pharmaceutical compositions according to one aspect of the invention may be prepared by conventional methods in the art.

The active ingredient of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Application dose determination based on these factors is within the level of those skilled in the art, and the single dose thereof may be for example 1 ng / kg to 10 mg / kg, specifically 10 ng / kg to 1 mg / kg, more specifically 0.1 μg / kg to 100 μg / kg, more specifically, 0.2 μg / kg to 20 μg / kg, but when the difference in effect depending on the dose can be adjusted appropriately. The pharmaceutical composition according to an aspect of the present invention may be administered once to three times a day, but is not limited thereto. When the difference in effect according to the dose is shown, it may be appropriately controlled, and the age of the subject to be administered, It can depend on many factors, including your health and complications.

In one aspect of the present invention, the composition comprises a peptide comprising an amino acid sequence of SEQ ID NO: 1, a therapeutic agent for multiple sclerosis comprising a peptide which is a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof as an active ingredient, and Provide a prophylactic food composition.

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be, for example, formulated into tablets, granules, powders, solutions, solid preparations, and the like. Each formulation may be appropriately selected and formulated by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and may be synergistic when applied simultaneously with other raw materials.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term without the number before the noun is not intended to limit the quantity but rather to the presence of one or more of the mentioned noun articles. The terms "comprising", "having", and "comprising" are interpreted as open terms (ie, meaning "including but not limited to").

Reference to a range of numbers is simply an easy way to substitute for referring to each individual number within the range individually, and unless stated otherwise, each individual number is referred to individually as if It is incorporated herein as is. The end values of all ranges fall within that range and can be combined independently.

All methods mentioned herein may be performed in the proper order unless otherwise specified or clearly contradicted by context. The use of one embodiment and all embodiments or example language (eg, “such as”) is merely intended to better describe the invention, unless it is within the scope of the claims, and covers the scope of the invention. It is not intended to be limiting. No language in the specification should be construed as essential to the practice of the invention as to any unclaimed elements. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferred embodiments of the invention include the most optimal mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to make appropriate use of such variations, and the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter referred to in the appended claims, as permitted by patent law. Moreover, any combination of the abovementioned elements within all possible variations is included in the invention unless expressly stated to the contrary or apparently contradictory in context. While the invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims .

In the following examples, the EAE mouse model was to determine the effect of preventing and treating multiple sclerosis of peptide RIA.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are provided only for the purpose of illustration in order to help the understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

Example 1 Synthesis of Peptides

The peptide of SEQ ID NO: 1 (hereinafter referred to as "RIA") was prepared according to the known solid phase peptide synthesis method. Specifically, peptides were synthesized by coupling amino acids one by one from the C-terminus through Fmoc solid phase synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). As follows, the first amino acid at the C-terminus of the peptides was attached to the resin. For example:

NH ₂ -Lys (Boc) -2-chloro-Trityl Resin

NH ₂ -Ala-2-chloro-Trityl Resin

NH ₂ -Arg (Pbf) -2-chloro-Trityl Resin

All amino acid feedstocks used for peptide synthesis were protected with N-term Fmoc and all residues removed from acid Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl) and the like were used. For example:

Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc- Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Met-OH, Fmoc -Asn (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

Coupling reagent is HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] / NMM [4-Methylmorpholine] It was. Fmoc removal was performed using piperidine in DMF in 20% of DMF. Isolation of the synthesized peptides from the resin and removal of the protecting groups of the residues include cleavage cocktail (TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O = 92.5 / 2.5 / 2.5 / 2.5 ] Was used.

Each peptide was synthesized by repeating a process of reacting the amino acids with each other, washing with a solvent, and then deprotecting the amino acid using the state in which the amino acid protecting group was bound to the solid support. The synthesized peptide was separated from the resin and then purified by HPLC, and confirmed by MS and lyophilized.

High performance liquid chromatography on the peptides used in this example showed that all peptides had a purity of at least 95%.

A detailed procedure for preparing peptide RIA is as follows.

1) coupling

Amino acid (8 equivalents) protected by NH ₂ -Lys (Boc) -2-chloro-Trityl Resin and coupling reagent HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were dissolved in DMF and added. Reaction was performed at room temperature for 2 hours, and washed with DMF, MeOH, and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF at 20% was added thereto, reacted twice at room temperature for 5 minutes, and washed in the order of DMF, MeOH, and DMF.

3) Repeated reaction of 1 and 2, peptide basic skeleton NH ₂ -E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -2-chloro-Trityl Resin).

4) Cleavage: The peptide cocktail was separated from the resin by adding a cleavage cocktail to the synthesized peptide resin.

5) Cooling diethyl ether is added to the obtained mixture, followed by centrifugation to precipitate the obtained peptide.

6) After purification by Prep-HPLC, the molecular weight was confirmed by LC / MS and frozen to prepare a powder (powder).

Example 2: Induction and Evaluation of EAE in Mice

EAE Induction Method

In this example, an EAE (experimental autoimmune encephalomyelitis) mouse model was used as the animal model of multiple sclerosis. Details on how to build an EAE mouse model as an animal model of multiple sclerosis are known in a number of prior publications (Baker et al., Review article, ACNR, Volume 6, 2007; Kim Dae-seung et al., Korean J Vet Res) , 51 (2), 139-149pp, 2011)

Animals used for EAE model construction were used female C57BL / 6 (B6) mice of 6-8 weeks of age. The experimental animals were bred and used at the National Cancer Center's Laboratory Animal Research Center according to the guidelines of the International Association of Laboratory Animal Management, and managed in a specific pathogen free (SPF) environment.

The EAE mouse model was tested divided into five groups as shown in Table 2 below.

TABLE 2

group	Explanation
A group	Control, EAE with control (PBS) treatment
B group	Healthy (negative) control with peptide RIA group at disease induction
Group C	Healthy (negative) control with peptide RIA group at disease onset
D group	EAE with peptide RIA group at disease induction (prophylactic treatment)
E group	EAE with peptide RIA group at disease onset (treatment of ongoing disease)

The method for preparing an EAE animal model according to the five groups classified above is as follows. In female C57BL / 6 (B6) mice (Orient Bio Inc., Korea), 6-8 weeks of age, 250 ㎍ of complete Freund's adjuvant (CFA), Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) ) 100 μg and emulsion containing MOG _35-55 peptide were injected subcutaneously, and additionally 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) was injected intraperitoneally on day 0 & 2. Control mice were injected with physiological saline only in the same manner as the experimental group. In the peptide RIA group, RIA was injected intraperitoneally (IP) or subcutaneously (SC) at defined doses.

Assessment Methods

Clinical symptoms of EAE mice were assessed and recorded daily using the following symptom scales.

EAE Symptom Scale

TABLE 3

0	normal
One	Loss of tail elasticity or hind limb paralysis
2	Loss of tail elasticity and hind limb paralysis
3	Hind limb paralysis
4	Hind limb paralysis
5	Immediately before death or death

For clinical symptom evaluation, the mice received the MOG- ₄₅ peptide and scored the symptoms appearing in the mice every day after 10 days. The clinical status of each control group and the experimental group was analyzed and body weight was measured at each reference date.

1000 mg / kg of ketamine (Ketamin) was injected intraperitoneally for serum collection, and then 1-2 ml of blood was collected by dissecting the rib cage and puncturing the left ventricle. The collected blood was centrifuged at 3000C (3000 rpm, 10 minutes) to separate serum.

For cell culture and T cell proliferation, blood was collected from the mice, and then the abdominal cavity was dissected to extract the inguinal lymph node and spleen. Single-cell suspensions were obtained from tissues using a wire-mesh, washed with DPBS (Dulbecco's Phosphate Buffered Saline), and centrifuged (1800 rpm, 3 minutes). This single cell suspension was subjected to 10% FBS (Fetal Bovine Serum, Sigma-Aldrich), 1.4 mM glutamine, 20 μM 2-ME (2-Hydroxyethylmercaptan, Sigma-Aldrich), 100 U / ml penicillin, 100 μg / ml streptomycin (Life Technologies / RPMI medium containing BRL, Grand Island, NY) was dispensed at 2.5 × 10 ⁵ cells / well in Roswell Park Memorial Institute medium, Gibco, Eggenstein, Germany. In vitro secondary antigenic (MOG _35-55 ) and non-antigenic (plate-bound anti-CD3 / soluble anti-CD28) stimulation was added to the culture and then incubated at 37 ℃, 5% CO ₂ / air. The supernatant was removed from the cultured T cells, and the culture solution added with tritium thymidine (3H-thymidine) was added thereto, followed by incubation for 18 hours, and the degree of thymidine incorporation was measured by a gamma-counter.

In addition, to obtain histopathological findings, the spinal cord and brain of the mouse were extracted and fixed in 4% paraformaldehyde for 6 hours. Then, paraffin embedded in 30% sucrose was maintained at 4 ℃ for one day. Hematoxylin and eosin stains were used to evaluate the perivascular leukocyte invasion of the lesions, and Luxol fast blue (LFB) staining was used to evaluate the degree of demyelination of the lesions. Was evaluated.

For statistical analysis in this example, all parameters were expressed in means +/- SD, and the comparison between each group used the Kruskal-Wallis test. Each group was analyzed using the Mann-Whitney test.

Example 3: Confirmation of Peptide Treatment Effects on EAE Mice

1. Comparison of Clinical Symptoms by Medication Route

Peptide RIA was administered at the appropriate concentration for each route to identify effective route of administration during intraperitoneal injection (IP) and subcutaneous injection (SC). RIA 200nmol (RIA 200nmol / kg) was used for intraperitoneal injection and RIA 50nmol (RIA 50nmol / kg) was used for subcutaneous injection.

Experiments were performed by subcutaneous and intraperitoneal injection into groups administered when EAE was induced (day 0) and those administered when EAE peaked (day 22).

Experiments in the group administered at the time of EAE induction (day 0) confirmed that EAE symptoms appeared faster and lasted longer than the control group in the two groups (200 nm intraperitoneal injection, 50 nm mol subcutaneous injection) administered RIA. The results are shown in FIG. In particular, the subcutaneous injection 50 nmol group was more severe and lasted longer than the intraperitoneal injection 200 nmol administration group. In the RIA-administered group, it was confirmed that abnormal movement of myoclonus pattern was observed as a side effect while EAE symptoms persisted. However, the control group did not show myoclonic pattern.

Next, the experimental results of the group treated with subcutaneous and intraperitoneal injection when EAE peaked (day 22) showed that the two groups with RIA (200 nmol intraperitoneal injection, 50 nmol subcutaneous injection) had more severe EAE symptoms than the control group. It was confirmed to last long. These results are shown in FIG. In particular, subcutaneous injection 50nmol group had more severe and longer lasting symptoms than intraperitoneal injection 200nmol group, and abnormal movement of myoclonus pattern was observed during EAE symptoms in RIA group as with RIA on day 0. . However, the control group did not show myoclonic pattern.

Based on the above results, the subcutaneous injection group was confirmed to show a similar effect even when administered a low dose of peptide RIA compared to the intraperitoneal injection group, and decided to use only the subcutaneous injection method.

2. Second EAE Experiment with Peptide RIA Dose

Secondary EAE experiments were performed according to the peptide RIA dose. As can be seen from the results of Example 3-1, the subcutaneous injection of 50 nmol group showed a worsening of the disease course in the intraperitoneal injection 200 nmol administration group, and an experiment was administered to a lower dose.

RIA50nmol, RIA25nmol and RIA10nmol were administered at EAE induction (day 0)

As shown in FIG. 3, RIA 50nmol, RIA 25nmol, and RIA 10nmol were administered at the time of EAE induction (day 0) by subcutaneous injection. As in the previous experiment, EAN symptoms were rapidly expressed at 50nmol / kg and 25nmol / kg for a long time. The results were sustained. However, at 10 nmol / kg, EAE symptoms were late and short-lived, and the intensity of symptoms was low.

At 50nmol / kg and 25nmol / kg, the myoclonic modality was remarkable, whereas at 10nmol / kg, the myoclonic modality was weak.

Peptide RIA was administered 50 days after RIA 50nmol, RIA 25nmol, RIA 10nmol, RIA 5nmol

As shown in FIG. 4, RIA 50nmol, RIA 25nmol, RIA 10nmol, and RIA 5nmol were administered when EAE symptoms peaked by subcutaneous injection (Day 22), and EAE at 50nmol / kg, 25nmol / kg, and 10nmol / kg. Long-term symptoms were observed, but at 5 nmol / kg, EAE symptoms were rapidly recovered after peptide RIA administration. At 50nmol / kg and 25nmol / kg, the myoclonic modality was remarkable, whereas at 10nmol / kg, the myoclonic modality was weak.

Additional experiments to identify side effects (clonic muscle spasms)

Note that (1) normal rats that did not induce EAE, (2) mice that received adjuvant used only for EAE induction, and (3) BBB ( To increase the blood-brain barrier permeability, 50 nmol / kg peptide RIA was injected into rats (BBB leaky mice) injected only with pertussis toxin used for EAE induction.

As a result, no abnormality was observed unlike EAE mice. In other words, myoclonic jerky movement was observed as a side effect only in rats with inflammatory lesions in the central nervous system.

3. Low Dose Peptide RIA Administration

Based on these results, concentrations lowered to 250 times the initial experimental dose to confirm the likelihood of effectiveness at low doses of peptide RIA and to demonstrate clinical effectiveness with a safety margin sufficient for the dose at which side effects occur. Confirmation experiment was carried out.

RIA 25nmol, RIA 5nmol, RIA 1nmol, RIA 0.2nmol are administered when EAE induction (day 0)

As shown in FIG. 5, RIA 25nmol, RIA 5nmol, RIA 1nmol, and RIA 0.2nmol were administered at the time of EAE induction (day 0) by subcutaneous injection (2 experiments under the same conditions). The symptoms appeared quickly, lasted a long time and were found to be severe.

However, improvement in the EAE score was observed in 5 nmol / kg treated mice at 1/10 of the initial experimental dose, and at 1 nmol / kg and 0.2 nmol / kg at 1/50, 1/250. Significant degradation and rapid recovery of symptoms were observed.

At 25 nmol / kg, myoclonic modality was also clearly observed as in the previous experiment, but at 1 nmol / kg and 0.2 nmol / kg, myoclonus was not observed.

Peptide RIA was administered 25 days after RIA 25nmol, RIA 5nmol, RIA 1nmol, RIA 0.2nmol

As shown in FIG. 6, peptide RIA was administered by subcutaneous injection (day 22) after RIA 25nmol, RIA 5nmol, RIA 1nmol, RIA 0.2nmol (2 experiments under the same conditions), EAE at 25nmol / kg Symptoms were observed to last longer than controls. On the other hand, at 5nmol / kg, 1nmol / kg and 0.2nmol / kg, EAE symptoms recovered rapidly after administration of peptide RIA, and the effect was more pronounced at low dose.

At 25 nmol / kg, there were also marked myofascial myopathy patterns, and at 1 nmol / kg and 0.2 nmol / kg, no significant myoclonus was observed.

Through the results of Example 3, it was confirmed that EAE symptoms worsen in both aspects of prophylactic treatment and treatment of symptoms when high concentration (10, 25, 50 nmol / kg) peptide RIA was administered, and low concentration (5, 1, 0.2 nmol / kg of peptide RIA showed significant clinical effects in both prophylactic treatment and ongoing disease treatment.

This is not only possible to prevent the onset of EAE symptoms through the administration of low concentration peptide RIA, but also has a significant effect on the rapid recovery of already advanced symptoms, namely disease modifying treatment and acute relapse treatment. It can be said that it can play a role. In particular, experiments on ongoing disease treatment show a significant effect despite the onset of symptoms after peaking, instead of the usual method of onset of symptoms at the same time. It can be said that it has shown the possibility of being a new option for the acute treatment.

However, myoclonic jerky movement was observed as a side effect when high doses of peptide RIA were administered. In particular, the administration of high concentrations of peptide RIA showed a clear myocardial spasms, and even at low concentrations, the myoclonus up to a certain concentration (5nmol / kg) was weak. This suggests that peptide RIA can cross the Brain-Brain Barrier (BBB) in the EAE model and act directly on the brains of already inflamed mice. Thus, if sufficient safety margins are secured, peptide RIA directly affects central nervous system lesions, whereas most existing MS drugs play a role in reducing peripheral nervous system infusion and immunomodulation in lymphocytes outside the central nervous system. It can be expected that acute treatment may be a useful drug in the future.

Example 4. T Cell Proliferation Experiment

As shown in Figure 7, after primary antigenic stimulation (MOG _35-45 ) under the same conditions as the induction of EAE, after administration of peptide RIA 1nmol / kg, 0.2nmo / kg or PBS, in draining lymph nodes T cell proliferation after in vitro secondary antigenic stimulation to the isolated cells was significantly decreased in the TIA 1nmol / kg and 0.2nmol / kg groups compared with the control group. There was no difference between RIA 1nmol / kg and 0.2nmol / kg doses.

Therefore, administration of the peptide RIA according to the present invention at a low dose (1 nmol / kg, 0.2 nmol / kg) reduces T-cell proliferation, which has a significant effect on the treatment of autoimmune diseases of central nervous system lesions including multiple sclerosis. It can be expected to be developed as a useful drug to treat the disease.

Based on the above experimental results, when the peptide RIA according to the present invention is administered at a low dose, unlike the high-dose administration, it does not show any side effect of myoclonus, but directly affects the central nervous system lesions, which is suitable for acute treatment. In addition, since T cell proliferation is reduced and shows a significant effect on the treatment of autoimmune diseases, it can be used as a useful drug for the treatment of central nervous system lesions including multiple sclerosis and there is a possibility of further development. .

SEQ ID NO 2: Human telomerase full length protein [1-1132aa]

Claims (11)

1. A composition for treating and preventing multiple sclerosis comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof.
2. The composition for treating and preventing multiple sclerosis according to claim 1, wherein the fragment is a fragment consisting of three or more amino acids.
3. The composition for treating and preventing multiple sclerosis according to claim 1, wherein the peptide comprises a low dose.
4. The composition for treating and preventing multiple sclerosis according to claim 3, comprising the peptide at a concentration of 10 nmol / kg or less.
5. The composition for treating and preventing multiple sclerosis according to claim 3, comprising the peptide at a concentration of 5 nmol / kg or less.
6. The method of claim 3. Multiple sclerosis treatment and prevention composition comprising the peptide in a concentration of 1 nmol / kg or less.
7. The method of claim 3. Multiple sclerosis treatment and prevention composition comprising the peptide in a concentration of 0.2nmol / kg or less.
8. The composition of claim 1, wherein the composition is a pharmaceutical composition.
9. The composition for treating and preventing multiple sclerosis according to claim 1, wherein the composition is a food composition.
10. A method for treating and preventing multiple sclerosis, wherein the composition according to any one of claims 1 to 9 is administered to a subject in need thereof.
11. The method of claim 10, wherein the composition comprises a low dose of peptide.

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