KR20210088443A - Composition for preventing or treating bone disease comprising VGF-derived peptide - Google Patents

Abstract

The present invention relates to a composition for preventing or treating bone diseases, including a VGF-derived peptide. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating bone diseases and a health functional food composition for preventing or alleviating bone diseases, all of which contain various VGF-derived peptides exhibiting cortical bone strengthening effect as an active component.

Description

Composition for preventing or treating bone disease comprising a VGF-derived peptide {Composition for preventing or treating bone disease comprising VGF-derived peptide}

The present invention relates to a composition for preventing or treating bone disease, including a VGF-derived peptide, and more particularly, to a pharmaceutical composition for preventing or treating bone disease and a bone disease comprising various VGF-derived peptides exhibiting cortical bone strengthening effect as an active ingredient. It relates to a health functional food composition for the prevention or improvement of.

The process of bone modeling and remodeling plays an important role in the development, growth and metabolism of bones. Bone formation starts from birth and continues until adolescence, when the skeleton matures and growth ends, and the maximum bone mass is formed from the 20s to the early 30s. For about 30 years after that, the bone remodeling process of removing the bone and replenishing it is repeated. At this time, the bone formation and bone resorption are paired to maintain the balance. After this period, bone formation cannot adequately follow the bone loss caused by bone resorption, resulting in a decrease in bone mass of about 0.3 to 0.5% per year. suffer a loss

Bone tissue constitutes the cartilage and skeletal system, plays a role of support and muscle attachment with mechanical functions, protects living organs and bone marrow, and preserves calcium and phosphorus ions to maintain homeostasis. Bone tissue consists of cell substrates such as collagen and glycoproteins, and various types of cells such as osteoblasts, osteoclasts, and osteocytes. Osteoblasts derived from bone marrow stromal cells play a major role in bone formation, and osteoclasts derived from hematopoietic stem cells are destroyed and are responsible for resorption of aged bone. It maintains bone remodeling by maintaining a balanced action. However, excessive activity of osteoclasts or decreased activity of osteoblasts causes imbalance in the remodeling process of bone formation, and the balance between osteoclasts and osteoblasts in vivo is broken, thereby causing bone disease.

As a representative example of bone disease, osteoporosis is an unavoidable symptom for the elderly, especially postmenopausal women, although there is a difference in degree, and interest in osteoporosis and its therapeutic agents is gradually increasing in developed countries as the population ages. In addition, it is known that there is a market of about $130 billion related to the treatment of bone disease worldwide, and it is expected to increase further in the future, so each research institute and pharmaceutical company around the world invests a lot in the development of a treatment for bone disease. have.

So far, several substances have been developed for the treatment of osteoporosis. Among them, estrogen, which is most often used as a treatment for osteoporosis, has a disadvantage in that its practical efficacy has not yet been verified, and it has to be taken for a lifetime. Alendronate also has a problem that its efficacy is not clear, absorption in the digestive tract is slow, and it causes inflammation in the stomach and esophageal mucosa. Calcium preparations are known to be effective with few side effects, but they are a preventive agent rather than a therapeutic agent. In addition, vitamin D preparations such as calcitonin are known, but their efficacy and side effects have not been sufficiently studied.

On the other hand, periodontal disease is divided into gingivitis and periodontitis according to the inflammatory state of the tooth supporting tissue, and advanced periodontitis causes tooth loss. Tooth loss due to periodontitis is a serious problem.

The destruction of periodontal ligaments and alveolar bone in periodontal disease is due to the decomposition of collagen, the main component of these tissues. Although the exact mechanism is not yet known, it is known that the degradation of collagen is due to the collagenase secreted by the host and the neutral proteolytic enzyme secreted from the periodontal disease-causing bacteria. Therefore, studies have been attempted to suppress the progression of periodontal disease by inhibiting the activities of collagenase enzymes and proteolytic enzymes. For the treatment of periodontal disease, the establishment of improved oral hygiene in the patient, non-surgical or surgical calculus removal, root planing, gingivectomy, and periodontal tissue regeneration using renal attachment have been used. However, this surgical treatment method is difficult to treat effectively due to the hassle of going to the dentist for treatment, and it is limited to treatment performed when the disease has progressed to some extent rather than prevention of the disease. Most of the time it goes on.

Although systemic antibiotics and topical sustained-release formulations have been used as additional treatments, too many drugs are delivered to unnecessary areas, resulting in side effects and isolated cases of periodontal disease bacteria showing resistance to recently used antibiotics. there is a serious problem. To compensate for the limitations of these surgical treatments and the problems with the use of antibiotics, the use of oral compositions, such as toothpaste and mouthwash containing antibacterial and anti-plaque agents, is an adjuvant for the prevention and treatment of bacterial oral diseases such as dental caries and periodontal disease. has been reported to be effective.

Fracture refers to a state in which bones are broken, and the treatment of fractures takes a considerable period of time, and as the population ages, the number of fractures in osteoporotic patients, one of the pathological fractures, is increasing significantly. Calcium, estrogen, and calcitonin, which are currently used as osteoporosis treatments, are (calcitonin), active vitamin D, and bisphosphonate preparations were used to treat fractures, which were expected to have a therapeutic effect on fractures. didn't This is due to the genetic or constitutional mechanism of osteoporosis, which is a state in which the bone maintains a negative equilibrium state and has delicate skeletal qualities, the bone quality is absorbed at a normal rate, but the bone formation is poor, and the bone is formed at a normal rate. This is because bone resorption is increased. As such, it cannot be inferred that osteoporosis and fractures can be treated with osteoporosis drugs because osteoporosis and fractures have completely different mechanisms of development.

Due to the difference in the mechanism of osteoporosis and fracture as described above, an agent that inhibits bone resorption and thus exhibits an osteoporosis therapeutic effect may also inhibit bone formation, thereby delaying fracture treatment, for example, a bisphosphonate agent. It has been reported that incadronate disodium delayed the fracture healing of the femur in rats when continuously administered for a long period of time (Li C et al., J. Bone Miner Res. 2001 Mar; 16 (Li C et al., J. Bone Miner Res. 2001 Mar; 16). 3):429-36), reported that pretreatment with incadronate did not affect fracture healing at 16 weeks after fracture, whereas continuous incadronate treatment increased callus but delayed the remodeling process during fracture healing. (Li J et al., J. Bone Miner Res. 1999 Jun; 14(6):969-79). In addition, it is reported that bFGF, an osteogenic biomarker known to be deeply related to osteoporosis, has no relation to fracture healing (Xu et al., Chin. J. Traumatol. 6, 160-166, 2003).

Therefore, there is a limit to the use of a therapeutic agent for osteoporosis in order to effectively promote healing to a normal state when a general fracture occurs, and there is an urgent need to develop a drug having a unique fracture healing effect.

Accordingly, the present inventors have sought to develop a osteogenesis-promoting agent that strengthens cortical bone by discovering a new therapeutic target for promoting bone formation and strengthening of cortical bone with a new mechanism of action, and VGF-derived peptide, which is a neurosecretory growth factor, of osteoblasts. The present invention was completed by confirming that the stimulatory Galphas-coupled GPCR was stimulated to promote cAMP, thereby promoting bone formation and also exhibiting cortical bone strengthening effect at the in vivo level.

Regarding the medicinal use of the VGF polypeptide, International Patent Publication No. WO 01/07477 discloses the therapeutic effect of the VGF polypeptide on obesity, infertility, cachexia, eating disorders, etc., but there is no disclosure on the therapeutic effect on bone diseases.

International Patent Publication WO 01/07477 (published on 2001.02.01)

SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition for preventing, improving, or treating bone disease, which has a cortical bone strengthening effect.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating bone disease and/or a health functional food composition for preventing or improving bone disease, comprising various types of VGF-derived peptides as an active ingredient.

According to a preferred embodiment of the present invention, the VGF-derived peptide is _{an amino acid sequence of X 1} X ₂ X ₃ X ₄ AEAEERRLQEQEELENYIEHVLX ₅ X ₆ X ₇ X ₈ (SEQ ID NO: 1) or X ₁ X ₂ X ₃ X ₄ ADAEERRLQEQEELENYIEHVLX ₅ It _{comprises the amino acid sequence of X 6} X ₇ X ₈ (SEQ ID NO: 2), wherein in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2,

X ₁ may be free of amino acids or may be alanine (A); X ₂ may be free of amino acids or may be glutamine (Q); X ₃ may be free of amino acids or may be glutamic acid (E); X ₄ may be free of amino acids or may be valine (V) or glutamic acid (E); X ₅ may be free of amino acids or may be leucine (L); X ₆ may be free of amino acids or may be arginine (R) or histidine (H); X ₇ may be free of amino acids or may be arginine (R); X ₈ may be free of amino acids or may be proline (P); Here, if there is no amino acid in _{X 4} , there may be no amino acid in _{X 1} to X _{3 ;} If there is no amino acid in X ₃ , there may also be no amino acid in _{X 1} and X _{2 ;} If there is no amino acid in X ₂ , there may also be no amino acid in _{X 1 ;} If there is no amino acid in X ₅ , there may be no amino acid in _{X 6} to X _{8 as well;} If there is no amino acid at X ₆ , there may also be no amino acid at _{X 7} and X _{8 ;} If there is no amino acid at X ₇ , there may also be no amino acid at _{X 8 .}

According to another preferred embodiment of the present invention, the peptide may comprise the amino acid sequence of AQEEAEAEERRLQEQEELENYIEHVLLRRP (SEQ ID NO: 3), AEAEERRLQEQEELENYIEHVLLRRP (SEQ ID NO: 4), VAEAEERRLQEQEELENYIEHVLLRRP (SEQ ID NO: 6) or AQEELEADAEERRLHVLLHRP (SEQ ID NO: 6).

According to another preferred embodiment of the present invention, the bone disease may be selected from the group consisting of osteoporosis, periodontal disease, fracture, and Paget's disease.

According to another preferred embodiment of the present invention, the fracture may be a non-vertebral fracture.

According to another preferred embodiment of the present invention, the peptide may be used in combination with a bone resorption inhibitor.

According to another preferred embodiment of the present invention, the bone resorption inhibitor may be a bisphosphonate.

According to another preferred embodiment of the present invention, the peptide may be administered by injection.

According to another preferred embodiment of the present invention, the injection may be a depot injection.

According to the present invention, AQEE30 and its various modified forms of VGF-derived peptides stimulate stimulatory Galphas-coupled GPCR to promote cAMP, thereby exhibiting a osteogenesis-promoting effect. Fundamental treatment is possible. In addition, most of the existing osteoporosis therapeutic agents only show a preventive effect on vertebral fractures made of trabecular bone, and the preventive effect of non-vertebral fractures made of cortical bone is insignificant, whereas the various VGF-derived peptides of the present invention have excellent cortical bone strengthening effects. It has the advantage of being excellent in preventing and treating vertebral fractures.

1 shows various VGF peptides in rats.
2 shows the 3D structure of AQEE30 using homology modeling.
3 is a result confirming the effect of VGF on osteocytes.
Figure 4 shows the osteoblast differentiation promoting effect of the VGF-derived peptide hAQEE30, A is alizarin red S (alizarin red S) staining after inducing calcification by treating the osteoblast differentiation promoting medium with AQEE11, AQEE19 and hAQEE30 peptides, respectively. to confirm the degree of calcification, B is the same method as A, and the total VGF, NERP1, NERP2, AQEE11, AQEE19, hAQEE30, TLQP21 and TLQP62 peptides were treated with 1 μM or 5 μM, respectively, to confirm the degree of calcification.
5 shows the cAMP-promoting effect of hAQEE30 in osteoblasts.
6 shows micro-CT results of VGF KO mice.
7 shows the quantitative measurement results of osteocalcin and C-telopeptide test results in VGF KO mice.
8 and 9 show the results of confirming the formation of cranial bones (Calvaria bone) in order to verify the in vivo therapeutic efficacy of hAQEE30. In the tables shown in FIGS. 8 and 9, a number with an auburn background means "p < 0.05 vs. PBS".
10 shows the osteoblast differentiation promoting effect of hAQEE30 and its modified peptides.
11 shows the results of confirming the osteoblast viability and proliferation promoting effect of mAQEE30.
12 shows the results of confirming the osteoblast differentiation promoting effect of mAQEE30.

As described above, the existing osteoporosis treatment exhibits various side effects and has a fatal disadvantage that it cannot effectively treat fractures caused by osteoporosis. Accordingly, the present inventors have sought to develop a osteogenesis-promoting agent that strengthens cortical bone by discovering a new therapeutic target for promoting bone formation and strengthening of cortical bone with a new mechanism of action, and VGF-derived peptide, which is a neurosecretory growth factor, of osteoblasts. By stimulating stimulatory Galphas-coupled GPCR to promote cAMP, it exhibits a osteogenesis-promoting action and also shows a cortical bone strengthening effect at the in vivo level, thereby seeking a solution to the above-mentioned problem.

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

The present invention relates to _{the amino acid sequence of X 1} X ₂ X ₃ X ₄ AEAEERRLQEQEELENYIEHVLX ₅ X ₆ X ₇ X ₈ (SEQ ID NO: 1) or X ₁ X ₂ X ₃ X ₄ ADAEERRLQEQEELENYIEHVLX ₅ X ₆ X ₇ X ₈ (SEQ ID NO: 2) It provides a pharmaceutical composition for preventing or treating bone disease, comprising a peptide comprising an amino acid sequence as an active ingredient.

The peptide comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 is a VGF-derived neurosecretory growth factor, and may be derived from human, rat or mouse.

Specifically, the amino acid sequence of human VGF peptide is known from NCBI Reference Sequence: NP_003369.2, and the nucleotide sequence encoding it is known from NCBI Reference Sequence: NM_003378.4. The amino acid sequence of rat VGF peptide is known from NCBI Reference Sequence: NP_112259.1, and the nucleotide sequence encoding it is known from NCBI Reference Sequence: NM_030997.1. The amino acid sequence of mouse VGF peptide is known from NCBI Reference Sequence: NP_001034474.1, and the nucleotide sequence encoding it is known from NCBI Reference Sequence: NM_001039385.1.

Here, the amino acid sequences of the human, rat and mouse AQEE30 peptides and the nucleotide sequences encoding them are as shown in Table 1.

peptide amino acid sequence SEQ ID NO: nucleotide sequence SEQ ID NO: Human AQEE30 AQEEAEAEER RLQEQEELEN YIEHVLLRRP 3 gcgcaggagg aggcggaggc ggaggagcgc cggctgcagg agcaggagga gctggagaat tacatcgagc acgtgctgct ccggcgcccg 7 Rat and Mouse AQEE30 AQEEADAEER RLQEQEELEN YIEHVLLHRP
6 gcgcaggagg aagcggacgc ggaggagcgc cggctgcagg agcaggagga gctggagaat tacattgagc acgtgctgct gcaccgcccg 8

In the pharmaceutical composition of the present invention, in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, X ₁ may be no amino acid or alanine (A); X ₂ may be free of amino acids or may be glutamine (Q); X ₃ may be free of amino acids or may be glutamic acid (E); X ₄ may be free of amino acids or may be valine (V) or glutamic acid (E); X ₅ may be free of amino acids or may be leucine (L); X ₆ may be free of amino acids or may be arginine (R) or histidine (H); X ₇ may be free of amino acids or may be arginine (R); X ₈ may be free of amino acids or may be proline (P); Here, if there is no amino acid in _{X 4} , there may be no amino acid in _{X 1} to X _{3 ;} If there is no amino acid in X ₃ , there may also be no amino acid in _{X 1} and X _{2 ;} If there is no amino acid in X ₂ , there may also be no amino acid in _{X 1 ;} If there is no amino acid in X ₅ , there may be no amino acid in _{X 6} to X _{8 as well;} If there is no amino acid at X ₆ , there may also be no amino acid at _{X 7} and X _{8 ;} If there is no amino acid at X ₇ , there may also be no amino acid at _{X 8 .}

For example, the peptide may comprise the amino acid sequence of AQEEAEAEERRLQEQEELENYIEHVLLRRP (SEQ ID NO: 3), AEAEERRLQEQEELENYIEHVLLRRP (SEQ ID NO: 4), VAEAEERRLQEQEELENYIEHVLLRRP (SEQ ID NO: 5) or AQEEADAEERRLQEQEELENYIEHVLLHRP (SEQ ID NO: 6).

Here, the amino acid sequence of SEQ ID NO: 3 is referred to as human AQEE30, and is referred to as hAQEE30 herein.

Here, the amino acid sequence of SEQ ID NO: 6 is referred to as mouse AQEE30, and herein referred to as mAQEE30.

The pharmaceutical composition of the present invention can be used for the prevention or treatment of metabolic bone diseases, and in particular, can be used without limitation in bone-related diseases caused by imbalance of osteoblasts and osteoclasts.

In the pharmaceutical composition of the present invention, the bone disease may be osteoporosis, periodontal disease, fracture or Paget's disease, but is not limited thereto.

As used herein, the term "prevention or treatment of bone diseases" includes the prevention and complete or partial treatment of metabolic bone diseases, in particular osteoporosis, periodontal disease, fractures and Paget's disease, and also reduction and improvement of symptoms of bone metabolic diseases. , any change in the patient that relieves the pain of its symptoms, reduces the incidence of metabolic bone disease, or increases the outcome of treatment.

The "osteoporosis" is a state in which the calcification of the bone tissue is reduced, the compactness of the bone is thinned, and thereby the bone marrow cavity is widened. easy to become

Bone mass is affected by several factors, such as genetic factors, nutritional intake, hormonal changes, and differences in exercise and lifestyle. The causes of osteoporosis include old age, lack of exercise, low weight, smoking, low-calcium diet, menopause, Ovariectomy and the like are known.

The "periodontal disease" refers to an inflammatory state of the dental support tissue caused by bacteria, and can be divided into gingivitis and periodontitis. The cause of the disease is the formation of dental plaque by oral bacteria caused by poor oral hygiene. Dental plaque refers to a mass of bacteria that proliferates after bacteria attach to the tooth surface by using the sticky substance in saliva as an adhesive. If the dental plaque is left unattended, it can become inflamed and sometimes bleed from the gums and bad breath. These symptoms are called gingivitis. As gingivitis progresses further, the gap between the teeth and the gums becomes deeper, forming a periodontal pocket, where bacteria that cause periodontal disease multiply and periodontitis occurs. As periodontitis progresses, the gums may bleed and swell even with mild stimulation such as brushing, and often turn into acute inflammation, causing pain. This inflammation lowers the bone-making function and increases the bone-resorbing action, causing the alveolar bone to gradually decrease, which leads to destruction of the alveolar bone and eventually the loss of teeth.

The "fracture" refers to a state in which the continuity of a bone, an epiphyseal end plate, or an articular surface is abnormally broken, and refers to a fracture of a bone. Causes of fractures include trauma such as traffic accidents, safety accidents that occur in industrial disorders, bone changes due to diseases such as osteoporosis, bone cancer, and metabolic disorders, and repetitive stress on bones due to sports or loads. In addition, the fracture status is based on the fracture line (a line along the end of the bone generated by the bone cut), such as a fissure fracture, a greenstick fracture, a transverse fracture, a filamentous fracture, a spiral fracture, a segmental fracture, a comminuted fracture, and a avulsion fracture. , compression fractures, and depression fractures.

Since the peptide contained in the pharmaceutical composition of the present invention has a cortical bone strengthening effect as a feature, it exhibits an excellent effect in preventing or treating non-vertebral fractures composed of cortical bone.

In the present invention, the term "pharmaceutical composition" refers to a mixture comprising various VGF-derived peptides of the present invention and modified forms thereof with pharmaceutically acceptable excipients such as diluents or carriers. Such pharmaceutical pharmaceutical compositions may optionally contain one or more additional therapeutically active agents. According to some embodiments, there is provided a method of administering a pharmaceutical composition comprising a peptide of the present invention to a subject as needed. In some embodiments, a composition of the present invention may be administered to a human.

While the description of pharmaceutical compositions provided herein relates principally to pharmaceutical compositions for administration to humans, the skilled person will understand that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions for administration to a variety of animals are well understood, and the skilled veterinary pharmacologist can, if necessary, design and/or carry out such modifications simply by routine experimentation.

The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology or developed hereinafter. In general, such methods for tableting comprise the step of associating the active ingredient with excipients and/or one or more other accessory ingredients, followed by shaping and/or packaging the product into the desired single- or multi-dose unit, if necessary or desired. includes steps.

The pharmaceutical compositions of the present invention may be prepared, packaged, and/or sold unpackaged as a single unit dose and/or a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of active ingredient is generally equal to the dosage of active ingredient administered to a subject and/or a convenient fraction of such dosage such as, for example, one-half or one-third of the dosage.

The relative amounts of active ingredients, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and/or disorder of the subject being treated and depending on the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w/w) active ingredient.

As used herein, pharmaceutically acceptable excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspending aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives suitable for the purpose of the particular dosage form. , solid binders, lubricants, and the like. Remington, The Science and Practice of Pharmacy, 21 ^st Edition, AR Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) describes various excipients used in the formulation of pharmaceutical compositions and known for their preparation. Discloses the description, except that any conventional carrier medium is incompatible with the substance or derivative thereof, for example, by providing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. , its use is considered to be within the scope of the present invention.

In some embodiments, the pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure.

In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia (EP).

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active and/or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and/or oils. doesn't happen

Such excipients may optionally be included in the formulations of the present invention. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, and flavoring agents may be present in the composition at the discretion of the formulator.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation-exchange resins , calcium carbonate, silicate, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethylstarch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose ( croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Vegum), sodium lauryl sulfate, quaternary ammonium compounds, and these including, but not limited to, combinations of

Exemplary surface active agents and/or emulsifiers include natural emulsifiers (eg, acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (such as bentonite [aluminum silicate] and veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (such as stearyl alcohol, cetyl alcohol, oleyl alcohol) , triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (such as carboxy polymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl) polymers), carrageenan, cellulose derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxy Ethylene Sorbitan Monolaurate [Tween 20], Polyoxyethylene Sorbitan [Tween 60], Polyoxyethylene Sorbitan Monooleate [Tween 80], Sorbitan Monopalmitate [Span 40], Sorbitan Monostearate [ span 60], sorbitan tristearate [span 65], glyceryl monooleate, sorbitan monooleate [span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [myrz 45]) , polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (eg Cremophor), polyoxyethylene ethers, ( For example polyoxyethylene lauryl ether [breeze 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl Laurate, Sodium Lauryl Sulfate, Pluronic F 68, Paul Roxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or combinations thereof.

Exemplary binders include starch (eg corn starch and starch paste); gelatin; sugars (eg sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwoer gum, shatty gum, isapol husks slime, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Vegum), and lachiarabogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylate; wax; water; Alcohol; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, bisulfite sodium sulfate, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. . Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetriimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluend (BHT), ethylenediamine, sodium lauryl sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), Sodium Bisulfite, Sodium Metabisulfite, Potassium Sulfite, Potassium Metabisulfite, Glydant Plus, Fenonib, Methylparaben, Low Molar 115, Germaben II, Neolon, Carton, and E including but not limited to In certain embodiments, the preservative is an anti-oxidant. In another embodiment, the preservative is a chelating agent.

Exemplary buffers include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluvionate, calcium gluceptate, calcium gluconate, D-gluconic acid, glycero Calcium phosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic Basic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen -including but not limited to free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

Exemplary oils are almond, apricot kernel, avocado, babassu palm, bergamot, black currant seed, borage, caid, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee. , corn, cottonseed, emu, eucalyptus, evening primrose, fish, flax seed, geraniol, pumpkin, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, lytsea cu Beva, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange-orange raffia, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower , sandalwood, sascana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil. , and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, sprout, olive, castor, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the peptides of the invention are admixed with solubilizing agents such as cremophor, alcohols, oils, denatured oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may also be formulated in accordance with the known art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by including a sterile agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. .

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This is done by using a liquid suspension of crystalline or amorphous material with low water solubility. The rate of absorption of the drug then depends on the rate of dissolution, which may in turn depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug is achieved by dissolving or suspending the drug in an oil vehicle.

In the pharmaceutical composition of the present invention, the injection may be a depot injection.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient may be administered in one or more inert pharmaceutically acceptable excipients or carriers such as sodium citrate or dicalcium phosphate and/or a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b ) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch , alginic acid, certain silicates, and sodium carbonate, e) dissolution delaying agents such as paraffin, f) absorption enhancers such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.

In the case of capsules, tablets and pills, dosage forms may contain buffers. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as such excipients. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may be of a composition that release only the active ingredient, or preferentially, a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as such excipients.

The active ingredient may be in micro-encapsulated form with one or more excipients described above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient may be admixed with one or more inert diluents such as sucrose, lactose or starch. Such dosage forms may contain additional substances that are not incompatible diluents as is normally practiced, such as tablet lubricants and other tablet adjuvants such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, dosage forms may also contain buffers. They may optionally contain opacifying agents and may be of a composition that release only the active ingredient, or preferentially, a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

The peptides of the invention described herein are typically prepared in dosage unit form for ease of administration and uniform administration. However, it will be understood that the total daily usage of the pharmaceutical composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. A particular therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disease, disorder, or disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the particular composition employed; the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the particular active ingredient employed; duration of treatment; drugs used in combination with or concurrently with the specific active ingredient employed; and factors well known in the medical arts.

The various VGF-derived peptides of the present invention, or a pharmaceutical composition thereof, may be administered by any route. In some embodiments, the peptide, or pharmaceutical composition thereof, is administered orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, subcutaneously, intraventricularly, transdermally, intradermally, rectal, intravaginally, intraperitoneally, topical (powder, by ointments, creams, and/or drops), mucosal, nasal, oral, enteral, sublingual; endotracheal instillation, bronchial instillation, and/or inhalation; and/or by various routes including oral spray, nasal spray, and/or aerosol. Routes specifically contemplated are invasive intravenous injection, local administration via blood and/or lymph supply, and/or administration directly to the affected site. In general, the most suitable route of administration will depend on a variety of factors, including the nature of the agent (eg, its stability in the environment of the gastrointestinal tract), and the disorder of the subject (eg, whether the subject can tolerate oral administration). will be. However, the present invention includes the delivery of the pharmaceutical composition according to the present invention by any suitable route taking into account advances in the field of drug delivery.

In certain embodiments, the peptides of the invention, or pharmaceutical compositions thereof, are from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about the subject's body weight daily. 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, or about 1 mg/kg to about 25 The desired therapeutic effect may also be achieved by administration at dosage levels sufficient to deliver mg/kg more than once per day. The desired dosage may be delivered three times a day, twice a day, every day, every two days, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered via multiple administrations (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). .

It will be understood that the dosage ranges described herein provide guidance for administration of a given pharmaceutical composition to adults. The amount administered to, for example, a child or adolescent may be determined by a medical professional or skilled in the art, and may be less than or equal to that administered to an adult. The exact amount of a peptide according to the invention required to achieve an effective amount will vary from subject to subject, for example depending on the species, age, and general disorder of the subject, the severity of the side effect or disorder, the mode of administration, and the like.

It will be understood that the peptides of the invention or pharmaceutical compositions thereof may be used in combination therapy. The particular combination of treatments (therapeutic agents or procedures) for use in combination therapy will take into account the desired therapeutic effect to be achieved and the suitability of the intended therapeutic agent and/or procedure.

The pharmaceutical compositions of the present invention may be administered alone or in combination with one or more therapeutically active agents. In the case of "combination," it is not intended to suggest that the agents must be administered at the same time and/or formulated to be delivered together, although the following methods of delivery are within the scope of the present invention. The composition may be administered concurrently with, prior to, or subsequent to one or more other therapeutic agents or medical procedures. In general, each agent will be administered at a dosage and/or time schedule defined for that agent. Further, the present invention relates to a pharmaceutical composition of the present invention in combination with an agent capable of improving its bioavailability, reducing and/or modifying its metabolism, inhibiting its secretion, and/or modifying its distribution in the body. encompasses conveying It will be further understood that the peptides of the invention and the therapeutically active agent used in this combination may be administered together in a single composition or separately in different compositions.

The particular combination used in combination therapy will take into account the desired therapeutic effect to be achieved and/or the suitability of the therapeutically active agent and/or procedure comprising the peptides of the invention. The combination used may achieve the desired effect for the same disorder (eg, a peptide of the invention may be administered in combination with another therapeutically active agent used to treat the same disorder), and/or these may achieve different effects (eg control of any side effects).

As used herein, "therapeutically active agent" refers to any substance used as a medicament for treating, preventing, delaying, reducing or ameliorating a disorder, and refers to a substance used in treatment, including prophylactic and therapeutic treatments. .

In some embodiments, the pharmaceutical composition of the present invention treats, alleviates, ameliorates, ameliorates, delays the onset, inhibits the progression, reduces the severity, and/or reduces the incidence of one or more symptoms of bone disease. It can be administered in combination with any therapeutically active agent or procedure useful for reducing (eg, surgery, radiation therapy).

In the pharmaceutical composition of the present invention, the peptide may be used in combination with a bone resorption inhibitor. Bone resorption inhibitors used in combination include, but are not limited to, bisphosphonates including zoledronic acid, SERM, or denosumab.

Various VGF-derived peptides and modified forms thereof of the present invention may be provided as a food composition, and the food composition may be used as a health functional food. When the peptide of the present invention is used as a food additive, it may be appropriately used according to a conventional method, such as adding the peptide as it is, or mixing it with other food or other food ingredients. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment).

The term "health functional food" in the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient in a food raw material for the purpose of health supplementation, It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc., by the above ingredients, and the composition for health food is used for prevention of diseases and prevention of diseases. It can perform functions related to recovery, etc.

In addition, there is no limitation on the type of health food in which the food composition of the present invention can be used. In addition, the composition comprising the peptide of the present invention as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

< Example 1> VGF's bone cells ( Osteocyte ) on the

In order to strengthen cortical bone, not only bone formation by osteoblasts and bone resorption by osteoclasts, but also osteocytes abundantly present in cortical bone must be healthy.

In order to observe the action on osteocytes at the living level, VGF-deficient mouse bones were fixed in 4% paraformaldehyde solution for 24 hours, and then decalcified with 0.5M EDTA for 14 days, followed by paraffin cutting ( paraffin section). The obtained section was subjected to H&E staining to count the number of osteocytes present in cortical bone.

In order to confirm the apoptosis inhibitory effect at the cell level, it was measured using a Tunnel staining kit (Roche) and a Tunnel assay kit (Millipore).

Specifically, MLO-Y4 cells, one of the osteocytic-like cell lines, were seeded in a 24 well plate at 2×10 ⁴ cells/well to stabilize the cells for 24 hours, and then treated with 5 μM of VGF hAQEE30 for 48 hours. At this time, serum was removed from the culture medium to induce apoptosis, and serum (2.5% FBS, 2.5% FCS) was added to the negative control group. The apoptosis inhibitory effect was confirmed using a commercially available Tunnel staining kit (Roche) and Tunnel assay kit (Millipore).

As a result, in VGF-deficient mice, cortical bone osteocytes (Osteocytes) decreased, which was confirmed because VGF hAQEE30 inhibits apoptosis of osteocytes (Osteocytes) (FIG. 3).

< Example 2> hAQEE30 Confirmation of osteoblast differentiation promoting effect

Osteoblasts originate from mesenchymal stem cells and develop into mature osteoblasts that form bones through differentiation. It also plays an important role in hormonal metabolism and calcium homeostasis.

Therefore, in this example, after stabilizing osteoblasts, various VGF-derived peptides of total VGF, NERP1, NERP2, AQEE19, hAQEE30, TLQP21 and TLQP62 were treated in a bone differentiation medium, respectively, and the osteoblast differentiation promoting effect of each VGF-derived peptide was confirmed.

Specifically, the cells inducing bone differentiation were washed twice with PBS, fixed with 70% ethanol solution for 2 minutes, and then stained with 2% alizarin red S solution for 30 minutes. Then, the non-specifically stained area was washed several times with sterile water. For decolorization, a 10% (w/v) cetylpyridinium chloride solution was reacted for 30 minutes at room temperature, and the absorbance of the supernatant was measured at 570 nm with a microplate reader.

As a result, as shown in FIG. 4A , the differentiation of osteoblasts in the group treated with hAQEE30 and TLQP62 was promoted, and it was confirmed that bone calcification was significantly increased compared to other groups.

In addition, as a result of confirming the effect of each concentration of each VGF peptide in the same manner as above, it was confirmed that only hAQEE30 promoted osteoblast differentiation at concentrations of 1 μM and 5 μM, as shown in FIG. 4B .

< Example 3> hAQEE30 cAMP Confirmation of the facilitation effect

After binding to the receptor, parathyroid hormone (PTH) activates PKA (Protein Kinase A) via adenylyl cyclase and cAMP increase, and cAMP/PKA signaling is an important signaling system in osteoblasts. It is known, and it is known to exhibit a calcium synergistic effect through continuous cAMP production.

Therefore, in this example, the cAMP promoting effect of hAQEE30 and PTH was confirmed.

Specifically, after treating osteoblasts with 5 μM of hAQEE30 and 100 nM of PTH, an increase in the amount of cAMP was measured with a cAMP assay kit (Amersham Pharmacia, RPN 225).

As a result, as shown in FIG. 5 , it was confirmed that hAQEE30 exhibited a cAMP promoting effect at the same level as that of PTH.

< Example 4> VGF Micro-CT (micro-CT) measurement of KO (knock-out) mice

The femurs were isolated from VGF KO mice and refrigerated in 80% ethanol, and then measured with a micro-CT scanner (Inveon preclinical CT, Siemens Healthcare, Hoffman Estates, IL) for micro-CT imaging.

As a result, as shown in FIG. 6 , cortical area (Ct.Ar), cortical bone area/total cross-sectional area inside the periosteum (Ct.Ar/Tt.Ar), cortical bone thickness (Ct.Th), and cortical cross-sectional area of VGF KO mice as shown in FIG. It was confirmed that the thickness (Ct.Cs.Th) was significantly reduced.

< Example 5> VGF in KO mice of osteocalcin Quantitative measurement and C- telopeptide (C- Telopeptide ) inspection

One. osteocalcin Measure

Osteocalin is the most important non-collagen protein in the bone matrix and is a bone-specific, vitamin K-dependent calcium-binding protein. Osteocalcin is defined as a marker of bone formation and is used for this purpose.

Therefore, in this example, the osteocalcin content in serum isolated from VGF KO mice was measured in ng/ml using an osteocalcin ELISA kit (Biomedical Tech. Inc) and an ELISA reader.

As a result, as shown in FIG. 7 , it was confirmed that the content of osteocalcin was significantly reduced in VGF KO mice.

2. C- telopeptide inspection

Elevated serum concentration of C-terminal telopeptide has been reported as a symptom of patients with increased bone loss, and as a monitoring method for antagonistic treatment (bisphosphonate or hormone replacement therapy) for osteoporosis and other bone-related diseases. Suitable.

Therefore, in this example, the activity of C-telopeptide, which is a bone resorption index, in VGF KO mice was measured with a RatLaps ELISA kit (Nordic Bioscience Diagnostics A/S, OsteoPark, Denmark).

As a result, as shown in FIG. 7 , no significant change in the activity of C-telopeptide was observed in KO VGF mice compared to the normal control group.

< Example 6> Mouse toxicity test

In this example, the toxicity (anaphylaxis) in mice following a single subcutaneous injection of hAQEE30 was confirmed.

In the case of body weight, as shown in Table 2, no significant change was observed before and after hAQEE30 administration at all doses.

Body weight before and after a single subcutaneous injection of hAQEE30 (g) control
(n = 5) 200 μg
(n = 4) 500 μg
(n = 5) 2,500 μg
(n = 4) 7,500 μg
(n = 4) P-value baseline 23.27±0.46 23.19±0.33 23.39±0.22 22.78±0.39 23.36±0.09 NS 1 day 23.68±0.36 23.36±0.42 23.56±0.20 22.58±0.62 24.20±0.20 NS 4.5 days 23.10±0.29 22.82±0.37 22.92±0.19 22.53±0.48 23.68±0.41 NS

In addition, no abnormal changes were observed in the brain, heart, lung, thymus, liver, kidney, spleen, stomach, small intestine, fat, muscle, cranial tube, femur, tibia and spine in the visual examination. (data not shown). Finally, as shown in Table 3 in the blood test, no significant deleterious changes were observed before and after administration.

control
(n = 5) 7,500 μg
(n = 4) P-value Mean SE Mean SE WBC (10³/μL) 2.94 0.27805 4.1325 0.87237 NS Hb (g/dL) 14.74 0.25612 14.8 0.21602 NS Hct (%) 49.64 0.72842 50.7 0.67946 NS Platelets (10³/μL) 731.36 226.13436 954 192.74638 NS Calcium (mg/dL) 8.892 0.07658 9.065 0.05909 NS Phosphate (mg/dL) 6.26 0.38417 6.255 0.18305 NS AST (U/L) 26.2 1.75983 28.1 2.30326 NS ALT (U/L) 93.66 18.55528 112.725 47.89411 NS ALP (U/L) 406.6 10.283 360.975 21.74517 NS BUN (mg/dL) 24.24 1.23919 25.425 0.83604 NS Creatinine (mg/dL) 0.498 0.0097 0.4625 0.00629 0.023 Glucose (mg/dL) 228.58 9.45862 210.725 9.35391 NS

< Example 7> hAQEE30 Confirmation of efficacy in vivo

In this example, the in vivo efficacy of hAQEE30 injected subcutaneously into a mouse model of osteoporosis was confirmed.

As shown in Table 4, it was confirmed that both BMD and BMC significantly increased after administration of 30 μg of hAQEE30.

control hAQEE30 SQ 1 μg 10 μg 30 μg BMD before treatment (mg/cm ² ) 0.0413±0.0003 0.0413±0.0005 0.0413±0.0003 0.0417±0.0003 BMC before treatment (mg) 0.3923±0.0053 0.3923±0.0059 0.4070±0.0099 0.3847±0.0022 BMD after treatment (mg/cm ² ) 0.0443±0.0004 0.0447±0.0005 0.0450±0.0002 0.0460±0.0004* BMC after treatment (mg) 0.4158±0.0068 0.4393±0.0025 0.4418±0.0010 0.4567±0.0112* Change in BMD % 7.25 ± 1.18 8.43±0.72 8.88±0.88 10.27±1.03 % change in BMC 5.98±0.42 12.13±1.25 8.78±2.49 18.7±2.90*

In addition, to verify the in vivo therapeutic efficacy of hAQEE30, it was attempted to confirm the formation of calvaria bone.

In the prevention of non-vertebral fractures, increasing the skeletal outer diameter and skeletal size is the most important. To confirm this, 3 μg and 10 μg of hAQEE30 were injected into the left periosteal membrane of the calvaria bone for 5 days, and then sacrificed 14 days after the last injection. The obtained cranial bones were fixed in 4% paraformaldehyde solution for 24 hours, decalcified with 0.5M EDTA for 10 days, and then paraffin sectioned. The obtained sections were subjected to Fast Green staining and then the thickness and area of the cranial bones were measured using the Image J program.

As a result, it was confirmed that in the hAQEE30 10 μg injection group, the thickness of the skull was significantly increased and the area of the skull at the injection site was significantly increased. Thus, it was demonstrated that AQEE30 can enable the prevention of non-vertebral fractures ( FIGS. 8 and 9 ).

< Example 8> deformed hAQEE30 Confirmation of osteoblast differentiation effect of peptide

1. Measurement of ALP Activity

In this Example, in order to confirm the effect of the peptide of hAQEE30 and its modified form of Table 5, alkaline phosphatase (ALP) activity of osteoblasts was measured.

peptide amino acid sequence SEQ ID NO: hAQEE30 AQEEAEAEERRLQEQEELENYIEHVLLRRP 3 hAQEE30_sh_01 AEAEERRLQEQEELENYIEHVLLRRP 4 hAQEE30_sh_02 VAEAEERRLQEQEELENYIEHVLLRRP 5

Specifically, after stabilizing the primary osteoblasts overnight in DMEM-HG, each of the peptides in Table 5 was treated in a bone differentiation medium to confirm their osteoblast differentiation promoting effect. ALP staining was reacted for 30 minutes with a Fast Violet B salt kit (Sigma Aldrich), and washed twice again with sterile water. ALP activity was measured by colorimetric assay, and after induction of differentiation for 5 days, the cells were washed twice with cold PBS and sampled with 0.5 ml 50 mM Tris-HCl (pH 7.6). Cells were sonicated and then centrifuged at 12000 rpm. The supernatant was collected separately and its activity was checked at 450 nm using p-nitrophenyl phosphate as a substrate, and the values divided based on the total protein concentration obtained using the BCA assay kit were compared. As a result, as shown in A and B of FIG. 10 , it was confirmed that all groups treated with the peptides of Table 5 exhibited significantly higher ALP activity than the control group.

2. Alizarin Red S staining

In addition, the osteoblast differentiation promoting effect of the peptides of Table 5 was confirmed through Alizarin Red S staining in the same manner as in Example 1.

As a result, as shown in FIG. 10C , it was confirmed that the differentiation of osteoblasts was promoted and bone calcification was significantly increased in all groups except the control group.

< Example 9> Mouse of mAQEE30 osteoblasts viability , proliferative capacity and pluripotency Confirmation of facilitation effect

The amino acid sequence of the mAQEE30 peptide used in the present invention is as follows.

AQEEADAEERRLQEQEELENYIEHVLLHRP (SEQ ID NO: 6)

One. of mAQEE30 osteoblasts viability Confirmation of promotion effect

To confirm the effect of mAQEE30, the viability of osteoblasts was measured.

Specifically, osteoblasts were aliquoted in a 96-well plate at 0.3×10 ⁴ cells/well to stabilize the cells for 24 hours, and then treated with mAQEE30 peptide at each concentration for 48 hours to Cell Counting Kit-8 (CCK-8, Dojindo) Viability was measured at 450 nm after 1 hour using

As a result, it was confirmed that the mAQEE30-treated group had a significant viability promotion effect than the control group (FIG. 11).

2. of mAQEE30 osteoblasts proliferative capacity Confirmation of facilitation effect

To confirm the effect of mAQEE30, the proliferation capacity of osteoblasts was measured.

Specifically, osteoblasts were dispensed in a 96-well plate at 0.3×10 ⁴ cells/well to stabilize the cells for 24 hours, then changed to a medium containing 1% FBS and treated with mAQEE30 peptide at each concentration for 24 hours, followed by Brd -U labeling detection kit (Roche) was used to measure the activity.

As a result, in the group treated with mAQEE30, a significantly higher proliferation promoting effect than the control group was confirmed (FIG. 11).

3. of mAQEE30 Confirmation of osteoblast differentiation promoting effect

To confirm the effect of mAQEE30, alkaline phosphatase (ALP) activity of osteoblasts was measured.

Specifically, osteoblast the rear seeded with 5 × 10 ⁴ cells / well in 24 well plate in which stabilize for 24 hours, cells, mAQEE30 peptide 17 days these osteoblasts was treated μ M in the general medium and bone differentiation medium The differentiation promoting effect was confirmed. ALP staining was performed using a Fast Violet B salt kit (Sigma Aldrich), and ALP activity was measured by a colorimetric assay. Cells were sampled with 50 mM Tris-HCl (pH7.6), sonicated, centrifuged, and the supernatant was collected and checked for activity at 450 nm using p-nitrophenyl phosphate as a substrate, at this time using the BCA assay kit. The values divided by the concentration of the total protein obtained using the method were compared.

As a result, it was confirmed that the group treated with mAQEE30 in the bone differentiation medium exhibited significantly higher ALP activity than the control group (FIG. 12).

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Composition for preventing or treating bone disease comprising VGF-derived peptide <130> ADP-2020-0621 <150> KR 10-2020-0001700 <151> 2020-01-06 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Modified hAQEE30 peptide <400> 1 Xaa Xaa Xaa Xaa Ala Glu Ala Glu Glu Arg Arg Leu Gln Glu Gln Glu 1 5 10 15 Glu Leu Glu Asn Tyr Ile Glu His Val Leu Xaa Xaa Xaa Xaa 20 25 30 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Modified mAQEE30 peptide <400> 2 Xaa Xaa Xaa Xaa Ala Asp Ala Glu Glu Arg Arg Leu Gln Glu Gln Glu 1 5 10 15 Glu Leu Glu Asn Tyr Ile Glu His Val Leu Xaa Xaa Xaa Xaa 20 25 30 <210> 3 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> hAQEE30 peptide <400> 3 Ala Gln Glu Glu Ala Glu Ala Glu Glu Arg Arg Leu Gln Glu Gln Glu 1 5 10 15 Glu Leu Glu Asn Tyr Ile Glu His Val Leu Leu Arg Arg Pro 20 25 30 <210> 4 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> hAQEE30_sh_01 peptide <400> 4 Ala Glu Ala Glu Glu Arg Arg Leu Gln Glu Gln Glu Glu Leu Glu Asn 1 5 10 15 Tyr Ile Glu His Val Leu Leu Arg Arg Pro 20 25 <210> 5 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> hAQEE30_sh_02 peptide <400> 5 Val Ala Glu Ala Glu Glu Arg Arg Leu Gln Glu Gln Glu Glu Leu Glu 1 5 10 15 Asn Tyr Ile Glu His Val Leu Leu Arg Arg Pro 20 25 <210> 6 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> mAQEE30 peptide <400> 6 Ala Gln Glu Glu Ala Asp Ala Glu Glu Arg Arg Leu Gln Glu Gln Glu 1 5 10 15 Glu Leu Glu Asn Tyr Ile Glu His Val Leu Leu His Arg Pro 20 25 30 <210> 7 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> hAQEE30 nucleotide <400> 7 gcgcaggagg aggcggaggc ggaggagcgc cggctgcagg agcaggagga gctggagaat 60 tacatcgagc acgtgctgct ccggcgcccg 90 <210> 8 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> mAQEE30 nucleotide <400> 8 gcgcaggagg aagcggacgc ggaggagcgc cggctgcagg agcaggagga gctggagaat 60 tacattgagc acgtgctgct gcaccgcccg 90

Claims (10)

The amino acid sequence of _{X 1 X 2 X 3 X 4} AEAEERRLQEQEELENYIEHVLX 5 X 6 X 7 amino acid sequence, or X ₁ X ₂ X of SEQ ID NO: 1 consisting of _{X 8 3 X 4 ADAEERRLQEQEELENYIEHVLX 5 X} 6 SEQ ID NO: 2 consisting of X ₇ X ₈ As a pharmaceutical composition for preventing or treating bone disease comprising a peptide comprising as an active ingredient,
In the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2,
X ₁ is no amino acid or is alanine (A);
X ₂ is no amino acid or glutamine (Q);
X ₃ is no amino acid or glutamic acid (E);
X ₄ is free of amino acids or is valine (V) or glutamic acid (E);
X ₅ is no amino acid or leucine (L);
X ₆ is no amino acid or is arginine (R) or histidine (H);
X ₇ is no amino acid or arginine (R);
X ₈ is no amino acid or proline (P);
here,
if there is no amino acid in X ₄ , there is no amino acid in X ₁ to X ₃ ;
If there is no amino acid at X ₃ , there are no amino acids at X ₁ and X ₂ either;
If there is no amino acid in X ₂ , there is also no amino acid in _{X 1 ;}
If there is no amino acid in X ₅ , there is also no amino acid in X ₆ to X ₈ ;
If there is no amino acid at X ₆ , there is also no amino acid at X ₇ and X ₈ ;
If there is no amino acid in X ₇ , there is also no amino acid in X ₈ , a pharmaceutical composition for preventing or treating bone disease. The method according to claim 1, wherein the peptide comprises an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence represented by SEQ ID NO: 6 A pharmaceutical composition for preventing or treating bone disease. According to claim 1, wherein the bone disease is osteoporosis, periodontal disease, fracture, and a pharmaceutical composition for the prevention or treatment of bone disease, characterized in that the disease selected from the group consisting of Paget's disease. The pharmaceutical composition for preventing or treating bone disease according to claim 3, wherein the fracture is a non-vertebral fracture. The pharmaceutical composition for preventing or treating bone disease according to claim 1, wherein the peptide is used in combination with a bone resorption inhibitor. The pharmaceutical composition for preventing or treating bone disease according to claim 5, wherein the bone resorption inhibitor is a bisphosphonate. The pharmaceutical composition for preventing or treating bone disease according to claim 1, wherein the peptide is administered as an injection. The pharmaceutical composition for preventing or treating bone disease according to claim 7, wherein the injection is a depot injection. The amino acid sequence of _{X 1 X 2 X 3 X 4} AEAEERRLQEQEELENYIEHVLX 5 X 6 X 7 amino acid sequence, or X ₁ X ₂ X of SEQ ID NO: 1 consisting of _{X 8 3 X 4 ADAEERRLQEQEELENYIEHVLX 5 X} 6 SEQ ID NO: 2 consisting of X ₇ X ₈ As a health functional food composition for preventing or improving bone disease comprising a peptide comprising as an active ingredient,
In the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2,
X ₁ is no amino acid or is alanine (A);
X ₂ is no amino acid or glutamine (Q);
X ₃ is no amino acid or glutamic acid (E);
X ₄ is free of amino acids or is valine (V) or glutamic acid (E);
X ₅ is no amino acid or leucine (L);
X ₆ is no amino acid or is arginine (R) or histidine (H);
X ₇ is no amino acid or arginine (R);
X ₈ is no amino acid or proline (P);
here,
if there is no amino acid in X ₄ , there is no amino acid in X ₁ to X ₃ ;
If there is no amino acid at X ₃ , there are no amino acids at X ₁ and X ₂ either;
If there is no amino acid in X ₂ , there is also no amino acid in _{X 1 ;}
If there is no amino acid in X ₅ , there is also no amino acid in X ₆ to X ₈ ;
If there is no amino acid at X ₆ , there is also no amino acid at X ₇ and X ₈ ;
If there is no amino acid in X ₇ , there is also no amino acid in X ₈ , a health functional food composition for preventing or improving bone disease. The health function for preventing or improving bone disease according to claim 9, wherein the peptide comprises the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, or the amino acid sequence represented by SEQ ID NO: 5 food composition.

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