Peptide for treating demyelinating diseases and use thereof

Abstract

The present invention relates to a peptide for treating demyelinating diseases and a use thereof. More specifically, a trimeric peptide according to the present invention has been found to inhibit demyelination and improve myelination, as well as improve nerve damage and symptoms caused by demyelination, in a chronic inflammatory demyelinating polyneuropathy (CIDP) animal model and an acquired peripheral nerve injury animal model. Thus, the peptide according to the present invention can be effectively used as an active ingredient of a composition for preventing or treating demyelinating diseases including CIDP.

Description

Peptides for treating demyelinating diseases and their uses

[0001] The present invention relates to a peptide for treating demyelinating diseases and its use, and more particularly, to the use of a peptide in the form of a trimer, composed of an amino acid represented by sequence number 1, as a demyelinating disease treatment agent.

[0002] Most nerve fibers in the nervous system are surrounded by many layers of tissue composed of a lipoprotein called myelin. Myelin acts as an insulator, playing a crucial role in transmitting neural impulses accurately and quickly along the nerve fibers. When this myelin is damaged or destroyed due to local injury, immune disorders, infection, nutritional deficiencies, drugs, or unknown causes, permanent damage is induced to the nerve fibers within. Therefore, when myelin is damaged, the body attempts to repair it through remyelination, but often remyelination fails and demyelination occurs. Diseases caused by this demyelination are called demyelinating diseases.

[0003] Demyelinating disorders occur when the myelin sheath is damaged without significant damage to the nerve cells themselves or their axons. They can occur in both the peripheral and central nervous systems. Symptoms of demyelinating disorders include sensory, motor, and autonomic symptoms, which are further divided into positive and negative symptoms. Negative motor symptoms are caused by conduction block or loss of motor nerve axons and manifest as muscle weakness. Positive motor symptoms, on the other hand, are caused by abnormal activity in the peripheral nerves and manifest as fasciculations, myokymia, tremors, and muscle cramps. Positive sensory symptoms include hypersensitivity, including pain, and dysesthesia, while negative sensory symptoms can include decreased sensation and numbness.

[0004] Currently, medications used for demyelinating diseases primarily address the underlying disease or manage neuropathic pain, a symptom of demyelinating diseases. Fundamental treatments are rare. For example, treatments for chronic inflammatory demyelinating polyneuropathy (CIDP), a type of demyelinating disease, include steroids (primarily prednisone), intravenous immunoglobulin (IVIg), and plasma exchange. However, some CIDP patients do not respond adequately to these treatments, requiring the trial of other treatments, such as immunosuppressants. However, immunosuppressants require periodic evaluation of their risks and benefits.

[0005] Accordingly, the present inventors have endeavored to develop a therapeutic agent with effective therapeutic effects while minimizing side effects as a therapeutic agent, and as a result, have manufactured a trimeric peptide composed of a very small peptide, which can minimize side effects caused by the administration of external substances. In addition, the inventors have confirmed the effects of the peptide in inhibiting demyelination and enhancing myelination in a CIDP animal model and an acquired peripheral nerve injury animal model, and improving nerve damage and symptoms caused by demyelination, thereby revealing that the peptide can be usefully utilized as an effective ingredient in a composition for preventing or treating demyelinating diseases including CIDP, thereby completing the present invention.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] Republic of Korea Publication Patent No. 10-2020-0138904

[0009] [Non-patent literature]

[0010] GGA van Lieverloo et al., Corticosteroids in chronic inflammatory demyelinating polyneuropathy, J Neurol. 2018; 265(9): 2052-2059.

[0011] Vanden Bergh et al., European Academy of Neurology / Peripheral Nerve Society guideline on diagnosis and treatment of chronic inflammatory demyelinating polyneuropathy: Report of a joint Task Force-Second revision, Eur J Neurol. 2021 Nov;28(11):3556-3583.

[0012] The purpose of the present invention is to provide a composition for preventing, treating or improving demyelinating disease, comprising as an active ingredient a peptide composed of an amino acid represented by sequence number 1 or a polynucleotide encoding the same.

[0013] In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a demyelinating disease, comprising as an active ingredient a peptide consisting of an amino acid represented by SEQ ID NO: 1 or a polynucleotide encoding the same; a use of the peptide or the polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating a demyelinating disease; a use of the peptide or the polynucleotide encoding the same for preparing a pharmaceutical composition for preventing or treating a demyelinating disease; and a method for preventing or treating a demyelinating disease, comprising administering to a subject a therapeutically effective amount of the peptide or the polynucleotide encoding the same.

[0014] In addition, the present invention provides a health functional food composition for preventing or improving demyelinating disease, comprising as an active ingredient a peptide consisting of an amino acid represented by SEQ ID NO: 1 or a polynucleotide encoding the same; a use of the peptide or the polynucleotide encoding the same for use as a health functional food composition for preventing or improving demyelinating disease; a use of the peptide or the polynucleotide encoding the same for manufacturing a health functional food composition for preventing or improving demyelinating disease; and a method for improving demyelinating disease, comprising administering to a subject the peptide or the polynucleotide encoding the same.

[0015] In the present invention, it was confirmed that the trimeric peptide according to the present invention has the effect of inhibiting demyelination and improving myelination in a chronic inflammatory demyelinating polyneuropathy (CIDP) animal model and an acquired peripheral nerve injury animal model, and improving nerve damage and symptoms due to demyelination. Therefore, the peptide of the present invention can be usefully used as an effective ingredient of a composition for preventing or treating demyelinating diseases including CIDP.

[0016] In addition, the trimer-shaped peptide according to the present invention is composed of peptides of very small size, so that side effects resulting from administration of external substances can be minimized.

[0017] Figure 1 is a diagram confirming changes in clinical scores following administration of the trimer form of the peptide KINE-101 according to the present invention in a chronic inflammatory demyelinating polyneuropathy (CIDP) animal model.

[0018] Figure 2 is a diagram confirming the degree of demyelination after administration of the trimer form of the peptide KINE-101 according to the present invention in a CIDP animal model.

[0019] Figure 3 is a diagram confirming the change in clinical score according to administration of the trimer form of peptide KINE-101 or intravenous immunoglobulin (IVIg) according to the present invention in a CIDP animal model (*: p<0.05, ***: p<0.001, ****: p<0.0001).

[0020] Figure 4 is a diagram confirming the change in body weight according to administration of the trimer form of peptide KINE-101 or IVIg according to the present invention in a CIDP animal model (*: p<0.05, **: P<0.01, ***: P<0.001, ****: p<0.0001).

[0021] FIG. 5 is a diagram confirming changes in motor nerve conduction velocity (MNCV) and compound muscle action potential (CMAP) following administration of the trimer form of peptide KINE-101 or IVIg according to the present invention in a CIDP animal model (*: p<0.05, **: P<0.01, ***: P<0.001, ****: p<0.0001).

[0022] Figure 6 is a diagram confirming changes in sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) following administration of the trimer form of peptide KINE-101 or IVIg according to the present invention in a CIDP animal model (*: p<0.05, **: P<0.01, ***: P<0.001).

[0023] Figure 7 is a diagram confirming the change in MNCV according to administration of the trimer form of peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0024] Figure 8 is a diagram confirming the change in CMAP according to administration of the trimer form of peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0025] Figure 9 is a diagram confirming the change in SNCV according to administration of the trimer form of peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0026] Figure 10 is a diagram confirming the change in SNAP following administration of the trimer form of the peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0027] Figure 11 is a diagram confirming behavioral changes following administration of the trimer form of the peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0028] Figures 12a and 12b are diagrams showing the degree of myelin deformity after administration of the trimer form of the peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0029] Figure 13 is a diagram confirming changes in the ratio and activity of Treg cells after administration of the trimer-type peptide KINE-101 according to the present invention in an animal model of acquired peripheral nerve damage.

[0030] Hereinafter, the present invention will be described in more detail.

[0031] The present invention provides a peptide composed of an amino acid represented by sequence number 1 or a polynucleotide encoding the same.

[0032] In the present invention, the "peptide" refers to a polymer composed of two or more amino acids linked by amide bonds (or peptide bonds). Despite various studies on peptide therapeutics, the peptide itself is too large, and thus has the disadvantage of reducing drug efficacy and causing side effects due to the generation of antibodies and induction of an immune response against the peptide drug itself. Therefore, the present invention is technically significant in that it has identified a peptide in the form of a trimer composed of 10 or fewer amino acids that has pharmaceutically effective activity.

[0033] The peptide of the present invention may be composed of an amino acid represented by SEQ ID NO: 1, and may include an amino acid sequence having a sequence identity of 75% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, with the amino acid sequence represented by SEQ ID NO: 1. More specifically, the peptide of the present invention may include an amino acid sequence having a sequence identity of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more with the amino acid sequence represented by SEQ ID NO: 1.

[0034] Additionally, the peptide of the present invention may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0035] Additionally, the peptide of the present invention can be obtained using various methods widely known in the art. For example, it can be produced using polynucleotide recombination and protein expression systems, in vitro synthesis through chemical synthesis such as peptide synthesis, and cell-free protein synthesis methods.

[0036] Additionally, a protecting group may be attached to the N- or C-terminus of the peptide to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), or reduced antigenicity. Examples of protecting groups include an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but any component that can modify the peptide, particularly enhance the stability of the peptide, may be included without limitation.

[0037] The term "stability" used in the present invention means not only in vivo stability that protects the peptide of the present invention from attack by in vivo protein cleavage enzymes, but also storage stability (e.g., room temperature storage stability).

[0038] The term "polynucleotide" used in the present invention refers to a polymer in which nucleotides are linked and serves to transmit genetic information. For the purposes of the present invention, it may include a sequence that encodes a peptide of SEQ ID NO: 1 and has a sequence homology of at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% to the polynucleotide sequence encoding the peptide.

[0039] The term "homology" used in the present invention is intended to indicate the degree of similarity with a wild-type amino acid sequence or polynucleotide sequence, and a comparison of such homology can be performed using a comparison program widely known in the art, and the homology between two or more sequences can be calculated as a percentage (%).

[0040] In addition, the present invention provides a pharmaceutical composition for preventing or treating a demyelinating disease, comprising as an active ingredient a peptide consisting of an amino acid represented by SEQ ID NO: 1 or a polynucleotide encoding the same; a use of the peptide or the polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating a demyelinating disease; a use of the peptide or the polynucleotide encoding the same for preparing a pharmaceutical composition for preventing or treating a demyelinating disease; and a method for preventing or treating a demyelinating disease, comprising administering to a subject a therapeutically effective amount of the peptide or the polynucleotide encoding the same.

[0041] The term “prevention” as used in the present invention means any act of suppressing a disease or delaying its onset by administering a pharmaceutical composition according to the present invention.

[0042] The term "treatment" as used in the present invention means any action in which the symptoms of a disease are improved or beneficially changed by administration of a pharmaceutical composition according to the present invention.

[0043] In the present invention, “subject” means a subject requiring treatment of a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0044] In the present invention, the demyelinating disease is Charcot-Marie-Tooth disease (CMT), Chronic inflammatory demyelinating polyneuropathy (CIDP), Idiopathic inflammatory demyelinating disease, Hereditary neuropathy, Anti-MAG peripheral neuropathy, Progressive inflammatory neuropathy, Optic neuropathy, Devic's disease, Central pontine myelinolysis (CPM), Extrapontine Myelinolysis (EPM), Tabes dorsalis, Leukoencephalopathies, Demyelinating These may include, but are not limited to, demyelinating leukodystrophy, neuromyelitis optica, demyelinating optic neuritis, acute disseminated demyelination, periaxial encephalitis, central demyelination of corpus callosum, acute transverse myelitis, subacute necrotizing myelitis, or concentric sclerosis.

[0045] Additionally, the peptide can prevent or treat demyelinating diseases by improving demyelination.

[0046] In the present invention, the peptide can prevent or treat the demyelinating disease by improving motor nerve and sensory nerve damage, specifically motor nerve and sensory nerve damage due to demyelination.

[0047] In addition, the peptide can prevent or treat demyelinating diseases by improving motor nerve conduction and sensory nerve conduction reduction, specifically, motor nerve conduction and sensory nerve conduction reduction caused by demyelination.

[0048] In addition, the peptide can prevent or treat demyelinating diseases by increasing the proportion of Treg cells in the lymph nodes of the peripheral nerve injury site and inducing Treg cell activation.

[0049] In a specific embodiment of the present invention, the inventors prepared a trimer-type peptide KINE-101 using a PSP fragment (PSP monomer), and confirmed that the peptide improved CIDP symptoms and demyelination in peripheral nerve tissue in a CIDP animal model, a demyelinating disease model. In addition, it was confirmed that the reduction in motor nerve conduction and sensory nerve conduction caused by the demyelination was improved.

[0050] In addition, the inventors of the present invention confirmed that the symptoms of motor and sensory nerve damage, such as muscle strength and sensory decline, were improved by the peptide in an animal model of acquired peripheral nerve damage as a demyelinating disease model, and that demyelination, which is a peripheral nerve damage, was improved.

[0051] Therefore, the inventors of the present invention have confirmed that the peptide of the present invention has the effect of inhibiting demyelination and improving myelination in a CIDP animal model and an acquired peripheral nerve injury animal model, and improving nerve damage and symptoms due to demyelination. Therefore, the peptide of the present invention can be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating demyelinating diseases including CIDP.

[0052] The peptide of the present invention or the polynucleotide encoding the same may be delivered in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipid, liposome, microspheres, or nano-spheres. They may be complexed or associated with a carrier, and may be delivered in vivo using carrier systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.

[0053] In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included in addition to the above ingredients.

[0054] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and body weight, the degree of disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art.

[0055] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0056] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and may increase or decrease depending on the route of administration, severity of obesity, sex, body weight, age, etc.

[0057] In addition, the present invention provides a health functional food composition for preventing or improving demyelinating disease, comprising as an active ingredient a peptide consisting of an amino acid represented by SEQ ID NO: 1 or a polynucleotide encoding the same; a use of the peptide or the polynucleotide encoding the same for use as a health functional food composition for preventing or improving demyelinating disease; a use of the peptide or the polynucleotide encoding the same for manufacturing a health functional food composition for preventing or improving demyelinating disease; and a method for improving demyelinating disease, comprising administering to a subject the peptide or the polynucleotide encoding the same.

[0058] As used herein, the term "improvement" means any action that at least reduces a parameter related to the condition being treated, for example, the severity of a symptom.

[0059] In the present invention, the content of the peptide, polynucleotide, and demyelinating disease is the same as described above, so the specific description is based on the above content, and only the unique composition of the health functional food composition is described below.

[0060] Meanwhile, the inventors of the present invention have confirmed that the peptide of the present invention has the effect of inhibiting demyelination and improving myelination in a CIDP animal model and an acquired peripheral nerve injury animal model, and improving nerve damage and symptoms due to demyelination. Therefore, the peptide of the present invention can be usefully used as an effective ingredient of a health functional food composition for preventing or improving demyelinating diseases including CIDP.

[0061] The health functional food composition of the present invention can be used simultaneously with or separately from a drug for treatment before or after the onset of the disease to prevent or improve the disease.

[0062] In the health functional food composition of the present invention, the active ingredient may be added directly to a food or used in combination with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the active ingredient may be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the composition of the present invention may be added in an amount of preferably 15% by weight or less, and preferably 10% by weight or less, relative to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0063] The health functional food composition of the present invention, in addition to containing the above-described effective ingredient, may contain other ingredients as essential ingredients without particular limitation. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-described natural carbohydrates include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, erythritol, etc. In addition to the above-described flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the above-described natural carbohydrates can be appropriately determined by those skilled in the art.

[0064] In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0065] Hereinafter, the present invention will be described in detail by examples.

[0066] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0067] <Example 1> Production of peptides

[0068] In this example, a trimer-type polymer KINE-101 was prepared using a PSP fragment (PSP monomer) as shown in [Table 1] below. Subsequently, the synthesized peptide was purified using high-performance liquid chromatography (SHIMADZU Prominence HPLC), and the column used was a Shiseido capcell pak C18 Column (4.6 x 50 mm). In addition, the mass of the synthesized peptide was confirmed using a mass spectrometer (AXIMA Assurance, MALDI-TOF, Shimadzu).

[0069] Peptide name Amino acid sequence Molecular weight (MW) KINE-101PSPPSPPSP (SEQ ID NO: 1) 861.9 g / mol

[0070] <Example 2> Confirmation of the effect of KINE-101 on improving chronic inflammatory demyelinating polyneuropathy (CIDP) in an animal model of CIDP.

[0071] <2-1> Creation of CIDP animal model and peptide administration

[0072] In order to investigate the effect of KINE-101 in demyelinating disease, an animal model of chronic inflammatory demyelinating polyneuropathy (CIDP), a form of demyelinating disease, was created as follows, and KINE-101 synthesized in <Example 1> was administered.

[0073] Seven-week-old female Lewis rats were provided, and after a one-week acclimatization period, solution A, which was prepared by dissolving peripheral myelin P0 protein (180-199) in 0.9% saline at 300 μg / μl, and solution B, which was prepared by dissolving Mycobacterium tuberculosis in Freund's incomplete adjuvant at 0.5 mg / μl, were prepared. After placing a sterile 50 ml tube in an icebox, solution A and solution B were mixed in a 1:1 ratio and homogenized using a homogenizer for 15 minutes to prepare an emulsion. The prepared emulsion was placed in a 1 ml syringe, the syringe was changed to a 25G, and stored in an icebox containing ice until use. Lewis rats were induced to become ill by subcutaneously (SC) injecting 200 μl of the emulsion into the tail tip. Because body weight decreased depending on the severity of CIDP, the rats were divided into six groups, as shown in Table 2 below. The groups were separated after matching the average body weight of the induction groups as closely as possible. Starting the day after disease induction, KINE-101 dissolved in DPBS was administered intravenously (IV) three times a week at doses of 1, 5, 25, and 50 mg / kg.

[0074] Group administered substances (administration method)Normal-EAN P0(180-199)Emulsion 200 ㎕ (SC) + DPBSKINE-101(1mg / kg)Emulsion 200 ㎕ (SC) + KINE-101 1 mg / kg (IV)KINE-101(5mg / kg)Emulsion 200 ㎕ (SC) + KINE-101 5 mg / kg (IV)KINE-101(25mg / kg)Emulsion 200 ㎕ (SC) + KINE-101 25 mg / kg (IV)KINE-101(50mg / kg)Emulsion 200 ㎕ (SC) + KINE-101 50 mg / kg (IV)

[0075] <2-2> Confirmation of the symptom improvement effect of peptides in CIDP animal models

[0076] To investigate the progression of CIDP according to KINE-101 administration, the degree of CIDP aggravation over time after KINE-101 administration to a CIDP animal model was measured by evaluating the clinical score.

[0077] Specifically, while administering KINE-101 in the manner described in Example <2-1>, symptoms were evaluated from 0 to 5 points as shown in [Table 3] below. The clinical scores were referenced from Brain Behav Immun. 2007 Jul;21(5):699-710. The average value of the evaluation results for each individual was calculated to quantify the severity of CIDP.

[0078] Score Symptoms 0 points: Does not limp, and does not curl when an observer places his or her hand on the tail 1 point: When the soles of the feet are turned upward, the hind legs point upward for more than 3 seconds 2 points: No strength in both hind legs and tail 3 points: One of the two hind legs is paralyzed and always points upward 4 points: Two of the two hind legs are paralyzed and always point upward 5 points: Moribund or dead

[0079] As a result, as shown in Fig. 1, except for the normal group, the remaining groups showed no change in clinical scores, and symptoms of weak tails began to appear from the 10th day. Meanwhile, the CIDP-induced group (EAN P0 (180-199)) showed persistent symptoms after the 14th day, whereas all KINE-101-administered groups showed symptoms that were alleviated after the 15th day, and in particular, the KINE-101 50 mg / kg administration group showed the lowest clinical score. Through the above results, it can be seen that the peptide of the present invention alleviates CIDP symptoms.

[0080] <2-3> Confirmation of the peptide's demyelination-improving effect in a CIDP animal model

[0081] Demyelination in peripheral nerve tissue was observed after administration of KINE-101 to a CIDP animal model.

[0082] Specifically, in the above Example <2-2>, the experimental animals of the KINE-101 (50 mg / kg) group, which had the best effect in alleviating CIDP symptoms, were sacrificed after the experiment was over, and the peripheral nerve tissue of the lower extremities was separated, and the degree of myelin loss was observed using a transmission electron microscope (TEM) in the tissue cross-section. In addition, comparisons were made with the normal group and the CIDP-induced group (EAN P0 (180-199)).

[0083] As a result, as shown in Fig. 2, demyelination and resulting structural changes were observed in the nerves of the CIDP-induced group compared to the normal group, whereas in the KINE-101 (50 mg / kg) group, demyelination was suppressed similarly to the normal group.

[0084] <Example 3> Comparison of the effects of KINE-101 and intravenous immunoglobulin (IVIg) in a CIDP animal model

[0085] <3-1> Creation of CIDP animal model and administration of peptide or intravenous immunoglobulin (IVIg)

[0086] In order to determine the effectiveness of KINE-101 compared to existing treatments, a CIDP animal model with long-term disease was created, and KINE-101 synthesized in <Example 1> or intravenous immunoglobulin (IVIg), used as a CIDP treatment, was administered.

[0087] Specifically, 7-week-old male Lewis rats were provided, and after a one-week acclimatization period, solution A was prepared by dissolving 2 mg / mL of s-palm P0, which is palmitic acid bound to cysteine, the 181st amino acid of the peripheral myelin P0 protein mentioned in <Example 2>, in physiological saline, and solution B was prepared by dissolving tuberculosis bacteria in Freund's adjuvant at 5 mg / mL. After placing a sterile 25 mL tube in an ice box, solution A and solution B were mixed in a 1:1 ratio and homogenized using a homogenizer for 15 minutes to prepare an emulsion, and then 200 μL was subcutaneously injected into the base of the tail of the rats to induce disease. Then, since body weight decreases depending on the severity of CIDP, the rats were divided into 7 groups as shown in [Table 4] below, and the groups were separated after matching the average body weight of the induction group as much as possible. The KINE-101 treatment group received intravenous (IV) injections of KINE-101 dissolved in DPBS at doses of 6.25, 12.5, 25, and 37.5 mg / kg, three times a week for 15 days, starting the day after disease induction. The IVIg treatment group received IVIg at a dose of 400 mg / kg, once daily for 5 days, starting the day after disease induction. In addition, the experiment was terminated on the 15th day after disease induction.

[0088] Group (group) Administration substance (administration method) Normal group (Control) - S-palmP0 (180-199) + saline emulsion 200 ㎕ (SC) + DPBSS-palmP0 (180-199) + KINE-101 6.25 mg / kg emulsion 200 ㎕ (SC) + KINE-101 6.25 mg / kg (IV) S-palmP0 (180-199) + KINE-101 12.5 mg / kg emulsion 200 ㎕ (SC) + KINE-101 12.5 mg / kg (IV) S-palmP0 (180-199) + KINE-101 25 mg / kg emulsion 200 ㎕ (SC) + KINE-101 25 mg / kg (IV) S-palmP0 (180-199) +KINE-101 37.5mg / kg emulsion 200 ㎕ (SC) +KINE-101 37.5 mg / kg (IV)S-palmP0(180-199) +IVIg 400mg / kg emulsion 200 ㎕ (SC) +IVIg 400mg / kg (IV)

[0089] <3-2> Comparison of the symptom improvement effects of peptides and IVIg in CIDP animal models

[0090] To determine the effectiveness of KINE-101 compared to existing treatments, the severity of CIDP over time after administration of KINE-101 or IVIg to a CIDP animal model was assessed using a clinical score.

[0091] Specifically, while administering KINE-101 or IVIg using the method described in Example <3-1>, the induction and progression of the disease were evaluated on a scale of 0 to 5 based on symptoms, as shown in [Table 5] below, by observing the movements of the tail and hind legs (PMID: 25595246). The mean value of the evaluation results for each individual was calculated to quantify the severity of CIDP.

[0092] Score Symptoms 0 points Normal 1 point Weak tail 2 points Abnormal gait 3 points Paralysis of one hind leg 4 points Paralysis of two hind legs 5 points Death

[0093] As a result, as shown in Fig. 3, the negative control group (S-palmP0 (180-199) + saline) showed symptoms of tail weakness on the 11th day of disease induction and abnormal gait on the 15th day, the final day of the experiment. This means that the disease was induced normally. In contrast, both KINE-101 administration groups (6.25 mg / kg ~ 37.5 mg / kg) showed lower clinical scores than the negative control group on the 15th day of disease induction, whereas the IVIg administration group, as a comparison group, tended to show clinical scores similar to or higher than the negative control group. In particular, the KINE-101 25 mg / kg administration group showed the lowest clinical score. Through the above results, it can be seen that the peptide of the present invention shows a superior CIDP symptom alleviation effect compared to IVIg in the treatment of CIDP.

[0094] <3-3> Comparison of the body weight improvement effects of peptides and IVIg in a CIDP animal model

[0095] Demyelinating diseases like CIDP are known to cause movement limitations due to peripheral muscle contraction and paralysis, leading to decreased weight gain due to inadequate food intake. Therefore, to investigate the efficacy of KINE-101 compared to existing treatments, we measured changes in body weight over time after administering KINE-101 or IVIg to CIDP animal models.

[0096] Specifically, body weight was measured while administering KINE-101 or IVIg using the method described in Example <3-1> above, and the change in body weight compared to the body weight before disease induction (Day 0) was calculated.

[0097] As a result, as shown in Fig. 4, the negative control group (S-palmP0 (180-199) + saline) showed a decrease in body weight gain by about 50% on the 8th day of disease induction compared to the normal group (Control), and a decrease in body weight compared to before disease induction (Day 0) on the 15th day of disease induction. On the other hand, the KINE-101 administration group showed a higher body weight gain compared to the negative control group, and maintained a significantly higher value than the body weight before disease induction even on the 15th day of disease induction. In addition, all KINE-101 administration groups maintained a similar or higher value than the IVIg administration group in body weight gain, except on the 14th and 15th days of disease induction. In particular, the body weight increase effect was the best in the KINE-101 12.5 mg / kg administration group. Through the above results, it can be seen that the peptide of the present invention exhibits a body weight improvement effect similar to or superior to IVIg.

[0098] <3-4> Comparison of the nerve conduction improvement effects of peptides and IVIg in CIDP animal models

[0099] Demyelination is known to cause reduced nerve conduction velocity and conduction block. Therefore, to investigate the efficacy of KINE-101 compared to existing treatments, we administered KINE-101 or IVIg to CIDP animal models and performed nerve conduction studies (NCS) using electrophysiological testing.

[0100] Specifically, KINE-101 or IVIg was administered using the method described in Example <3-1> above, and after the experiment was completed, 1.5% isoflurane was used for anesthesia and examination was performed. The fur was removed from the distal to the upper part of the lower extremities, and a Nicolet VikingQuest device was used as an electrophysiological examination device to perform nerve conduction tests to measure motor and sensory nerves, respectively. For the nerve conduction test, needle electrodes were placed on the motor or sensory nerves, the reference electrode was placed on the gastrocnemius muscle, and the stimulating cathode was placed close to the recording electrode in the middle of the posterior thigh to stimulate the distal and proximal parts, respectively. The motor nerve conduction velocity (MNCV) and compound muscle action potential (CMAP) of the motor nerves and the sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) of the sensory nerves were measured.

[0101] As a result, as shown in Fig. 5, the negative control group (S-palmP0(180-199) + saline) showed decreased MNCV and CMAP compared to the normal group (Control). On the other hand, all KINE-101-administered groups showed higher MNCV and CMAP compared to the negative control group, and confirmed effects similar to or superior to the IVIg-administered group. In particular, the KINE-101 12.5 mg / kg administration group showed the best effect.

[0102] In addition, as shown in Figure 6, the negative control group (S-palmP0(180-199) + saline) showed decreased SNCV and SNAP compared to the normal group (Control). On the other hand, both KINE-101-administered groups showed higher SNCV and SNAP compared to the negative control group, confirming effects similar to those of the IVIg-administered group. In particular, the KINE-101 12.5 mg / kg administration group showed the best effect.

[0103] The above results show that the peptide of the present invention exhibits a nerve conduction improvement effect that is superior to (motor nerve conduction) or similar to (sensory nerve conduction) IVIg.

[0104] In addition, when the results of the above examples <3-2> to <3-4> are summarized, it can be seen that the peptide of the present invention exhibits a superior therapeutic effect compared to IVIg in the treatment of CIDP.

[0105] <Example 4> Confirmation of the nerve damage improvement effect of KINE-101 in an animal model of acquired peripheral nerve damage.

[0106] <4-1> Creation of an animal model of acquired peripheral nerve damage and peptide administration

[0107] Demyelination disease occurs when the myelin sheath that makes up nerves is damaged, and can occur in both the peripheral and central nervous systems. Therefore, to investigate the effects of KINE-101 on demyelinating disease, an animal model of acquired peripheral nerve injury was created as described below, and KINE-101, synthesized in <Example 1>, was administered.

[0108] Specifically, by damaging the nerves coming out of the spinal cord at a predetermined intensity for a set period of time, a unified nerve injury model can be obtained, and since the intensity and time can be controlled, a nerve injury model that lasts for a desired period of time can be produced. Accordingly, C57BL / 6 mice were supplied, and after a one-week acclimatization period, they were divided into five groups as shown in [Table 6] below. The disease-induced group produced an animal model of acquired peripheral nerve injury by incising the border area between the gluteal muscles and the thigh muscles of the mice, exposing the sciatic nerve, and then using a surgical tool, a Macjam, to injure the nerve at one of the intensities of 1, 2, or 3 (with 3 seconds as the standard) for a set period of time, and then suturing. From the day after disease induction, KINE-101 dissolved in DPBS was administered intravenously (IV) at 10, 50, or 100 mg / kg three times a week for a total of 4 weeks.

[0109] Group Administration Substance (Administration Method)WT Ctr-Injury CtrNerve injury + DPBSInjury KINE-101 (10 mg / kg)Nerve injury + KINE-101 10 mg / kg (IV)Injury KINE-101 (50 mg / kg)Nerve injury + KINE-101 50 mg / kg (IV)Injury KINE-101 (100 mg / kg)Nerve injury + KINE-101 100 mg / kg (IV)

[0110] <4-2> Confirmation of the peptide's nerve damage-improving effect in an animal model of acquired peripheral nerve damage

[0111] To investigate the effect of KINE-101 in demyelinating diseases, nerve conduction tests were performed using electrophysiological tests after administering KINE-101 to an animal model of acquired peripheral nerve injury.

[0112] Specifically, mice were anesthetized with 1.5% isoflurane at 0, 3, and 6 weeks of KINE-101 administration using the method described in Example <4-1> above, and MNCP, CMAP, SNCV, and SNAP were measured using the same method described in Example <3-4> above to confirm damage to motor nerves and sensory nerves.

[0113] As a result, as shown in Fig. 7, the MNCV was measured as 25.8 ± 9.4 m / s in the acquired peripheral nerve injury group (Injury Ctr), electrophysiologically confirming that the motor nerves were damaged compared to the normal group (WT Ctr, 57.9 ± 9.5 m / s). On the other hand, in the third week of administering KINE-101 three times a week for four weeks after acquired peripheral nerve injury, the MNCV of the KINE-101 50 mg / kg administration group was 46.7 ± 7.4 m / s (p = 0.030), which was statistically significantly increased compared to the acquired peripheral nerve injury group (36.3 ± 7.2 m / s). In addition, in the 6th week of administering KINE-101 3 times a week for 4 weeks, the MNCV of the KINE-101 administration group increased, and in particular, the MNCV of the KINE-101 50 mg / kg administration group (63.9 ± 7.6 m / s, p = 0.008) and the KINE-101 100 mg / kg administration group (59.0 ± 4.5 m / s, p = 0.013) increased statistically significantly compared to the acquired peripheral nerve injury group.

[0114] In addition, as shown in Fig. 8, CMAP was measured as 2.5 ± 0.87 mV in the acquired peripheral nerve injury group, electrophysiologically confirming that the motor nerves were damaged compared to the normal group (39.3 ± 2.4 mV). On the other hand, in the 3rd and 6th weeks of KINE-101 administration 3 times a week for 4 weeks after acquired peripheral nerve injury, the CMAP of the KINE-101 administration group increased compared to the acquired peripheral nerve injury group. In particular, in the third week of administering KINE-101 three times a week for four weeks after acquired peripheral nerve damage, statistically significant increases were observed in the KINE-101 10 mg / kg administration group (11.0 ± 2.3 mV, p = 0.044) and the KINE-101 50 mg / kg administration group (12.3 ± 3.5 mV, p = 0.039) compared to the acquired peripheral nerve damage group, and in the sixth week of administering KINE-101 three times a week for four weeks after acquired peripheral nerve damage, statistically significant increases were observed in the KINE-101 50 mg / kg administration group (32.9 ± 4.0 mV, p = 0.007) compared to the acquired peripheral nerve damage group.

[0115] In addition, as shown in Figure 9, SNCV was measured as 20.2 ± 1.9 m / s in the acquired peripheral nerve damage group, electrophysiologically confirming that the sensory nerves were damaged compared to the normal group (25.6 ± 2.6 m / s). On the other hand, in the third week of administering KINE-101 three times a week for four weeks after acquired peripheral nerve injury, SNCV was statistically significantly increased in the KINE-101 10 mg / kg group (27.7 ± 2.8 m / s, p = 0.000), KINE-101 50 mg / kg group (27.6 ± 2.2 m / s, p = 0.000), and KINE-101 100 mg / kg group (28.6 ± 3.7 m / s, p = 0.001) compared to the acquired peripheral nerve injury group (21.1 ± 1.2 m / s). After 6 weeks of administering KINE-101 3 times a week for 4 weeks after acquired peripheral nerve injury, the SNCV of the KINE-101 50 mg / kg administration group was 27.9 ± 3.1 m / s (p = 0.038), which was statistically significantly higher than that of the acquired peripheral nerve injury group (23.4 ± 3.6 m / s).

[0116] In addition, as shown in Fig. 10, SNAP was electrophysiologically confirmed to be 7.8 ± 2.2 μV in the acquired peripheral nerve injury group, which was higher than that in the normal group (8.5 ± 2.6 μV). On the other hand, in the 3rd and 6th weeks of KINE-101 administration 3 times a week for 4 weeks after acquired peripheral nerve injury, SNAP in the KINE-101 administration group increased compared to the acquired peripheral nerve injury group. In particular, in the third week of administering KINE-101 three times a week for four weeks after acquired peripheral nerve damage, a statistically significant increase was observed in the KINE-101 50 mg / kg group (11.2 ± 3.1 μV, p = 0.050) compared to the acquired peripheral nerve damage group (7.9 ± 1.1 μV), and in the sixth week of administering KINE-101 three times a week for four weeks after acquired peripheral nerve damage, a statistically significant increase was observed in the KINE-101 50 mg / kg group (12.3 ± 2.9 μV, p = 0.046) compared to the acquired peripheral nerve damage group (8.3 ± 3.5 μV).

[0117] The above results show that the peptide of the present invention improves damage to the peripheral nervous system.

[0118] <4-3> Confirmation of symptom improvement by peptide in an animal model of acquired peripheral nerve damage

[0119] Demyelination disorders are known to be accompanied by symptoms such as muscle weakness and impaired balance. Therefore, a rotarod test was performed in an animal model of acquired peripheral nerve injury after administering KINE-101.

[0120] Specifically, the phenotypes of experimental animals were analyzed using Rotarod, grip strength, and tail suspension. The Rotarod test was performed up to three times with a cut-off time of 3 minutes to assess muscle strength. Using the Rotarod device (ROTA ROD, LE8205, Panlab), the time it took the subject animal to fall from the rod was measured during a 2-minute and 30-second trial at a fixed speed of 8 rpm. For each test, three trials were performed, and the maximum duration was analyzed.

[0121] As a result, as shown in Fig. 11, it was confirmed that both the KINE-101 administration groups showed improvement in muscle strength and sense of balance compared to the acquired peripheral nerve injury group (Injury Ctr) in the 3rd and 6th weeks of administering KINE-101 3 times a week for 4 weeks after acquired peripheral nerve injury.

[0122] <4-4> Confirmation of improvement in demyelination by peptides in an animal model of acquired peripheral nerve damage

[0123] After administering KINE-101 to an animal model of acquired peripheral nerve injury, nerve tissue pathology examination was performed.

[0124] Specifically, KINE-101 was administered to an animal model of acquired peripheral nerve damage for 4 weeks using the method described in Example <4-1>, and the experimental animals were sacrificed 6 weeks after the start of administration. Then, the peripheral nerve tissue of the lower extremities was isolated and fixed with a 4% paraformaldehyde solution containing 2.5% glutaraldehyde. After dehydration with ethanol, it was embedded in epoxy resin, and semi-thin sections (1 μm) were performed. The semi-thin sections were stained with toluidine blue for 5 to 10 seconds and observed under a light microscope. In addition, ultra-thin sections (65 nm) were performed, and the tissue sections were observed for myelin malformations (demyelination, dysmyelination, hypermyelination) using a transmission electron microscope (TEM). In the nerves of neuropathic mice, nerves with abnormal thickness and shape were observed, and malformed myelin sheaths due to axonal abnormalities were observed under an electron microscope.

[0125] As a result, as shown in Figures 12a and 12b, the KINE-101 administration group showed improved myelination compared to the acquired peripheral nerve injury group (Injury Ctr). In addition, the number of large myelinated fibers increased and the number of small unmyelinated fibers decreased. In particular, compared to the acquired peripheral nerve injury group, the KINE-101 50 mg / kg administration group and the KINE-101 100 mg / kg administration group showed a clear increase in myelinated axons and a decrease in unmyelinated fibers.

[0126] <4-5> Confirmation of Treg cell ratio and activity in an animal model of acquired peripheral nerve injury

[0127] After administering KINE-101 to an animal model of acquired peripheral nerve injury, the proportion of Treg cells present in the lymph nodes at the site of nerve injury and Treg cell activity were confirmed.

[0128] Specifically, KINE-101 was administered to an animal model of acquired peripheral nerve injury for 4 weeks using the method described in Example <4-1> above, and the experimental animals were sacrificed 6 weeks after the start of administration. Next, immune cells were isolated from the lymph nodes at the site of nerve injury in the experimental animals. The isolated immune cells were subjected to fluorescent staining for CD4 and Foxp3 proteins, and the ratio of Treg cells among CD4 T cells was confirmed through flow cytometry. Fluorescent staining for CTLA4 protein, an activation marker of Treg cells, was performed, and the degree of CTLA4 expression in Treg cells was compared and the degree of activation was confirmed through flow cytometry.

[0129] As a result, as shown in Fig. 9, the ratio of Treg cells present in the lymph nodes of the peripheral nerve injury site of the acquired peripheral nerve injury model was compared with the normal group, and it was confirmed that the ratio of Treg cells increased in the KINE-101 administration group. In particular, the ratio of Treg cells increased to a statistically significant level in the KINE-101 50 mg / kg administration group.

[0130] In addition, the expression level of CTLA4, one of the activation markers of Treg cells, was compared, and it was confirmed that CTLA4 expression increased in the KINE-101 administration group. In particular, CTLA4 expression increased to a statistically significant level in the KINE-101 50 mg / kg administration group, confirming that Treg cell activation was induced by KINE-101 administration.

[0131] Therefore, through the results of the above <Example 2> to <Example 4>, it was confirmed that the peptide of the present invention has the effect of inhibiting demyelination and improving myelination in a CIDP animal model and an acquired peripheral nerve injury animal model, and improving nerve damage and symptoms due to demyelination. Therefore, the peptide of the present invention can be used for the prevention or treatment of demyelinating diseases including CIDP.

[0132] The trimer-shaped peptide according to the present invention exhibits effects of inhibiting demyelination and enhancing myelination in a chronic inflammatory demyelinating polyneuropathy (CIDP) animal model and an acquired peripheral nerve injury animal model, and improving nerve damage and symptoms due to demyelination, and thus can be usefully used as an effective ingredient of a composition for preventing or treating demyelinating diseases including CIDP.

Claims

1. A pharmaceutical composition for preventing or treating demyelinating disease, comprising as an active ingredient a peptide composed of an amino acid represented by sequence number 1 or a polynucleotide encoding the same.

2. A pharmaceutical composition for preventing or treating demyelinating disease, wherein the N- or C-terminus of the peptide in claim 1 is bonded to a protecting group selected from the group consisting of an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).

3. In paragraph 1, the demyelinating disease is Charcot-Marie-Tooth disease (CMT), Chronic inflammatory demyelinating polyneuropathy (CIDP), Idiopathic inflammatory demyelinating disease, Hereditary neuropathy, Anti-MAG peripheral neuropathy, Progressive inflammatory neuropathy, Optic neuropathy, Devic's disease, Central pontine myelinolysis (CPM), Extrapontine Myelinolysis (EPM), Tabes dorsalis, Leukoencephalopathies, Demyelinating A pharmaceutical composition for preventing or treating a demyelinating disease, wherein the demyelinating disease is selected from the group consisting of demyelinating leukodystrophy, neuromyelitis optica, demyelinating optic neuritis, acute disseminated demyelination, periaxial encephalitis, central demyelination of corpus callosum, acute transverse myelitis, subacute necrotizing myelitis, and concentric sclerosis.

4. A pharmaceutical composition for preventing or treating a demyelinating disease, wherein the peptide in claim 1 improves demyelination.

5. A pharmaceutical composition for preventing or treating demyelinating disease, wherein the peptide in claim 1 improves damage to motor nerves and sensory nerves.

6. A pharmaceutical composition for preventing or treating demyelinating disease, wherein the peptide in paragraph 1 improves reduction in motor nerve conduction and sensory nerve conduction.

7. A pharmaceutical composition for preventing or treating demyelinating disease, wherein the composition in claim 1 increases the ratio of Treg cells in the lymph nodes at the site of peripheral nerve damage and induces Treg cell activation.

8. A pharmaceutical composition for preventing or treating demyelinating disease, wherein the composition in claim 1 is formulated for oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration, or topical administration.

9. A health functional food composition for preventing or improving demyelinating disease, comprising as an active ingredient a peptide composed of an amino acid represented by sequence number 1 or a polynucleotide encoding the same.

10. A method for treating a demyelinating disease, comprising administering to a subject a therapeutically effective amount of a peptide consisting of an amino acid represented by sequence number 1 or a polynucleotide encoding the same.

11. A method for preventing or improving a demyelinating disease, comprising administering to a subject a peptide consisting of an amino acid represented by sequence number 1 or a polynucleotide encoding the same.

12. Use of a peptide consisting of an amino acid represented by sequence number 1 or a polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating demyelinating diseases.

13. Use of a peptide consisting of an amino acid represented by sequence number 1 or a polynucleotide encoding the same for use as a health functional food composition for preventing or improving demyelinating disease.

14. Use of a peptide consisting of an amino acid represented by sequence number 1 or a polynucleotide encoding the same for the manufacture of a pharmaceutical composition for preventing or treating demyelinating disease.

15. Use of a peptide composed of an amino acid represented by sequence number 1 or a polynucleotide encoding the same for manufacturing a health functional food composition for preventing or improving demyelinating disease.

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