WO2023272658A1 - Polypeptide for enhancing osteoblast activity, and application thereof in treatment of orthopedic disease - Google Patents

Abstract

Provided is a polypeptide for enhancing osteoblast activity. The polypeptide has stable chemical properties and no toxicity, can promote bone formation, and can be used for prevention and treatment of orthopedic diseases.

Description

Polypeptides that enhance osteoblast activity and their application in the treatment of orthopedic diseases technical field

The invention belongs to the field of biomedicine, and relates to a polypeptide for enhancing osteoblast activity and its application in treating orthopedic diseases.

Background technique

Now, with the rapid development of my country's transportation and industry, there are millions of patients with bone defects caused by trauma and disease every year. Its treatment and rehabilitation are difficult, causing different degrees of loss of labor ability of patients, causing extreme physical and mental pain to the families of patients, and bringing great harm to the society. Bringing serious economic burden is an urgent problem to be solved.

The development and therapeutic 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. Clinically, anti-osteoporosis drugs are also divided into anti-bone resorption drugs based on these two points, such as bisphosphonates, calcitonin, etc., and bone-promoting drugs such as teriparatide; however, the former can inhibit bone resorption. At the same time, it also inhibits bone formation, which significantly increases the incidence of adverse reactions in its long-term application; while the latter promotes bone formation and bone resorption, it may increase the incidence of fractures after 2 years of use. Until now, there is no anti-resorptive drug that only inhibits bone resorption but not bone formation, and no bone-promoting drug that only promotes bone formation but not bone resorption, which greatly limits the effectiveness of clinical drugs and scope of application. At present, bone remodeling drugs mainly include bisphosphonates, calcitonin, selective estrogen receptor modulators, parathyroid hormone analogs, RANKL inhibitors, etc., of which only parathyroid hormone analogs are a bone remodeling agent. Regulatory polypeptide drugs, which are derived from a hormone that itself has a bone remodeling regulatory effect: parathyroid hormone. At present, there are no non-hormone-based peptide drugs at home and abroad.

Contents of the invention

The first object of the present invention is to provide a polypeptide that enhances osteoblast activity with high activity and low toxicity.

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

To achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a polypeptide, the core sequence of which comprises proline, glycine and alanine.

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

Further, 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;

Further, in a specific embodiment of the present invention, n is 1-5.

The invention also provides a nucleic acid encoding a polypeptide of the invention; a vector comprising a nucleic acid encoding the polypeptide; and a host cell comprising the vector.

The invention also provides pharmaceutical compositions comprising the polypeptides of the invention. The pharmaceutical composition can be used in a method for treating, improving or preventing orthopedic diseases, wherein the method includes administering a therapeutically effective amount of the polypeptide, nucleic acid or pharmaceutical composition of the present invention to a patient.

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

The present invention also provides the application of the polypeptide, nucleic acid, vector, host cell and pharmaceutical composition of the present invention.

These and other aspects of the invention, including other features, advantages and embodiments, are described and illustrated in further detail in the following detailed description and claims of the invention.

Description of drawings

Figure 1 shows the activity detection diagram of polypeptides of different lengths;

Figure 2 shows the activity detection diagram after extending irrelevant amino acids at both ends of the polypeptide;

Figure 3 shows the activity detection diagram of the polypeptide incorporated with non-natural D-type amino acids;

Figure 4 shows the activity detection diagram of the modified polypeptide;

Figure 5 shows the activity detection diagram of the polypeptide conjugate;

Figure 6 shows the activity detection diagram of the polypeptide sustained-release dosage form;

Figure 7 shows the HE staining image and Micro-CT scanning results of wild-type mice, where A: trabecular bone and cortical bone scanning image; B: HE staining image of trabecular bone; C: number of trabecular bone; D: trabecular bone beam thickness;

Figure 8 shows the results of double fluorescent labeling experiments after wild-type mice were given polypeptides;

Figure 9 shows the results of Von Kossa staining after administration of polypeptides to wild-type mice;

Figure 10 shows the staining results of the mineralization indicator Dmp-1 in wild-type mice after administration of polypeptides;

Figure 11 shows the results of Micro-CT scanning of OVX mice after administration of polypeptides, wherein A: the overall scan of the fifth lumbar vertebra; B: the scan of the trabecular bone of the fifth lumbar vertebra;

Figure 12 shows the results of the double fluorescent labeling experiment after the OVX mice were given polypeptides;

Figure 13 shows the results of the double-labeled fluorescence experiment after the mice with fractures were given polypeptides;

Figure 14 shows the results of Micro-CT scans after the administration of polypeptides to mice with fractures;

Figure 15 shows the results of the ELISA experiment used to evaluate the biological safety of the polypeptide, wherein, A: BUN; B: CK; C: ALT;

Figure 16 shows H&E staining plots for evaluating the biosafety of polypeptides.

detailed description

The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are for better understanding of the present application, and are only used to illustrate the present invention and not limit the scope of the present invention. However, those skilled in the art can readily recognize that some of the features can be omitted in different situations, or can be replaced by other elements, materials, and methods. For the experimental methods that do not indicate specific conditions in the examples, usually according to the conditions described in the conventional conditions, or according to the conditions suggested by the manufacturer, the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial way to get.

polypeptide

In some embodiments, the polypeptide of the present invention includes a variant of the polypeptide, the variant has 80% or more homology with the amino acid sequence of the above-mentioned polypeptide of the present invention, and the function of the variant is the same as that of the above-mentioned polypeptide or resemblance. For example, the amino acid sequence of the variant may be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the above-mentioned polypeptide. Sequences with % homology. Hereinafter, the polypeptide of the present invention is referred to as wild-type polypeptide.

The term "variant" refers to a polypeptide that is "substantially similar" to a wild-type polypeptide. If two molecules have substantially similar structure (i.e., they are at least 50% similar in amino acid sequence as determined by BLASTp alignment at default parameters settings) and are substantially similar in at least one related function, then A molecule is said to be "substantially similar" to another molecule. A variant differs from a 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 an amino acid is replaced with a different naturally occurring or non-naturally encoded amino acid residue. Some substitutions may be classified as "conservative," in which case an amino acid residue contained in a polypeptide is replaced by another naturally occurring amino acid with similar properties related to polarity, side chain functionality, or size . As described herein, variants may also include substitutions that may be "non-conservative," wherein amino acid residues present in the peptide are replaced with amino acids having different properties (e.g., uncharged or hydrophilic amino acids for charged ones). or hydrophobic amino acids), or alternatively, wherein a naturally occurring amino acid is replaced by a non-naturally encoded amino acid.

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

A non-naturally encoded amino acid can typically be of any structure with any substituent side chains different from those used in the 20 natural amino acids. Since the non-naturally encoded amino acids of the invention typically differ only in side chain structure from natural amino acids, the non-naturally encoded amino acids can be compared with other amino acids, including but not limited to, naturally or non-naturally encoded amino acids, and natural polypeptides. The amide bond is formed in the same way as the amide bond is formed. However, non-naturally encoded amino acids have side chain groups that differ from natural amino acids. For example, R optionally can comprise alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether , thiol, seleno, sulfonyl, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone ), imines, aldehydes, esters, thioacids, hydroxylamines, amino groups, etc., or any combination thereof. Other interesting unnatural amino acids that may be suitable for use in the present invention include, but are not limited to, amino acids comprising photoactivatable cross-linkers, 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 covalently or non-covalently interacting with other molecules, photocaged and/or photoisomerizable amino acids, containing biotin or biotin Analogous amino acids, glycosylated amino acids such as sugar substituted serine, other sugar modified amino acids, ketogenic amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and/or Photocleavable amino acids, amino acids having 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), carbon-containing Amino acids with linked sugars, redox active amino acids, amino acids containing aminothiocarbonic acid, and amino acids containing one or more toxic moieties.

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

Further, the variant is obtained by adding 1 to 3 unrelated amino acids to the amino-terminal and/or carboxyl-terminal of the aforementioned polypeptide.

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

In a specific embodiment of the present invention, the variant is obtained by adding 1 to 3 glycines to the amino-terminal or carboxy-terminal of the PGAPGP polypeptide.

In a specific embodiment of the present invention, the variant is obtained by simultaneously adding 1 to 2 glycines to the amino terminus and carboxyl terminus of the PGAPGP polypeptide.

Further, the variant is obtained by adding an oligopeptide consisting of 3 amino acids to the amino-terminal or carboxyl-terminal of the aforementioned polypeptide.

In a specific embodiment of the present invention, the variant is obtained by adding RGD oligopeptide to the amino-terminal or carboxyl-terminal of the aforementioned polypeptide.

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

In some embodiments, the invention also discloses derivatives comprising the polypeptides of the invention. The polypeptide derivatives include products obtained by routine modification of the polypeptide of the present invention, fusion proteins formed by linking polypeptides with heterologous peptides, and conjugates formed by linking polypeptides with other compounds.

The conventional modification is amination, methylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization, biotin Chemical modification, fluorescent group modification, polyethylene glycol PEG modification, myristoylation, non-metal chemical element modification, immobilization modification, etc.

In some embodiments, a polypeptide of the 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, a heterologous peptide is fused to the amino terminus of a polypeptide of the invention. In additional or alternative embodiments, a heterologous polypeptide is fused to the carboxyl terminus of a polypeptide of the invention. For affinity purification, relevant matrices for affinity chromatography can be used, eg glutathione, alpha amylase. Heterologous peptides can be selected to facilitate detection of the polypeptides of the invention. Examples of assays include various fluorescent proteins such as GFP, as well as "epitope tags" such as His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.

In some embodiments, polypeptides of the invention are linked to other compounds. The other compounds include bisphosphonates and iridoids. The bisphosphonate drugs include alendronic acid, ibandronate and zoledronic acid. The iridoid compounds include genipenic acid, genipin-gentiobioside, geniposide, geniposide.

Polypeptides useful in the present invention may be prepared by any suitable means known in the art. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by combinations of these methods. Means and methods for preparing such polypeptides are well known in the art.

Nucleic acids, vectors and host cells

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

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

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, and the like. Both prokaryotic and eukaryotic expression systems are available. In some embodiments, the expression system is a mammalian cell expression system, such as a CHO cell expression system. In some embodiments, nucleic acids can be codon-optimized for expression in the desired host cell.

Non-viral vectors and systems include plasmids and episomal vectors (typically containing 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 are known in the art for expressing other proteins. Useful viral vectors include, but are not limited to, adenovirus, adeno-associated virus, herpes virus-based vectors, SV40, papilloma virus, HBP Epstein-Barr virus-based vectors, fowl pox virus vectors, vaccinia virus vectors, and Semliki forest virus (SFV) vector.

The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (eg, enhancers) operably linked to a polynucleotide encoding a polypeptide of the invention. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence under conditions other than inducible 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 to improve expression of nucleic acids encoding polypeptides of the invention, such as enhancers, ribosomal binding sites, transcription termination sequences, and the like.

In some embodiments, a nucleic acid encoding a 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. The sequence may be provided by the vector, or as part of the nucleic acid of the polypeptide of the invention present in the vector.

Methods for introducing expression vectors containing polynucleotide sequences of interest may vary depending on the host cell type. For example, calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment or electroporation can be used in other cellular hosts. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, bio-bombardment methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, Artificial virions, fusion to the herpesvirus structural protein VP22, reagent-enhanced DNA uptake, and ex vivo transduction. For long-term high-yield production of recombinant proteins, stable expression is often desired. For example, a cell line stably expressing a polypeptide of the present invention can be prepared using an expression vector of the present invention containing a viral origin of replication or an endogenous expression element and a selectable marker gene.

In some embodiments, nucleic acids encoding polypeptides of the invention may be delivered to patients for the treatment of orthopedic disorders. The nucleic acid can be delivered using any means known in the art, but delivery is typically achieved using direct injection. In some embodiments, the DNA is delivered as naked DNA by direct injection. In some embodiments, viral vectors are used, including, but not limited to, adenoviral or adeno-associated viral vectors, herpes viral vectors, fowl pox virus or vaccinia viral vectors.

pharmaceutical composition

The polypeptide of the present invention or its encoding nucleic acid can be administered together with suitable pharmaceutical excipients as needed. Compositions will vary depending on the mode of administration and dosage unit, as will be appreciated by those skilled in the art.

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

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

In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a carrier as described hereinabove.

In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a host cell as described hereinabove.

Compositions typically include conventional pharmaceutical carriers or excipients, and may additionally include other agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, and the like. Preferably, the composition will contain from about 0.01% to about 90%, from about 0.1% to about 75%, from about 0.1% to 50%, or from about 0.1% to 10% by weight of the composition of the present invention or a combination thereof, and the rest are composed of suitable pharmaceutical carriers and/or excipients. Suitable excipients can be tailored for a particular composition and route of administration by methods well known in the art. See, eg, REMINGTON'SPHARMACEUTICALSCIENCES, 18th Edition, Mack Publishing Co., Easton, Pa. (1990).

Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose and polyacrylic acid such as Carbopol, eg Carbopol 941, Carbopol 980, Carbopol 981 and the like. The composition may additionally include lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives such as methylparaben, ethylparaben and propylparaben ( Namely, parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; coloring agents; The composition may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

For oral administration, the compositions can be in the form of tablets, troches, capsules, emulsions, suspensions, solutions, syrups, sprays, powders and sustained release formulations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical composition is in the form of a pill, tablet or capsule, and the composition contains any of the following: diluents, such as lactose, sucrose, dicalcium phosphate, etc.; disintegrants, such as starch or derivatives thereof; lubricants, such as magnesium stearate, etc.; and binders, such as starch, acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. The conjugates can also be formulated as suppositories, eg, in polyethylene glycol (PEG) carriers.

The liquid composition 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 saline solution (for example, 0.9% w/v sodium chloride), dextro Aqueous sugar solutions, glycerol, ethanol and the like are prepared to form solutions or suspensions, for example, for oral, topical or intravenous administration.

For topical administration, the compositions of the invention may be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments and transdermal patches. For delivery by inhalation, the composition can be delivered by a nebuliser in dry powder or liquid form. For parenteral administration, the compositions can be in the form of sterile injectable solutions and sterile packaged powders. Preferably, injectable solutions are formulated at a pH of from about 4.5 to about 7.5.

Compositions of the invention may also be provided in lyophilized form. Such compositions may include a buffer, such as bicarbonate, for reconstitution prior to administration, or in lyophilized compositions may include a buffer for reconstitution, for example, with water. The lyophilized composition may additionally contain suitable pharmaceuticals, such as other drugs for the treatment of orthopedic diseases. The lyophilized composition may optionally be packaged in combination with a reconstitution buffer for presentation in a syringe so that the reconstituted composition can be administered immediately to the patient.

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

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

The dosage of the pharmaceutical composition of the present invention for the treatment of orthopedic diseases is variable depending on the mode of administration, the age and/or weight of the individual, and the condition of the individual being treated, and is ultimately determined by the attending physician or veterinarian . The dose administered to an individual should, in the case of the present invention, be sufficient to elicit a beneficial response in the individual over a period of time. This dose is a "therapeutically effective amount".

method

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

The present invention provides a method for enhancing bone formation or promoting bone mineralization in a subject in need, the method comprising administering the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned The aforementioned carrier, the aforementioned host cell, and the aforementioned pharmaceutical composition.

The present invention provides a method for inducing bone deposition in a subject in need, the method comprising administering the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned carrier, The aforementioned host cell, the aforementioned pharmaceutical composition.

application

therapeutic and prophylactic applications

The aforementioned polypeptides, the aforementioned nucleic acids, the aforementioned vectors, the aforementioned host cells, and the aforementioned pharmaceutical compositions of the present invention are used in the preparation of treatment or prevention of diseases or disorders in subjects in need. Application in pharmaceutical preparations. In the present invention the disease or condition is an orthopedic disease.

Those skilled in the art will understand that the aforementioned polypeptides, the aforementioned nucleic acids, the aforementioned vectors, the aforementioned host cells, and the aforementioned pharmaceutical compositions of the present invention can be used for the treatment or prevention of orthopedic Other therapeutic agents for the disease are co-administered. Co-administration can be simultaneous, eg, in a single pharmaceutical composition or separate compositions. A composition of the invention may also be administered separately from another therapeutic agent, eg, on an independent dosing schedule.

Applications in changing the properties of cells

Use of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned carrier, the aforementioned host cell, and the aforementioned pharmaceutical composition of the present invention in the preparation of a drug for promoting bone formation.

Use of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned carrier, the aforementioned host cell, and the aforementioned pharmaceutical composition of the present invention in the preparation of a drug for promoting bone mineralization.

Use of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned carrier, the aforementioned host cell, and the aforementioned pharmaceutical composition of the present invention in the preparation of a drug for inducing bone deposition.

Use of the aforementioned polypeptide, the aforementioned nucleic acid, the aforementioned carrier, the aforementioned host cell, and the aforementioned pharmaceutical composition of the present invention in the preparation of a drug for enhancing the activity of osteoblasts.

A "patient" or "subject" refers herein to any individual who is administered a polypeptide of the invention, or a nucleic acid encoding it, or a pharmaceutical composition comprising the same. The present invention contemplates that the polypeptides, compositions and methods of the present invention are useful in the treatment of mammals. As used herein, "individual" refers to any mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals such as cattle (e.g., cows), horses, dogs, sheep, pigs, rabbits, goats, Cats, mice, rats, monkeys, etc. In some embodiments of the invention, the individual is a human. In some embodiments, the individual is a mouse.

As used herein, the term "D-amino acid" refers to the dextro-stereoisomer of an amino acid. The letters D and L are commonly used in the art to designate stereoisomers of amino acids. D-amino acids are those amino acids that can be synthesized from the dextro-isomer of glyceraldehyde, ie D-glyceraldehyde. Similarly, L-amino acids are those amino acids that can be synthesized from the L-isomer of glyceraldehyde, ie, L-glyceraldehyde.

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

As used in the present invention, the expression "osteopathic disease" refers to any of those diseases causing various abnormalities or deformities of one or more bones and/or bone cells.

Preferably, said orthopedic disease comprises 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, loss of bone mass, frail bones throughout the body, joint degeneration, non-union fractures, caused by diabetes Orthopedic and Dental Problems, Implantable Periodontitis, Adverse Reactions to Bone Grafts/Implants/Bone Replacement Materials, Periodontal Disease, Bone Aging, Fractures, Bone Defects, Bone Grafts, Bone Grafts, Bone Cancer , joint replacement, joint repair, fusion, facet joint repair, bone degeneration, dental implants and restoration, bone marrow defect, 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, said osteoporosis includes postmenopausal osteoporosis, male and female Senile osteoporosis, glucocorticoid-induced osteoporosis, immobilized osteoporosis, weightlessness-induced osteoporosis, post-transplant osteoporosis, migratory osteoporosis, idiopathic osteoporosis osteoporosis, juvenile osteoporosis.

Embodiment 1 polypeptide preparation

Carry out synthesis by liquid phase synthesis method, concrete steps are as follows:

1. Synthetic raw materials and related reagents/instruments

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

2) Amino acid: purchased from Chengdu Chengnuo, >99%;

3) Synthetic reagents: DMF (origin South Korea), DCM (origin South Korea), MEOH (origin Japan), DIEA (Xinde Chemical, 99%), HBTU (Haofan Biotechnology, 99%);

4) Deprotection reagent: piperidine (Sinopharm Shanghai Chemical Reagent Company, 99%);

5) Detection reagents: phenol reagent (self-made), pyridine reagent (self-made), ninhydrin reagent (self-made);

6) Lysis reagent: 95% cutting fluid: TFA (J.T.Baker, 99%), TIS (Shanghai Darui Fine Chemicals, 98%), EDT (Shanghai Darui Fine Chemicals, 98%), anhydrous ether (Shanghai Experimental, Measured 99.7%);

7) Nitrogen: (Xinlian Gas);

8) Instrument: ① 12-channel semi-automatic peptide synthesizer, a semi-automatic peptide synthesizer independently designed and patented by Shanghai Qiangyao Biotechnology Co., Ltd., the patent number is 201020226529.2 ② SHIMADZU high performance liquid chromatography (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).

2. The peptide synthesis steps are as follows: the synthesis sequence is from C-terminal to N-terminal.

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

2) Connect 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-terminal, add DMF to dissolve, then add 10 times molar excess of DIEA, shake for 60 minutes; block with methanol.

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

4) Detection: Take out the piperidine solution, take a dozen resins, wash them three times with ethanol, add detection reagents for detection, heat at 105°C-110°C for 5min, and turn dark blue to indicate a positive reaction.

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

6) Condensation: Three-fold excess of protected amino acids and three-fold excess of HBTU were dissolved with as little DMF as possible, added to the reaction tube, and ten-fold excess of DIEA was added immediately, and reacted for 30 minutes.

7) Detection: Take more than a dozen resins, wash them three times with ethanol, add detection reagents for detection, heat at 105°C-110°C for 5min, if colorless, it is a negative reaction.

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

9) Repeat operations 3) to 6), and connect the amino acids in the sequence sequentially from right to left.

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

11) Cut the polypeptide from the resin: prepare a cutting solution (10/g) TFA 95%; water 1%; EDT 2%; TIS 2%; cutting time: 120min.

12) Drying and washing: blow the lysate as dry as possible with nitrogen, wash with ether six times, and evaporate to dry at room temperature.

13) Analysis and purification: Purify the crude product by high performance liquid chromatography.

14) Freeze-drying: the target polypeptide solution is collected, concentrated in a freeze dryer, and freeze-dried into a white powder.

15) Send the polypeptide to the Quality Inspection Department for confirmation.

Example 2 Activity Detection of Polypeptides and Derivatives thereof

1. Activity detection of polypeptides of different lengths

1.1 Synthesis of polypeptides of different lengths

Synthesize according to the method for embodiment 1.

1.2 Cell experiments

The osteoblast cell line MC-3T3E1 cells were cultured, and after incubation with polypeptides of different lengths at a final concentration of 2 μg/ml for 24 hours, the expression level of ALP gene was detected by QPCR.

1.3 Results

The results are shown in Figure 1. The combination of six amino acids of PGAPGP is a better combination to enhance the activity of osteoblasts. When 2-3 PGAPGPs are connected in series, they all have good activity, and when 4 or more PGAPGPs are connected in series, the activity begins to weaken.

2. Peptide activity detection after amino acid extension at both ends of the polypeptide

2.1 Synthesis of polypeptides of different lengths

Synthesize according to the method for embodiment 1.

2.2 Cell experiments

The osteoblast cell line MC-3T3E1 cells were cultured, and after incubation with polypeptides of different lengths at a final concentration of 2 μg/ml for 24 hours, the expression level of ALP gene was detected by QPCR.

2.3 Results

The results are shown in Figure 2. When PGAPGP was unilaterally extended by one unrelated amino acid, it had no effect on its activity, when it was unilaterally extended by two unrelated amino acids, it had little effect on its activity, and when it was extended by more than three unrelated amino acids, the activity decreased. There is no effect on the activity when one unrelated amino acid is extended on both sides, but the activity begins to decrease when two or more unrelated amino acids are extended on both sides. Glycine (G) is representative of an unrelated amino acid.

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

3.1 Synthesis of polypeptides incorporating non-natural D-amino acids

According to conventional methods, the distribution of unnatural D-type amino acids is shown in Table 1.

Table 1 Distribution of unnatural D-type amino acids

serial number	Unnatural D-amino acid distribution
P1	PGAPGP
P2	PGAPGPD
P3	PGAPG D P
P4	PGAP D GP
P5	PGA D PGP
P6	PG D APGP
P7	P D GAPGP

3.2 Cell experiments

The osteoblast cell line MC-3T3E1 cells were cultured, and after incubation with polypeptides of different lengths at a final concentration of 2 μg/ml for 24 hours, the expression level of ALP gene was detected by QPCR.

3.3 Results

The results are shown in Figure 3, the activity of the amino acid at any position of the PGAPGP peptide was replaced by a non-natural D-type amino acid, and the activity decreased.

4. Detection of variant activity after polypeptide modification

4.1 Synthesis of modified variants

According to conventional methods, polypeptide modifications are shown in Table 2.

Table 2 Differently modified polypeptides

serial number	Modification method
Pep1	N-methylation
Pep2	N-myristoylation
Pep3	N-PEG modification
Pep4	C-fluorine modification
Pep5	C-biotin modification
Pep6	C-FAM fluorescent labeling
Pep7	unmodified-PGAPGP

4.2 Cell experiments

The osteoblast cell line MC-3T3E1 cells were cultured, and after incubation with polypeptides of different lengths at a final concentration of 2 μg/ml for 24 hours, the expression level of ALP gene was detected by QPCR.

4.3 Results

The results are shown in Figure 4, the effect of common polypeptide terminal modifications on the activity of PGAPGP. Methylation, myristylation, PEG modification, fluorine element modification, biotin modification, and FAM fluorescently labeled PGAPGP are all active.

5. Activity detection of peptide conjugates

5.1 Synthesis of polypeptide conjugates

According to conventional methods, the polypeptide conjugates are shown in Table 3.

Table 3 Polypeptide Conjugates

serial number	Modification method
PC1	unmodified-PGAPGP
PC2	N-coupled sodium alendronate
PC3	C-coupled alendronate sodium

PC4	N-Coupled RGD Peptides
PC5	C-coupled RGD peptide

5.2 Cell experiments

The osteoblast cell line MC-3T3E1 cells were cultured, and after incubation with the polypeptide conjugate at a final concentration of 5 μg/ml for 24 hours, the expression level of ALP gene was detected by QPCR.

5.3 Results

The results are shown in Fig. 5, and sodium alendronate as the representative PGAPGP has activity after coupling bisphosphonate. Represented by RGD, PGAPGP is active after coupling oligopeptides.

6. Activity detection of polypeptide sustained-release dosage forms

6.1 Sustained-release dosage form of synthetic microspheres

Steps: After stirring and dissolving PLGA, add the porogenic solution dropwise, and ultrasonically emulsify to form an emulsion; wherein the molar ratio of LA and GA of PLGA is 50:50; then add the emulsion dropwise to the stirred external water phase, And add a preset volume of deionized water, change the speed and continue stirring until the solvent is completely evaporated. Wherein the external water phase is water-soluble surfactant added to deionized water, and then the solution after the organic solvent is completely volatilized is centrifuged, washed with deionized water, and the supernatant is removed, thereby obtaining PLGA microspheres. The NaOH solution containing the polypeptide is added to the PLGA microspheres, mixed evenly, and placed on a shaker to continue the reaction, and the reacted PLGA microspheres are repeatedly centrifuged and washed with deionized water, and then freeze-dried for later use.

6.2 Cell experiments

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

6.3 Results

The results are shown in Figure 6. Represented by PLGA 50:50 microspheres, the sustained-release dosage form of PGAPGP has significant biological activity.

The effect of embodiment 3 polypeptide on mouse bone formation

1. Materials

Source of wild-type mice: All mice were of SPF grade, purchased from Beijing Weitong Lihua (Charles River) Experimental Animal Technology Co., Ltd.

2. Polypeptide solution preparation: Weigh 1.000 g of sodium hydroxide in a beaker, add a small amount of normal saline to dissolve, then pour it into a 1000 ml volumetric flask, wash the beaker for 3 times, pour all the solution into the volumetric flask, and finally use physiological Dilute with saline to the mark. After shaking well, a 1000mg/mL polypeptide stock solution was obtained. Dilute in equal proportions according to the required concentration when using.

3. Drug treatment

The mice were randomly divided into two groups, the control group and the 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; femur samples were collected after 12 injections for Micro- CT scan and H&E staining.

4. Micro-CT scanning

The collected femur samples were scanned by Micro-CT of trabecular bone and cortical bone, and the number and thickness of trabecular bone were analyzed.

4.1 steps

(1) After the femur sample was fixed in 4% paraformaldehyde for 24 hours, it was transferred to 0.5% paraformaldehyde solution to prevent the formation of crystals in the bone tissue and affect the scanning of the sample.

(2) Place the sample in a Micro-CT dedicated scanning tube with a diameter of 14mm. The sample is placed horizontally, and the horizontal axis is perpendicular to the Micro-CT scanning axis. The scanning tube is placed in the sample tray of the Micro-CT scanning case.

(3) The relevant parameters of the scanning program are as follows: the scanning voltage is 70kVp, the scanning energy power is 14W, the scanning current is 200μA, the exposure time is 300ms, the scanning BH is 1200mg HA/cc, the scanning accuracy is 10μm, and the scanning filter (Filter) is 0.5mm AI Filter. Later image data were reconstructed and analyzed using Mimics 13.0 software.

(4) When femur data is reconstructed, the trabecular bone data analysis selects all the trabecular bone in the inner layer of cortical bone starting 1mm below the femoral growth plate; the cortical bone data analysis selects the cortical bone at 5mm-6mm below the growth plate.

4.2 Results

Micro-CT scanning results are shown in Figure 7, after the application of peptides, the bone density of the mice was significantly increased (Figure 7A), the thickness of the trabecular bone was significantly increased (Figure 7C), and the number of trabecular bone was significantly increased (Figure 7D), indicating that The degree of mineralization of mouse femur was enhanced.

5. H&E staining

5.1 Steps

1) Slicing: making paraffin sections of tissues in a conventional manner;

2) Baked slices: put the slices in an oven at 65°C for 30 minutes;

3) Dewaxing and hydration: Dewaxing the tissue sections sequentially, xylene I for 30 minutes, xylene II for 30 minutes. Then the sections were hydrated with absolute ethanol I for 5 min, absolute ethanol II for 5 min, 90% alcohol for 5 min, 75% alcohol for 5 min, and distilled water for 5 min.

4) Staining: wash with hematoxylin for 5 minutes, soak in PBS for 5 minutes, and turn the cell nuclei to blue. Stain with eosin for 2min and wash with water.

5) Dehydration and transparency: tissue dehydration with graded ethanol, 80% alcohol, 90% alcohol, and 95% alcohol for 10 seconds respectively, and then air-dried at room temperature. Absolute ethanol I 5min, absolute ethanol II 5min, and dry at room temperature. After soaking in xylene for 5min to become transparent, seal the slide with neutral resin.

6) Observation: observe and take pictures under the upright microscope.

5.2 Results

H&E staining was performed on the femur samples above, and the results showed that the trabecular bone under the growth plate was denser after the mice were injected with the polypeptide (Fig. 7B).

6. Mouse double fluorescent labeling experiment

6.1 Steps

(1) The above-mentioned femur samples (intraperitoneal injection of xylenol orange and calcein at a dose of 80 mg/kg, with an interval of 2 weeks) were sliced into hard tissues, and the thickness of the slices was about 15 μm.

(2) DAPI staining: DAPI staining solution diluted 1:1000 was added dropwise to hard tissue sections, and stained for 5 minutes. Soak and rinse in PBS 3 times, each time 5min. Mount the slides with anti-fade mounting medium. The red and green two-color fluorescent markers were observed under an inverted fluorescence microscope, and the bone deposition rate was counted.

6.2 Results

The results are shown in Figure 8, the red mark and the green mark are separated by 2 weeks, and the interval is new bone deposition. Compared with the control group, the amount of femoral bone deposition in the experimental group was significantly increased. The results of the analysis showed that the bone formation was significantly enhanced in mice injected with the polypeptide.

7. Von Kossa staining

7.1 Steps

(1) Staining with silver nitrate staining solution: Prepare 0.2% silver nitrate solution, drop it on the femoral hard tissue section, cover the femoral tissue, irradiate with strong light for 15 minutes, and rinse the section with running water for 2-3 seconds.

(2) Sodium thiosulfate incubation: Prepare 5% sodium thiosulfate solution, drop it on the slices, cover the femoral tissue, 5s, wash the slices with running water for 2-3s.

(3) Methyl green counter-dyeing: counter-dye with methyl green dye solution for 5 minutes, rinse with running water for 5 seconds, wash away floating colors, and dry in an oven at 65°C for 30 minutes.

(4) Sealing and observation: After the sections were transparent with xylene for 5 minutes, the slides were sealed with neutral resin. Observe and photograph under a stereomicroscope.

7.2 Results

The replacement of calcium ions and silver ions in bone tissue is black, indicating the density of mineralized bone and the level of bone mineralization. The results of Von Kossa staining are shown in Figure 9. The density of trabecular bone under the growth plate in the polypeptide injection group increased, indicating that the degree of bone mineralization was significantly enhanced.

8. Detection of bone mineralization related index Dmp-1

8.1 Immunohistochemical staining

(1) Section: after decalcification of femoral tissue, conventional paraffin tissue section;

(2) Baked slices: put the slices in a 65°C oven for 30 minutes;

(3) Dewaxing and hydration: dewax the tissue sections sequentially, xylene I 30min, xylene II 30min. Then slices were hydrated, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 90% alcohol for 5 min, and 75% alcohol for 5 min. Distilled water for 5 minutes. Immerse in PBS and rinse three times, each time for 3 minutes.

(4) Antigen restoration: According to the requirements of the antibody, the tissue antigen is repaired accordingly.

(5) Inactivation of endogenous peroxidase: drop an appropriate amount of peroxidase blocking solution on the slice, and incubate at room temperature for 10 min (to block the activity of endogenous peroxidase). PBS was added dropwise to wash three times, each time for 5 min.

(6) Tissue sealing: drop an appropriate amount of normal non-immune animal serum on the slice, and incubate at room temperature for 10 min.

(7) Incubate primary antibody: shake off the serum on the tissue, drop an appropriate amount of primary antibody on the tissue, and incubate at room temperature for 60 min or overnight at 4°C. PBS was added dropwise for 3 min to rinse three times.

(8) Secondary antibody incubation: Add an appropriate amount of biotin-labeled secondary antibody dropwise to the slices, incubate at room temperature for 10 minutes, wash with PBS three times, 3 minutes each time.

(9) Remove PBS, add 1 drop or 50 μl of streptomycin anti-biotin-labeled secondary antibody (reagent D) to each slice, incubate at room temperature for 10 min, wash with PBS three times, each time for 3 min.

(10) DAB color development: DAB color development solution (prepared and used now) was added dropwise, and observed under a microscope for 3-10 minutes. After the color development is over, rinse with tap water to stop the color development.

(11) Counterstaining of cell nuclei: counterstaining with methyl green staining solution, and PBS turns blue.

(12) Dehydration and transparency: Dehydrate the tissues with graded ethanol, 80% alcohol, 90% alcohol, and 95% alcohol for 10 seconds respectively, and then dry at room temperature. Absolute ethanol I 5min, absolute ethanol II 5min and dry at room temperature. After being transparent in xylene for 5min. Cover and observe.

8.2 Results

Results As shown in Figure 10, the degree of immunohistochemical staining in the polypeptide injection group increased, indicating that the expression of the mouse mineralization protein Dmp1 was significantly enhanced.

Example 3 Effect of Polypeptide on Bone Mass in Osteoporosis Mice

1. Construction of osteoporosis mouse model

(1) Eighteen 12-week-old female WT mice were selected and divided into 3 groups (OVX group, OVX+peptide group, OVX+PTH group).

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

(3) Prepare the skin on the lower abdomen of the mouse, disinfect with iodophor, wipe off the iodophor with 75% alcohol, and make a 0.5 cm long incision at 1 cm below the ribs and 0.5 cm to the left of the midline of the abdomen.

(4) Cut 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, and go up the uterus to find the fallopian tubes and ovaries.

(5) Cut off the ovaries and fallopian tubes after ligation of the fallopian tubes. After bilateral resection, place the uterus and fat into the abdomen.

(6) The skin and muscles are sutured in layers.

(7) Three months after the establishment of osteoporosis model mice, after the model tended to be stable, the polypeptide was injected intravenously, a total of 12 injections, once a week, with a dose of 10 mg/kg; the PTH administration method was Subcutaneous administration, the dose is 0.1μg/kg, injection for 4 weeks, 3 times a week.

2. Micro-CT scanning

Step is with embodiment 1.

The results are shown in Figure 11. Compared with the OVX group, the bone mass of the mice in the polypeptide group and the PTH group increased significantly, indicating that the polypeptide can increase the bone mass of the mice with osteoporosis.

3. Fluorescence double standard experiment

Step is with embodiment 1.

The results are shown in Figure 12, the amount of bone deposition in the polypeptide group and the PTH group was significantly higher than that in the OVX group, indicating that the bone formation function of the mice was improved after polypeptide treatment.

Example 4 Effect of Polypeptide on Fracture Healing

1. Construction of mouse fracture model

A mouse fracture model was constructed according to the method described in [Glycosylation of dentin matrix protein 1 is critical for fracture healing via promoting chondrogenesis. Frontiers of Medicine. 2019 Oct; 13(5):575-589]. After 1 week of modeling, mice were injected with 10 mg/kg polypeptide once a week, and samples were collected after 6 injections to evaluate the formation of callus and the density of callus at the fracture end of the mouse.

2. Fluorescence double standard experiment

Step is with embodiment 1.

The results are shown in Figure 13, the bone formation ability of the mice in the polypeptide group was enhanced.

3. Micro-CT scanning

The steps are basically the same as in Example 1. The difference is that when the fracture sample data is reconstructed, the display site of the callus is 500 μm above and below the broken end of the fracture, and the data analysis is selected from the part of the callus outside the cortical bone at the corresponding site.

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

The biosafety evaluation of embodiment 5 polypeptide

The biosafety of peptides was explored through the evaluation of serum biochemical indicators and the analysis of pathological slices of important organs.

1. ELISA detection

ELISA was used to detect BUN (blood urea nitrogen), CK (creatine kinase), and ALT (alanine aminotransferase) in the serum of mice injected with polypeptides, and to evaluate the impact of small molecule polypeptides on biochemical indicators.

2.1 steps

2.1.1 Mouse serum collection

(1) Blood collection from the retro-orbital venous plexus of mice:

Take a glass capillary with an inner diameter of 1.0-1.5 mm, break it into a 2-2.5 cm long capillary section before use, immerse it in 1% heparin solution, and use it after drying. When taking blood, grasp the skin on the back of the neck between the two ears of the rat with the left hand to fix the head, and gently press down on both sides of the neck, causing difficulty in the venous return of the head and congesting the orbital venous plexus. Insert the conjunctiva, then gently push toward the fundus of the eye, gently rotate the capillary to cut the venous plexus, let the blood flow out along the capillary, and receive it into the pre-prepared container. After blood collection, lightly press the gauze on the eye to stop the bleeding.

(2) After the blood is obtained by the above method, let it stand in the EP tube at room temperature for more than one hour (or 37°C water bath for 1 hour, 4°C refrigerator for 2 hours or overnight) and centrifuge at 3000rpm for 10 minutes, and the supernatant is the serum . Can be stored at -80°C.

2.1.2. Enzyme-linked immunosorbent assay (ELISA)

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

(2) Dilute the washing solution: dilute the 20X washing solution to 1X with dd H ₂ O.

(3) Serum preparation: After blood was collected in a test tube free of pyrogens and endotoxins (need to avoid any cell stimulation during the subsequent experimental operation), centrifuge at 3000 rpm for 10 minutes at room temperature, carefully draw, and separate serum and red blood cells.

(4) Adding samples: Take out the required number of orifice plates and put them on the 96-well bottom plate, and mark the order of adding samples on the plate. Add 20 μl of Matrix solution to the blank and standard wells, and add 20 μl each of 0, 0.025, 0.05, 0.1, 0.2, and 0.4 ng/ml standards for the standard. Add 10 μl of diluted serum from mice in the control group and polypeptide group to the sample wells, then add 40 μl of the sample diluent, and do not add to the blank wells.

(5) Manual plate washing: slowly add the plate washing solution along the well wall, let it stand for 1 min, discard the plate washing solution, and pat it on absorbent paper. Repeat 5 times. When adding different samples, replace the tip in time to avoid contamination.

Automatic plate washing: Add 350 μl of washing solution to each reaction well, the program is soaking for 1 min, and washing the plate 5 times.

(6) In addition to the blank wells, add 100 μl of horseradish peroxidase (HRP)-labeled detection antibody to the standard reaction wells and sample reaction wells. Incubate for 60min.

(7) Plate washing: wash the plate 5 times in the above-mentioned manner.

(8) Color development: add 50 μl each of substrate A and B to each reaction well, and incubate at 37° C. in the dark for 15 minutes.

(9) Measurement: 50 μl of stop solution was added to each well, and after 10 min, the absorbance was read with a microplate reader at a wavelength of 450 nm. After making the standard curve, use the corresponding formula to calculate the content of each protein in the sample.

2.2 Results

The results are shown in Figure 15. The mice were given intravenous injection of 10 mg/ml polypeptide every week, and blood was drawn for detection at the 2nd, 4th, 6th, 8th, 10th, and 12th weeks. The circle marks the control group, and the square mark represents the polypeptide group.

3. H&E staining

The mice were given intravenous injection of 10 mg/ml polypeptide every week, and were sacrificed after the 12th week for testing. H&E staining was performed on important organs for pathological evaluation to explore whether the polypeptide has chronic toxicological effects on internal organs. Paraffin sections of heart, lung, liver, spleen, kidney and other tissues were performed, and H&E staining was performed. The results are shown in Figure 16. Compared with the control group, no significant pathological changes such as tumor or inflammatory infiltration occurred in important organs (heart, lung, liver, spleen, kidney) after injection of polypeptide. The results of this example show that the polypeptide of the present invention has better biological safety.

The above-mentioned embodiments are only used to explain the present invention, but should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: without departing from the principle and purpose of the present invention, various changes, modifications, replacements and modifications can be made to these embodiments, and the scope of the present invention is defined by the claims and their equivalents .

Claims (10)

1. A polypeptide, characterized in that the core sequence of the polypeptide comprises proline, glycine, and alanine;

   Preferably, the core sequence of the polypeptide contains 3-60 amino acids, and the number ratio of proline, glycine and alanine is (1-3):(1-8):1;

   Preferably, 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;

   Preferably, n is 1-5.
2. The polypeptide according to claim 1, wherein the polypeptide comprises any of the following:

   1) A variant obtained by one or more amino acid deletions, additions, or substitutions on the basis of the core sequence;

   2) The amino acid sequence has 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the core sequence Variants;

   Preferably, the polypeptide is obtained by adding 1 to 3 amino acids to the amino-terminal and/or carboxy-terminal of the core sequence;

   Preferably, the polypeptide is obtained by adding 1 to 3 unrelated amino acids to the amino-terminal and/or carboxy-terminal of the core sequence;

   Preferably, the unrelated amino acid is glycine;

   Preferably, the polypeptide is obtained by adding an oligopeptide consisting of 3 amino acids to the amino-terminal or carboxyl-terminal of the core sequence;

   Preferably, the oligopeptide is RGD;

   Preferably, the substitution refers to the substitution of an amino acid at any position of the core sequence with a non-natural D-type amino acid.
3. A polypeptide derivative, characterized in that the polypeptide derivative comprises the product obtained by routine modification of the polypeptide described in claim 1 or 2, a fusion protein formed by linking the polypeptide with a heterologous peptide, the polypeptide and other Conjugates formed by linking compounds;

   Preferably, the conventional modification is amination, methylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization , biotinylation, fluorescent group modification, polyethylene glycol PEG modification, myristoylation, non-metal chemical element modification, immobilization modification, etc.

   Preferably, the heterologous peptides include human serum albumin, immunoglobulin heavy chain constant region, glutathione S transferase, thioredoxin, protein A, protein G, mannose binding protein, glutathione , Alpha Amylase, Fluorescent Protein, His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, ProfinityeXact.

   Preferably, the other compounds include bisphosphonate drugs and iridoid compounds; preferably, the bisphosphonate drugs include alendronic acid, ibandronate, zoledronic acid; preferably Specifically, the iridoid compounds include genipenic acid, genipin-gentiobioside, geniposide, and geniposide.
4. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the polypeptide according to claim 1 or 2.
5. A vector, characterized in that the vector comprises the nucleic acid molecule of claim 4.
6. A host cell, characterized in that it comprises the nucleic acid molecule of claim 4 or the vector of claim 5.
7. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polypeptide described in claim 1 or 2, the polypeptide derivative described in claim 3, the nucleic acid molecule described in claim 4, the polypeptide described in claim 5 carrier, the host cell described in claim 6; preferably, the pharmaceutical composition also includes conventional pharmaceutical carriers or excipients; preferably, the pharmaceutical composition also includes other medicaments, carriers, adjuvants, diluents, tissue Penetration enhancer, solubilizer.
8. A method, characterized in that the method comprises any of the following methods:

   1) A method for enhancing the activity of osteoblasts, the method comprising administering the polypeptide according to claim 1 or 2, the polypeptide derivative according to claim 3, the nucleic acid molecule according to claim 4, or the nucleic acid molecule according to claim 5. The carrier described in claim 6, the host cell described in claim 6, the pharmaceutical composition described in claim 7;

   2) A method for preventing or treating orthopedic diseases, the method comprising administering the polypeptide according to claim 1 or 2, the polypeptide derivative according to claim 3, or the polypeptide according to claim 4 to a subject in need Nucleic acid molecule, the carrier according to claim 5, the host cell according to claim 6, the pharmaceutical composition according to claim 7;

   3) A method for promoting bone formation in a subject in need, the method comprising administering the polypeptide according to claim 1 or 2, the polypeptide derivative according to claim 3, The nucleic acid molecule of claim 4, the carrier of claim 5, the host cell of claim 6, and the pharmaceutical composition of claim 7;

   4) A method of inducing bone deposition in a subject in need, the method comprising administering the polypeptide according to claim 1 or 2, the polypeptide derivative according to claim 3, The nucleic acid molecule of claim 4, the carrier of claim 5, the host cell of claim 6, and the pharmaceutical composition of claim 7;

   5) A method for promoting bone mineralization in a subject in need, the method comprising administering the polypeptide according to claim 1 or 2, or the polypeptide derivative according to claim 3 to a subject in need , the nucleic acid molecule of claim 4, the carrier of claim 5, the host cell of claim 6, and the pharmaceutical composition of claim 7.
9. The use of the polypeptide according to claim 1 or 2, characterized in that the use includes the use described in any of the following:

   1) the purposes in preparing the polypeptide derivative described in claim 3;

   2) purposes in the preparation of the pharmaceutical composition described in claim 7;

   3) Use in the preparation of drugs for promoting bone formation;

   4) Use in the preparation of drugs that enhance the activity of osteoblasts;

   5) Use in the preparation of drugs for promoting bone mineralization;

   6) Application in the preparation of drugs for inducing bone deposition;

   7) Use in the preparation of medicines for the prevention or treatment of orthopedic diseases;

   Preferably, said orthopedic disease comprises 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, loss of bone mass, frail bones throughout the body, joint degeneration, non-union fractures, caused by diabetes Orthopedic and Dental Problems, Implantable Periodontitis, Adverse Reactions to Bone Grafts/Implants/Bone Replacement Materials, Periodontal Disease, Bone Aging, Fractures, Bone Defects, Bone Grafts, Bone Grafts, Bone Cancer , joint replacement, joint repair, fusion, facet joint repair, bone degeneration, dental implants and restoration, bone marrow defect, 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, said osteoporosis includes postmenopausal osteoporosis, male and female Senile osteoporosis, glucocorticoid-induced osteoporosis, immobilized osteoporosis, weightlessness-induced osteoporosis, post-transplant osteoporosis, migratory osteoporosis, idiopathic osteoporosis osteoporosis, juvenile osteoporosis.
10. The use of the polypeptide derivative according to claim 3, characterized in that the use includes the use described in any of the following:

    1) the purposes in the preparation of the pharmaceutical composition described in claim 7;

    2) Application in the preparation of drugs for promoting bone formation;

    3) Use in the preparation of drugs that enhance the activity of osteoblasts;

    4) purposes in the preparation of the medicine that promotes bone mineralization;

    5) purposes in the preparation of the medicine that induces bone deposition;

    6) Use in the preparation of medicines for the prevention or treatment of orthopedic diseases;

    Preferably, said orthopedic disease comprises 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, loss of bone mass, frail bones throughout the body, joint degeneration, non-union fractures, caused by diabetes Orthopedic and Dental Problems, Implantable Periodontitis, Adverse Reactions to Bone Grafts/Implants/Bone Replacement Materials, Periodontal Disease, Bone Aging, Fractures, Bone Defects, Bone Grafts, Bone Grafts, Bone Cancer , joint replacement, joint repair, fusion, joint repair, bone degeneration, dental implants and restorations, bone marrow defects, bone disease in patients with acromegaly, cystic fibrosis-related bone disease, adynamic bone disease, and Renal osteodystrophy associated with chronic kidney disease, bone disease associated with cystinosis, and bone disease associated with hyperoxaluria; preferably, said osteoporosis includes postmenopausal osteoporosis, osteoporosis in men and women Senile osteoporosis, glucocorticoid-induced osteoporosis, immobilized osteoporosis, weightlessness-induced osteoporosis, post-transplant osteoporosis, migratory osteoporosis, idiopathic Osteoporosis, Juvenile Osteoporosis.

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