Peptides that enhance osteoblast activity

Abstract

The present invention discloses a polypeptide for enhancing osteoblast activity. The polypeptide of the present invention has stable chemical properties, is easy to synthesize on a large scale at low cost, has high biocompatibility, is non-toxic, and can be widely used in the prevention and treatment of orthopedic diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a polypeptide for improving osteoblast activity. Background Art

[0002] Now, with the rapid development of transportation and industry in my country, there are millions of patients suffering from bone defects due to trauma and disease every year. The treatment and rehabilitation of these patients are difficult, resulting in varying degrees of loss of working ability for the patients, causing extreme physical and mental pain to the patients' families, and bringing serious economic burden to the society. This is an urgent problem that needs to be solved.

[0003] The development and treatment targets of existing anti-osteoporosis drugs are almost all focused on bone remodeling (the dynamic balance between bone formation and bone resorption), that is, how to inhibit bone resorption or promote bone formation. Based on these two points, clinical anti-osteoporosis drugs are divided into anti-resorptive drugs, such as bisphosphonates and calcitonin, and pro-osteoporotic drugs, such as teriparatide. However, the former inhibit bone resorption while also inhibiting bone formation, which significantly increases the incidence of adverse reactions with long-term use. The latter promotes bone resorption while also promoting bone formation, which may increase the incidence of fractures after two years of use. To date, there is no anti-resorptive drug that only inhibits bone resorption without inhibiting bone formation, nor is there any pro-osteoporotic drug that only promotes bone formation without promoting bone resorption, which greatly limits the effectiveness and application range of clinical drugs. Currently, bone remodeling-regulating drugs primarily include bisphosphonates, calcitonin, selective estrogen receptor modulators, parathyroid hormone analogs, and RANKL inhibitors. Of these, only parathyroid hormone analogs are peptide drugs that regulate bone remodeling. These drugs are derived from parathyroid hormone, a hormone that itself regulates bone remodeling. Currently, no non-hormonal peptide drugs have been developed, either domestically or internationally. Summary of the Invention

[0004] The first object of the present invention is to provide a polypeptide that has high activity and low toxicity for enhancing osteoblast activity.

[0005] The second object of the present invention is to provide the use of the above polypeptide in promoting bone mineralization, promoting bone formation, and preventing and / or treating orthopedic diseases.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a polypeptide, wherein the core sequence of the polypeptide comprises proline, glycine and alanine.

[0008] Furthermore, the core sequence of the polypeptide contains 3-60 amino acids, and the ratio of the number of proline, glycine, and alanine is (1-3): (1-8):1.

[0009] Furthermore, the core sequence of the polypeptide has the following general formula: (PGAPGP, PGAPG, PGAP, PGA, GAPGP, APGP, PGP, or APG)n, wherein n is a natural number;

[0010] Furthermore, in a specific embodiment of the present invention, n is 1 to 5.

[0011] The present invention also provides nucleic acids encoding the polypeptides of the present invention; vectors comprising the nucleic acids encoding the polypeptides; and host cells comprising the vectors.

[0012] The present invention also provides a pharmaceutical composition comprising the polypeptide of the present invention. The pharmaceutical composition can be used in a method for treating, ameliorating or preventing orthopedic diseases, wherein the method comprises administering to a patient a therapeutically effective amount of the polypeptide, nucleic acid or pharmaceutical composition of the present invention.

[0013] The present invention also provides methods for treating an individual, comprising administering a therapeutically effective amount of a polypeptide, nucleic acid, or pharmaceutical composition of the present invention. The provided methods include treating an individual suffering from or at risk of suffering from an orthopedic disease, comprising administering to the individual a therapeutically effective amount of one or more polypeptides, nucleic acids, or pharmaceutical compositions of the present invention. The present invention also provides methods for promoting bone formation and bone mineralization. The present invention also provides methods for enhancing osteoblast activity, promoting differentiation, proliferation, maturation, and calcification of osteoblasts or their precursor cells, comprising contacting the cells with an effective amount of a polypeptide, nucleic acid, or pharmaceutical composition of the present invention.

[0014] The present invention also provides applications of the polypeptide, nucleic acid, vector, host cell, and pharmaceutical composition of the present invention.

[0015] These and other aspects of the present invention, including other features, advantages, and embodiments, are further described and explained in detail in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Activity assay graph showing peptides of different lengths;

[0017] Figure 2 Activity assay diagram showing the extension of irrelevant amino acids at both ends of the peptide;

[0018] Figure 3 A graph showing the activity assay of peptides incorporating non-natural D-amino acids;

[0019] Figure 4 Shows the activity assay diagram of the modified peptide;

[0020] Figure 5 Shows activity detection diagram of peptide conjugates;

[0021] Figure 6Activity assay diagram showing sustained-release peptide dosage form;

[0022] Figure 7 Shows HE staining and Micro-CT scan results of wild-type mice, where A: trabecular and cortical bone scan; B: trabecular HE staining; C: trabecular number; D: trabecular thickness;

[0023] Figure 8 A diagram showing the results of a double fluorescence labeling experiment after wild-type mice were administered with the peptide;

[0024] Figure 9 The figure shows the results of Von Kossa staining after wild-type mice were administered with the peptide;

[0025] Figure 10 The figure shows the results of Dmp-1 staining, a mineralization indicator, after wild-type mice were given the peptide;

[0026] Figure 11 Shown are the Micro-CT scan results of OVX mice after peptide administration, where A: overall scan of the fifth lumbar vertebra; B: scan of the trabeculae of the fifth lumbar vertebra;

[0027] Figure 12 A diagram showing the results of a double fluorescence labeling experiment after the peptide was administered to OVX mice;

[0028] Figure 13 A diagram showing the results of a fluorescence double-labeling experiment after administering peptides to mice with fractures;

[0029] Figure 14 Figure showing the Micro-CT scan results of mice with fractures after administration of peptides;

[0030] Figure 15 ELISA test results for evaluating the biosafety of peptides are shown, where A: BUN; B: CK; C: ALT;

[0031] Figure 16 Shows H&E staining images used to evaluate the biosafety of the peptides. DETAILED DESCRIPTION

[0032] Below by specific embodiment, the present invention is described in further detail in conjunction with accompanying drawing.In the following embodiment, many detailed descriptions are in order to make this application can be better understood, are only used to illustrate the present invention and are not used to limit the scope of the invention.However, those skilled in the art can recognize without difficulty that wherein some features can be omitted under different circumstances, or can be replaced by other elements, materials, methods.The experimental method of not indicating specific conditions in the embodiment, usually according to the conditions described in the normal conditions, or according to the conditions recommended by the manufacturer, the materials, reagents etc. used in the embodiment, if not otherwise specified, all can be obtained from commercial sources.

[0033] peptides

[0034] In some embodiments, the polypeptides of the present invention include variants of the polypeptides, wherein the variants have 80% or greater homology to the amino acid sequence of the polypeptides of the present invention, and the functions of the variants and the polypeptides are the same or similar. For example, the amino acid sequence of the variants can be a sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the polypeptides of the present invention. Hereinafter, the polypeptides of the present invention are referred to as wild-type polypeptides.

[0035] The term "variant" refers to a polypeptide that is "substantially similar" to a wild-type polypeptide. If two molecules have a substantially similar structure (i.e., as determined by BLASTp alignment set under default parameters, they are at least 50% similar in amino acid sequence) and are substantially similar in at least one related function, then one molecule is considered to be "substantially similar" to another molecule. The variant is different from the wild-type polypeptide in one or more amino acid deletions, additions, substitutions or side chain modifications, but retains one or more specific functions or biological activities of the wild-type molecule. Amino acid substitutions include changes in which amino acids are replaced by different naturally occurring or non-naturally encoded amino acid residues. Some substitutions can be classified as "conservative," in which case the amino acid residues contained in the polypeptide are replaced by another naturally occurring amino acid with similar properties related to polarity, side chain functionality or size. As described herein, the substitutions included in the variant can also be "non-conservative," in which the amino acid residues present in the peptide are replaced by amino acids with different properties (e.g., replacing charged or hydrophobic amino acids with uncharged or hydrophilic amino acids), or alternatively, wherein naturally occurring amino acids are replaced by non-naturally encoded amino acids.

[0036] "Non-naturally encoded amino acid" refers to an unconventional amino acid or an amino acid that is pyrrolysine, pyrroline-carboxyl-lysine, or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid," "non-natural amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated forms. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that are formed by modification (e.g., post-translational modification) of naturally encoded amino acids (including but not limited to the 20 conventional amino acids or pyrrolysine, pyrroline-carboxyl-lysine, and selenocysteine) but that cannot themselves be naturally incorporated into a growing peptide chain by the translation complex. Examples of such non-natural amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine.

[0037] Non-naturally encoded amino acids can typically be any structure having any substituent side chain that is different from the substituent side chains used in the 20 natural amino acids. Since the non-naturally encoded amino acids of the present invention are typically different from natural amino acids only in the side chain structure, the non-naturally encoded amino acids can form amide bonds with other amino acids, including but not limited to, natural or non-naturally encoded amino acids, in the same manner as the amide bond formation in natural polypeptides. However, non-naturally encoded amino acids have side chain groups that are different from natural amino acids. For example, R optionally can include alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, thiol, seleno, sulfonyl, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, etc., or any combination thereof. Other non-natural amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and / or photoisomerizable amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids such as sugar-substituted serine, other sugar-modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with extended side chains compared to natural amino acids (including but not limited to, polyethers or long-chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons), amino acids containing carbon-linked sugars, redox-active amino acids, aminothiocarboxylic acid-containing amino acids, and amino acids containing one or more toxic moieties.

[0038] Furthermore, the variant is obtained by adding 1 to 3 amino acids to the amino terminus and / or carboxyl terminus of the aforementioned polypeptide.

[0039] Furthermore, the variant is obtained by adding 1 to 3 unrelated amino acids to the amino terminus and / or carboxyl terminus of the aforementioned polypeptide.

[0040] Unrelated amino acids useful in the present invention include glycine, tryptophan, tyrosine, cysteine, methionine, glutamine, and threonine.

[0041] In a specific embodiment of the present invention, the variant is obtained by adding 1 to 3 glycine residues to the amino terminus or carboxyl terminus of the PGAPGP polypeptide.

[0042] In a specific embodiment of the present invention, the variant is obtained by adding 1 to 2 glycine residues to both the amino and carboxyl termini of the PGAPGP polypeptide.

[0043] Furthermore, the variant is obtained by adding an oligopeptide consisting of three amino acids to the amino terminus or carboxyl terminus of the aforementioned polypeptide.

[0044] In a specific embodiment of the present invention, the variant is obtained by adding RGD oligopeptide to the amino terminus or carboxyl terminus of the aforementioned polypeptide.

[0045] In a specific embodiment of the present invention, the variant is obtained by replacing an amino acid at any position of the PGAPGP polypeptide with a non-natural D-amino acid.

[0046] In some embodiments, the present invention also discloses derivatives containing the polypeptide of the present invention. The polypeptide derivatives include products obtained by conventional modification of the polypeptide of the present invention, fusion proteins formed by connecting the polypeptide with a heterologous peptide, and conjugates formed by connecting the polypeptide with other compounds.

[0047] The conventional modifications include amination, methylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, myristoylation, non-metallic chemical element modification, immobilization modification, etc.

[0048] In some embodiments, the polypeptide of the present invention is fused to a heterologous peptide. Examples of heterologous peptides include, but are not limited to, human serum albumin (HAS), immunoglobulin heavy chain constant region (Fc), polyhistidine, glutathione S-transferase (GST), thioredoxin, protein A, protein G, mannose binding protein (MBP), or fragments of any of the above heterologous polypeptides. In some embodiments, the heterologous peptide is fused to the amino terminus of the polypeptide of the present invention. In other or alternative embodiments, the heterologous peptide is fused to the carboxyl terminus of the polypeptide of the present invention. For affinity purification, relevant matrices for affinity chromatography, such as glutathione and α-amylase, can be used. The heterologous peptide can be selected to facilitate detection of the polypeptide of the present invention. Examples of detection include various fluorescent proteins (e.g., GFP) and "epitope tags," including His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityXact.

[0049] In some embodiments, the polypeptides of the present invention are linked to other compounds. These other compounds include bisphosphonates and iridoid compounds. These bisphosphonates include alendronic acid, ibandronate, and zoledronic acid. These iridoid compounds include genipinenic acid, genipine-gentiobioside, geniposide, and geniposide acid.

[0050] The polypeptides useful in the present invention can be prepared using any suitable means known in the art. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means and methods for preparing such polypeptides are well known in the art.

[0051] Nucleic acids, vectors and host cells

[0052] In some embodiments, the present invention also provides nucleic acids encoding polypeptides of the present invention, as well as expression vectors and host cells for expressing the polypeptides. In other aspects, the present invention provides polynucleotides encoding polypeptides of the present invention, as well as expression vectors and host cells comprising the polynucleotides. In some embodiments, the polynucleotides are optimized for expression in host cells.

[0053] The polypeptides of the present invention can be expressed using conventional techniques in the field of recombinant genetics. Basic textbooks that disclose general methods that can be used in the present invention include Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, which are known in the art; and the series Ausubel et al. eds. (2007, updated to 2010) Current Protocols in Molecular Biology, etc.

[0054] Expression can use any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. Prokaryotic and eukaryotic expression systems are all available. In some embodiments, the expression system is a mammalian cell expression system, such as a CHO cell expression system. In some embodiments, the nucleic acid can be codon optimized to facilitate expression in the desired host cell.

[0055] Non-viral vectors and systems include plasmids and episomal vectors (typically comprising expression cassettes for expressing proteins or RNA), and artificial human chromosomes. For example, non-viral vectors that can be used to express polypeptides of the invention in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.I / His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include, but are not limited to, vectors based on adenovirus, adeno-associated virus, herpes virus, vectors based on SV40, papilloma virus, HBP Epstein-Barr virus, fowlpox virus vectors, vaccinia virus vectors, and Semliki Forest Virus (SFV) vectors.

[0056] The selection of expression vector depends on the expected host cell of the vector to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) that are operatively connected to the polynucleotide encoding the polypeptide of the present invention. In some embodiments, an inducible promoter is used to prevent the insertion sequence from being expressed under conditions outside the inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, glucocorticoid promoters, or heat shock promoters. In addition, other regulatory elements may also be incorporated into the expression of nucleic acids encoding the polypeptide of the present invention, such as enhancers, ribosome binding sites, transcription termination sequences, and the like.

[0057] In some embodiments, the nucleic acid encoding the polypeptide of the present invention may also include a sequence encoding a secretion signal sequence so that the polypeptide can be secreted from the host cell. This sequence can be provided by the vector or as part of the nucleic acid of the polypeptide of the present invention present in the vector.

[0058] The method for introducing the expression vector that contains the purpose polynucleotide sequence can change according to the host cell type.For example, calcium chloride transfection is generally used for prokaryotic cells, and calcium phosphate treatment or electroporation can be used for other cell hosts.Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated conversion, injection and microinjection, bio-bombardment method, virion, immunoliposome, polycationic acid: nucleic acid conjugate, naked DNA, artificial virion, merge with herpes virus structural protein VP22, DNA uptake and the in vitro transduction that reagent strengthens.For the long-term high yield production of recombinant protein, usually expect stable expression.For example, can use the expression vector of the present invention that contains viral replication origin or endogenous expression element and selective marker gene, prepare the cell line of stably expressing polypeptide of the present invention.

[0059] In some embodiments, nucleic acids encoding polypeptides of the present invention can be delivered to patients to treat orthopedic diseases. The nucleic acid can be delivered using any means known in the art, but typically delivery is achieved using direct injection. In some embodiments, the DNA is delivered by direct injection in the form of naked DNA. In some embodiments, viral vectors are used, including but not limited to adenovirus or adeno-associated virus vectors, herpes virus vectors, fowlpox virus, or vaccinia virus vectors.

[0060] Pharmaceutical composition

[0061] The polypeptide of the present invention or its encoding nucleic acid can be administered together with a suitable pharmaceutical excipient as needed. It will be appreciated by those skilled in the art that the composition will vary depending on the mode of administration and dosage unit.

[0062] In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide of the present invention.

[0063] In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a nucleic acid encoding a polypeptide of the present invention.

[0064] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned vector of the present invention.

[0065] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the host cell described above.

[0066] The composition generally includes conventional pharmaceutical carriers or excipients, and can additionally include other medicaments, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizing agents, etc. Preferably, the composition will contain approximately 0.01 % by weight to approximately 90 % by weight, approximately 0.1 % by weight to approximately 75 % by weight, approximately 0.1 % by weight to 50 % by weight, or approximately 0.1 % by weight to 10 % by weight of the composition of the present invention or its combination, and all the other are made up of suitable pharmaceutical carriers and / or excipients. Suitable excipients can be customized for particular compositions and route of administration by methods well known in the art. Referring to, for example, REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition, Mack Publishing Co., Easton, Pa. (1990).

[0067] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginates, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, and polyacrylic acid such as Carbopol, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The composition may further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives such as methyl hydroxybenzoate, ethyl hydroxybenzoate, and propyl hydroxybenzoate (i.e., parabens); pH adjusting agents such as inorganic and organic acids and bases; sweeteners; colorants; and flavoring agents. The composition may also include biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0068] For oral administration, the composition can be in the form of tablets, lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays, powders and sustained release formulations. Suitable excipients for oral administration include pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate and the like.

[0069] In some embodiments, the pharmaceutical composition is in the form of a pill, tablet, or capsule, and the composition contains any one of the following: a diluent, such as lactose, sucrose, dicalcium phosphate, etc.; a disintegrant, such as starch or its derivatives; a lubricant, such as magnesium stearate, etc.; and a binder, such as starch, gum arabic, polyvinyl pyrrolidone, gelatin, cellulose, and its derivatives. The conjugate can also be formulated as a suppository, for example, in a polyethylene glycol (PEG) carrier.

[0070] Liquid compositions can be prepared by dissolving or dispersing the polypeptide of the present invention or its encoding nucleic acid and optionally one or more pharmaceutically acceptable adjuvants in a carrier such as saline solution (e.g., 0.9% w / v sodium chloride), dextrose solution, glycerol, ethanol, etc. to form a solution or suspension for oral, topical or intravenous administration.

[0071] For topical administration, the compositions of the present invention may be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For delivery by inhalation, the compositions may be delivered by sprayers in dry powder or liquid form. For parenteral administration, the compositions may be in the form of sterile injectable solutions and sterile packaged powders. Preferably, the injectable solution is formulated at a pH of about 4.5 to about 7.5.

[0072] The compositions of the present invention can also be provided in lyophilized form. Such compositions can include a buffer (e.g., bicarbonate) for reconstitution prior to administration, or a buffer can be included in the lyophilized composition for reconstitution, for example, with water. The lyophilized composition can additionally contain a suitable agent, such as other drugs for treating orthopedic diseases. The lyophilized composition can optionally be packaged in combination with a reconstitution buffer and provided in a syringe so that the reconstituted composition can be administered immediately to the patient.

[0073] In some embodiments, the peptide-containing compositions of the present invention are administered to a subject at a specific dose of peptide or are formulated for administering a peptide to a subject in a unit dose. In some embodiments, the dose administered to a subject is about 0.001 to about 1000 mg per day. In some embodiments, the dose administered to a subject is about 0.1 to about 500 mg per day. In some embodiments, the dose administered to a subject is about 0.5 to about 100 mg per day. In some embodiments, the compositions of the present invention are formulated for unit dose administration, wherein the unit dose is about 0.001 to about 1000 mg per day. In some embodiments, the compositions of the present invention are formulated for unit dose administration, wherein the unit dose is about 0.1 to about 500 mg per day. In some embodiments, the compositions of the present invention are formulated for unit dose administration, wherein the unit dose is about 0.5 to about 100 mg per day.

[0074] In some embodiments, the polypeptide of the present invention or the nucleic acid encoding the polypeptide of the present invention can be prepared into a controlled release or sustained release formulation, such as injectable microspheres, bioerodible particles, polymeric compounds, beads, or liposomes or other biocompatible matrices, and the formulation can then be delivered by injection. For example, the polypeptide of the present invention or the nucleic acid encoding the polypeptide of the present invention can be encapsulated in liposomes, or formulated into microparticles or microcapsules, or can be incorporated into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins, poly (lactic acid-co-glycolic acid) (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or delivered via proteinaceous carriers or using conjugates.

[0075] The dosage of the pharmaceutical compositions of the present invention for treating orthopedic conditions is variable and depends on the mode of administration, the age and / or weight of the individual, and the condition of the individual being treated. Ultimately, it is determined by the attending physician or veterinarian. The dosage administered to an individual, in the context of the present invention, should be sufficient to elicit a beneficial response in the individual over a period of time. This dosage is a "therapeutically effective amount."

[0076] method

[0077] The present invention provides a method for preventing or treating orthopedic diseases, comprising administering the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, or the aforementioned pharmaceutical composition to a subject in need thereof.

[0078] The present invention provides a method for enhancing bone formation or promoting bone mineralization in a subject in need thereof, comprising administering the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, or the aforementioned pharmaceutical composition to the subject in need thereof.

[0079] The present invention provides a method for inducing bone deposition in a subject in need thereof, comprising administering the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, or the aforementioned pharmaceutical composition to the subject in need thereof.

[0080] application

[0081] Therapeutic and preventive applications

[0082] The present invention relates to the use of the aforementioned polypeptide, nucleic acid, vector, host cell, and pharmaceutical composition in the preparation of a pharmaceutical preparation for treating or preventing a disease or condition in a subject in need thereof. In the present invention, the disease or condition is an orthopedic disease.

[0083] Those skilled in the art will appreciate that the polypeptides, nucleic acids, vectors, host cells, and pharmaceutical compositions of the present invention described above can be co-administered with other therapeutic agents for treating or preventing orthopedic diseases. Co-administration can be simultaneous, for example, in the form of a single pharmaceutical composition or separate compositions. The compositions of the present invention can also be administered separately from another therapeutic agent, for example, on separate dosing schedules.

[0084] Applications in changing cell properties

[0085] The present invention provides uses of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of drugs for promoting bone formation.

[0086] The present invention provides uses of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of drugs for promoting bone mineralization.

[0087] The present invention relates to use of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of a drug for inducing bone deposition.

[0088] The present invention relates to use of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of a drug for enhancing osteoblast activity.

[0089] "Patient" or "subject" herein refers to any individual to whom a polypeptide of the present invention or its encoding nucleic acid, or a pharmaceutical composition comprising the same, is administered. The present invention contemplates that the polypeptides, compositions, and methods of the present invention can be used to treat mammals. As used herein, "subject" refers to any mammal, including humans, livestock and farm animals, and zoo, sports, or pet animals, such as cattle (e.g., cows), horses, dogs, sheep, pigs, rabbits, goats, cats, mice, rats, monkeys, and the like. In some embodiments of the present invention, the subject is a human. In some embodiments, the subject is a mouse.

[0090] As used herein, the term "D-amino acid" refers to the dextrorotatory stereoisomer of an amino acid. The letters D and L are commonly used in the art to refer to stereoisomers of amino acids. D-amino acids are those that can be synthesized from the dextrorotatory isomer of glyceraldehyde, i.e., D-glyceraldehyde. Similarly, L-amino acids are those that can be synthesized from the L-isomer of glyceraldehyde, i.e., L-glyceraldehyde.

[0091] As used herein, the term "treatment" refers to any type of treatment that is beneficial to a subject suffering from a disease, including improving the patient's condition (e.g., in one or more symptoms), delaying the progression of the disease, etc.

[0092] As used herein, the expression "orthopedic disease" refers to any of those diseases that cause various abnormalities or deformities of one or more bones and / or bone cells.

[0093] Preferably, the orthopedic diseases include osteoporosis, rickets, osteomalacia, osteogenesis imperfecta, marble bone disease, fibrous dysplasia, Paget's disease, chronic hyperparathyroidism, hyperthyroidism, rheumatoid arthritis, Gorham-Stout disease, McCune-Albright syndrome, osteolytic metastases of various cancers or multiple myeloma, bone loss, systemic bone fragility, joint degeneration, non-healing fractures, orthopedic and dental problems caused by diabetes, implant-induced periodontitis, adverse reactions to bone grafts / implants / bone substitute materials, periodontal disease, skeletal aging, fractures, bone defects, bone transplantation , bone grafting, bone cancer, joint replacement, joint repair, fusion, small joint repair, bone degeneration, dental implants and repair, bone marrow defects, bone disease in patients with acromegaly, cystic fibrosis-related bone disease, adynamic bone disease, renal osteodystrophy associated with chronic kidney disease, bone disease associated with cystinosis and bone disease associated with hyperoxaluria; preferably, the osteoporosis includes postmenopausal osteoporosis, senile osteoporosis in men and women, glucocorticoid-induced osteoporosis, immobility osteoporosis, osteoporosis caused by weightlessness, post-transplantation osteoporosis, migratory osteoporosis, idiopathic osteoporosis, and juvenile osteoporosis.

[0094] Example 1 Polypeptide Preparation

[0095] The synthesis is carried out by liquid phase synthesis method, and the specific steps are as follows:

[0096] 1. Synthetic raw materials and related reagents / instruments

[0097] 1) Resin: 2-Chlorotrityl Chloride Resin with a degree of substitution of 1.03 mmol / g (Tianjin Nankai Synthetic Technology Co., Ltd.);

[0098] 2) Amino acids: purchased from Chengdu Chengnuo, >99%;

[0099] 3) Synthesis reagents: DMF (originating from South Korea), DCM (originating from South Korea), MEOH (originating from Japan), DIEA (Xinde Chemical, 99%), HBTU (Haofan Biotechnology, 99%);

[0100] 4) Deprotection reagent: piperidine (Sinopharm Shanghai Chemical Reagent Co., Ltd., 99%);

[0101] 5) Detection reagents: phenol reagent (self-prepared), pyridine reagent (self-prepared), ninhydrin reagent (self-prepared);

[0102] 6) Lysis reagents: 95% cutting solution: TFA (JTBaker, 99%), TIS (Shanghai Darui Fine Chemicals, 98%), EDT (Shanghai Darui Fine Chemicals, 98%), anhydrous ether (Shanghai Laboratory, measured 99.7%);

[0103] 7) Nitrogen: (Xinlian Gas);

[0104] 8) Instruments: ① Twelve-channel semi-automatic peptide synthesizer. This semi-automatic peptide synthesizer was independently designed and patented by Shanghai Qiangyao Biotechnology Co., Ltd., with patent number 201020226529.2. ② SHIMADZU high performance liquid chromatograph (model: preparative, analytical, software: Class-VP. Sevial System, manufacturer: SHIMADZU). ③ Centrifuge (Shanghai Anting Scientific Instrument Factory, model: TDL-40B). ④ LABCONCO freeze dryer (model: Freezone.Plus.6, manufacturer: LABCONCO).

[0105] 2. The steps for peptide synthesis are as follows: the synthesis order is from C-terminus to N-terminus.

[0106] 1) Resin swelling: Add 2-Chlorotrityl Chloride Resin to a reaction tube, add DCM (15 ml / g), and shake for 30 min.

[0107] 2) Add the first amino acid: Filter the solvent through a sand core, add 3 times the molar mass of the first amino acid at the C-terminus, add DMF to dissolve, then add 10 times the molar excess of DIEA, shake for 60 minutes; block with methanol.

[0108] 3) Deprotection: remove DMF, add 20% piperidine DMF solution (15 ml / g), 5 min, remove and add 20% piperidine DMF solution (15 ml / g), 15 min.

[0109] 4) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add detection reagent for detection, heat at 105℃-110℃ for 5 minutes, and a dark blue color indicates a positive reaction.

[0110] 5) Washing: DMF (10 ml / g) twice, DCM (10 ml / g) twice, DMF (10 ml / g) twice.

[0111] 6) Condensation: Dissolve the protected amino acid in a three-fold excess and HBTU in a three-fold excess in as little DMF as possible, add them to the reaction tube, and immediately add a ten-fold excess of DIEA. Let the reaction proceed for 30 min.

[0112] 7) Detection: Take a dozen resin pellets, wash them three times with ethanol, add detection reagent for detection, heat at 105℃-110℃ for 5 minutes, and colorless is a negative reaction.

[0113] 8) Washing: DMF (10 ml / g) once, DCM (10 ml / g) twice, DMF (10 ml / g) twice.

[0114] 9) Repeat steps 3) to 6) to connect the amino acids in the sequence from right to left.

[0115] 10) Drain and wash the resin: DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice, DCM (10 ml / g) twice, drain for 10 min.

[0116] 11) Cleavage of the polypeptide from the resin: prepare a cleavage solution (10 / g): TFA 95%, water 1%, EDT 2%, TIS 2%, cleavage time: 120 min.

[0117] 12) Drying and washing: Dry the lysate as much as possible with nitrogen, wash it with ether six times, and then evaporate it at room temperature.

[0118] 13) Analysis and purification: The crude product was purified by high performance liquid chromatography.

[0119] 14) Freeze-drying: Collect the target polypeptide solution and concentrate it in a freeze dryer, then freeze-dry it into a white powder.

[0120] 15) Send the peptide to the quality inspection department for confirmation.

[0121] Example 2 Activity detection of polypeptides and their derivatives

[0122] 1. Activity detection of peptides of different lengths

[0123] 1.1 Synthesis of peptides of different lengths

[0124] Synthesized according to the method of Example 1.

[0125] 1.2 Cell experiments

[0126] Osteoblastic cell line MC-3T3E1 cells were cultured and incubated with peptides of different lengths at a final concentration of 2 μg / ml for 24 hours. The ALP gene expression level was then detected by QPCR.

[0127] 1.3 Results

[0128] The results are as follows Figure 1 As shown, the combination of six amino acids of PGAPGP is a better combination for enhancing osteoblast activity. When 2-3 PGAPGPs are connected in series, good activity is observed. When 4 or more PGAPGPs are connected in series, the activity begins to weaken.

[0129] 2. Peptide activity detection after amino acid extension at both ends of the peptide

[0130] 2.1 Synthesis of peptides of different lengths

[0131] Synthesized according to the method of Example 1.

[0132] 2.2 Cell experiments

[0133] Osteoblastic cell line MC-3T3E1 cells were cultured and incubated with peptides of different lengths at a final concentration of 2 μg / ml for 24 hours. The ALP gene expression level was then detected by QPCR.

[0134] 2.3 Results

[0135] The results are as follows Figure 2 As shown, unilateral extension of PGAPGP by one unrelated amino acid had no effect on its activity, unilateral extension by two unrelated amino acids had a minor effect, and extension by three or more unrelated amino acids decreased its activity. Bilateral extension by one unrelated amino acid each had no effect on its activity, but extension by two or more unrelated amino acids each began to decrease its activity. Glycine (G) is a representative unrelated amino acid.

[0136] 3. Activity detection of peptides incorporated with non-natural D-amino acids

[0137] 3.1 Synthesis of peptides incorporating non-natural D-amino acids

[0138] The conventional method was followed, and the distribution of non-natural D-amino acids is shown in Table 1.

[0139] Table 1 Distribution of non-natural D-amino acids

[0140] serial number Distribution of unnatural D-amino acids P1 PGAPGP P2 <![CDATA[PGAPGP D ]]> P3 <![CDATA[PGAPG D P]]> P4 <![CDATA[PGAP D GP]]> P5 <![CDATA[PGA D PGP]]> P6 <![CDATA[PG D APGP]]> P7 <![CDATA[P D GAPGP]]>

[0141] 3.2 Cell experiments

[0142] Osteoblastic cell line MC-3T3E1 cells were cultured and incubated with peptides of different lengths at a final concentration of 2 μg / ml for 24 hours. The ALP gene expression level was then detected by QPCR.

[0143] 3.3 Results

[0144] The results are as follows Figure 3 As shown, the activity of PGAPGP peptide decreased after the amino acid at any position was replaced by non-natural D-amino acid.

[0145] 4. Activity detection of modified peptide variants

[0146] 4.1 Synthesis of modified variants

[0147] The conventional method was followed and the polypeptide modifications were as shown in Table 2.

[0148] Table 2 Different modified peptides

[0149] serial number Modification Pep1 N-methylation Pep2 N-myristoylation Pep3 N-PEG modification Pep4 C-fluorine element modification Pep5 C-biotin modification Pep6 C-FAM fluorescent labeling Pep7 Unmodified PGAPGP

[0150] 4.2 Cell experiments

[0151] Osteoblastic cell line MC-3T3E1 cells were cultured and incubated with peptides of different lengths at a final concentration of 2 μg / ml for 24 hours. The ALP gene expression level was then detected by QPCR.

[0152] 4.3 Results

[0153] The results are as follows Figure 4 The figure shows the effects of common peptide terminal modifications on PGAPGP activity. PGAPGP modified with methylation, myristylation, PEG, fluorine, biotin, and FAM fluorescence all showed activity.

[0154] 5. Activity detection of peptide conjugates

[0155] 5.1 Synthesis of Peptide Conjugates

[0156] The conventional method was followed, and the polypeptide conjugates were shown in Table 3.

[0157] Table 3 Peptide conjugates

[0158] serial number Modification PC1 Unmodified PGAPGP PC2 N-coupled sodium alendronate PC3 C-coupled sodium alendronate PC4 N-coupled RGD peptide PC5 C-coupled RGD peptide

[0159] 5.2 Cell experiments

[0160] Osteoblastic cell line MC-3T3E1 cells were cultured and incubated with the peptide conjugate at a final concentration of 5 μg / ml for 24 hours, and the ALP gene expression level was detected by QPCR.

[0161] 5.3 Results

[0162] The results are as follows Figure 5 As shown, PGAPGP is active when coupled to bisphosphonates, represented by alendronate, and is active when coupled to oligopeptides, represented by RGD.

[0163] 6. Activity testing of peptide sustained-release dosage forms

[0164] 6.1 Synthetic Microsphere Sustained-Release Dosage Form

[0165] The process involves stirring and dissolving PLGA, adding a porogenic solution dropwise, and ultrasonically emulsifying the solution to form an emulsion. The molar ratio of LA to GA in the PLGA is 50:50. The emulsion is then added dropwise to a stirred external aqueous phase, followed by a preset volume of deionized water. The stirring is continued at varying speeds until the solvent is completely evaporated. The external aqueous phase comprises a water-soluble surfactant added to deionized water. The solution, after complete evaporation of the organic solvent, is then centrifuged and washed with deionized water. The supernatant is removed to obtain PLGA microspheres. A NaOH solution containing the polypeptide is added to the PLGA microspheres, mixed evenly, and placed on a shaker to continue the reaction. The reacted PLGA microspheres are repeatedly washed by centrifugation with deionized water, freeze-dried, and set aside.

[0166] 6.2 Cell experiments

[0167] Osteoblastic cell line MC-3T3E1 cells were cultured and incubated with microspheres at a final concentration of 2 μg / ml for 1-6 days, and the ALP gene expression level was detected by QPCR.

[0168] 6.3 Results

[0169] The results are as follows Figure 6 As shown, the PGAPGP sustained-release formulation, represented by PLGA 50:50 microspheres, has significant biological activity.

[0170] Example 3 Effect of polypeptide on bone formation in mice

[0171] 1. Materials

[0172] Source of wild-type mice: All mice were SPF grade and purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd.

[0173] 2. Preparation of polypeptide solution: Weigh 1.000 g of sodium hydroxide into a beaker and dissolve it in a small amount of saline. Pour the solution into a 1000 ml volumetric flask. Rinse the beaker three times, pour the entire solution into the volumetric flask, and dilute to the mark with saline. Shake well to obtain a 1000 mg / mL polypeptide stock solution. Dilute to the desired concentration when using.

[0174] 3. Drug treatment

[0175] Mice were randomly divided into two groups, a control group and an experimental group. The mice in the control group were injected with normal saline once a week, and the mice in the experimental group were injected with 10 mg / kg once a week. After 12 injections, femoral samples were collected for Micro-CT scanning and H&E staining.

[0176] 4. Micro-CT Scanning

[0177] The collected femoral samples were subjected to Micro-CT scanning of trabecular and cortical bones, and the trabecular number and trabecular thickness were analyzed.

[0178] 4.1 Steps

[0179] (1) The femoral specimens were fixed in 4% paraformaldehyde for 24 hours and then transferred to 0.5% paraformaldehyde solution to prevent crystals from forming in the bone tissue and affecting the specimen scanning.

[0180] (2) Place the sample in a 14 mm diameter Micro-CT dedicated scanning tube. Place the sample horizontally with the horizontal axis perpendicular to the Micro-CT scanning axis. Place the scanning tube in the sample tray of the Micro-CT scanning chassis.

[0181] (3) Scanning parameters were as follows: scanning voltage 70 kVp, scanning energy power 14 W, scanning current 200 μA, exposure time 300 ms, scanning BH 1200 mg HA / cc, scanning precision 10 μm, and scanning filter 0.5 mm AI filter. Image data were reconstructed and analyzed using Mimics 13.0 software.

[0182] (4) When reconstructing femoral data, trabecular bone data analysis selects all trabeculae in the inner layer of the cortical bone starting from 1 mm below the femoral growth plate; cortical bone data analysis selects the cortical bone 5 mm to 6 mm below the growth plate.

[0183] 4.2 Results

[0184] Micro-CT scan results Figure 7 As shown, the bone density of mice increased significantly after the application of peptide ( Figure 7 A), trabecular thickness increased significantly ( Figure 7 C), the number of trabeculae increased significantly ( Figure 7 D), showing enhanced mineralization of mouse femur.

[0185] 5. H&E staining

[0186] 5.1 Steps

[0187] 1) Sectioning: Prepare paraffin sections of tissue using conventional methods;

[0188] 2) Baking: Place the slices in a 65°C oven for 30 minutes;

[0189] 3) Dewaxing and hydration: Dewax the tissue sections in xylene I for 30 minutes, then xylene II for 30 minutes. Then, hydrate the sections in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 90% ethanol for 5 minutes, 75% ethanol for 5 minutes, and distilled water for 5 minutes.

[0190] 4) Staining: Wash with hematoxylin for 5 minutes, soak in PBS for 5 minutes to turn the cell nuclei blue, stain with eosin for 2 minutes, and wash with water.

[0191] 5) Dehydration and Transparency: Dehydrate the tissue using a gradient of ethanol: 80% ethanol, 90% ethanol, and 95% ethanol for 10 seconds each, then air-dry at room temperature. Then, use anhydrous ethanol I for 5 minutes, and anhydrous ethanol II for 5 minutes, then air-dry at room temperature. After immersion in xylene for 5 minutes to achieve transparency, mount the slides with a neutral resin.

[0192] 6) Observation: Observe and take pictures under an upright microscope.

[0193] 5.2 Results

[0194] The femoral samples were stained with H&E. The results showed that after the mice were injected with the peptide, the trabeculae under the growth plate became denser ( Figure 7 B).

[0195] 6. Mouse dual fluorescence labeling experiment

[0196] 6.1 Steps

[0197] (1) The femur samples (xylenol orange and calcein were injected intraperitoneally at a dose of 80 mg / kg at an interval of 2 weeks) were sliced into hard tissue sections with a thickness of approximately 15 μm.

[0198] (2) DAPI staining: Add 1:1000 dilution of DAPI stain to hard tissue sections and stain for 5 min. Rinse in PBS three times, 5 min each time. Mount with anti-fluorescence quenching mounting medium. Observe the red and green fluorescence labeling under an inverted fluorescence microscope, and calculate the bone apposition rate.

[0199] 6.2 Results

[0200] The results are as follows Figure 8 As shown, the red and green markers are separated by two weeks, and the distance between them represents new bone deposition. Compared to the control group, the experimental group showed a significant increase in femoral bone deposition. This analysis indicates that the peptide injection significantly enhanced bone formation in mice.

[0201] 7. Von Kossa dyeing

[0202] 7.1 Steps

[0203] (1) Silver nitrate staining: Prepare 0.2% silver nitrate solution and add it dropwise to the femoral hard tissue slices to cover the femoral tissue. Irradiate with strong light for 15 minutes and rinse the slices with running water for 2-3 seconds.

[0204] (2) Sodium thiosulfate incubation: Prepare 5% sodium thiosulfate solution and add it dropwise to the slices, covering the femoral tissue, for 5 seconds. Rinse the slices with running water for 2-3 seconds.

[0205] (3) Methyl green dyeing: Dye with methyl green solution for 5 minutes, rinse with running water for 5 seconds to remove floating color, and dry in an oven at 65℃ for 30 minutes.

[0206] (4) Sealing and Observation: After the sections were transparentized by adding xylene for 5 minutes, they were sealed with neutral resin. The sections were observed and photographed under a stereo microscope.

[0207] 7.2 Results

[0208] The calcium ions in the bone tissue are replaced by silver ions and appear black, indicating the density of mineralized bone and the level of bone mineralization. Figure 9 As shown in the figure, the density of trabecular bone under the growth plate increased in the polypeptide injection group, indicating that the degree of bone mineralization was significantly enhanced.

[0209] 8. Detection of bone mineralization-related indicators Dmp-1

[0210] 8.1 Immunohistochemical staining

[0211] (1) Sectioning: After decalcification of the femoral tissue, routine paraffin tissue sections were performed;

[0212] (2) Baking: Place the slices in a 65°C oven for 30 min;

[0213] (3) Dewaxing and hydration: Dewax the tissue sections in xylene I for 30 minutes, then xylene II for 30 minutes. Then, hydrate the sections in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 90% ethanol for 5 minutes, and 75% ethanol for 5 minutes. Then, rinse in distilled water for 5 minutes. Rinse the sections three times in PBS, each time for 3 minutes.

[0214] (4) Antigen repair: Repair tissue antigens accordingly according to the requirements of the antibodies.

[0215] (5) Inactivation of endogenous peroxidase: Add an appropriate amount of peroxidase blocking solution to the slices and incubate at room temperature for 10 minutes (to block the activity of endogenous peroxidase). Rinse with PBS three times, each time for 5 minutes.

[0216] (6) Tissue blocking: Add an appropriate amount of normal non-immune animal serum to the slice and incubate at room temperature for 10 minutes.

[0217] (7) Incubation with primary antibody: Shake off the serum on the tissue, add an appropriate amount of primary antibody to the tissue, and incubate at room temperature for 60 minutes or at 4°C overnight. Rinse three times with PBS for 3 minutes.

[0218] (8) Incubation with secondary antibody: Add appropriate amount of biotin-labeled secondary antibody to the sections, incubate at room temperature for 10 min, and rinse three times with PBS, each time for 3 min.

[0219] (9) Remove PBS and add 1 drop or 50 μl of streptavidin-labeled secondary antibody (reagent D) to each section. Incubate at room temperature for 10 min and rinse three times with PBS for 3 min each time.

[0220] (10) DAB color development: Add DAB color development solution (freshly prepared) and observe under a microscope for 3-10 minutes. After the color development is complete, rinse with tap water to terminate the color development.

[0221] (11) Counterstaining of cell nuclei: counterstain with methyl green solution and return to blue with PBS.

[0222] (12) Dehydration and clearing: Dehydrate the tissue using graded ethanol: 80% ethanol, 90% ethanol, and 95% ethanol for 10 seconds each, then air dry at room temperature. Dehydrate in anhydrous ethanol I for 5 minutes, then in anhydrous ethanol II for 5 minutes, then air dry at room temperature. Clear in xylene for 5 minutes. Mount the slides and observe.

[0223] 8.2 Results

[0224] result Figure 10 As shown in the figure, the immunohistochemical staining level of the peptide injection group increased, indicating that the expression of mouse mineralization protein Dmp1 was significantly enhanced.

[0225] Example 3 Effect of polypeptide on bone mass in osteoporosis mice

[0226] 1. Construction of osteoporosis mouse model

[0227] (1) Eighteen 12-week-old female WT mice were selected and divided into three groups (OVX group, OVX+peptide group, and OVX+PTH group).

[0228] (2) Mice were anesthetized with 35 mg / kg sodium pentobarbital.

[0229] (3) Prepare the skin of the mouse's lower abdomen, disinfect it with iodine, wipe off the iodine with 75% alcohol, and make a 0.5 cm incision 1 cm below the ribs and 0.5 cm to the left of the abdominal midline.

[0230] (4) Cut open the fascia and muscles, and bluntly widen the surgical field of view. Find the white adipose tissue in the lower abdomen and pull it out of the body. Follow the uterus upward to find the fallopian tubes and ovaries.

[0231] (5) After tubal ligation, the ovaries and fallopian tubes are removed, and after bilateral resection, the uterus and fat are returned to the abdomen.

[0232] (6) Suture the skin and muscles in layers.

[0233] (7) Three months after the osteoporosis model mice were established, after the model became stable, the peptide was injected intravenously for a total of 12 times, once a week, at a dose of 10 mg / kg; PTH was administered subcutaneously at a dose of 0.1 μg / kg, for 4 weeks, 3 times a week.

[0234] 2. Micro-CT Scanning

[0235] The steps are the same as in Example 1.

[0236] The results are as follows Figure 11 As shown in the figure, compared with the OVX group, the bone mass of mice in the polypeptide group and PTH group was significantly increased, indicating that polypeptide can increase the bone mass of osteoporosis mice.

[0237] 3. Fluorescence double labeling experiment

[0238] The steps are the same as in Example 1.

[0239] The results are as follows Figure 12 As shown in the figure, the amount of bone deposition in the peptide group and PTH group was significantly higher than that in the OVX group, indicating that the bone formation function of mice was improved after peptide treatment.

[0240] Example 4 Effect of polypeptide on fracture healing

[0241] 1. Establishment of Mouse Fracture Model

[0242] A mouse fracture model was established using the method described in the literature [Glycosylation of dentin matrix protein 1 is critical for fracture healing via promoting chondrogenesis. Frontiers of Medicine. 2019 Oct; 13(5): 575-589]. One week after modeling, mice were injected with 10 mg / kg of the peptide once weekly. After six injections, bone samples were collected to evaluate callus formation and callus density at the fracture ends of the mice.

[0243] 2. Fluorescence double labeling experiment

[0244] The steps are the same as in Example 1.

[0245] The results are as follows Figure 13 As shown, the bone formation ability of the mice in the polypeptide group was enhanced.

[0246] 3. Micro-CT Scanning

[0247] The steps are basically the same as those in Example 1. The difference is that when reconstructing the fracture sample data, the callus display area is 500 μm above and below the fracture end, and the data analysis is selected from the callus part outside the cortical bone at the corresponding location.

[0248] The results are as follows Figure 14 As shown, the fracture site healing of the fracture model mice in the peptide injection group was accelerated. The callus reconstruction results showed that the callus after peptide injection was denser than that in the control group, and the bone density in the peptide group was significantly higher than that in the control group.

[0249] Example 5 Biosafety Evaluation of Polypeptides

[0250] The biosafety of the peptide was explored through evaluation of serum biochemical indicators and analysis of pathological sections of important organs.

[0251] 1. ELISA test

[0252] After the mice were injected with peptides, the serum levels of BUN (blood urea nitrogen), CK (creatine kinase), and ALT (alanine aminotransferase) were detected by ELISA to evaluate the effects of small molecule peptides on biochemical indicators.

[0253] 2.1 Steps

[0254] 2.1.1 Mouse serum collection

[0255] (1) Blood collection from the retroorbital venous plexus of mice:

[0256] Take a glass capillary tube with an inner diameter of 1.0-1.5mm and break it into 2-2.5cm long segments immediately before use. Immerse the tube in 1% heparin solution and allow it to dry before use. To draw blood, grasp the skin on the back of the neck between the ears with your left hand to stabilize the head. Gently press down on both sides of the neck to obstruct venous return in the head and cause congestion of the orbital venous plexus. Hold the capillary tube with your right hand and insert it into the conjunctiva from the medial canthus. Gently push the tube toward the fundus of the eye. Gently rotate the tube to rupture the venous plexus, allowing the blood to flow out through the capillaries. Collect the blood into a prepared container. After blood collection, gently apply gauze to the eye to stop the bleeding.

[0257] (2) After obtaining blood using the above method, place it in an EP tube at room temperature for at least one hour (or in a 37°C water bath for one hour, or in a 4°C refrigerator for two hours or overnight). Centrifuge at 3000 rpm for 10 minutes. The supernatant is serum. It can be stored at -80°C.

[0258] 2.1.2 Enzyme-linked immunosorbent assay (ELISA)

[0259] (1) Equilibration: Take out CK (creatine kinase), BUN (blood urea nitrogen), ALS (aldosterone), ALT (alanine aminotransferase), and ELISA kit and equilibrate them at room temperature for 20 minutes.

[0260] (2) Dilute the washing solution: dilute the 20X washing solution to 1X with dd H2O.

[0261] (3) Serum preparation: After collecting blood using a test tube that does not contain pyrogens or endotoxins (any cell stimulation should be avoided during subsequent experimental operations), centrifuge at 3000 rpm for 10 minutes at room temperature, and carefully aspirate to separate the serum and red blood cells.

[0262] (4) Sample loading: Take the required number of well plates and place them on a 96-well plate. Mark the order of sample loading on the plate. Add 20 μl of Matrix solution to the blank and standard wells. Add 20 μl of each of the 0, 0.025, 0.05, 0.1, 0.2, and 0.4 ng / ml standards to the standard wells. Add 10 μl of diluted serum from the control and peptide groups to the sample wells, followed by 40 μl of sample diluent. Do not add anything to the blank wells.

[0263] (5) Manual plate washing: Slowly add plate washing solution along the well wall. After standing for 1 minute, discard the plate washing solution and tap on absorbent paper. Repeat 5 times. Change the pipette tip promptly when adding different samples to avoid contamination.

[0264] Automatic plate washing: Add 350 μl of washing solution to each reaction well, soak for 1 min, and wash the plate 5 times.

[0265] (6) Except for the blank wells, add 100 μl of horseradish peroxidase (HRP)-labeled detection antibody to the standard reaction wells and sample reaction wells. Seal the reaction wells with a sealing film and incubate in a 37°C water bath or incubator for 60 min.

[0266] (7) Washing the plate: Wash the plate 5 times in the above manner.

[0267] (8) Color development: Add 50 μl of substrate A and B to each reaction well and incubate at 37°C in the dark for 15 min.

[0268] (9) Measurement: Add 50 μl of stop solution to each well. After 10 min, read the absorbance at 450 nm using a microplate reader. After creating a standard curve, calculate the protein content in the sample using the corresponding formula.

[0269] 2.2 Results

[0270] The results are as follows Figure 15 As shown, mice were intravenously injected with 10 mg / ml of peptide weekly, and blood was drawn for testing at weeks 2, 4, 6, 8, 10, and 12. Circles represent the control group, and squares represent the peptide group.

[0271] 3. H&E staining

[0272] Mice were intravenously injected with 10 mg / ml of the peptide weekly and sacrificed after 12 weeks to collect samples for testing. H&E staining was performed on important organs for pathological evaluation to investigate whether the peptide had chronic toxic effects on internal organs. Paraffin sections of tissues such as the heart, lungs, liver, spleen, and kidneys were sectioned and H&E stained. The results are shown in Figure 2. Figure 16 As shown, compared with the control group, no obvious pathological changes such as tumors or inflammatory infiltration occurred in important organs (heart, lungs, liver, spleen, and kidneys) after polypeptide injection. The results of this example show that the polypeptide of the present invention has good biosafety.

[0273] The above embodiments are intended to illustrate the present invention only and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and intent of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A polypeptide, characterized in that The sequence of the polypeptide is PGAP or PGA.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide of claim 1.

3. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 2.

4. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 2 or the vector of claim 3.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3, and the host cell according to claim 4.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further includes a conventional pharmaceutical carrier.

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