CN115322245A - Polypeptide for improving osteoblast activity - Google Patents

Abstract

The invention discloses a polypeptide for improving osteoblast activity. The polypeptide of the invention has stable chemical property, easy low-cost large-scale synthesis, high biocompatibility and no toxicity, and can be widely used for preventing and treating orthopedic diseases.

Description

Polypeptide for increasing osteoblast activity

Technical Field

The invention belongs to the field of biological medicines, and relates to a polypeptide for improving osteoblast activity.

Background

At present, with the rapid development of traffic and industry in China, millions of patients with bone defects caused by wounds and diseases are treated and recovered difficultly every year, the labor capacity of the patients is injured to different degrees, the family members of the patients suffer from extreme physical and mental pains, and serious economic burden is brought to the society, so that the problems are urgently needed to be solved.

The development and treatment targets of the existing anti-osteoporosis drugs almost focus on bone reconstruction (dynamic balance between bone formation and bone resorption), namely how to inhibit bone resorption or promote bone formation. Clinical anti-osteoporosis drugs are also classified into anti-bone resorption drugs such as diphosphonates and calcitonin, and bone formation promoting drugs such as teriparatide, based on these two points; however, the former can inhibit bone formation while inhibiting bone resorption, so that the incidence of adverse reaction of the long-term application of the bone-resorption inhibitor is obviously increased; the latter promotes bone resorption while promoting bone formation, and may increase fracture incidence after 2 years of use. Until now, no bone absorption resisting medicine can only inhibit bone absorption but not bone formation, and no bone formation promoting medicine can only promote bone formation but not bone absorption, so that the effectiveness and application range of clinical medicines are greatly limited. At present, bone remodeling regulation drugs mainly comprise diphosphonate, calcitonin, selective estrogen receptor regulator, parathyroid hormone analogue, RANKL inhibitor and the like, wherein only parathyroid hormone analogue is a bone remodeling regulation polypeptide drug which is derived from hormone with bone remodeling regulation function: parathyroid hormone. At present, no non-hormone-based polypeptide medicine appears at home and abroad.

Disclosure of Invention

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

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

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a polypeptide, wherein a core sequence of the polypeptide comprises proline, glycine and alanine.

Furthermore, the core sequence of the polypeptide contains 3-60 amino acids, and the number proportion 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 to 5.

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

The invention also provides a pharmaceutical composition comprising the polypeptide of the invention. The pharmaceutical composition may be used in a method of treating, ameliorating or preventing an orthopedic disorder, wherein the method comprises administering to a patient a therapeutically effective amount of a polypeptide, nucleic acid or pharmaceutical composition of the invention.

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

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

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

Drawings

FIG. 1 shows activity detection maps for polypeptides of different lengths;

FIG. 2 shows activity detection maps of polypeptides after extension of unrelated amino acids at both ends;

FIG. 3 shows a graph of the activity of polypeptides incorporating an unnatural D-form amino acid;

FIG. 4 shows a map of modified polypeptide activity;

fig. 5 shows activity detection maps of polypeptide conjugates;

FIG. 6 shows activity detection profiles of polypeptide sustained release dosage forms;

FIG. 7 shows graphs of HE staining and Micro-CT scan results for wild-type mice, where A: trabecular and cortical bone scans; b: bone trabecula HE staining pattern; c: number of trabeculae; d: trabecular bone thickness;

FIG. 8 is a graph showing the results of a double-fluorescence labeling experiment after administration of a polypeptide to wild-type mice;

FIG. 9 is a graph showing the result of Von Kossa staining after administration of polypeptide to wild-type mice;

FIG. 10 is a graph showing the results of staining with mineralization index Dmp-1 in wild-type mice with the polypeptide;

FIG. 11 is a graph showing the results of Micro-CT scans after OVX mice were administered with polypeptide, wherein A: a global scan of the fifth lumbar vertebra; b: a fifth lumbar trabecular scan;

FIG. 12 is a graph showing the results of a double-fluorescent-labeling experiment after OVX mouse administration of a polypeptide;

FIG. 13 is a graph showing the results of a fluorescent dual-label experiment after administration of a polypeptide to a fractured mouse;

FIG. 14 is a graph showing the results of Micro-CT scans after administration of polypeptides to fractured mice;

fig. 15 shows a graph of the results of an ELISA experiment for evaluating the biosafety of polypeptides, wherein a: BUN; b: CK; c: ALT;

figure 16 shows H & E staining patterns used to evaluate the biosafety of polypeptides.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention, which is set forth in order to illustrate, but not to limit the scope of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. The experimental methods not specified in the examples are generally commercially available according to the conditions described in the conventional conditions or according to the conditions recommended by the manufacturers, and the materials, reagents and the like used in the examples, unless otherwise specified.

Polypeptides

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

The term "variant" refers to a polypeptide that is "substantially similar" to a wild-type polypeptide. A molecule is considered "substantially similar" to another molecule if the two molecules have substantially similar structures (i.e., they are at least 50% similar in amino acid sequence as determined by a BLASTp alignment set at default parameters) and are substantially similar in at least one related function. Variants differ from the wild-type polypeptide in one or more amino acid deletions, additions, substitutions or side chain modifications, but retain 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 with another naturally occurring amino acid of similar nature, either in relation to polarity, side chain functionality, or size. As described herein, variants include substitutions that are also "non-conservative," wherein an amino acid residue present in a peptide is substituted with an amino acid having different properties (e.g., a charged or hydrophobic amino acid is substituted with an uncharged or hydrophilic amino acid), or alternatively, wherein a naturally occurring amino acid is substituted with 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 "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-carboxy-lysine, and selenocysteine), but that are not inherently incorporated into a 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.

The non-naturally encoded amino acid 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 acid of the invention typically differs from a natural amino acid only in side chain structure, the non-naturally encoded amino acid can form an amide bond with other amino acids, including, but not limited to, naturally or non-naturally encoded amino acids, in the same manner as an amide bond in a natural polypeptide. However, non-naturally encoded amino acids have side chain groups that differ from the natural amino acids. For example, R optionally can comprise an alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine (hydrazine), cyano, halo, hydrazide (hydrazine), alkenyl, alkynyl, ether, thiol (thiol), seleno, sulfonyl, boronic acid (borate), boronate (boronate), phospho (phosphine), phosphono (phosphono), phosphine (phosphine), heterocycle, enone (enone), imine (imine), aldehyde, ester, thioacid (thioacid), hydroxylamine, amino groups, and the like, 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 crosslinker, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with new functional groups, amino acids that interact covalently or non-covalently with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serines, other sugar-modified amino acids, ketone-containing amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with extended side chains compared to the 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-linked sugar amino acids, redox active amino acids, amino acids containing thionocarboxylic acids, and amino acids containing one or more toxic moieties.

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

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

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

In a particular embodiment of the invention, the variant is obtained by adding 1 to 3 glycines at the amino-or carboxy-terminus of the PGAPGP polypeptide.

In a particular embodiment of the invention, the variant is obtained by the simultaneous addition of 1 to 2 glycines at the amino-and carboxy-termini of the PGAPGP polypeptide.

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

In a specific embodiment of the invention, the variant is obtained by adding an RGD oligopeptide to the amino-terminus or the carboxy-terminus of the aforementioned polypeptide.

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

In some embodiments, the invention also discloses derivatives comprising the polypeptides of the invention. The polypeptide derivative comprises a product obtained by conventional modification of the polypeptide, a fusion protein formed by connecting the polypeptide with a heterologous peptide, and a conjugate formed by connecting the polypeptide with other compounds.

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, nonmetal chemical element modification, immobilization modification and the like.

In some embodiments, the polypeptide of the invention is fused to a heterologous peptide. Examples of heterologous peptides include, but are not limited to: human serum albumin (HAS), an immunoglobulin heavy chain constant region (Fc), polyhistidine, glutathione S Transferase (GST), thioredoxin, protein A, protein G, mannose Binding Protein (MBP), or a fragment of any of the above heterologous polypeptides. In some embodiments, the heterologous peptide is fused at the amino terminus of the polypeptide of the invention. In other or alternative embodiments, the heterologous polypeptide is fused to the carboxy terminus of the polypeptide of the invention. For affinity purification, relevant matrices for affinity chromatography, such as glutathione, alpha amylase, may be used. Heterologous peptides may be selected to facilitate detection of the polypeptides of the invention. Examples of assays include various fluorescent proteins (e.g., GFP) and "epitope tags" including His6, GST, EGFP, MBP, nus, HA, igG, FLAG, c-Myc, or ProfinityXact.

In some embodiments, the polypeptides of the invention are linked to other compounds. The other compounds comprise bisphosphonate medicines and iridoid compounds. The bisphosphonate medicines comprise alendronate, ibandronate and zoledronic acid. The iridoid compounds include geniposide, genipin-gentiobioside, geniposide, and geniposidic acid.

The polypeptides useful in the present invention may 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.

Nucleic acids, vectors and host cells

In some embodiments, the invention also provides nucleic acids encoding the polypeptides of the 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, the polynucleotide is optimized for expression in a host cell.

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

Expression may be by 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, the nucleic acid may be codon optimized to facilitate expression in a desired host cell.

Non-viral vectors and systems include plasmids and episomal vectors (typically containing expression cassettes for expression of proteins or RNA), and artificial human chromosomes. For example, non-viral vectors that may be used to express a polypeptide of the invention in a mammalian (e.g., human) cell include pThioHis A, B & C, pcDNA3.I/His, pEBVHis A, B & C (Invitrogen, sanDiego, calif.), MPSV vectors, and many other vectors known in the art for the expression of other proteins. Useful viral vectors include, but are not limited to, adenovirus, adeno-associated virus, herpes virus-based vectors, SV40, papilloma virus, HBP EB virus-based vectors, fowlpox virus vectors, vaccinia virus vectors, and Semliki Forest Virus (SFV) vectors.

The choice of expression vector will depend on the intended host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the polypeptide of the present invention. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence under conditions other than the inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoter, glucocorticoid promoter, or heat shock promoter. In addition, other regulatory elements may also be incorporated to improve the expression of the nucleic acid encoding the polypeptide of the present invention, such as enhancers, ribosome 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, such that the polypeptide may be secreted from a host cell. The sequence may be provided by a vector, or as part of a nucleic acid for a polypeptide of the invention present in a vector.

The method used to introduce the expression vector containing the polynucleotide sequence of interest may vary depending on the host cell type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cellular hosts. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, biolistic methods, virosomes, immunoliposomes, polycations nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22, agent-enhanced DNA uptake, and ex vivo transduction. For long-term high-yield production of recombinant proteins, stable expression is often desired. For example, cell lines stably expressing a polypeptide of the invention can be prepared using an expression vector of the invention containing a viral origin of replication or endogenous expression elements and a selectable marker gene.

In some embodiments, a nucleic acid encoding a polypeptide of the invention can be delivered to a patient to treat an orthopedic disorder. The nucleic acid may 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, adenovirus or adeno-associated virus vectors, herpes virus vectors, fowlpox or vaccinia virus vectors.

Pharmaceutical composition

The polypeptide of the invention or a nucleic acid encoding it may be administered with a suitable pharmaceutical excipient as desired. Those skilled in the art will appreciate that the compositions will vary depending upon the mode of administration and the dosage unit.

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 invention provides pharmaceutical compositions comprising a therapeutically effective amount of a carrier of the invention as described previously.

In some embodiments, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of a host cell of the invention as described above.

The 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 invention or a combination thereof, the remainder consisting of suitable pharmaceutical carriers and/or excipients. Suitable excipients may be tailored for the particular composition and route of administration by methods well known in the art. See, e.g., REMINGTON' S SPHARMACEMENT SCIENCES, 18 th edition, mackPublishingCo., easton, pa. (1990).

Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids, such as Carbopol, e.g., carbopol941, carbopol980, carbopol981, and the like. The composition may additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; a humectant; an emulsifier; a suspending agent; preservatives such as methyl hydroxybenzoate, ethyl hydroxybenzoate, and propyl hydroxybenzoate (i.e., p-hydroxybenzoate); pH adjusters such as inorganic and organic acids and bases; a sweetener; a colorant; and a flavoring agent. The composition may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

For oral administration, the compositions may be in the form of tablets, troches, capsules, emulsions, suspensions, solutions, syrups, sprays, powders, and sustained release preparations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, 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, the composition containing any one of the following: diluents such as lactose, sucrose, dicalcium phosphate, and the like; disintegrants, such as starch or derivatives thereof; lubricants, such as magnesium stearate and the like; and binding agents such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose, and derivatives thereof. Conjugates may also be formulated as suppositories, for example, formulated in polyethylene glycol (PEG) carriers.

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

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

The compositions of the present invention may also be provided in lyophilized form. Such compositions may include a buffer (e.g., bicarbonate) for reconstitution prior to administration, or a buffer in a lyophilized composition for reconstitution, e.g., with water. The lyophilized composition may additionally comprise suitable pharmaceutical agents, such as other drugs for the treatment of orthopedic disorders. The lyophilized composition can optionally be packaged in combination with a reconstitution buffer to be provided in a syringe such that the reconstituted composition can be immediately administered to a patient.

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

In some embodiments, a polypeptide of the invention or a nucleic acid encoding a polypeptide of the invention may be prepared as a controlled or sustained release formulation, such as an injectable microsphere, bioerodible particle, polymeric compound, bead, or liposome or other biocompatible matrix, and the formulation may then be delivered by injection. For example, the polypeptides of the invention or nucleic acids encoding the polypeptides of the invention may be encapsulated in liposomes, or formulated as microparticles or microcapsules, or may be incorporated into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins, poly (lactic-co-glycolic acid) (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or delivered via a proteinaceous carrier or with a conjugate.

The dosage of the pharmaceutical composition of the invention for the treatment of orthopaedic diseases is variable, depending on the mode of administration, the age and/or weight of the individual and the condition of the subject, and is ultimately at the discretion of the attendant physician or veterinarian. The dose administered to the subject, in the context of the present invention, should be sufficient for a period of time to elicit a beneficial response in the subject. The dose is a "therapeutically effective amount".

Method

The present invention provides a method for the prevention or treatment of an orthopaedic disease, comprising administering to a subject in need thereof a polypeptide as described above, a nucleic acid as described above, a vector as described above, a host cell as described above, a pharmaceutical composition as described above.

The present invention provides a method for enhancing bone formation or promoting bone mineralization in a subject in need thereof, said method comprising administering to a subject in need thereof a polypeptide as described above, a nucleic acid as described above, a vector as described above, a host cell as described above, a pharmaceutical composition as described above.

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

Applications of

Therapeutic and prophylactic applications

Use of a polypeptide as hereinbefore described, a nucleic acid as hereinbefore described, a vector as hereinbefore described, a host cell as hereinbefore described, a pharmaceutical composition as hereinbefore described for the manufacture of a pharmaceutical formulation for the treatment or prophylaxis of a disease or condition in a subject in need thereof. In the present invention the disease or condition is an orthopaedic disease.

It will be appreciated by those skilled in the art that the aforementioned polypeptides, the aforementioned nucleic acids, the aforementioned vectors, the aforementioned host cells, the aforementioned pharmaceutical compositions of the invention may be co-administered with other therapeutic agents for the treatment or prevention of orthopaedic disorders. The co-administration may be simultaneous, e.g. in the form of a single pharmaceutical composition or separate compositions. The compositions of the invention may also be administered separately from the other therapeutic agent, for example in a separate dosing schedule.

Application of changing cell property

Use of a polypeptide as defined hereinbefore, a nucleic acid as defined hereinbefore, a vector as defined hereinbefore, a host cell as defined hereinbefore, a pharmaceutical composition as defined hereinbefore for the manufacture of a medicament for the promotion of bone formation.

The polypeptide, the nucleic acid, the carrier, the host cell and the pharmaceutical composition are used for preparing the medicines for promoting bone mineralization.

Use of a polypeptide as hereinbefore described, a nucleic acid as hereinbefore described, a vector as hereinbefore described, a host cell as hereinbefore described, a pharmaceutical composition as hereinbefore described in the manufacture of a medicament for inducing bone deposition.

The use of a polypeptide as described hereinbefore, a nucleic acid as described hereinbefore, a vector as described hereinbefore, a host cell as described hereinbefore, a pharmaceutical composition as described hereinbefore for the manufacture of a medicament for enhancing osteoblast activity.

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

The term "D-amino acid" as used herein 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.

As used herein, the term "treatment" as used herein refers to any type of treatment that is beneficial to a subject having a disease, including improving the condition of the patient (e.g., in one or more symptoms), delaying the progression of the disease, and the like.

As used herein, the expression "orthopaedic disease" refers to any of those diseases which cause various abnormalities or abnormalities of one or more bones and/or bone cells.

Preferably, the orthopedic disease comprises osteoporosis, rickets, osteomalacia, osteogenesis imperfecta, marble bone disease, fibrodysplasia, paget's disease, chronic hyperparathyroidism, hyperthyroidism, rheumatoid arthritis, gorham-Stout disease, mccube-Albright syndrome, osteolytic metastasis or multiple myeloma of various cancers, bone mass loss, systemic skeletal fragility, joint degeneration, non-healing fracture, orthopedic and dental problems caused by diabetes, implanted periodontitis, adverse effects on bone graft/implant/bone substitute materials, periodontal disease, bone aging, fracture, bone defect, bone graft, bone cancer, joint replacement, joint repair, fusion, facet joint repair, bone degeneration, dental and repair, bone marrow defect, bone disease of acromegaly patient, cystic fibrosis related bone disease, renal osteodystrophy related to chronic kidney disease, bone disease related to alanine disease, and bone disease related to hyperuricemia; preferably, the osteoporosis comprises postmenopausal osteoporosis, senile osteoporosis in both men and women, glucocorticoid-induced osteoporosis, arrested osteoporosis, weight loss induced osteoporosis, post-transplant osteoporosis, migratory osteoporosis, idiopathic osteoporosis, juvenile osteoporosis.

EXAMPLE 1 preparation of the polypeptide

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

1. synthetic feedstock and related reagents/apparatus

1) Resin: 2-Chlorotrityl Chloride Resin (Nankai Synthesis science and technology, tianjin) with a degree of substitution of 1.03 mmol/g;

2) Amino acid (b): purchased from Chengduchinuo, >99%;

3) Synthesizing a reagent: DMF (korea origin), DCM (korea origin), MEOH (japan origin), DIEA (new chemical industry, 99%), HBTU (haohan biotechnology, 99%);

4) Deprotection reagents: piperidine (Shanghai chemical reagent, national drug group, 99%);

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

6) And (3) a cracking reagent: 95% of cutting fluid: TFA (j.t. baker, 99%), TIS (shanghai degarei fine chemical, 98%), EDT (shanghai degarei fine chemical, 98%), anhydrous ether (shanghai experiment, found 99.7%);

7) Nitrogen gas: (New Union gas);

8) The instrument comprises: (1) twelve-channel semi-automatic peptide synthesizer A semi-automatic peptide synthesizer was independently designed and patented by Shanghai Qianyao Biotechnology Co., ltd, and its patent No. 201020226529.2 (2) SHIMADZU high performance liquid chromatograph (model: preparative type, analytical type, software: class-VP. Sevical System, manufacturer: SHIMADZU) (3) centrifuge (Shanghai Anting scientific instruments factory, model: TDL-40B) (4) LABCONCO freeze dryer (model: freezone. Plus.6, manufacturer: LABCONCO).

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

1) Swelling resin: 2-Chlorotrityl Chloride Resin was added to the reaction tube, DCM (15 ml/g) was added, and shaking was carried out for 30min.

2) Grafting with the first amino acid: filtering off the solvent by a sand core, adding 3 times of molar mass of the first amino acid at the C terminal, adding DMF for dissolving, adding 10 times of molar excess DIEA, and oscillating for 60min; blocking with methanol.

3) Deprotection: DMF was removed and 20% piperidine DMF solution (15 ml/g) was added for 5min and 20% piperidine DMF solution (15 ml/g) was added for 15min.

4) And (3) detection: and (3) pumping out the piperidine solution, taking dozens of particles of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.

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

6) Condensation: and (3) dissolving protected amino acid with triple excess and HBTU with triple excess by using DMF as little as possible, adding into a reaction tube, immediately adding DIEA with ten-fold excess, and reacting for 30min.

7) And (3) detection: taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and taking colorless negative reaction.

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

9) Repeating the operations 3) to 6), and connecting the amino acids in the sequence from right to left.

10 Suction dry, resin wash: DMF (10 ml/g) twice, methanol (10 ml/g) twice, DMF (10 ml/g) twice, DCM (10 ml/g) twice, and suction-dried for 10min.

11 Cleavage of the polypeptide from the resin: preparing cutting fluid (10/g) TFA 95%; 1% of water; EDT 2%; 2% of TIS; cutting time: and (4) 120min.

12 Blow-dry washing: the lysate is blown dry as much as possible with nitrogen, washed with ether six times, and then evaporated to dryness at normal temperature.

13 Analysis and purification: the crude product is purified by high performance liquid chromatography.

14 Freeze-drying: collecting the target polypeptide solution, concentrating in a freeze dryer, and freeze-drying to obtain white powder.

15 The polypeptide is sent to a quality inspection part to be qualified.

EXAMPLE 2 Activity assays of Polypeptides and derivatives thereof

1. Activity assays for polypeptides of varying lengths

1.1 Synthesis of Polypeptides of varying lengths

Synthesized according to the method of example 1.

1.2 cell assay

The ALP gene expression level was measured by QPCR after culturing osteoblast line MC-3T3E1 cells and incubating them for 24 hours with different length polypeptides at a final concentration of 2. Mu.g/ml.

1.3 results

The results are shown in fig. 1, the combination of six amino acids of PGAPGP is a better combination for enhancing osteoblast activity, the activity is better when the combination is connected in series to 2-3 PGAPGP, and the activity begins to weaken when the combination is connected in series to 4 PGAPGP or more.

2. Polypeptide activity detection after polypeptide two-end amino acid extension

2.1 Synthesis of Polypeptides of varying lengths

Synthesized according to the method of example 1.

2.2 cell assay

The osteoblast line MC-3T3E1 cells were cultured and QPCR measured ALP gene expression levels after 24 hours incubation using different length polypeptides at a final concentration of 2. Mu.g/ml.

2.3 results

As shown in FIG. 2, PGAPGP showed no effect on the activity when the independent amino acid was extended by 1, little effect on the activity when the independent amino acid was extended by 2, and decreased activity when the independent amino acid was extended by 3 or more. Double-sided extension of 1 unrelated amino acid had no effect on its activity, and double-sided extension of more than 2 unrelated amino acids began to decrease in activity. Glycine (G) is a representative of unrelated amino acids.

3. Detection of the Activity of a polypeptide incorporating an unnatural D-amino acid

3.1 Synthesis of Polypeptides incorporating unnatural D-amino acids

Following conventional procedures, the unnatural D amino acid profile is shown in Table 1.

TABLE 1 unnatural D amino acid profile

Numbering	Unnatural D-amino acid profile
		P1	PGAPGP
P2	PGAPGP D
		P3	PGAPG D P
P4	PGAP D GP
		P5	PGA D PGP
P6	PG D APGP
		P7	P D GAPGP

3.2 cell assay

The ALP gene expression level was measured by QPCR after culturing osteoblast line MC-3T3E1 cells and incubating them for 24 hours with different length polypeptides at a final concentration of 2. Mu.g/ml.

3.3 results

As a result, as shown in FIG. 3, the activity was decreased by substituting an unnatural D-form amino acid for an amino acid at any position of the PGAPGP peptide.

4. Detection of variant Activity after modification of Polypeptides

4.1 Synthesis of modified variants

Following conventional procedures, polypeptide modifications are shown in table 2.

TABLE 2 Polypeptides with different modifications

Number of	Modification mode
		Pep1	N-methylation
Pep2	N-myristoylation
		Pep3	N-PEG modification
Pep4	Modification with C-fluorine
		Pep5	C-biotin modification
Pep6	C-FAM fluorescent labels
		Pep7	Unmodified PGAPGP

4.2 cell assay

The ALP gene expression level was measured by QPCR after culturing osteoblast line MC-3T3E1 cells and incubating them for 24 hours with different length polypeptides at a final concentration of 2. Mu.g/ml.

4.3 results

The results are shown in fig. 4, the effect of common polypeptide end modifications on PGAPGP activity. Methylation, myristoylation, PEG modification, fluorine modification, biotin modification and FAM fluorescence labeled PGAPGP all have activity.

5. Activity assays for polypeptide conjugates

5.1 synthetic polypeptide conjugates

Following conventional procedures, polypeptide conjugates are shown in table 3.

TABLE 3 polypeptide conjugates

Numbering	Modification mode
		PC1	Unmodified PGAPGP
PC2	N-coupled sodium Allen phosphate
		PC3	C-coupled sodium Allen phosphate
PC4	N-coupled RGD polypeptides
		PC5	C-coupled RGD polypeptides

5.2 cell experiments

ALP gene expression levels were measured by QPCR after culturing osteoblast line MC-3T3E1 cells and incubating with a final polypeptide conjugate concentration of 5. Mu.g/ml for 24 hours.

5.3 results

The results are shown in fig. 5, where the coupling of PGAPGP to bisphosphonates is represented by sodium alendronate. RGD is used as a representative, and the PGAPGP has activity after being coupled with the oligopeptide.

6. Activity detection of polypeptide sustained release dosage forms

6.1 synthetic microsphere sustained Release dosage forms

The method comprises the following steps: stirring and dissolving PLGA, dripping a pore-forming solution, and performing ultrasonic emulsification to form an emulsion; wherein the molar ratio of LA to GA of PLGA is 50; the emulsion is then added dropwise to the stirred external aqueous phase, and a predetermined volume of deionized water is added, with the stirring being continued at a varying rate until the solvent has completely evaporated. And adding the water-soluble surfactant serving as the external water phase into deionized water, centrifuging the solution after the organic solvent is completely volatilized, washing with the deionized water, and removing the supernatant to obtain the PLGA microspheres. And adding NaOH solution containing polypeptide into the PLGA microspheres, uniformly mixing, putting the mixture into a shaking table for continuous reaction, repeatedly centrifuging and washing the reacted PLGA microspheres by using deionized water, and freeze-drying for later use.

6.2 cell assay

The ALP gene expression level was measured by QPCR after culturing osteoblast line MC-3T3E1 cells and incubating them for 1-6 days with microspheres at a final concentration of 2. Mu.g/ml.

6.3 results

The results are shown in fig. 6, and are represented by PLGA 50 microspheres, the PGAPGP sustained release dosage forms have significant biological activity.

EXAMPLE 3 Effect of Polypeptides on mouse bone formation

1. Material

Wild type mouse origin: all mice were SPF grade purchased from experimental animal technology limited, viton, beijing (Charles River).

2. Preparing a polypeptide solution: weighing 1.000 g of sodium hydroxide into a beaker, adding a small amount of normal saline to dissolve the sodium hydroxide, pouring the solution into a 1000 ml volumetric flask, washing the beaker by 3 times, pouring the solution into the volumetric flask completely, and finally diluting the solution to a scale mark by using the normal saline. Shaking up to obtain 1000mg/mL polypeptide storage mother liquor. When in use, the medicine is diluted according to the required concentration in equal proportion.

3. Drug treatment

Randomly dividing the mice into two groups, namely a control group and an experimental group, wherein the mice in the control group are injected with physiological saline 1 time per week, and the mice in the experimental group are injected with 10mg/kg 1 time per week; femoral samples were collected after 12 injections for Micro-CT scanning and H & E staining.

4. Micro-CT scanning

Collected femoral samples were subjected to Micro-CT scanning of trabecular and cortical bone and analyzed for trabecular number and trabecular thickness.

4.1 step

(1) After being fixed in 4% paraformaldehyde for 24 hours, the femur sample is transferred to 0.5% paraformaldehyde solution to prevent the formation of crystals in bone tissues from influencing sample scanning.

(2) The method comprises the following steps of placing a sample in a Micro-CT special scanning tube with the tube diameter of 14mm, placing the sample horizontally, enabling a transverse shaft to be perpendicular to a Micro-CT scanning shaft, and placing the scanning tube in a Micro-CT scanning case sample disc.

(3) The parameters related to the scanning procedure are as follows: the scanning voltage is 70kVp, the scanning energy power is 14W, the scanning current is 200 muA, the exposure time is 300ms, the scanning BH 1200mg HA/cc, the scanning precision is 10μm, and the scanning Filter (Filter) is 0.5mm AI Filter. And reconstructing and analyzing later-period image data by using Mimics 13.0 software.

(4) When the femoral data are reconstructed, analyzing and selecting the trabecular bone data from all trabeculae which start 1mm below the femoral growth plate and are arranged in the inner layer of cortical bone; cortical bone data analysis cortical bone 5mm-6mm below the growth plate was selected.

4.2 results

The Micro-CT scanning results are shown in fig. 7, and the bone density of the mice is significantly increased after the application of the polypeptide (fig. 7A), the trabecular bone thickness is significantly increased (fig. 7C), and the trabecular bone number is significantly increased (fig. 7D), indicating that the mineralization degree of the femurs of the mice is enhanced.

5. H & E staining

5.1 step

1) Slicing: paraffin sections of the tissues were made in a conventional manner;

2) Baking slices: placing the slices in a 65 ℃ oven for 30min;

3) Dewaxing and hydrating: and sequentially dewaxing the tissue section by using xylene I for 30min and xylene II for 30min. Then carrying out hydration treatment on the slices, wherein the absolute ethyl alcohol I is 5min, the absolute ethyl alcohol II is 5min, the 90% alcohol is 5min, the 75% alcohol is 5min, and the distilled water is 5min.

4) Dyeing: washing with hematoxylin for 5min, and soaking in PBS for 5min to turn nucleus blue. Eosin staining for 2min, washing with water.

5) And (3) dehydrating and transparency: gradient ethanol is used for tissue dehydration, 80% ethanol, 90% ethanol and 95% ethanol are respectively used for 10s, and then the tissue is dried at room temperature. And (3) drying the mixture in the air at room temperature after 5min of absolute ethanol I and 5min of absolute ethanol II. Immersing in xylene for 5min, and sealing with neutral resin.

6) And (4) observation: and (5) observing and photographing under an upright microscope.

5.2 results

The femoral samples were H & E stained 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-fluorescence labeling experiment

6.1 step

(1) The above femur samples (xylenol orange and calcein were injected intraperitoneally at a dose of 80mg/kg, with an interval of 2 weeks) were subjected to hard tissue sectioning, and the thickness of the sections was about 15 μm.

(2) DAPI staining: hard tissue section dropwise addition 1:1000 diluted DAPI staining solution, 5min. The cells were washed 3 times with 5min each time in PBS. The encapsulated tablets were encapsulated using an anti-fluorescence quenching. And (5) observing the red and green double-color fluorescent markers under an inverted fluorescent microscope, and counting the bone deposition rate.

6.2 results

The results are shown in FIG. 8, with the red and green markers separated by 2 weeks, at a distance of new bone deposition. Compared with the control group, the femoral bone deposition amount of the experimental group is obviously increased. The analysis result shows that the bone formation is obviously enhanced after the mouse is injected with the polypeptide.

7. Von Kossa staining

7.1 step

(1) Dyeing with silver nitrate dye liquor: preparing 0.2% silver nitrate solution, dripping on femur hard tissue slice, covering femur tissue, irradiating with strong light for 15min, and washing slice with running water for 2-3s.

(2) And (3) incubation with sodium thiosulfate: 5% sodium thiosulfate solution is prepared and dripped on the slices to cover the femoral tissues for 5s, and the slices are washed by running water for 2-3s.

(3) Methyl green padding: carrying out back dyeing on the methyl green dye liquor for 5min, washing for 5s by running water, washing away floating color, and drying in a 65 ℃ oven for 30min.

(4) Mounting and observing: and (5) adding dimethylbenzene dropwise into the slices, and sealing the slices by using neutral resin after the slices are transparent for 5min. And observing and photographing under a body type microscope.

7.2 results

The replacement of calcium and silver ions in bone tissue appears black, indicating the density and level of mineralized bone. Von Kossa staining results are shown in fig. 9, with increased trabecular bone density under the growth plates of the polypeptide-injected group, indicating a significant increase in bone mineralization.

8. Bone mineralization related index Dmp-1 detection

8.1 immunohistochemical staining

(1) Slicing: after femoral tissue decalcification, slicing normal paraffin tissue;

(2) Baking slices: putting the slices in a 65 ℃ oven for 30min;

(3) Dewaxing and hydrating: sequentially dewaxing the tissue slices, namely, 30min of dimethylbenzene I and 30min of dimethylbenzene II. Then carrying out hydration treatment on the slices, wherein the absolute ethyl alcohol I is 5min, the absolute ethyl alcohol II is 5min, the 90% alcohol is 5min, and the 75% alcohol is 5min. Distilled water for 5min. Rinsed three times in PBS for 3min each time.

(4) Antigen retrieval: according to the requirements of the antibody, the tissue antigen is repaired correspondingly.

(5) Inactivation of endogenous peroxidase: the sections were added dropwise with an appropriate amount of peroxidase blocking solution and incubated at room temperature for 10min (to block the activity of endogenous peroxidase). PBS was added dropwise and washed three times for 5min each time.

(6) Tissue sealing: and (3) dropwise adding a proper amount of serum of the normal non-immune animal to the section, and incubating for 10min at room temperature.

(7) Incubation of primary antibody: the serum was removed from the tissue, an appropriate amount of primary antibody was added dropwise to the tissue, and the tissue was incubated at room temperature for 60min or overnight at 4 ℃. PBS was added dropwise for 3min and washed three times.

(8) Incubation of secondary antibody: the sections were added drop wise with appropriate biotin-labeled secondary antibody, incubated at room temperature for 10min, washed three times with PBS, 3min each time.

(9) PBS was removed and 1 drop or 50. Mu.l of streptavidin-labeled secondary antibody (reagent D) was added to each section, incubated at room temperature for 10min and washed three times with PBS for 3min each.

(10) DAB color development: dripping DAB color development solution (used as prepared) and observing under microscope for 3-10min. And after the color development is finished, washing with tap water, and stopping the color development.

(11) Cell nucleus counterstaining: and (4) carrying out counterstaining by using methyl green staining solution and returning blue by using PBS.

(12) And (3) dehydrating and transparency: gradient ethanol is used for tissue dehydration, 80% ethanol, 90% ethanol and 95% ethanol are respectively used for 10s, and then the tissue is dried at room temperature. And (5) drying the dehydrated alcohol I for 5min and the dehydrated alcohol II for 5min at room temperature. After 5min of transparency in xylene. Mounting and observing.

8.2 results

Results shown in fig. 10, the immunohistochemical color development degree of the injected polypeptide group is increased, which indicates that the expression of the mouse mineralization protein Dmp1 is obviously enhanced.

EXAMPLE 3 Effect of Polypeptides on bone Mass in osteoporotic mice

1. Construction of osteoporosis mouse model

(1) 18 female WT mice of 12 weeks old were selected and divided into 3 groups (OVX group, OVX + polypeptide group, OVX + PTH group).

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

(3) The lower abdomen of the mouse was prepared for skin preparation, sterilized with iodophor, wiped with 75% alcohol, and a 0.5cm length incision was made 1cm below the rib, 0.5cm left of the midline of the abdomen.

(4) The fascia and the muscle are cut open, and the operation visual field is widened bluntly. White adipose tissues are found in the lower abdomen and pulled out of the body, and the oviduct and the ovary are found along the ascending of the uterus.

(5) After ligation of the oviduct, the ovary and oviduct were cut off, and after bilateral resection, the uterus and fat were placed in the abdomen.

(6) The skin and the muscle are sewed in layers.

(7) Three months after the osteoporosis model mouse self-builds a model, after the model tends to be stable, injecting polypeptide through vein, total 12 times, 1 time per week, the dosage is 10mg/kg; PTH was administered subcutaneously at a dose of 0.1. Mu.g/kg for 4 weeks, 3 times per week.

2. Micro-CT scanning

The procedure is as in example 1.

The results are shown in fig. 11, and the bone mass of mice in the group of polypeptides and PTH was significantly increased compared to the OVX group, indicating that the polypeptides can increase the bone mass of mice with osteoporosis.

3. Fluorescent double-label experiment

The procedure is as in example 1.

The results are shown in fig. 12, the bone deposition amount of the polypeptide group and PTH group is significantly increased compared with that of OVX group mice, which indicates that the bone formation function of the mice is increased after the polypeptide treatment.

EXAMPLE 4 Effect of Polypeptides on fracture healing

1. Construction of mouse fracture model

According to the literature [ Glycosylation of protein matrix 1 is critical for a reactive chemistry of a stimulating hormone production. Frontiers of medicine.2019 Oct;13 (5) 575-589] the mouse fracture model was constructed. After modeling for 1 week, the mice are injected with 10mg/kg of polypeptide 1 time per week, and the callus formation and callus density at the fracture broken ends of the mice are evaluated after 6 times of injection.

2. Fluorescent double-label experiment

The procedure is as in example 1.

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

3. Micro-CT scanning

The procedure is essentially the same as in example 1. The difference lies in that: when the fracture sample data is reconstructed, the callus display parts are 500 micrometers above and below the fractured end of the fracture respectively, and the callus parts outside the cortical bone of the corresponding parts are selected by data analysis.

The results are shown in FIG. 14, which shows that the fracture site of the group of injected polypeptides was accelerated in the fracture model mouse. The callus part reconstruction result shows that the callus is denser than the control group after the polypeptide injection, and the bone density of the polypeptide group is obviously higher than that of the control group.

Example 5 evaluation of biological safety of Polypeptides

The biological safety of the polypeptide is discussed by methods such as serum biochemical index evaluation and pathological section analysis of important organs.

1. ELISA detection

After the mice are injected with the polypeptide, the serum of the mice is tested for BUN (blood urea nitrogen), CK (creatine kinase) and ALT (glutamic-pyruvic transaminase) by ELISA, and the influence of the small molecular polypeptide on biochemical indexes is evaluated.

2.1 step

2.1.1 mouse serum Collection

(1) Blood collection of the retroorbital venous plexus of the mouse:

taking a glass capillary tube with the inner diameter of 1.0-1.5mm, breaking the glass capillary tube into a capillary tube section with the length of 2-2.5 cm before use, immersing the capillary tube section into 1% heparin solution, and drying the capillary tube for use. When blood is taken, the left hand grasps the skin of the back and neck part between two ears of a mouse to fix the head, gently presses two sides of the neck part downwards to cause difficulty in backflow of the blood of the head vein, so that the orbital vein Cong Chongxie is difficult to return, the right hand holds the capillary tube to be inserted into a conjunctiva from the inner canthus part, then the capillary tube is gently pushed towards the fundus part, the capillary tube is gently rotated to lacerate a venous plexus, and the blood flows along the capillary tube and is received into a container prepared in advance. After blood sampling, the gauze slightly presses the eyes to stop bleeding.

(2) After blood was obtained by the above method, the supernatant was centrifuged at 3000rpm for 10 minutes in an EP tube at room temperature for more than one hour (or in a 37 ℃ water bath for 1 hour, in a 4 ℃ refrigerator for 2 hours or overnight), and the supernatant was serum. Can be stored at-80 deg.C.

2.1.2 enzyme-linked immunosorbent assay (ELISA)

(1) Balancing: CK (creatine kinase), BUN (blood urea nitrogen), ALS (aldosterone) ALT (glutamic pyruvic transaminase) were removed and ELISA kit equilibrated for 20min at room temperature.

(2) Diluting the washing solution: dd H ₂ Dilute 20X wash to 1X.

(3) Serum preparation: after blood collection using a tube without heat source and endotoxin (needed to avoid any cell irritation during subsequent experimental manipulations), serum and red blood cells were separated by careful aspiration after centrifugation at 3000rpm for 10 minutes at room temperature.

(4) Sample adding: the desired number of plates are removed and placed on a 96-well plate, and the plate is marked with the order of the sample application. To the blank and standard wells, 20. Mu.l of Matrix solution was added, and to the standard 20. Mu.l each of 0, 0.025, 0.05, 0.1, 0.2, 0.4ng/ml standard was added. The diluted sera of mice in the control group and the polypeptide group were added to each sample well in an amount of 10. Mu.l, and then 40. Mu.l of the diluted sample was added to each sample well, but no blank well was added.

(5) Manually washing the plate: slowly adding plate washing solution along the hole wall, standing for 1min, discarding the plate washing solution, and patting on absorbent paper. Repeat 5 times. And the gun head is replaced in time when different samples are added, so that pollution is avoided.

Automatic plate washing: add 350. Mu.l of washing solution to each reaction well, program for 1min, wash plate 5 times.

(6) In addition to blank wells, 100. Mu.l of detection antibody labeled with horseradish peroxidase (HRP) was added to the standard reaction wells and the sample reaction wells, and after the reaction wells were sealed with a sealing plate film, the wells were incubated in a 37 ℃ water bath or incubator for 60min.

(7) Washing the plate: the plate was washed 5 times in the above manner.

(8) Color development: 50. Mu.l each of the substrates A and B was added to each reaction well, and incubated at 37 ℃ for 15min in the absence of light.

(9) Measurement: adding 50 mul of stop solution into each hole, and reading the absorbance at the wavelength of 450nm by using an enzyme-labeling instrument after 10min. After a standard curve is made, the content of each protein in the sample is calculated by using a corresponding formula.

2.2 results

The results are shown in FIG. 15, mice were administered with 10mg/ml of the polypeptide weekly and intravenously, and the mice were tested by drawing blood at 2,4,6,8, 10, 12 weeks. Circles are labeled as control and squares are labeled as polypeptide.

3. H & E staining

Mice were dosed and intravenously with 10mg/ml of polypeptide weekly and sacrificed for testing after week 12. H & E staining was performed on important organs for pathological evaluation, and whether the polypeptides have chronic toxicological effects on the organs of the internal organs was investigated. Tissues such as heart, lung, liver, spleen, kidney, etc. were paraffin sectioned and H & E stained. The results are shown in fig. 16, and compared with the control group, the important organs (heart, lung, liver, spleen and kidney) after the polypeptide injection have no obvious pathological changes such as tumor or inflammatory infiltration. The results of this example show that the polypeptides of the invention have better biological safety.

The above-described embodiments are only for illustrating the present invention and are not to be construed as limiting the present invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims (10)

1. A polypeptide, wherein the core sequence of the polypeptide comprises PGAP, PGA, or PGP.

2. The polypeptide of claim 1, wherein the polypeptide comprises any one of:

1) Variants obtained by deletion, addition, substitution of one or more amino acids on the basis of the core sequence;

2) A variant having an amino acid sequence 85%,90%,91%,92%,93%,94%,95%,96%,97%, 98%, or 99% homologous to the amino acid sequence of said core sequence;

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

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

preferably, the oligopeptide is RGD;

preferably, the substitution is obtained after replacing an amino acid at any position of the core sequence with a non-natural D-amino acid.

3. A polypeptide derivative, wherein the polypeptide derivative comprises a product obtained by conventional modification of the polypeptide of claim 1 or 2, a fusion protein formed by linking the polypeptide to a heterologous peptide, and a conjugate formed by linking the polypeptide to another compound;

preferably, the conventional modification is amination, methylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, sulfation, esterification, glycosylation, cyclization, biotinylation, fluorophore modification, polyethylene glycol (PEG) modification, myristoylation, nonmetal chemical element modification, immobilization modification and the like;

preferably, the heterologous peptide comprises human serum albumin, an 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, profinityexcact;

preferably, the other compounds include bisphosphonates, iridoid compounds; preferably, the bisphosphonate drug comprises alendronate, ibandronate, zoledronic acid; preferably, the iridoid compounds include geniposide, genipin-gentiobioside, geniposide and geniposidic acid.

4. A nucleic acid molecule encoding the polypeptide of claim 1 or 2.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A pharmaceutical composition comprising the polypeptide of claim 1 or 2, the polypeptide derivative of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6; preferably, the pharmaceutical composition further comprises a conventional pharmaceutical carrier or excipient; preferably, the pharmaceutical composition further comprises other agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers.

8. A method, characterized in that the method comprises any of the following methods:

1) A method of enhancing osteoblast activity, the method comprising administering the polypeptide of claim 1 or 2, the polypeptide derivative of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, the pharmaceutical composition of claim 7;

2) A method of preventing or treating an orthopedic disorder, the method comprising administering to a subject in need thereof the polypeptide of claim 1 or 2, the polypeptide derivative of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, the pharmaceutical composition of claim 7;

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

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

5) A method of promoting bone mineralization in a subject in need thereof, said method comprising administering to a subject in need thereof the polypeptide of claim 1 or 2, the polypeptide derivative of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, the pharmaceutical composition of claim 7.

9. Use of the polypeptide according to claim 1 or 2, characterized in that it comprises the use according to any one of the following:

1) Use in the preparation of a polypeptide derivative according to claim 3;

2) Use in the preparation of a pharmaceutical composition according to claim 7;

3) Use in the manufacture of a medicament for promoting bone formation;

4) Use in the manufacture of a medicament for enhancing osteoblast activity;

5) The use in the preparation of a medicament for promoting bone mineralization;

6) The use in the manufacture of a medicament for inducing bone deposition;

7) The application in preparing the medicine for preventing or treating orthopedic diseases;

preferably, the orthopedic disease comprises osteoporosis, rickets, osteomalacia, osteogenesis imperfecta, marble bone disease, fibrodysplasia, paget's disease, chronic hyperparathyroidism, hyperthyroidism, rheumatoid arthritis, gorham-Stout disease, mccube-Albright syndrome, osteolytic metastasis or multiple myeloma of various cancers, bone mass loss, systemic skeletal fragility, joint degeneration, non-healing fracture, orthopedic and dental problems caused by diabetes, implanted periodontitis, adverse effects on bone graft/implant/bone substitute materials, periodontal disease, bone aging, fracture, bone defect, bone graft, bone cancer, joint replacement, joint repair, fusion, facet joint repair, bone degeneration, dental and repair, bone marrow defect, bone disease of acromegaly patient, cystic fibrosis related bone disease, renal osteodystrophy related to chronic kidney disease, bone disease related to alanine disease, and bone disease related to hyperuricemia; preferably, the osteoporosis comprises postmenopausal osteoporosis, senile osteoporosis in both men and women, glucocorticoid-induced osteoporosis, arrested osteoporosis, weight loss induced osteoporosis, post-transplant osteoporosis, migratory osteoporosis, idiopathic osteoporosis, juvenile osteoporosis.

10. The use of the polypeptide derivative of claim 3, wherein the use comprises any one of the following:

1) Use in the preparation of a pharmaceutical composition according to claim 7;

2) Use in the manufacture of a medicament for promoting bone formation;

3) Use in the manufacture of a medicament for enhancing osteoblast activity;

4) The use in the preparation of a medicament for promoting bone mineralization;

5) Use in the manufacture of a medicament for inducing bone deposition;

6) The application in preparing the medicine for preventing or treating orthopedic diseases;

preferably, the orthopedic disease comprises osteoporosis, rickets, osteomalacia, osteogenesis imperfecta, marble bone disease, fibrodysplasia, paget's disease, chronic hyperparathyroidism, hyperthyroidism, rheumatoid arthritis, gorham-Stout disease, mcCune-Albright syndrome, osteolytic metastasis or multiple myeloma of various cancers, bone mass loss, systemic skeletal fragility, joint degeneration, non-healing fracture, orthopedic and dental problems caused by diabetes, implanted periodontitis, adverse effects on bone graft/implant/bone substitute material, periodontal disease, bone aging, fracture, bone defect, bone graft, bone cancer, joint replacement, joint repair, fusion, joint repair, bone degeneration, dental and repair, bone marrow defect, bone disease in acromegaly patients, cystic fibrosis related bone disease, oxalate bone disease, renal osteodystrophy related bone dystrophy, bone disease related to cystinosis and disease related to hyperuria; preferably, the osteoporosis comprises postmenopausal osteoporosis, senile osteoporosis in men and women, glucocorticoid-induced osteoporosis, immobilization osteoporosis, weight loss induced osteoporosis, post-transplant osteoporosis, metastatic osteoporosis, idiopathic osteoporosis, juvenile osteoporosis.

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