KR101921085B1 - Pharmaceutical composition for treating muscle atrophy comprising glucagon like-peptide-1, GLP-1 derived peptide, or GLP-1 degradation inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscular dystrophy or myopenia comprising glucagon-like peptide-1 (GLP-1), a GLP-1 derived peptide, or a GLP-1 decomposition inhibitor and a pharmaceutical composition for preventing or treating muscular dystrophy or myopenia ≪ / RTI > When the pharmaceutical composition provided by the present invention is administered to a subject suffering from myopenia or myopathy, the level of expression of the genes involved in myopenia or muscular dystrophy, weight of skeletal muscle, Therefore, it can be widely used for the development of a therapeutic agent for effective myopenia or muscular dystrophy.

Description

Pharmaceutical composition for treating muscle atrophy comprising glucagon like-peptide-1, GLP -1 derived peptide, or GLP-1 degradation inhibitor}

The present invention comprises a glucagon-like peptide-1 (GLP-1, Glucagon-like peptide-1), a GLP-1 derived peptide, or a pharmaceutical composition for preventing or treating muscular dystrophy or sarcopenia and the pharmaceutical composition comprising a GLP-1 degradation inhibitor It relates to a method of treating muscular dystrophy or sarcopenia by using.

Sarcopenia caused by degeneration of the spinal nerve, motor nerve or skeletal muscle fibers is one of the representative refractory diseases for which the cause of the disease has not been identified. According to the studies so far, the motor nerve that induces the contraction of the skeletal muscle is degenerated so that the contraction of the skeletal muscle does not proceed, or the expression of the protein involved in the contraction of the muscle in the skeletal muscle is reduced or the protein is modified to cause normal skeletal muscle contraction. This does not proceed, and in the long term, it is known that the motor nerve or skeletal muscle is transformed into a fibrous tissue. Since the underlying cause of such sarcopenia has not yet been identified, and a method for preventing or recovering degeneration of motor nerves or skeletal muscles has not been developed, at present, a method of slowing the progression of sarcopenia has been developed. Research for this is being actively conducted.

Currently, as a treatment method for sarcopenia, a method of inhibiting muscle atrophy caused by degeneration or progressive mutation of muscle cells, which is a type of sarcopenia, is mainly used. For example, WO 2007/088123 discloses a therapeutic agent for muscular dystrophy comprising a nitroxy derivative as an active ingredient, and WO 2006/081997 discloses a therapeutic agent for muscular atrophy comprising atlaric acid or a derivative thereof as an active ingredient. However, these therapeutic agents containing the compound as an active ingredient act not only on the skeletal muscles in which muscular dystrophy has occurred, but also on visceral muscles or myocardium that are not related to muscular dystrophy, and thus, various side effects, large and small, have not been used for practical treatment. . On the other hand, hormone preparations have significantly reduced side effects compared to compound preparations, and because the characteristics of hormone preparations are biocompatible, the development of drugs for treating muscular dystrophy or sarcopenia using hormone preparations is accelerating.

On the other hand, muscular atrophy is a disease in which the muscles of the limbs are atrophy, and amyotrophic lateral sclerosis and spinal progressive muscular atrophy are typical, and this is known as a disease caused by progressive degeneration of motor nerve fibers and cells in the spinal cord.

Specifically, the spinal muscular dystrophy is a genetic disorder caused by degeneration of motor neurons of the spinal cord and is known as one of neuromuscular diseases. In addition, amyotrophic lateral sclerosis is characterized by intractable and irreversible neurodegenerative changes due to the death of upper and lower motor neurons in the cerebral and spinal cord, and lack of neuronal growth factors and neuroinflammation are known to be the main causes.

Under this background, the present inventors have made intensive research efforts to develop an agent capable of effectively treating muscular dystrophy or sarcopenia. As a result, glucagon-like peptide-1, a peptide derived therefrom, and a decomposition inhibitor thereof, can alleviate the symptoms of muscular atrophy or sarcopenia. It was confirmed that it could be, and the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating muscular atrophy or sarcopenia.

Another object of the present invention is to provide a method of treating muscular dystrophy or sarcopenia using the pharmaceutical composition.

While conducting various studies to develop a hormone formulation that can effectively treat sarcopenia or slow its progression, the present inventors have been paying attention to hormone formulations with few side effects. Since the hormone preparation is a pharmaceutical composition that minimizes side effects through a certain level of preclinical and clinical trials, if an agent capable of treating or slowing sarcopenia among these hormone preparations can be selected, the problem caused by the onset of side effects can be solved. I expected to be able to. Accordingly, as a result of screening a formulation exhibiting a treatment or slowing effect of sarcopenia with the previously disclosed hormone formulation, it was confirmed that GLP-1 or its receptor stimulating agent had such an effect.

Specifically, exendin-4 is a type of glucagon-like peptide-1 receptor agonist, and is a proteinaceous hormone preparation that plays a similar role to GLP-1. Exendin-4 was developed for the treatment of diabetes and is known to have an increased half-life in blood compared to GLP-1.

In the present invention, as a result of administering the exendin-4 to an animal artificially inducing muscular dystrophy, and analyzing the effect on the disease, muscle characteristics such as body weight, skeletal muscle weight, and grip strength reduced due to muscular atrophy are recovered, and muscle protein It was confirmed that the expression of the gene that destroys the was decreased, and the expression of the gene that produced the muscle protein was increased.

In addition, as with GLP-1, exendin-4 is known to have similar activity in the body via the GLP-1 receptor, and the present inventors found that from the effect of exendin-4 on the treatment of muscular atrophy or sarcopenia, GLP- 1 is also expected to exhibit the same level of effect, and muscular atrophy caused by administration of sitagliptin, which is known to inhibit the degradation of GLP-1 derived peptides GLP-1(32-36)amide and GLP-1, or As a result of confirming the therapeutic effect of sarcopenia, like Exendin-4, muscle characteristics such as body weight, skeletal muscle weight, and grip strength decreased due to muscle atrophy are restored, the expression of genes that destroy muscle protein is reduced, and muscle protein is produced. It was confirmed that the expression of the gene was increased.

As such, it was newly identified that GLP-1, a peptide derived therefrom, and a decomposition inhibitor exhibit an effect of preventing or treating muscular dystrophy or sarcopenia, apart from the previously known diabetes treatment effect, and such a new effect has not been known at all until now. , Was first identified by the inventors.

In order to achieve the above object, the present invention is a glucagon-like peptide-1 (GLP-1, Glucagon-like peptide-1), a GLP-1 derived peptide, a GLP-1 degradation inhibitor, and a combination thereof as an embodiment. It provides a pharmaceutical composition for preventing or treating muscular atrophy or sarcopenia comprising any one selected from the group consisting of.

The term “glucagon-like peptide-1” of the present invention is an incretin derived from the transcription product of the proglucagon gene, which is a prohormone. It is a hormone that is secreted by stimulation by the concentration of internal nutrients or blood sugar. Its main function is known to stimulate insulin secretion. Therefore, research and development are currently being conducted as drugs that can effectively lower blood sugar in the treatment of diabetes.

GLP-1 is composed of 30 amino acids, and the amino acid sequence of GLP-1 is known to be 100% identical in all mammals. It is known that glucagon is produced in islet α cells and GLP-1 is produced in L cells of the ileum and large intestine by a post-transcriptional process from proglucagon. Specifically, the GLP-1 may have the sequence of SEQ ID NO: 2, but is not limited thereto.

The protein and the gene sequence encoding the protein can be obtained from a known database, and examples thereof include NCBI's GenBank, but are not limited thereto.

The protein of the present invention includes not only the amino acid sequence described in SEQ ID NO: but also an amino acid sequence having 80% or more, specifically 90% or more, more specifically 95% or more, particularly, 97% or more homology with the sequence. It may include. As a sequence having such a homology, it includes without limitation any amino acid sequence that represents a protein that is substantially the same as each of the proteins or exhibits a corresponding efficacy. In addition, in the case of an amino acid sequence having such homology, it is obvious that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

In addition, the gene encoding each protein of the present invention is not only the base sequence encoding the amino acid described in each of the sequence numbers, but also 80% or more, specifically 90% or more, more specifically 95% or more. , More specifically, a nucleotide sequence showing homology of 98% or more, and most specifically 99% or more, and a gene sequence encoding a protein that is substantially the same as or corresponding to each protein is included without limitation. In addition, in the case of a nucleotide sequence having such homology, it is obvious that a nucleotide sequence in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

The term "homology" used in the present invention refers to the amino acid sequence or nucleic acid sequence of a gene encoding a protein, after aligning both sequences so that they match as much as possible in a specific comparison region, and then the base or amino acid residue between the sequences It means the same degree. If the homology is sufficiently high, the expression product of the corresponding gene may have the same or similar activity. The percent of sequence identity may be determined using a known sequence comparison program, and examples include BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), and the like.

The term "GLP-1 derived peptide" of the present invention is derived from the amino acid sequence of GLP-1 and prepared through any one of substitution, addition, removal, and modification of some amino acids in GLP-1, or a combination of these methods. Refers to the amino acid sequence. The GLP-1-derived peptide can be prepared by a method known to those skilled in the art. For the purposes of the present invention, it may be included in the "GLP-1 derived peptide" of the present invention as long as it has a muscular atrophy or sarcopenia effect derived from GLP-1. The GLP-1 derived peptide may include at least 5 or more, at least 10 or more, at least 15 or more, or at least 20 or more amino acids of the GLP-1 sequence. Specifically, the GLP-1-derived peptide may be GLP-1(28-36)amide, GLP-1(32-36)amide, and more specifically, the GLP-1-derived peptide is 5 of GLP-1. It may be GLP-1 (32-36) amide (LVKGR amide) prepared from amino acids, but is not limited thereto.

According to one specific aspect of the present invention, the GLP-1(32-36)amide may be composed of the sequence of SEQ ID NO: 3 (LVKGR).

The present inventors found that GLP-1(32-36)amide has a therapeutic effect on muscular atrophy or sarcopenia through an increase in body weight, muscle weight, and grip strength, decrease in Myostatin expression, and increase in MyoD expression in dexamethasone-treated mice. As a result, it was confirmed that the GLP-1(32-36)amide treatment effect could be obtained for muscular dystrophy or sarcopenia (FIGS. 6 to 9).

The term "GLP-1 receptor (GLP-1R, Glucagon-like peptide-1 receptor)" of the present invention means GLP-1 (Glucagon-like peptide-1), a type of gastrointestinal hormone derived from the transcript of the glucagon gene. It refers to a receptor protein that can bind to and can play a role in reducing blood sugar levels. Specifically, the receptor increases the transcription and expression of insulin genes through upregulation of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) by binding to GLP-1.

As used herein, the term "GLP-1 receptor agonist (GLP-1R agonist)" refers to a substance or drug that binds to the GLP-1 receptor and acts similar to GLP-1, or a molecule that increases the activity of a receptor site. , Also called operator. As the effect of increasing insulin secretion by receptor binding of GLP-1 was revealed, the agonist is currently being used as a therapeutic agent for type 2 diabetes.

Examples of the receptor agonist include, as natural agonists, GLP-1 and glucagon, and other liraglutide, exendin-4, lixisenatide, and the like. have.

For the purposes of the present invention, the GLP-1 receptor agonist includes a substance that binds to the GLP-1 receptor and has a similar activity through a signal transduction pathway similar to that of GLP-1, and increases muscle mass. And it may have a therapeutic effect of muscular atrophy or sarcopenia through strengthening of muscle strength.

The term "Exendin-4" of the present invention refers to a peptide composed of a sequence of 39 amino acids that serves as an agonist of the GLP-1 receptor and has a molecular weight of about 4kDa. Exendin-4 rapidly regulates blood sugar levels, reduces insulin resistance and glucagon levels, and has an effect of stimulating the growth of pancreatic beta cells that stimulate insulin production, so it is a major therapeutic agent for diabetes that exhibits insulin resistance. Is being used. The amino acid sequence of exendin-4 is not particularly limited thereto, but may be composed of the amino acid sequence of SEQ ID NO: 1.

In the present invention, the exendin-4 is used as an active ingredient of a pharmaceutical composition for the treatment of muscular dystrophy or sarcopenia, and the exendin-4 is a weight gain, skeletal muscle weight increase, muscle protein with respect to the diseased animal. It may exhibit effects such as suppressing the expression of genes that destroy cells, and increasing the expression of genes that produce muscle proteins. At this time, the gene that destroys the muscle protein is not particularly limited thereto, but as an example, proteins such as Myostatin, Atrogin-1, and MuRF1 (Muscle RING-finger protein-1) are used. It may be a gene encoding, and a gene that generates muscle protein is not particularly limited thereto, but may be, for example, a gene encoding a protein such as MyoD or Myogenin.

According to an embodiment of the present invention, as an animal model, a vehicle (PBS) or exendin-4 is treated respectively in a mouse in which sarcopenia or atrophy is induced by dexamethasone and a mouse in which the disease is induced, and exendin-4 is treated by using the same. As a result of verifying the effect of -4 on the mouse model, when exendin-4 was treated in the mouse, the reduced body weight was recovered (Figure 1), and the reduced skeletal muscle weight was recovered (Figure 2), Reduced grip strength is restored (Fig. 3), genes that destroy muscle proteins increased by dexamethasone (Myostatin, Atrogin-1, and MuRF1) have decreased expression levels, while genes that produce muscle proteins (MyoD and Myogenin) It was confirmed that the expression level of was increased (FIG. 4).

From this, the present inventors confirmed that the exendin-4 can be used for the prognosis and treatment of muscular dystrophy or sarcopenia.

On the other hand, the exendin-4 is an example of a GLP-1 receptor agonist, the same as GLP-1, since it has a similar in vivo activity through GLP-1 receptor binding, the GLP-1 is the exendin-4 Equally, it can be used as an active ingredient in a pharmaceutical composition for preventing or treating sarcopenia or muscular atrophy.

The term "GLP-1 degradation inhibitor" of the present invention means an agent capable of maintaining the action of GLP-1 by inhibiting the degradation of GLP-1. Although not particularly limited thereto, DPP-4 inhibitors and the like are known.

According to a specific embodiment of the present invention, the inhibitor of GLP-1 degradation may be a DPP-4 (dipeptidyl peptidase-4) inhibitor.

The term "DPP-4 (dipeptidyl peptidase-4) inhibitor" of the present invention is an enzyme that inhibits DPP-4 enzyme known to degrade GLP-1, and inhibits GLP-1 degradation through inhibition of DPP-4. , It is possible to keep the concentration of GLP-1 high. Specifically, the inhibitor may be vildagliptin, sitagliptin, saxagliptin, and more specifically sitagliptin, but is not limited thereto.

In the present invention, the inhibitor of GLP-1 degradation acts to maintain the concentration of GLP-1 by inhibiting the degradation of GLP-1, and as a result, the inhibitor of GLP-1 degradation may also have a therapeutic effect on muscular atrophy or sarcopenia. have.

The present inventors confirmed that sitagliptin increases body weight, muscle weight, and grip strength, decreases the expression of Myostatin, and increases the expression of MyoD in dexamethasone-treated animals with muscular dystrophy. It was confirmed that the treatment effect was shown (FIGS. 6 to 9).

The term “muscle atrophy” of the present invention refers to a general term for a disease in which the muscles of the limbs are gradually atrophy in an almost symmetrical symmetric manner. When inducing cancer, aging, kidney disease, inherited disease, and various chronic diseases It may be accompanied by, and is represented by amyotrophic lateral sclerosis (Lou Gehrig's disease), spinal progressive muscular atrophy, and the like.

The term "sarcopenia" of the present invention means that the density and function of the muscles are gradually weakened, and the cause is known as the progressive degeneration and destruction of motor neurons or muscle cells in the spinal nerve or the brain. In particular, muscle loss due to aging is called age-related sarcopenia.

As a method of slowing the progression of sarcopenia, a method of suppressing muscle atrophy, a type of sarcopenia, is mainly used. The present inventors also confirmed the increase in body weight, muscle mass, and grip strength of exendin-4, GLP-1(32-36)amide and sitagliptin in a mouse model of dexamethasone-induced muscular atrophy, decrease in Myostatin expression, and increase MyoD expression. In addition to the treatment effect of muscular dystrophy, the effect of treating sarcopenia was confirmed.

The present inventors have confirmed that the muscular dystrophy and sarcopenia can be treated using a glucagon-like peptide-1, a GLP-1 derived peptide, or a GLP-1 degradation inhibitor.

The composition of the present invention may be prepared in the form of a pharmaceutical composition for the prevention or treatment of muscular dystrophy or sarcopenia disease, further comprising an appropriate carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions. Specifically, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. I can. In the present invention, the carrier, excipient, and diluent that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate. , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the extract and its fractions, It is prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include various excipients, such as humectants, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are simple diluents commonly used for suspensions, liquid solutions, emulsions, syrups, etc. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The content of the GLP-1, GLP-1 derived peptide, or GLP-1 degradation inhibitor contained in the pharmaceutical composition of the present invention is not particularly limited thereto, but as an example, 0.0001 to 10% by weight based on the total weight of the final composition, As another example, it may be included in an amount of 0.01 to 3% by weight.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention is used to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. It means a sufficient amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion. The duration, factors including drugs used in combination or co-use with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or may be administered in combination with a known agent for treating muscular dystrophy or sarcopenia. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, weight, sex, history, or type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention can be administered in an amount of about 0.1 ng to about 100 mg/kg, preferably 1 ng to about 10 mg/kg per adult, and the frequency of administration of the composition of the present invention is specifically Although not limited, it may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

In another aspect, the present invention provides a method for treating sarcopenia or muscular atrophy comprising administering the pharmaceutical composition in a pharmaceutically effective amount to an individual with or at risk of developing sarcopenia or muscular atrophy. .

The term "individual" of the present invention may include, without limitation, mammals, farmed fish, etc., including mice, livestock, humans, etc. that are likely to develop sarcopenia or muscular dystrophy.

The route of administration of the pharmaceutical composition for the treatment of muscular dystrophy or sarcopenia of the present invention may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but the route of intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. Can be administered through. However, when administered orally, the GLP-1, GLP-1 derived peptide, or GLP-1 degradation inhibitor may be denatured by gastric acid, so the oral composition should be coated with an active agent or formulated to protect against degradation in the stomach. do. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.

In another aspect, the present invention provides a use of the pharmaceutical composition in the manufacture of a medicament for the prevention or treatment of muscular dystrophy or sarcopenia.

When the pharmaceutical composition for preventing or treating muscular dystrophy or sarcopenia provided in the present invention is administered to an individual suffering from sarcopenia or muscular dystrophy, the reduced body weight due to sarcopenia or muscular atrophy, the weight of skeletal muscle, grip strength, and genes involved in muscle production Since the expression level of can be restored to a normal state, it can be widely used in the development of effective treatments for sarcopenia or muscular atrophy.

FIG. 1 is a graph showing changes in body weight measured while rearing control mice, control mice, mice in which muscular atrophy is induced, and mice whose muscular atrophy is improved by exendin-4 for 20 days.
FIG. 2 is a graph showing the results of comparing the weight of each skeletal muscle obtained in a control mouse, a control group, a mouse induced with muscular dystrophy, and a mouse whose muscular atrophy was improved by exendin-4.
3 is a graph showing the results of comparing the measured grip strength in a control mouse, a control group mouse, a mouse induced with muscular dystrophy, and a mouse whose muscular atrophy was improved by exendin-4.
Figure 4 is a control mouse, a control mouse, a muscle dystrophy-induced mouse, and the muscle of the mouse whose muscular atrophy is improved by exendin-4 genes (Myostatin, Atrogin-1, and MuRF1) and muscle protein are generated in the muscle. This is a graph showing the results of comparing the expression levels of the genes (MyoD and Myogenin).
5 is a control muscle cell, a muscle cell in which muscle damage was induced by treatment with dexamethisone, a control group muscle cell, and a gene related to muscular atrophy in muscle cells in which muscle damage was recovered by treatment with dexamethasone and exendin-4 (Myostatin, It is a graph showing the result of comparing the expression level of Atrogin-1 and MuRF1).
6 is a diagram showing changes in the weight of mice by sitagliptin and GLP-1(32-36)amide.
7 is a diagram showing changes in muscle weight of mice by sitagliptin and GLP-1(32-36)amide.
Figure 8 is a diagram showing the change in grip strength of the mouse by sitagliptin and GLP-1(32-36)amide.
9 is a diagram showing changes in the expression of myostatin and MyoD by sitagliptin and GLP-1(32-36)amide.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Effect of Exendin-4 on the body weight of an animal in which muscular dystrophy is induced

Muscular dystrophy is caused by chronic diseases (chronic renal failure, chronic heart failure, chronic obstructive disease, etc.), and it is known that it is caused when a drug such as dexamethasone is administered at a high dose, so dexamethasone is used as an animal model (C57BL/ 6J male mice) to create a model of muscular dystrophy, and to verify the effect of exendin-4 when muscular atrophy occurs.

Specifically, control mice that did not receive dexamethasone or exendin-4, mice in which muscle atrophy was induced by intraperitoneal injection of dexamethasone (200 mg/kg) for 8 days, exendin-4 (100 ng/mouse) for 12 days Control mice injected intraperitoneally and dexamethasone (200mg/kg) were injected intraperitoneally for 8 days, and then exendin-4 (100 ng/mouse) was injected into the intraperitoneal cavity for 12 days, respectively. Set and, while raising each of these mice for 20 days, the change in body weight was measured (FIG. 1).

1 is a graph showing changes in body weight measured while rearing control mice, control mice, mice induced with muscular atrophy, and mice treated with muscular atrophy for 20 days. As shown in FIG. 1, it was confirmed that the weight of mice treated with dexamethasone and exendin-4 in combination was increased compared to mice treated with dexamethasone alone from the time point 11 days had elapsed.

Therefore, it was found that exendin-4 exhibits an effect of improving the symptoms of the mouse caused by muscular dystrophy.

Example 2: Effect of Exendin-4 on the muscle weight of an animal in which muscular atrophy is induced

Control mice set in Example 1, mice in which muscular atrophy was induced by treatment with dexamethasone, control mice treated with exendin-4, and mice treated with exendin-4 after dexamethasone treatment were treated with After sacrifice, the total weight of each mouse skeletal muscle was measured, and the quadratus muscle, gastrocnemus muscle, tibialis anterior muscle, soleus muscle, and extensor extension muscle constituting the skeletal muscle were measured. digitorum longus muscle) was separated by region, and the weights of each of them were compared (FIG. 2).

FIG. 2 is a graph showing the results of comparing the weight of each skeletal muscle obtained in a control mouse, a control group, a mouse induced with muscular dystrophy, and a mouse treated with muscular atrophy. As shown in FIG. 2, it was confirmed that the muscle weight of the mice caused by muscular dystrophy in all types of skeletal muscles decreased compared to the control and control groups, but the muscle weight of the mice treated with muscular atrophy increased to a level similar to that of the control or control group. I did.

Example 3: Effect of Exendin-4 on the grip strength of an animal caused by muscular dystrophy

Control mice set in Example 1, mice in which muscular atrophy was induced by treatment with dexamethasone, control mice treated with exendin-4, and mice treated with exendin-4 after dexamethasone treatment were treated with By measuring the grip strength as a target, it was attempted to confirm whether the function of the muscle was restored (FIG. 3). At this time, the force measured when all the feet of the mouse were put on and pulled with the same force using a grip strength machine was regarded as the grip strength.

3 is a graph showing the results of comparing the measured grip strength in a control mouse, a control group, a muscle atrophy-induced mouse, and a muscle atrophy-treated mouse. As shown in FIG. 3, it was confirmed that the mouse in which muscular dystrophy was induced showed the lowest level of grip strength, while the grip strength was significantly higher than that of the mouse in which the muscular dystrophy was induced. The recovery was confirmed.

Example 4: Effect of Exendin-4 on the Expression Level of Genes Related to Muscle Protein Production and Destruction in Muscular Atrophy-Induced Animals

When muscular dystrophy occurs, it is known that the expression of genes that destroy muscle proteins (Myostatin, Atrogin-1, and MuRF1) increases, and the expression of genes that produce muscle proteins (MyoD and Myogenin) decreases, so we tried to confirm this. That is, a control mouse set in Example 1, a mouse in which muscular atrophy was induced by treatment with dexamethasone, a control mouse treated with exendin-4, and a treatment with exendin-4 after dexamethasone treatment to treat muscular atrophy Total RNA was obtained from the muscle tissue extracted from the mouse, cDNA was synthesized therefrom, and each gene was amplified from the synthesized cDNA using a PCR method to compare the changes in their expression levels (FIG. 4).

4 shows genes (Myostatin, Atrogin-1, and MuRF1) that destroy muscle proteins and genes (MyoD and Myogenin) that destroy muscle proteins in the muscles of control mice, control mice, mice induced with muscular dystrophy, and mice treated with muscular dystrophy. ) Is a graph showing the result of comparing the expression level. As shown in Figure 4, in the muscle tissue of the mouse in which muscular dystrophy is induced, genes that destroy muscle proteins (Myostatin, Atrogin-1, and MuRF1) were expressed at a relatively high level, and genes that generate muscle proteins (MyoD and Myogenin ) Was found to be expressed at the relatively lowest level. On the other hand, in mice treated with muscular dystrophy, genes that destroy muscle proteins (Myostatin, Atrogin-1, and MuRF1) were expressed at relatively low levels, and genes that produce muscle proteins (MyoD and Myogenin) were expressed at relatively high levels. Confirmed that it is.

Example 5: Effect of Exendin-4 on the Expression Level of Genes Related to Muscular Atrophy at the Cell Level

C2C12 cells, a myoblast line, were cultured for 5 days in a medium containing 2% (v/v) horse serum to differentiate into muscle cells. Control muscle cells without any treatment on the differentiated muscle cells (control), muscle cells in which muscle atrophy symptoms were induced by treatment with 1 μM dexamethasone for 12 hours, and control muscle cells treated with 20 nM exendin-4 for 6 hours And 1 μM of dexamethasone for 12 hours, and then 20 nM of exendin-4 was treated for 6 hours to produce muscle cells in the experimental group in which the symptoms of muscular dystrophy were treated, respectively, and target each of the produced muscle cells related to muscular atrophy. The expression levels of genes (Myostatin, Atrogin-1, and MuRF1) were compared (FIG. 5).

Figure 5 shows the results of comparing the expression levels of genes (Myostatin, Atrogin-1, and MuRF1) related to muscular atrophy in control muscle cells, muscle cells in which the symptoms of muscular atrophy are induced, muscle cells in the control group, and muscle cells treated with symptoms of muscular atrophy. It is a graph. As shown in FIG. 5, the genes (Myostatin, Atrogin-1, and MuRF1) were expressed at a relatively high level in muscle cells in which the symptoms of muscular dystrophy were induced, whereas the genes (Myostatin, Atrogin) were expressed at a relatively high level in muscle cells treated with symptoms of muscular atrophy. It was confirmed that the expression level of -1 and MuRF1) was remarkably reduced, indicating a level similar to that of the control muscle cells.

When the results of Examples 1 to 5 are summarized above, it was found that exendin-4 has an effect of treating, improving, or recovering muscle atrophy or sarcopenia caused by dexamethasone.

Furthermore, the present inventors sought to confirm the therapeutic effect of GLP-1 degradation inhibitors and GLP-1 derived peptides having in vivo activity similar to exendin-4 in addition to exendin-4 on muscular atrophy or sarcopenia. Thus, it was confirmed that the body weight, muscle, grip strength, and genetic changes caused by sitagliptin, which is one of the inhibitors of GLP-1 degradation, and GLP-1 (32-36)amide, a GLP-1 derived peptide in an animal model. .

Example 6: Effect of sitagliptin and GLP-1(32-36)amide on the body weight and muscle mass of an animal with muscular dystrophy

The present inventors treated dexamethasone (20 mg/kg/ip) to C57BL/6J male mice (n=10/group) to reduce the muscles of the mice, and sitagliptin (300 mg /kg/oral gavage) and GLP-1(32-36)amide (5 μg/kg/ip).

As a result, it was confirmed that the body weight (FIG. 6) and muscle mass (FIG. 7), which were reduced by dexamethasone, slightly increased by sitagliptin or GLP-1(32-36)amide, but the muscle mass was significantly increased.

Example 7: Effects of sitagliptin and GLP-1(32-36)amide on grip strength of animals with muscular dystrophy

The present inventors confirmed that through Example 6, the body weight and muscle mass of the animal causing muscular dystrophy was increased by sitagliptin, and thus, to confirm whether the actual muscle function was strengthened due to the increased body weight and muscle mass, It was also investigated whether the mouse's grip strength was increased.

Specifically, the grip strength of the mouse when all the feet of the mouse of Example 6 were put on and pulled with the same force using a grip strength machine was confirmed.

As a result, it was confirmed that the grip strength reduced by dexamethasone was increased again by sitagliptin and GLP-1(32-36)amide (FIG. 8).

Example 8: Effect of sitagliptin and GLP-1(32-36)amide on the expression level of genes related to muscular atrophy in an animal model of muscular dystrophy

As described above, muscular dystrophy occurs due to increased expression of genes that destroy proteins constituting muscle or suppression of expression of genes that produce the protein.

Accordingly, the present inventors confirmed changes in the expression of myostatin, which is a factor of muscular dystrophy, and MyoD, which is a muscle development factor, caused by sitagliptin and GLP-1(32-36)amide.

As a result, the expression of myostatin was increased by dexamethasone and then decreased by sitagliptin and GLP-1(32-36)amide, whereas the expression of MyoD was decreased by dexamethasone, followed by sitagliptin and GLP-1. It was confirmed that it was increased again by (32-36)amide (FIG. 9).

From the results of Examples 6 to 8, the present inventors found that not only GLP-1, but also sitagliptin, which is a decomposition inhibitor of GLP-1, or GLP-1(32-36)amide, which is a GLP-1 fragment, also prevents sarcopenia or muscular atrophy. It was confirmed that it has a therapeutic effect on. In addition, all the results were checked for significance through statistical processing. **p<0.01, *p<0.05 are marks that show statistical significance compared to control, and ##p<0.01, #p<0.05 are marks that show statistical significance compared to Dex.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

<110> GIL MEDICAL CENTER Gachon University of Industry-Academic cooperation Foundation <120> Pharmaceutical composition for treating muscle atrophy comprising glucagon like-peptide-1, GLP-1 derived peptide, or GLP-1 degradation inhibitor <130> KPA160869-KR-D1 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> recombinant Exendin-4 <400> 1 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 2 <211> 36 <212> PRT <213> Homo sapiens <400> 2 His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg 35 <210> 3 <211> 5 <212> PRT <213> Homo sapiens <400> 3 Leu Val Lys Gly Arg 1 5

Claims (6)

A pharmaceutical composition for the prevention or treatment of muscular dystrophy or myopenia comprising any one selected from the group consisting of peptides derived from glucagon-like peptide-1 (GLP-1), sitagliptin and combinations thereof. Wherein the GLP-1 derived peptide is composed of the amino acid sequence of SEQ ID NO: 3.
The composition according to claim 1, wherein the composition is selected from the group consisting of body weight gain, skeletal muscle weight gain, increase in expression of a muscle protein-producing gene, inhibition of expression of a muscle protein-destroying gene, and combinations thereof / RTI &gt;
The composition according to claim 2, wherein the gene that disrupts the muscle protein is a gene encoding Myostatin, Atrogin-1 or MuRF1 (Muscle RING-finger protein-1) .
3. The composition according to claim 2, wherein the gene for producing the muscle protein is a gene encoding MyoD or Myogenin.
The composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
A method of treating a condition in which the pharmaceutical composition of any one of claims 1 to 5 is administered to a subject other than a human suffering from a myelopathy or a muscular dystrophy in a pharmaceutically effective amount.

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