WO2022110092A1 - 一种促骨形成多肽及其应用 - Google Patents

一种促骨形成多肽及其应用 Download PDF

Info

Publication number
WO2022110092A1
WO2022110092A1 PCT/CN2020/132556 CN2020132556W WO2022110092A1 WO 2022110092 A1 WO2022110092 A1 WO 2022110092A1 CN 2020132556 W CN2020132556 W CN 2020132556W WO 2022110092 A1 WO2022110092 A1 WO 2022110092A1
Authority
WO
WIPO (PCT)
Prior art keywords
bone
polypeptide
osteoporosis
disease
modification
Prior art date
Application number
PCT/CN2020/132556
Other languages
English (en)
French (fr)
Inventor
魏化伟
张伦
杨承刚
Original Assignee
北京泽勤生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京泽勤生物医药有限公司 filed Critical 北京泽勤生物医药有限公司
Priority to PCT/CN2020/132556 priority Critical patent/WO2022110092A1/zh
Publication of WO2022110092A1 publication Critical patent/WO2022110092A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

  • the invention belongs to the field of biomedicine, and relates to a bone-forming polypeptide and its application.
  • Bone is an active organ that continuously remodels bone and maintains serum calcium concentration through repeated formation and resorption/destruction in bone morphogenesis.
  • bone formation by osteoblasts and bone resorption by osteoclasts are usually in balance. Bone mass can be maintained at a constant level through a mechanism of interaction between these cells.
  • Metabolic bone disease occurs when the balance is disrupted due to menopause, aging, inflammation, etc., due to disruption caused by osteoporosis or rheumatoid arthritis. The occurrence of this metabolic bone disease is a serious problem in the current aging society. Therefore, it is an urgent task to elucidate the pathological mechanism of this disease at the molecular level and develop effective therapeutic drugs.
  • bone diseases including metabolic bone diseases include osteoporosis, osteopenia, osteogenesis imperfecta, osteonecrosis, low bone mass, Paget's disease, bone metastases, loosening of sterile prostheses, periodontitis, Metastatic bone disease, rheumatoid arthritis, lupus arthritis, periodontal disease, alveolar bone loss, post-osteotomy, childhood idiopathic bone loss, spinal curvature, height loss and prosthetic surgery, etc.
  • metabolic bone diseases include osteoporosis, osteopenia, osteogenesis imperfecta, osteonecrosis, low bone mass, Paget's disease, bone metastases, loosening of sterile prostheses, periodontitis, Metastatic bone disease, rheumatoid arthritis, lupus arthritis, periodontal disease, alveolar bone loss, post-osteotomy, childhood idiopathic bone loss, spinal curvature, height loss and prosthetic surgery, etc.
  • bone resorption inhibitors that inhibit the process of bone resorption, rather than enhance bone formation, have been used as therapeutic agents for bone metabolic diseases exhibiting bone loss, such as osteoporosis.
  • drugs that inhibit bone resorption and are used or suggested for the treatment of osteoporosis include estrogens, selective estrogen receptor modulators (SERMs), ipriflavone, vitamin K2, calcium, calcitriol, Calcins and bisphosphonates such as alendronate.
  • SERMs selective estrogen receptor modulators
  • ipriflavone ipriflavone
  • vitamin K2 calcium
  • Calcins Calcins
  • bisphosphonates such as alendronate
  • Teriparatide is the only drug that promotes bone formation. Clinical data show that it can significantly increase the bone mineral density of many bones in the body of postmenopausal women, and it can also increase the bone density of lumbar and femoral neck in men. Therefore, it is widely used in various guidelines. is recommended. The FDA approves teriparatide for the treatment of persistent, systemic severe osteoporosis. Teriparatide is indicated for the treatment of osteoporosis in postmenopausal women at high risk of fractures. Significantly reduces the risk of vertebral and nonvertebral fractures in postmenopausal women, but has not been demonstrated to reduce the risk of hip fractures.
  • Teriparatide is a molecular fragment of the parathyroid hormone PTH (1-34). Patients can only receive one treatment for a maximum of 24 months in their lifetime. Common side effects are nausea, leg cramps, dizziness, and increased risk of osteosarcoma. The average monthly cost of treatment is more than 6,000 yuan, and it takes at least 3 months to take effect, which brings great economic pressure to patients. Therefore, it is urgent to develop a new osteogenesis-promoting drug to break the monopoly of teriparatide.
  • the purpose of the present invention is to provide a polypeptide and its derivatives for promoting bone formation.
  • the present invention provides a polypeptide, the polypeptide sequence is composed of two amino acids, serine, glutamic acid or aspartic acid, the polypeptide contains 2-50 amino acids, the serine, The number ratio of glutamic acid or aspartic acid is (0.25 ⁇ 2):1;
  • polypeptide has the following general formula: (XSSXS)n, wherein X is glutamic acid or aspartic acid, S is serine, and n is a natural number;
  • n 1 to 8.
  • n 1, 2, 3, 4;
  • n is 1.
  • the present invention provides a polypeptide derivative, the polypeptide derivative includes a modified product of the aforementioned polypeptide, one or more amino acids added and/or substituted for the aforementioned polypeptide.
  • Variants obtained are conjugates of the aforementioned polypeptides with other substances.
  • modification in the present invention refers to any change made to a peptide, such as a change in peptide length, a change in amino acid sequence, a change in chemical structure, a co-translational modification or a post-translational modification of the peptide.
  • the peptides of the invention comprise one or more modified amino acid residues. Types of modifications are well known in the art.
  • Modifications that can be used in the present invention include methylation, myristoylation, PEG modification, fluorine modification, biotin modification, fluorescent label modification, cyclization, carboxylation, fatty acid modification, acetylation modification, phosphorylation modification, glycosylation modification, amidation modification, or other known polypeptide modifications.
  • the modification includes methylation, myristoylation, PEG modification, fluorine modification, biotin modification, fluorescent label modification, and cyclization.
  • the above-mentioned modification of amino acids serves to largely improve the stability of the peptides of the present invention.
  • stability used in the present invention means not only “in vivo” stability, but also storage stability (eg, room temperature storage stability).
  • the above-mentioned protecting group functions to protect the peptide of the present invention from attack by a protein cleaving enzyme in vivo.
  • amino acid additions and/or substitutions of the present invention include additions and/or substitutions of amino acids at the amino terminus, carboxyl terminus or any position within the aforementioned polypeptide sequence.
  • the added and/or replaced amino acids include natural amino acids or unnatural amino acids.
  • the variant is obtained by adding 1 to 3 unrelated amino acids to the amino- or carboxy-terminus of the aforementioned polypeptide.
  • Unrelated amino acids useful in the present invention include glycine, tryptophan, tyrosine, cysteine, methionine, glutamine and threonine.
  • the variant is obtained by adding 1 to 3 glycines to the amino-terminus or carboxyl-terminus of an ESSES or DSSDS polypeptide.
  • the variant is obtained by simultaneously adding 1 to 2 unrelated amino acids to the amino-terminus and carboxyl-terminus of the aforementioned polypeptide.
  • Unrelated amino acids useful in the present invention include glycine, tryptophan, tyrosine, cysteine, methionine, glutamine and threonine.
  • the variant is obtained by the simultaneous addition of 1 to 2 glycines to the amino terminus and carboxyl terminus of an ESSES or DSSDS polypeptide.
  • the variant is obtained by replacing 1 amino acid at the carboxyl terminus of the aforementioned polypeptide; preferably, the variant is obtained by replacing the carboxyl terminus of the aforementioned polypeptide with an unnatural amino acid of D-type amino acid obtained from a natural amino acid; more preferably, the variant is obtained by replacing the natural amino acid with a non-natural D-form amino acid of serine at the carboxy terminus of the aforementioned polypeptide.
  • the variant is obtained by replacing a natural amino acid with a non-natural D-form amino acid of serine at the carboxy terminus of the ESSES or DSSDS polypeptide.
  • polypeptides of the present invention include, but are not limited to, carrier proteins, exogenous polypeptides, and drugs.
  • carrier proteins include serum albumin, hemocyanin, and chicken ovalbumin.
  • exogenous polypeptide includes an Fc region, a signal peptide, and a polypeptide marker.
  • the Fc region is selected from the group consisting of: IgA Fc region, IgD Fc region, IgG Fc region, IgE Fc region, IgM Fc region.
  • the polypeptide tag is selected from the following group: Flag tag, strep tag, polyhistidine tag, VSV-G tag, influenza virus hemagglutinin tag, c-Myc tag.
  • the drugs include bisphosphonates and iridoids.
  • the bisphosphonates include alendronate, ibandronate and zoledronic acid.
  • Iridoid compounds include geniponiac acid, genipin-gentiobiside, geniposide, geniposide.
  • the present invention provides a pharmaceutically acceptable salt or ester of the aforementioned polypeptide or polypeptide derivative.
  • pharmaceutically acceptable salts include acetate and hydrochloride.
  • the present invention provides a method for preparing the aforementioned polypeptide.
  • Polypeptides useful in the techniques of the present invention can be prepared using any suitable means known in the art.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means and methods for preparing such polypeptides are well known in the art.
  • the present invention provides a pharmaceutical composition comprising an effective amount of the aforementioned polypeptide or polypeptide derivative; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable accepted vector.
  • the pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is a carrier commonly used as a formulation, and the pharmaceutically acceptable carrier includes diluents, fillers, excipients, binders, wetting agents Agents, disintegrants, effervescent agents, surfactants, absorption enhancers, lubricants, adsorption carriers, sustained-release microspheres, implants, in situ microspheres, liposomes, microemulsions, in situ hydrogels , nanoparticles, protease inhibitors, bioadhesives.
  • the sustained-release microspheres can be prepared from the following sustained-release excipients: racemic polylactic acid (D, L-PLA), racemic polylactic acid/glycolic acid copolymer (D, L-PLGA), monomethyl polyethylene Glycol/polylactic acid (MPEG-PLA), monomethyl polyethylene glycol/polylactic acid copolymer (MPEG-PLGA), polyethylene glycol/polylactic acid (PLA-PEG-PLA), polyethylene glycol/polyethylene glycol Lactic acid copolymer (PLGA-PEG-PLGA), carboxyl-terminated polylactic acid (PLA-COOH), carboxyl-terminated polylactic acid/glycolic acid copolymer (PLGA-COOH), polystyrene, difatty acid and sebacic acid copolymer ( PFAD-SA), poly(erucic acid dimer-sebacic acid) [P(EAD-SA)], poly(fumaric acid-sebacic acid) [P(FA-
  • the sustained release microspheres are PLGA sustained release microspheres.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, via skin administration, etc.
  • the appropriate dosage of the pharmaceutical composition of the present invention depends on factors such as the formulation method, the administration method, the patient's age, body weight, sex, morbidity, diet, administration time, administration route, excretion rate, and reaction sensitivity.
  • a variety of doses can generally be readily determined and prescribed to be administered therapeutically or prophylactically as desired by the skilled physician.
  • the pharmaceutical composition of the present invention can be formulated according to a method that can be easily implemented by those of ordinary skill in the art to which the present invention belongs, using a pharmaceutically acceptable carrier and/or excipient, so that it can be prepared into a unit volume form or Prepare by filling into multi-volume containers.
  • the dosage form may also be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet, capsule or gel (eg, hydrogel), and may also contain Dispersant or stabilizer.
  • the pharmaceutical composition of the present invention also includes the combination of the polypeptide of the present invention or its derivatives and other drugs for promoting bone formation.
  • the polypeptide of the present invention or its derivatives and other drugs can be formulated separately or combined in the same preparation for joint administration drug, which is determined by the nature of the polypeptide to be administered and the formulation compatibility of the polypeptide of the present invention or its derivative. Co-administration can be sequential, including on the same day or staggered on different days.
  • a suitable dosing regimen should be suitable for the effective administration of the polypeptides of the invention or derivatives thereof with other pharmaceutical or therapeutic ingredients.
  • drugs that promote bone formation include teriparatide, raloxifene.
  • the present invention provides nucleic acid molecules encoding the aforementioned polypeptides or polypeptide derivatives.
  • the present invention provides a vector comprising the aforementioned nucleic acid molecule.
  • a vector refers to a molecule that can carry an inserted polynucleotide into a host cell.
  • carriers are liposomes, biocompatible polymeric micelles, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses, such as Baculoviruses, adenoviruses and retroviruses, phages, cosmids, plasmids, fungal vectors and others commonly used in the art for expression in various eukaryotic and prokaryotic hosts, potentially for gene therapy, and for simple protein Expression recombinant vector.
  • the present invention provides a host cell comprising the aforementioned nucleic acid molecule, or the aforementioned vector.
  • a method of promoting the differentiation, proliferation, maturation or calcification of osteoblasts or cells capable of differentiating into osteoblasts comprising administering the polypeptide or polypeptide as described above Derivatives, or pharmaceutical compositions as previously described.
  • the methods include non-diagnostic and therapeutic purposes.
  • the methods of the invention may be used to promote the differentiation, proliferation, maturation or calcification of osteoblasts or cells capable of differentiating into osteoblasts in vitro. Using this method can be used to study the molecular mechanisms of differentiation, proliferation, maturation and calcification. In addition, this method can also be used to prepare mature osteoblasts.
  • the cells that can differentiate into osteoblasts include osteoblast precursor cells, mesenchymal stem cells, stromal cells, and myoblasts.
  • the present invention provides a method for preventing or treating a bone disease, the method comprising administering the aforementioned polypeptide or polypeptide derivative, or the aforementioned medicament to a subject in need thereof combination.
  • the present invention provides a method of enhancing bone formation in a subject in need thereof, the method comprising administering to the subject in need thereof the aforementioned polypeptide or polypeptide derivative, or the aforementioned pharmaceutical composition.
  • the present invention provides a method of inducing bone deposition in a subject in need thereof, the method comprising administering to the subject in need thereof the aforementioned polypeptide or polypeptide derivative, or the aforementioned pharmaceutical composition.
  • the present invention provides a method of inducing bone maturation in a subject in need thereof, the method comprising administering to the subject in need thereof the aforementioned polypeptide or polypeptide derivative, or the aforementioned pharmaceutical composition.
  • the subject of the present invention includes a mammal, and further, the subject of the present invention is a human.
  • the present invention provides a method for expanding stem cells in vitro, the method comprising combining the aforementioned polypeptide or polypeptide derivative, or the aforementioned pharmaceutical composition with a contact with stem cells.
  • the present invention provides the use of the aforementioned polypeptide, the use comprising the use according to any one of the following:
  • the bone diseases include osteoporosis, rickets, osteomalacia, osteogenesis imperfecta, marbled bone disease, fibrous dysplasia, postmenopausal osteoporosis, senile osteoporosis in men and women, glucocorticoids Hormone-induced osteoporosis, immobilized osteoporosis, weightlessness-induced osteoporosis, post-transplant osteoporosis, migratory osteoporosis, idiopathic osteoporosis, juvenile osteoporosis disease, Paget's disease, chronic hyperparathyroidism, hyperthyroidism, rheumatoid arthritis, Gorham-Stout disease, McCune-Albright syndrome, osteolytic metastases of various cancers, or multiple myeloma , bone mass loss, systemic bone fragility, joint degeneration, non-union fractures, orthopedic and dental problems caused by diabetes, implanted periodontitis, adverse reactions to bone grafts/implants/bone
  • the present invention provides the use of the aforementioned polypeptide derivatives, the use comprising the use according to any one of the following:
  • the bone disease includes osteoporosis, rickets, osteomalacia, osteogenesis imperfecta, marbled bone disease, fibrous dysplasia, Paget's disease, chronic hyperparathyroidism, hyperthyroidism, Rheumatoid arthritis, Gorham-Stout disease, McCune-Albright syndrome, osteolytic metastases of various cancers or multiple myeloma, bone loss, generalized bone fragility, joint degeneration, non-healing fractures, caused by diabetes Orthopedic and dental problems, implanted periodontitis, adverse reactions to bone grafts/implants/bone substitutes, 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 repair, bone marrow defects, bone disease in patients with acromegaly, cystic fibrosis-related bone disease, adynamic bone disease, Renal osteodyst
  • peptide refers to a compound consisting of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids linked by peptide bonds.
  • peptides are about 2 to 50 amino acids in length.
  • the peptides of the present invention are about 2 to about 20 amino acids in length, more preferably 5 to 20 amino acids, and most preferably 5 amino acids in length.
  • amino acid refers to natural, non-natural, and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids.
  • natural amino acids refers to those amino acids encoded by the genetic code, as well as those amino acids that have been modified later, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Naturally occurring alpha-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine ( Gly), Histidine (His), Isoleucine (Ile), Arginine (Arg), Lysine (Lys), Leucine (Leu), Methionine (Met), Asparagine (Asn) , Proline (Pro), Glutamine (Gln), Serine (Ser), Threonine (Thr), Valine (Val), Tryptophan (Trp), Tyrosine (Tyr) and combinations thereof .
  • Stereoisomers of natural amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamine Acid (D-Glu), D-Phenylalanine (D-Phe), D-Histidine (D-His), Dileucine (D-Ile), D-Arginine (D-Arg) , D-Lysine (D-Lys), D-Leucine (D-Leu), D-Methionine (D-Met), D-Asparagine (D-Asn).
  • unnatural amino acid includes, but is not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, M'-modified lysines, and L- or D-configurations that function in a manner similar to natural amino acids of methyl amino acids.
  • Unnatural amino acids are not encoded by the genetic code and may, but need not, have the same basic structure as natural amino acids.
  • the unnatural amino acid is selected from: 2-amino fatty acid, 3-amino fatty acid, ⁇ -alanine, ⁇ -aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidinecarboxylic acid, 6-amino acid Caproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, Chaintin, 2,2'-diaminoheptanoic acid Diacid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, isohydroxylysine, 3-hydroxyproline, 4-hydroxyproline , Isosin, isoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, nor
  • amino acid analog refers to a compound that has the same basic chemical structure as a natural amino acid, ie a carbon to which hydrogen, carboxyl, amino and R groups are bound, eg homoserine, norleucine, methylsulfide amino acid sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the natural amino acid.
  • amino acid mimetic refers to a compound whose structure differs from the general chemical structure of an amino acid, but which functions similarly to a natural amino acid.
  • Suitable amino acid mimetics include, but are not limited to, beta-amino acids and gamma-amino acids.
  • beta-amino acids the amino group is bonded to the beta-carbon atom of the carboxyl group so that there are two carbon atoms between the amino group and the carboxyl group.
  • gamma-amino acids the amino group is bonded to the gamma-carbon atom of the carboxyl group so that there are three carbon atoms between the amino group and the carboxyl group.
  • Suitable R groups for beta- or gamma-amino acids include, but are not limited to, side chains found in natural and unnatural amino acids.
  • D-amino acid refers to the dextrorotatory stereoisomer of an amino acid.
  • the letters D and L are commonly used in the art to refer to stereoisomers of amino acids.
  • D-amino acids are those amino acids that can be synthesized from the dextroisomer of glyceraldehyde, D-glyceraldehyde.
  • L-amino acids are those amino acids that can be synthesized from the levorotatory isomer of glyceraldehyde, ie, L-glyceraldehyde.
  • treatment refers to any type of treatment that is beneficial to a subject suffering from a disease, including improving the patient's condition (eg, in one or more symptoms), delaying the disease progress, etc.
  • Subjects generally refer to human subjects and are used interchangeably.
  • Subjects can be male or female, and can be of any race or ethnicity, including but not limited to Caucasian, African American, African, Asian, Hispanic, Indian, and the like.
  • Subjects can be of any age, including neonates, infants, toddlers, children, adolescents, adults, and the elderly.
  • the subject has a bone disease.
  • Subjects may also include animal subjects, particularly mammalian subjects, such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (eg rats and mice), lagomorphs, primates (including non-human primates), etc., for the purpose of veterinary drug or medical drug development.
  • animal subjects particularly mammalian subjects, such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (eg rats and mice), lagomorphs, primates (including non-human primates), etc.
  • bone disease refers to any of those diseases that cause various abnormalities or deformities of one or more bones and/or bone cells.
  • the present invention reports two pentapeptides (ESSES or DSSDS) for the first time.
  • Said pentapeptides have osteogenesis activity and can increase bone mass in various models.
  • the curative effect is no less than that of PTH. Its chemical properties are stable and easy to be synthesized on a large scale at low cost. .
  • the polypeptide B5P of the present invention exhibits excellent biocompatibility in animal experiments, and acute toxicity and reproductive toxicity have not been observed for the time being.
  • the polypeptide B5P of the present invention has a wide range of applications, and can not only be used for the treatment of osteoporosis. It can also promote fracture repair, osseointegration of dental implants and other diseases.
  • Figure 1 shows the activity detection chart of different length polypeptides, wherein, A: ESSES; B: DSSDS;
  • Figure 2 shows the activity detection diagram after extending irrelevant amino acids at both ends of the polypeptide, wherein A: ESSES; B: DSSDS;
  • Figure 3 shows the detection chart of polypeptide activity incorporating unnatural D-type amino acids, wherein A: ESSES; B: DSSDS;
  • Figure 4 shows the modified polypeptide activity detection chart, wherein, A: ESSES; B: DSSDS;
  • Figure 5 shows the activity detection chart of the polypeptide conjugate, wherein, A: ESSES; B: DSSDS;
  • Figure 6 shows the activity detection chart of the polypeptide sustained-release dosage form, wherein, A: ESSES; B: DSSDS;
  • Figure 7 shows the results of Micro-CT scan of wild-type mice, wherein A: scan of trabecular bone; B: scan of local trabecular bone; C: scan of cortical bone cross-section; D: bone volume fraction of cortical bone; E: Femoral bone volume fraction; F: trabecular number; G: trabecular separation; H: bone material density;
  • Figure 8 shows the H&E staining pattern of wild-type mice
  • Figure 9 shows the results of the wild-type mouse double fluorescent labeling experiment, wherein, A: staining chart; B: statistical chart;
  • Figure 10 shows the results of Von Kossa staining of wild-type mice, wherein A: staining chart; B: statistical chart;
  • Figure 11 shows the results of detection of Dmp-1, a bone mineralization-related index in wild-type mice, wherein A: staining chart; B: statistical chart;
  • Figure 12 shows the results of Micro-CT scan of OVX mice, wherein A: scan of trabecular bone in OVX+ctrl group; B: scan of trabecular bone in OVX+B5P group; C: scan of trabecular bone in OVX+PTH group; D: femoral bone volume fraction; E: number of trabecular bone; F: trabecular bone thickness; G: bone material density;
  • Figure 13 shows the results of cortical bone in OVX mice, wherein, A: scan of cortical bone in OVX+ctrl group; B: scan of cortical bone in OVX+B5P group; C: scan of cortical bone in OVX+PTH group; D: cortical bone Bone volume fraction;
  • Figure 14 shows the results of the double fluorescent labeling experiment of OVX mice, wherein, A: OVX+ctrl group staining; B: OVX+ctrl group staining; C: OVX+PTH group staining; D: MAR;
  • Figure 15 shows the H&E staining chart of OVX mice, wherein A: staining chart of OVX+ctrl group; B: staining chart of OVX+ctrl group; C: staining chart of OVX+PTH group;
  • Figure 16 shows the Von Kossa staining chart of OVX mice, wherein A: staining chart of OVX+ctrl group; B: staining chart of OVX+ctrl group; C: staining chart of OVX+PTH group;
  • FIG 17 shows the results of the fluorescent double-labeling experiment in mice with fractures, wherein A: Fracture+ctrl; B: Fracture+B5P;
  • Figure 18 shows the results of H&E staining of fractured mice, wherein A: Fracture+ctrl; B: Fracture+B5P;
  • Figure 19 shows the results of Micro-CT scanning of fractured mice, wherein A: Fracture+ctrl; B: Fracture+B5P;
  • FIG 20 shows the results of detection of bone mineralization-related index Dmp-1 in fracture mice, wherein A: Fracture+ctrl; B: Fracture+B5P;
  • Fig. 21 shows the results of Micro-CT scan of the femoral titanium nail implantation model mouse, wherein, A: implant+ctrl scan; B: implant+B5P scan; C: bone volume fraction; D: bone trabecular thickness;
  • Figure 22 shows the results of VanGieson staining in the femoral titanium nail implantation model mouse, wherein, A: implant+ctrl staining; B: implant+B5P scan; C: bone surface area/titanium nail surface area;
  • Figure 23 shows the results of a fluorescent double-labeling experiment in a mouse model of femoral titanium nail implantation, wherein A: implant+ctrl staining; B: implant+B5P scan; C: MAR;
  • Figure 24 shows the results of Von Kossa staining and bone formation in femoral titanium nail implantation model mice, wherein, A: Von Kossa staining of implant+ctrl; B: Von Kossa staining of implant+B5P; C: implant Bone formation staining diagram of +ctrl; D: bone formation staining diagram of implant+B5P; E: statistical diagram;
  • Figure 25 shows the results of Micro-CT scan of aged osteoporotic rhesus monkeys, wherein, A: scan chart of control group; B: scan chart of B5P group; C: BV/TV; D: TB.n; E: TB.th;
  • Figure 26 shows the results of Q-CT scan of aged osteoporotic rhesus monkeys, wherein, A: the bone density of the hip; B: the bone density of the second to fourth lumbar vertebrae;
  • Figure 27 shows the H&E staining diagram of aged osteoporotic rhesus monkeys, wherein, A: ctrl; B: B5P;
  • Figure 28 shows the results of the three-point bending experiment, where A: flexural strength; B compressive strength:
  • Figure 29 shows the results of QPCR experiments, wherein A: Runx2; B: Osx; C: Alpl; D: Ocn;
  • Figure 30 shows a mineralization staining map
  • Figure 31 shows the results of the signal pathway, wherein, A: western blot; B: ALP staining; C: Alizarin red staining;
  • Figure 32 shows the results of the hemagglutination experiment for evaluating the biosafety of polypeptide B5P, wherein A: control; B: polypeptide;
  • Figure 33 shows the results of ELISA experiments for evaluating the biosafety of polypeptide B5P, wherein A: BUN; B: CK; C: ALT; D: ALS;
  • Figure 34 shows the H&E staining chart for evaluating the biosafety of polypeptide B5P
  • Figure 35 shows the effect of polypeptide B5P on serum biochemical indexes of aged rhesus monkeys, wherein, A: CK; B: ALT; C: CKM; D: ALS; E: blood glucose; F: cholesterol; G: BUN; H: HDLC.
  • Detection reagents phenol reagent (self-preparation), pyridine reagent (self-preparation), ninhydrin reagent (self-preparation);
  • Cleavage reagent 95% cutting solution: TFA (J.T. Baker, 99%), TIS (Shanghai Darui Fine Chemicals, 98%), EDT (Shanghai Darui Fine Chemicals, 98%), anhydrous ether (Shanghai Experiment, Measured 99.7%);
  • polypeptide synthesis is as follows: the synthesis sequence is from C-terminus to N-terminus.
  • Detection remove the piperidine solution, take more than ten resins, wash with ethanol three times, add detection reagent for detection, heat at 105°C-110°C for 5 minutes, and turn dark blue as a positive reaction.
  • Cleavage polypeptide from resin prepare cutting solution (10/g) TFA 95%; water 1%; EDT 2%; TIS 2%; cutting time: 120min.
  • Freeze-drying collect the target polypeptide solution, concentrate in a freeze-drying machine, and freeze-dry it into a white powder.
  • the osteoblast cell line MC-3T3E1 cells were cultured and incubated with polypeptides of different lengths at a final concentration of 1 ⁇ g/ml for 24 hours, and then the ALP gene expression levels were detected by QPCR.
  • the osteoblast cell line MC-3T3E1 cells were cultured and incubated with polypeptides of different lengths at a final concentration of 1 ⁇ g/ml for 24 hours, and then the ALP gene expression levels were detected by QPCR.
  • DSSDS had no effect on its activity when 1 unrelated amino acid was extended on one side, and had little effect on its activity when 2 unrelated amino acids were extended on one side, and the activity decreased significantly when it was extended by more than 3 unrelated amino acids.
  • the activity had no effect, and the activity began to decrease when the two sides were extended by more than 2 irrelevant amino acids.
  • Glycine (G) is a representative of unrelated amino acids.
  • the osteoblast cell line MC-3T3E1 cells were cultured and incubated with polypeptides of different lengths at a final concentration of 1 ⁇ g/ml for 24 hours, and then the ALP gene expression levels were detected by QPCR.
  • the osteoblast cell line MC-3T3E1 cells were cultured and incubated with polypeptides of different lengths at a final concentration of 1 ⁇ g/ml for 24 hours, and then the ALP gene expression levels were detected by QPCR.
  • polypeptide conjugates are shown in Table 3.
  • the osteoblast cell line MC-3T3E1 cells were cultured and incubated with the polypeptide conjugate at a final concentration of 2 ⁇ g/ml for 24 hours, and the ALP gene expression level was detected by QPCR.
  • ESSES represented by sodium alendronate
  • ESSES has activity after coupling with bisphosphonates.
  • Geniposide as the representative ESSES has activity after coupling with Chinese medicine monomers.
  • Represented by RGD ESSES has activity after coupling with oligopeptide.
  • DSSDS represented by sodium alendronate
  • Geniposide as the representative of DSSDS has activity after coupling with Chinese medicine monomers.
  • RGD as the representative DSSDS, it has activity after coupling with oligopeptide.
  • the outer 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 to obtain PLGA microspheres.
  • the NaOH solution containing the polypeptide was added to the PLGA microspheres, mixed evenly, and placed on a shaker to continue the reaction.
  • the reacted PLGA microspheres were repeatedly centrifuged and washed with deionized water, and freeze-dried for later use.
  • the osteoblast cell line MC-3T3E1 cells were cultured and incubated with microspheres with a final concentration of 2 ⁇ g/ml for 1-7 days, and the ALP gene expression level was detected by QPCR.
  • mice were SPF grade, purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd.
  • polypeptide B5P solution Weigh 1.000 grams of sodium hydroxide in a beaker, add a small amount of physiological saline to dissolve, then pour it into a 1000 ml volumetric flask, wash the beaker three times, pour all the solution into the volumetric flask, and finally use Normal saline was diluted to the mark. After shaking, 1000 mg/mL of polypeptide storage stock solution was obtained. When using, it should be diluted in equal proportion to the desired concentration.
  • mice were randomly divided into two groups, the control group and the experimental group.
  • the mice in the control group were injected with saline three times a week, and the mice in the experimental group were injected with 20 mg/kg polypeptide B5P three times a week; femur samples were collected one month later.
  • the collected femur samples were scanned by Micro-CT to analyze bone volume fraction (BV/TV); trabecular bone number (TB.N); trabecular bone separation (TB.Sp); bone material density (BMD).
  • BV/TV bone volume fraction
  • T.N trabecular bone number
  • T.Sp trabecular bone separation
  • BMD bone material density
  • the scanning voltage is 70kVp
  • the scanning energy power is 14W
  • the scanning current is 200 ⁇ A
  • the exposure time is 300ms
  • the scanning BH is 1200mg HA/cc
  • the scanning accuracy is 10 ⁇ m
  • the scanning filter (Filter) is 0.5mm AI Filter. Later image data were reconstructed and analyzed using Mimics 13.0 software.
  • the trabecular bone data analysis was selected from 1mm below the femoral growth plate and all the trabecular bone in the inner layer of cortical bone; the cortical bone data analysis was selected from the cortical bone 5mm-6mm below the growth plate.
  • Micro-CT scan results are shown in Figure 7.
  • the density of trabecular bone in mice was significantly increased, the thickness of trabecular bone was significantly increased, and the thickness of cortical bone was increased.
  • the bone volume fraction (BV/TV) and the number of trabecular bone (TB.N) in the femur of mice were significantly increased, and the degree of trabecular bone separation (TB.Sp) was decreased, indicating that the bone mineral density of the mouse femur was increased and the distribution of trabecular bone was increased. denser; the bone material density (BMD) index increased, indicating that the degree of mineralization of the mouse femur was enhanced.
  • BMD bone material density
  • Sectioning paraffin sections of the tissue are prepared in a conventional manner
  • Dewaxing and hydration Deparaffinize the tissue sections in sequence, xylene I for 30 min, xylene II for 30 min. Then, the slices 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.
  • DAPI staining 1:1000 diluted DAPI staining solution was added dropwise to hard tissue sections for staining for 5 minutes. Soak and rinse 3 times in PBS for 5 min each time. Mount slides with anti-fluorescence quenching mounting medium. Red and green two-color fluorescent markers were observed under an inverted fluorescence microscope, and the bone deposition rate was counted.
  • Staining with silver nitrate staining solution configure 0.2% silver nitrate solution, drip it on the femoral hard tissue slices, cover the femoral tissue, irradiate with strong light for 15 minutes, and rinse the slices with running water for 2-3 s.
  • Methyl green lining dyeing lining dyeing with methyl green dye solution for 5 minutes, rinse with running water for 5 s, rinse off the floating color, and dry in an oven at 65°C for 30 minutes.
  • Tissue sealing drop an appropriate amount of normal non-immune animal serum onto the slices, and incubate at room temperature for 10 min.
  • DAB color development add DAB color development solution (currently used) dropwise, and observe under the microscope for 3-10min. After the color development is over, rinse with tap water to stop the color development.
  • mice were anesthetized with sodium pentobarbital at a dose of 35 mg/kg.
  • mice (3) The skin of the lower abdomen of the mice was prepared, sterilized with iodophor, and the iodophor was wiped off with 75% alcohol.
  • the osteoporosis model mice were injected with polypeptide B5P through the tail vein three months after modeling, and after the model became stable, for a total of 4 weeks, 3 times a week, at a dose of 20 mg/kg; PTH was administered The way is subcutaneous administration, the dose is 0.1 ⁇ g/kg, injected for 4 weeks, 3 times a week.
  • mice Compared with the control group, the bone mass of the mice was significantly increased under the action of polypeptide B5P and PTH, and the bone mineralization degree of the mice in the B5P group and the PTH group was significantly increased.
  • mice were injected with 20 mg/kg polypeptide B5P three times a week, and two weeks later, the callus formation and callus density at the fractured ends of the mice were evaluated.
  • 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 ⁇ m above and below the fracture end, and the data analysis is selected from the callus outside the cortical bone of the corresponding part.
  • the results are shown in Fig. 19.
  • the fracture healing of the fracture site was accelerated in the B5P injection group in the fracture model mice.
  • the results of callus reconstruction showed that the callus after the polypeptide B5P injection was denser than that in the control group, and the callus density in the B5P group was significantly higher than that in the control group.
  • Example 5 The effect of polypeptide B5P on implant osseointegration
  • mice Twelve 8-week-old female WT mice were selected and divided into two groups (control group, titanium nail+B5P group). Mice were anesthetized with sodium pentobarbital at a dose of 35 mg/kg.
  • a 1mm drill was installed on the handpiece at a slow speed, and the lateral cortical bone of the middle femur was drilled.
  • mice were injected with drugs at the second week after operation, and the injection was carried out for 3 weeks in total, 3 times a week, at a dose of 20 mg/kg, and samples were taken at the fourth week after operation.
  • the steps are basically the same as in Example 1. The difference is: when the titanium nail data is reconstructed, the analysis of the bone volume data around the titanium nail is selected from the inner diameter of the titanium nail with an inner diameter of 1 mm and an outer diameter of 1.1 mm.
  • the bone threshold is 212-535, and the titanium nail threshold is 535-1000.
  • VanGieson staining was performed according to conventional methods.
  • the results are shown in Figure 23.
  • the left side is a titanium nail.
  • the surface of the titanium nail is covered with bone tissue.
  • the calcein and xylenol orange markers are separated by one week. After the polypeptide B5P injection, the bone deposition rate on the surface of the titanium nail is significantly increased.
  • Example 6 The effect of polypeptide B5P on bone mass in senile osteoporotic rhesus monkeys
  • Steps Use the WD-T1 animal bone three-point bending strength testing machine of Shanghai Qixiang Testing Instrument Co., Ltd. for super-testing.
  • the bone specimen is placed on two supporting points with a certain distance, and a downward load is applied to the specimen at the midpoint of the two supporting points.
  • Three-point bending occurs when the three contact points of the bone specimen form two equal moments. .
  • Example 7 The effect of polypeptide B5P on the osteogenic differentiation of stem cells
  • polypeptide B5P By adding polypeptide B5P to the bone marrow mesenchymal stem cells (BMSCs) cultured in vitro and conducting mineralization induction, the expression of osteogenesis-related genes and mineralization-related staining were detected, and the ability of polypeptide B5P to promote mineralization in vitro was verified.
  • BMSCs bone marrow mesenchymal stem cells
  • BMSCs bone marrow mesenchymal stem cells
  • Cell RNA The above cells were taken, the cell culture medium was aspirated, and the cells were washed three times with PBS. Add 1ml of Trizol reagent, pipetting gently, and transfer it to an RNase-inactivated 1.5ml EP tube.
  • the primer sequences are shown in Table 5.
  • polypeptide B5P By culturing mouse BMSCs, 0.5 ⁇ g/ml or 1 ⁇ g/ml polypeptide B5P was added, and mineralization was induced. ALP staining was performed on the 7th day after mineralization induction to observe the osteogenic differentiation effect of BMSCs. On the 21st day after mineralization induction, Alizarin red and Von kossa staining were performed to observe the mineralization effect of BMSCs. The results are shown in Figure 30. ALP staining was enhanced after 7 days of BMSCs mineralization induction, and the formation of mineralized nodules in BMSCs was significantly increased. It is suggested that the polypeptide B5P can promote the osteogenic differentiation of BMSCs.
  • integrin receptor an important receptor in the FAK-AKT pathway, by inhibiting the integrin receptor (integrin ⁇ 5) of the FAK-AKT pathway to study its effect on the osteopromoting effect of B5P
  • Steps Digest when the cells grow to occupy 90% of the bottom of the bottle, and inoculate 200,000 cells per well in a 6-well culture plate. When the cells grow to 40% of the bottom of the plate, group them for transfection according to the instructions of Lipofectamine 3000. Control group The sequence of transfected NC siRNA was CGGAAGCTGACCCTGAAGT (SEQ ID NO. 11), and the sequence of integrin ⁇ 5 siRNA transfected in the experimental group was GGACCAGGAAGCTATTTCT (SEQ ID NO. 12). Cells were harvested 48 hours after transfection for assays.
  • FIG. 31B The results of ALP staining (FIG. 31B) and Alizarin red staining (FIG. 31C) showed that the osteopromoting effect of B5P was blocked after inhibition of integrin ⁇ 5. It is suggested that B5P may promote the osteogenic differentiation of stem cells by activating integrin ⁇ 5 and then activating the FAK-AKT signaling pathway.
  • biosafety of polypeptide B5P was investigated by means of hemagglutination, serum biochemical index evaluation and pathological section analysis of important organs.
  • the serum BUN blood urea nitrogen
  • CK creatine kinase
  • ALT alanine aminotransferase
  • ALS aldosterone
  • the right hand holds the capillary from the inner canthus Insert the conjunctiva, then gently push it towards the bottom of the eye, gently rotate the capillary to cut through the venous plexus, and allow the blood to flow out along the capillary and receive it into a pre-prepared container. After blood collection, apply gauze to the eye to stop bleeding.
  • Serum preparation use a test tube without pyrogen and endotoxin (need to avoid any cell stimulation during subsequent experimental operations) after collecting blood, centrifuge at 3000 rpm for 10 minutes at room temperature, carefully pipette, and separate serum and red blood cells.
  • HRP horseradish peroxidase
  • 80 pregnant rats were screened by pairing male and female SPF Wistar rats, and divided into solvent control group and 4 groups of low, medium and high doses, with 20 rats in each group. Uniform intravenous peptide injection, 2 times a week, for 2 weeks. The rats were sacrificed on the 18.5th day of gestation. Immediately after dissection, the number of uterine implantation and the number of live births and stillbirths, the weight of the uterus, the weight of the placenta, and the number of corpora lutea were measured after ovary HE sectioning.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种促骨形成多肽及其应用，所述多肽能增强成骨细胞或能分化为成骨细胞的细胞的分化、成熟或钙化。另外，用于治疗或预防骨疾病的药物组合物，该药物组合物包含作为有效成分的促骨形成多肽。促骨形成多肽化学性质稳定，容易低成本大规模合成，且生物相容性高，无毒性，可广泛用于骨疾病的预防和治疗。

Description

一种促骨形成多肽及其应用 技术领域
本发明属于生物医药领域，涉及一种骨形成多肽及其应用。
背景技术
骨是通过在骨形态发生中重复形成和吸收/破坏而不断进行骨的重建并维持血清钙浓度的活动器官。一般而言，由成骨细胞引起的骨形成和由破骨细胞引起的骨吸收通常处于平衡状态。骨量可通过这些细胞间的相互反应的机制而维持在恒定水平上。当平衡状态因绝经、衰老、炎症等而遭到破坏时，就会因骨质疏松症或类风湿性关节炎导致破坏而引起代谢性骨病。这种代谢性骨病的发生在目前的老龄化社会中是一个严重问题。因此，在分子水平上阐明这种疾病的病理机制并开发有效治疗药是迫切任务。
包括代谢性骨疾病在内的骨疾病的实例包括质疏松症、骨质减少、成骨不全、骨坏死、低骨量、佩吉特病、骨转移、无菌假体松动、牙周炎、转移性骨病、类风湿性关节炎、狼疮关节炎、牙周病、牙槽骨丢失、截骨术后、儿童特发性骨质流失、脊柱弯曲、身高下降和假体手术等。
迄今为止，抑制骨吸收过程、而不是增强骨形成的骨吸收抑制剂已用作表现出骨丢失的骨代谢疾病(例如骨质疏松症)的治疗药。能抑制骨吸收并用于或建议用于治疗骨质疏松症的药物实例包括雌激素、选择性雌激素受体调节剂(SERM)、依普黄酮、维生素K2、钙剂、骨化三醇、降钙素和二膦酸盐例如阿仑膦酸盐。然而，采用以上药物的治疗方法在疗效和结果上并不总是令人满意的。因此，一直都在期待进一步开发安全有效的新治疗药。
特立帕肽作为仅有的促进骨形成药物，临床数据显示其可显著增加绝经后女性全身多处骨骼的骨密度，在男性中也可增加腰稚和股骨颈骨密度，因此在各类指南被推荐使用。FDA批准特立帕肽用于持久性、全身性重度骨质疏松治疗。特立帕肽适用于有骨折高发风险的绝经后妇女骨质疏松症的治疗。可显著降低绝经 后妇女椎骨和非椎骨骨折风险，但对降低髋骨骨折风险的效果尚未证实。
特立帕肽属于甲状旁腺激素PTH的分子片段(1-34)。病人终身仅可接受一次最长为24个月的治疗。常见副作用恶心、腿抽筋、眩晕，增加骨肉瘤机率。治疗费用月均6000元以上，至少3个月起效，给患者带来了极大的经济压力。因此亟待开发一种新的促骨形成药物，打破特立帕肽的垄断地位。
发明内容
为了解决现有技术存在的问题，本发明的目的在于提供了一种用于促骨形成的多肽及其衍生物。
根据本发明的一个方面，本发明提供了一种多肽，所述多肽序列由丝氨酸，和谷氨酸或天冬氨酸两种氨基酸组成，所述多肽含有2-50个氨基酸，所述丝氨酸，和谷氨酸或天冬氨酸的个数比为(0.25～2)：1；
进一步，所述多肽具有如下通式：(XSSXS)n，其中，X为谷氨酸或天冬氨酸，S为丝氨酸，n为自然数；
进一步，在本发明的具体实施方案中，n为1至8。
优选地，n为1、2、3、4；
更优选地，n为1。
根据本发明的另一个方面，本发明提供了一种多肽衍生物，所述多肽衍生物包括前面所述的多肽的修饰产物、前面所述的多肽经一个或多个氨基酸添加和/或替换后获得的变体，前面所述的多肽与其他物质的偶联物。
在本发明中的术语“修饰”是指对肽作出的任何改变，例如肽长度的改变、氨基酸序列的改变、化学结构的改变、肽的共翻译修饰或翻译后修饰。在一些情况下，本发明的肽包含一个或多个经修饰的氨基酸残基。修饰的类型在本领域中是公知的。
可用于本发明的修饰包括甲基化、豆蔻酰化、PEG修饰、氟元素修饰、生物素修饰、荧光标记修饰、环化、羧化、脂肪酸修饰、乙酰化修饰、磷酸化修饰、糖基化修饰、酰胺化修饰或其他已知的多肽修饰。
在本发明的具体实施方案中，所述修饰包括甲基化、豆蔻酰化、PEG修饰、氟元素修饰、生物素修饰、荧光标记修饰、环化。
上述氨基酸的修饰起到很大程度上改善本发明的肽的稳定性的作用。在本发明中所使用的术语“稳定性”不仅意味着“体内”稳定性，而且还意味着储存稳定性(例如，常温储存稳定性)。上述保护基起到从生物体内的蛋白质切割酶的攻击保护本发明的肽的作用。
本发明所述的氨基酸添加和/或替换包括在前面所述的多肽序列的氨基端、羧基端或内部任意位置进行添加和/或替换氨基酸。
进一步，所述添加和/或替换氨基酸包括天然氨基酸或非天然氨基酸。
更进一步，所述变体是在前面所述的多肽的氨基端或羧基端添加1至3个无关氨基酸获得的。
可用于本发明的无关氨基酸包括甘氨酸、色氨酸、酪氨酸、半胱氨酸、蛋氨酸、谷氨酰胺和苏氨酸。
在本发明的具体实施例中，所述变体是在ESSES或DSSDS多肽的氨基端或羧基端添加1至3个甘氨酸获得的。
更进一步，所述变体是在前面所述的多肽的氨基端和羧基端同时添加1至2个无关氨基酸获得的。
可用于本发明的无关氨基酸包括甘氨酸、色氨酸、酪氨酸、半胱氨酸、蛋氨酸、谷氨酰胺和苏氨酸。
在本发明的具体实施例中，所述变体是在ESSES或DSSDS多肽的氨基端和羧基端同时添加1至2个甘氨酸获得的。
更进一步，所述变体是在前面所述的多肽的羧基端替换1个氨基酸获得的；优选地，所述变体是在前面所述的多肽的羧基端用D型氨基酸的非天然氨基酸替换天然氨基酸获得的；更优选地，所述变体是在前面所述的多肽的羧基端用丝氨酸的非天然D型氨基酸替换天然氨基酸获得的。
在本发明的具体实施例中，所述变体是在ESSES或DSSDS多肽的羧基端用用丝氨酸的非天然D型氨基酸替换天然氨基酸获得的。
可与本发明的前面所述的多肽偶联的其他物质包括但不限于载体蛋白、外源多肽、药物。
进一步，载体蛋白包括血清白蛋白、血蓝蛋白、鸡卵白蛋白。
进一步，所述外源多肽包括Fc区、信号肽、多肽标记。
Fc区选自以下组：IgA Fc区，IgD Fc区，IgG Fc区，IgE Fc区，IgM Fc区。
多肽标记选自以下组：Flag标记、strep标记、聚组氨酸标记、VSV-G标记、流感病毒血凝素标记、c-Myc标记。
进一步，所述药物包括双膦酸盐类药物、环烯醚萜化合物。
所述双膦酸盐类药物包括阿仑膦酸、伊班膦酸盐、唑来膦酸。
环烯醚萜化合物包括京尼平尼酸、京尼平-龙胆双糖苷、京尼平苷、京尼平苷酸。
根据本发明的又一个方面，本发明提供了前面所述的多肽或多肽衍生物的可药用盐或酯。
进一步，可药用盐包括醋酸盐、盐酸盐。
根据本发明的又一个方面，本发明提供了前面所述的多肽的制备方法。
可以使用本领域已知的任何合适的方式制备可用于本发明技术的多肽。此类多肽包括分离的天然存在的多肽、重组产生的多肽、合成产生的多肽、或通过这些方法的组合产生的多肽。用于制备此类多肽的手段和方法是本领域熟知的。
根据本发明的又一个方面，本发明提供了一种药物组合物，所述药物组合物包括有效量的前面所述的多肽或多肽衍生物；优选地，所述药物组合物还包括药学上可接受的载体。
包含于本发明的药物组合物的药学上可接受的载体是作为制剂时通常利用的载体，包含所述药学上可接受的载体包括稀释剂、填充剂、赋形剂、粘合剂、润湿剂、崩解剂、泡腾剂、表面活性剂、吸收促进剂、润滑剂、吸附载 体、缓释微球、埋植剂、原位微球、脂质体、微乳、原位水凝胶、纳米粒、蛋白酶抑制剂、生物黏附剂。
缓释微球可以由以下缓释辅料制备而成：外消旋聚乳酸(D，L-PLA)、外消旋聚乳酸/乙醇酸共聚物(D，L-PLGA)、单甲基聚乙二醇/聚乳酸(MPEG-PLA)、单甲基聚乙二醇/聚乳酸共聚物(MPEG-PLGA)、聚乙二醇/聚乳酸(PLA-PEG-PLA)、聚乙二醇/聚乳酸共聚物(PLGA-PEG-PLGA)、端羧基聚乳酸(PLA-COOH)、端羧基聚乳酸/乙醇酸共聚物(PLGA-COOH)、聚苯丙生、双脂肪酸与癸二酸共聚物(PFAD-SA)、聚(芥酸二聚体-癸二酸)[P(EAD-SA)]、聚(富马酸-癸二酸)[P(FA-SA)]、乙烯乙酸乙烯酯共聚物(EVAc)、聚乳酸(PLA)、聚乙醇酸和羟基乙酸的共聚物(PLGA)、木糖醇、低聚糖、软骨素、甲壳素、透明质酸、胶原蛋白、明胶、蛋白胶之一或其组合。
在本发明的具体实施方案中，所述缓释微球是PLGA缓释微球。
本发明的药物组合物能够以口服或非口服的方式进行给药，在以非口服的方式给药的情况下，可通过静脉内注射、皮下注射、肌肉注射、腹腔注射、局部给药、经皮给药等的方式进行给药。
本发明的药物组合物的适当的给药量根据制剂化方法、给药方式、患者的年龄、体重、性别、病态、饮食、给药时间、给药途径、排泄速度及反应灵敏度等的因素而多样，一般可容易确定及处方对熟练的医生希望的治疗或预防有效的给药量。
并且，本发明的药物组合物根据本发明所属技术领域的普通技术人员可容易实施的方法，利用药学上可接受的载体和/或赋形剂来进行制剂化，从而可制备成单位容量形态或装入多容量容器内来制备。此时，剂型还可以为油性或水性介质中的溶液、悬浮液或乳化液形态或浸膏剂、粉剂、颗粒剂、片剂、胶囊剂或凝胶(例如，水凝胶)形态，还可包含分散剂或稳定剂。
另外，本发明的药物组合物还包括本发明的多肽或其衍生物与其他促进骨形成药物的药物组合，本发明的多肽或其衍生物和其他药物可以分别制剂或合并在同一制剂里联合给药，这根据给药的多肽性质和本发明的多肽或其衍生物的制剂相容性决定。联合给药可以是按顺序给药，包括在同一天或交错在不同天。本领 域的技术人员应理解适宜的给药方案是应适于本发明的多肽或其衍生物与其它药物或治疗成分的有效给药。促进骨形成药物的实例包括特立帕肽、雷洛昔芬。
根据本发明的又一个方面，本发明提供了编码前面所述的多肽或多肽衍生物的核酸分子。
根据本发明的又一个方面，本发明提供了包含前面所述的核酸分子的载体。
载体是指可以携带插入的多核苷酸进入宿主细胞的分子。载体实例有，脂质体、生物相容性聚体胶束、包括天然聚体和合成聚体；脂蛋白；多肽；多糖；脂多糖；人工病毒包膜；金属颗粒；以及细菌、病毒，如杆状病毒、腺病毒和逆转录病毒、噬菌体、粘粒、质粒、真菌载体和其它通常用在本领域的各种真核和原核宿主表达、可能被用于基因治疗、以及用于简单的蛋白表达的重组载体。
根据本发明的又一个方面，本发明提供了包含前面所述的核酸分子，或前面所述的载体的宿主细胞。
根据本发明的又一个方面，本发明提供了一种促进成骨细胞或能分化成成骨细胞的细胞的分化、增殖、成熟或钙化的方法，所述方法包括施用前面所述的多肽或多肽衍生物、或前面所述的药物组合物。
进一步，所述方法包括非诊治目的和诊疗目的。
作为非诊治目的的实例，本发明的方法可以用于体外促进成骨细胞或能分化成成骨细胞的细胞的分化、增殖、成熟或钙化。利用该方法可用于研究分化、增殖、成熟和钙化的分子机制。另外，利用该方法还可用于制备成熟的成骨细胞。
进一步，所述能分化成成骨细胞的细胞包括成骨细胞前体细胞、间充质干细胞、基质细胞、成肌细胞。
根据本发明的又一个方面，本发明提供了一种预防或治疗骨疾病的方法，所述方法包括给有需要的受试者施用前面所述的多肽或多肽衍生物、或前面所述的药物组合物。
根据本发明的又一个方面，本发明提供了一种在有需要的受试者中增强骨形成的方法，所述方法包括给有需要的受试者施用前面所述的多肽或多肽衍生物、或前面所述的药物组合物。
根据本发明的又一个方面，本发明提供了一种在有需要的受试者中诱导骨沉积的方法，所述方法包括给有需要的受试者施用前面所述的多肽或多肽衍生物、或前面所述的药物组合物。
根据本发明的又一个方面，本发明提供了一种在有需要的受试者中诱导骨成熟的方法，所述方法包括给有需要的受试者施用前面所述的多肽或多肽衍生物、或前面所述的药物组合物。
进一步，本发明的受试者包括哺乳动物，更进一步，本发明的受试者是人类。
根据本发明的又一个方面，本发明提供了一种在体外扩增干细胞的方法，所述方法包括将前面所述的多肽或多肽衍生物，或前面所述的药物组合物与来自受试者的干细胞接触。
根据本发明的又一个方面，本发明提供了前面所述的多肽的用途，所述用途包括以下任一项所述的用途：
1)在制备前面所述的多肽衍生物中的用途；
2)在制备前面所述的药物组合物中的用途；
3)在制备促进骨形成的药物中的用途；
4)在制备促进成骨细胞分化、增殖、成熟或钙化的药物中的用途；
5)在制备促进干细胞成骨向分化的药物中的用途；
6)在制备预防或治疗骨疾病的药物中的用途。
进一步，所述骨疾病包括骨质疏松症、佝偻病、骨软化症、成骨不全、大理石骨病、纤维异常增生、绝经后骨质疏松症、男性和女性的老年性骨质疏松症、糖皮质激素诱导的骨质疏松症、制动性骨质疏松症、失重引起的骨质疏松症、移植后骨质疏松症、迁移性骨质疏松症、特发性骨质疏松症、青少年骨质疏松症、佩吉特病、慢性甲状旁腺功能亢进症、甲状腺功能亢进症、类风湿性 关节炎、Gorham-Stout病、McCune-Albright综合征、各种癌症的溶骨性转移或多发性骨髓瘤、骨量丢失、全身骨骼脆弱、关节退变、非愈合性骨折、由糖尿病引起的骨科和牙科问题、植入性牙周炎、对骨移植物/植入物/骨替代性材料的不良反应、牙周病、骨骼老化、骨折、骨缺损、骨移植、植骨、骨癌、关节置换、关节修复、融合、小关节修复、骨质退变、牙种植体和修复、骨髓缺损、肢端肥大症患者的骨病、囊性纤维化相关骨病、无动力性骨病、与慢性肾病相关的肾性骨营养不良、与胱氨酸病相关的骨病和与高草酸尿相关的骨病。
根据本发明的又一个方面，本发明提供了前面所述的多肽衍生物的用途，所述用途包括以下任一项所述的用途：
1)在制备前面所述的药物组合物中的用途；
2)在制备促进骨形成的药物中的用途；
3)在制备促进成骨细胞分化、增殖、成熟或钙化的药物中的用途；
4)在制备促进干细胞成骨向分化的药物中的用途；
5)在制备预防或治疗骨疾病的药物中的用途；
优选地，所述骨疾病包括骨质疏松症、佝偻病、骨软化症、成骨不全、大理石骨病、纤维异常增生、佩吉特病、慢性甲状旁腺功能亢进症、甲状腺功能亢进症、类风湿性关节炎、Gorham-Stout病、McCune-Albright综合征、各种癌症的溶骨性转移或多发性骨髓瘤、骨量丢失、全身骨骼脆弱、关节退变、非愈合性骨折、由糖尿病引起的骨科和牙科问题、植入性牙周炎、对骨移植物/植入物/骨替代性材料的不良反应、牙周病、骨骼老化、骨折、骨缺损、骨移植、植骨、骨癌、关节置换、关节修复、融合、小关节修复、骨质退变、牙种植体和修复、骨髓缺损、肢端肥大症患者的骨病、囊性纤维化相关骨病、无动力性骨病、与慢性肾病相关的肾性骨营养不良、与胱氨酸病相关的骨病和与高草酸尿相关的骨病；优选地，所述骨质疏松症包括绝经后骨质疏松症、男性和女性的老年性骨质疏松症、糖皮质激素诱导的骨质疏松症、制动性骨质疏松症、失重引起的骨质疏松症、移植后骨质疏松症、迁移性骨质疏松症、特发性骨质疏松症、青少年骨质疏松症。
如本文所用，术语“肽”是指由D-或L-氨基酸的单链或由肽键连接的D-和L-氨基酸的混合物组成的化合物。通常，肽的长度约为2至50个氨基酸。优选地，本发明的肽长度为约2至约20个氨基酸，更优选5至20个氨基酸，最优选5个氨基酸。
如本文所用，术语“氨基酸”是指天然、非天然和合成的氨基酸，以及以类似于天然氨基酸的方式起作用的氨基酸类似物和氨基酸模拟物。
如本文所用，术语“天然氨基酸”是指那些由遗传密码编码的氨基酸，以及那些后来被修饰的氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。天然产生的α-氨基酸包括但不限于丙氨酸(Ala)、半胱氨酸(Cys)、天冬氨酸(Asp)、谷氨酸(Glu)、苯丙氨酸(Phe)、甘氨酸(Gly)、组氨酸(His)、异亮氨酸(Ile)、精氨酸(Arg)、赖氨酸(Lys)、亮氨酸(Leu)、蛋氨酸(Met)、天冬酰胺(Asn)、脯氨酸(Pro)、谷氨酰胺(Gln)、丝氨酸(Ser)、苏氨酸(Thr)、缬氨酸(Val)、色氨酸(Trp)、酪氨酸(Tyr)及其组合。天然氨基酸的立体异构体包括但不限于D-丙氨酸(D-Ala)、D-半胱氨酸(D-Cys)、D-天冬氨酸(D-Asp)、D-谷氨酸(D-Glu)、D-苯丙氨酸(D-Phe)、D-组氨酸(D-His)、二亮氨酸(D-Ile)、D-精氨酸(D-Arg)、D-赖氨酸(D-Lys)、D-亮氨酸(D-Leu)、D-蛋氨酸(D-Met)、D-天冬酰胺(D-Asn)。
如本文所用，术语“非天然氨基酸”包括但不限于氨基酸类似物、氨基酸模拟物、合成氨基酸、M’-修饰赖氨酸和以类似于天然氨基酸的方式起作用的L-或D-构型的甲基氨基酸。非天然氨基酸不是由遗传密码编码的，可以但不一定具有与天然氨基酸相同的基本结构。
所述非天然氨基酸选自：2-氨基脂肪酸、3-氨基脂肪酸、β-丙氨酸、β-氨基丙酸、2-氨基丁酸、4-氨基丁酸、哌啶羧酸、6-氨基己酸、2-氨基庚酸、2-氨基异丁酸、3-氨基异丁酸、2-氨基庚二酸、2,4-二氨基丁酸、锁链素、2,2'-二氨基庚二酸、2,3-二氨基丙酸、N-乙基甘氨酸、N-乙基天冬酰胺、羟赖氨酸、异羟赖氨酸、3-羟脯氨酸、4-羟基脯氨酸、异锁链素、异-异亮氨酸、N-甲基甘氨酸、N-甲基异亮氨酸、6-N-甲基赖氨酸、N-甲基缬氨酸、正缬氨酸、正亮氨酸或鸟氨酸。
如本文所用，术语“氨基酸类似物”是指具有与天然氨基酸相同的基本化学结构的化合物，即与氢、羧基、氨基和R基团结合的碳，例如高丝氨酸、正亮 氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这种类似物具有修饰的R基团(如正亮氨酸)或修饰的肽主链，但保留了与天然氨基酸相同的基本化学结构。
如本文所用，术语“氨基酸模拟物”是指其结构不同于氨基酸的一般化学结构，但其功能类似于天然氨基酸的化合物。合适的氨基酸模拟物包括但不限于β-氨基酸和γ-氨基酸。在β-氨基酸中，氨基键合到羧基的β-碳原子上，使得氨基和羧基之间有两个碳原子。在γ-氨基酸中，氨基键合到羧基的γ-碳原子上，使得氨基和羧基之间有三个碳原子。适用于β-或γ-氨基酸的R基团包括但不限于天然氨基酸和非天然氨基酸中存在的侧链。
如本文所用，术语“D-氨基酸”是指氨基酸的右旋立体异构体。字母D和L在本领域中通常用于指代氨基酸的立体异构体。D-氨基酸是那些可以从甘油醛的右旋异构体，即D-甘油醛合成的氨基酸。类似地，左旋氨基酸是那些可以从甘油醛的左旋异构体，即左旋甘油醛合成的氨基酸。
如本发明所用，本发明所用的术语“治疗”均指对患有疾病的受试者有益的任何类型的治疗，包括改善患者的状况(例如，在一种或多种症状中)、延迟疾病的进展等。
如本发明所用，术语“受试者”和“患者”通常涉及人类受试者并且可互换使用。受试者可以是男性或女性，可以是任何人种或种族，包括但不限于白种人、非裔美国人、非洲人、亚洲人、西班牙人、印度人等。受试者可以是任何年龄，包括新生儿、婴儿、幼儿、儿童、青少年、成人和老年人。在一些实施方案中，受试者患有骨病。受试者还可包括动物受试者，特别是哺乳动物受试者，例如犬科动物、猫科动物、牛科动物、山羊科动物、马科动物、绵羊科动物、猪科动物、啮齿动物(例如大鼠和小鼠)、兔类动物、灵长类动物(包括非人灵长类动物)等，以用于兽医药物或医疗药物开发的目的。
如本发明所用，表述“骨疾病”是指引起一种或多种骨骼和/或骨细胞的各种异常或畸形的那些疾病中的任何一种。
本发明的优点和有益效果：
本发明首次报道了两种五肽(ESSES或DSSDS)，所述五肽具有促骨形成活性可在多种模型中增加骨量疗效不亚于PTH，其化学性质稳定，容易低成本大规模合成。
本发明的多肽B5P在动物实验中表现出优越的生物相容性，暂未观察到急性毒性、生殖毒性发生。
本发明的多肽B5P应用范围广，不仅可用于骨质疏松症治疗。亦可促进骨折修复、牙种植体的骨结合等疾病。
附图说明
图1显示不同长度多肽的活性检测图，其中，A：ESSES；B：DSSDS；
图2显示多肽两端延长无关氨基酸后的活性检测图，其中，A：ESSES；B：DSSDS；
图3显示掺入非天然D型氨基酸的多肽活性检测图，其中，A：ESSES；B：DSSDS；
图4显示经修饰的多肽活性检测图，其中，A：ESSES；B：DSSDS；
图5显示多肽偶联物的活性检测图，其中，A：ESSES；B：DSSDS；
图6显示多肽缓释剂型的活性检测图，其中，A：ESSES；B：DSSDS；
图7显示野生型小鼠Micro-CT扫描结果图，其中A：骨小梁扫描图；B：骨小梁局部扫描图；C：皮质骨截面扫描图；D：皮质骨骨体积分数；E：股骨骨体积分数；F：骨小梁数目；G：骨小梁分离度；H：骨材料密度；
图8显示野生型小鼠H&E染色图；
图9显示野生型小鼠双荧光标记实验结果图，其中，A：染色图；B：统计图；
图10显示野生型小鼠Von Kossa染色结果图，其中，A：染色图；B：统计图；
图11显示野生型小鼠骨矿化相关指标Dmp-1检测结果图，其中，A：染色图；B：统计图；
图12显示OVX小鼠Micro-CT扫描结果图，其中A：OVX+ctrl组骨小梁扫描图；B：OVX+B5P组骨小梁扫描图；C：OVX+PTH组骨小梁扫描图；D：股骨骨体积分数；E：骨小梁数目；F：骨小梁厚度；G：骨材料密度；
图13显示OVX小鼠皮质骨结果图，其中，A：OVX+ctrl组皮质骨扫描图；B： OVX+B5P组皮质骨扫描图；C：OVX+PTH组皮质骨扫描图；D：皮质骨骨体积分数；
图14显示OVX小鼠双荧光标记实验结果图，其中，A：OVX+ctrl组染色图；B：OVX+ctrl组染色图；C：OVX+PTH组染色图；D：MAR；
图15显示OVX小鼠H&E染色图，其中，A：OVX+ctrl组染色图；B：OVX+ctrl组染色图；C：OVX+PTH组染色图；
图16显示OVX小鼠Von Kossa染色图，其中，A：OVX+ctrl组染色图；B：OVX+ctrl组染色图；C：OVX+PTH组染色图；
图17显示骨折小鼠荧光双标实验结果图，其中，A：Fracture+ctrl；B：Fracture+B5P；
图18显示骨折小鼠H&E染色结果图，其中，A：Fracture+ctrl；B：Fracture+B5P；
图19显示骨折小鼠Micro-CT扫描结果图，其中，A：Fracture+ctrl；B：Fracture+B5P；
图20显示骨折小鼠骨矿化相关指标Dmp-1检测结果图，其中，A：Fracture+ctrl；B：Fracture+B5P；
图21显示股骨钛钉植入模型小鼠Micro-CT扫描结果图，其中，A：implant+ctrl扫描图；B：implant+B5P扫描图；C：骨体积分数；D：骨小梁厚度；
图22显示股骨钛钉植入模型小鼠VanGieson染色结果图，其中，A：implant+ctrl染色图；B：implant+B5P扫描图；C：骨表面积/钛钉表面积；
图23显示股骨钛钉植入模型小鼠荧光双标实验结果图，其中，A：implant+ctrl染色图；B：implant+B5P扫描图；C：MAR；
图24显示股骨钛钉植入模型小鼠Von Kossa染色和骨形成结果图，其中，A：种植体+ctrl的Von Kossa染色图；B：种植体+B5P的Von Kossa染色图；C：种植体+ctrl的骨形成染色图；D：种植体+B5P的骨形成染色图；E：统计图；
图25显示老年骨质疏松猕猴Micro-CT扫描结果图，其中，A：对照组扫描图；B：B5P组扫描图；C：BV/TV；D：TB.n；E：TB.th；
图26显示老年骨质疏松猕猴Q-CT扫描结果图，其中，A：髋部骨密度；B：第 二到第四腰椎骨密度；
图27显示老年骨质疏松猕猴H&E染色图，其中，A：ctrl；B：B5P；
图28显示三点弯曲实验结果图，其中，A：抗折能力；B抗压能力：
图29显示QPCR实验结果图，其中，A：Runx2；B：Osx；C：Alpl；D：Ocn；
图30显示矿化染色图；
图31显示信号通路结果图，其中，A：westernblot；B：ALP染色；C：茜素红染色；
图32显示用于评价多肽B5P的生物安全性的红细胞凝集实验结果图，其中，A：对照；B：多肽；
图33显示用于评价多肽B5P的生物安全性的ELISA实验结果图，其中，A：BUN；B：CK；C：ALT；D：ALS；
图34显示用于评价多肽B5P的生物安全性的H&E染色图；
图35显示多肽B5P对老年猕猴血清生化指标的影响，其中，A：CK；B：ALT；C：CKM；D：ALS；E：血糖；F：胆固醇；G：BUN；H：HDLC。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本申请能被更好的理解，仅用于说明本发明而不用于限制本发明的范围。然而，本领域技术人员可以毫不费力地认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。实施例中未注明具体条件的实验方法，通常按照常规条件中所述的条件，或按照制造厂商所建议的条件，实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1多肽制备
通过液相合成法进行合成，具体步骤如下：
1、合成原料及相关试剂/仪器
1)树脂：取代度为1.03mmol/g的2-Chlorotrityl Chloride Resin(天津市南开合成科技有限公司)；
2)氨基酸：Fmoc-Ser-OH(成都诚诺，>99％)，Fmoc-Glu(OTbu)-OH(成都诚诺，>99％)；
3)合成试剂：DMF(原产地韩国)，DCM(原产地韩国)，MEOH(原产地日本)，DIEA(新德化工，99％)，HBTU(昊帆生物科技，99％)；
4)脱保护试剂：哌啶(国药集团上海化学试剂公司，99％)；
5)检测试剂：苯酚试剂(自配)，吡啶试剂(自配)，茚三酮试剂(自配)；
6)裂解试剂：95％切割液：TFA(J.T.Baker，99％)，TIS(上海达瑞精细化工，98％)，EDT(上海达瑞精细化工，98％)，无水乙醚(上海实验，实测99.7％)；
7)氮气：(新联气体)；
8)仪器：①十二通道半自动多肽合成仪上海强耀生物科技有限公司自主设计并申请专利的半自动多肽合成仪，专利号为201020226529.2②SHIMADZU高效液相色谱仪(型号：制备型，分析型，软件：Class-VP.Sevial System，厂商：SHIMADZU)③离心机(上海安亭科学仪器厂，型号：TDL-40B)④LABCONCO冻干机(型号：Freezone.Plus.6，厂商：LABCONCO)。
2、多肽合成步骤如下：合成顺序为从C端到N端。
1)树脂溶涨：将2-Chlorotrityl Chloride Resin加至反应管中，加DCM(15ml/g)，振荡30min。
2)接第一个氨基酸：通过沙芯抽滤掉溶剂，加入3倍摩尔质量的Fmoc-Glu(OTbu)-OH氨基酸，加入DMF溶解，再加入10倍摩尔过量的DIEA，振荡60min；用甲醇封闭。
3)脱保护：去掉DMF，加20％哌啶DMF溶液(15ml/g)，5min，去掉再加20％哌啶DMF溶液(15ml/g)，15min。
4)检测：抽掉哌啶溶液，取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，变深蓝色为阳性反应。
5)洗涤：DMF(10ml/g)两次，DCM(10ml/g)两次，DMF(10ml/g)两次。
6)缩合：保护氨基酸三倍过量，HBTU三倍过量，均用尽量少DMF溶解，加入反应管，立刻加入DIEA十倍过量，反应30min。
7)检测：取十几粒树脂，用乙醇洗三次，加入检测试剂检测，105℃-110℃加热5min，无色为阴性反应。
8)洗涤：DMF(10ml/g)一次，DCM(10ml/g)两次，DMF(10ml/g)两次。
9)重复3)至6)操作，从右到左依次连接序列中的氨基酸。
10)抽干，洗树脂：DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次，DCM(10ml/g)两次，抽干10min。
11)从树脂上切割多肽：配制切割液(10/g)TFA 95％；水1％；EDT 2％；TIS 2％；切割时间：120min。
12)吹干洗涤：将裂解液用氮气尽量吹干，用乙醚洗六次，然后常温挥干。
13)分析提纯：用高效液相色谱将粗品提纯。
14)冻干：收集目标多肽溶液于冻干机中进行浓缩，冻干成白色粉末。
15)将多肽送到质检部确认合格。
实施例2骨科五肽及其衍生物的活性检测
1、不同长度多肽的活性检测
1.1合成不同长度的多肽
按照实施例1的方法合成。
1.2细胞实验
培养成骨细胞系MC-3T3E1细胞，使用终浓度为1μg/ml的不同长度的多肽孵育24小时后，QPCR检测ALP基因表达水平。
1.3结果
结果如图1A所示，ESSES五个氨基酸的组合是增强成骨细胞活性的较好组合，串联至2-4个ESSES时均存在较好活性，串联至5个及以上时活性开始减弱。
结果如图1B所示，DSSDS五个氨基酸的组合是增强成骨细胞活性的最佳单元，串联至2-4个DSSDS时均存在较好活性，串联至5个及以上时活性开始减弱。
2、多肽两端氨基酸延长后多肽活性检测
2.1合成不同长度的多肽
按照实施例1的方法合成。
2.2细胞实验
培养成骨细胞系MC-3T3E1细胞，使用终浓度为1μg/ml的不同长度的多肽孵育24小时后，QPCR检测ALP基因表达水平。
2.3结果
结果如图2A所示，ESSES单侧延长1个无关氨基酸时对其活性无影响，单侧延长2个无关氨基酸时对其活性有较小影响，延长3个以上无关氨基酸时活性下降。双侧延长各1个无关氨基酸时对其活性无影响，双侧各延长2个以上无关氨基酸时活性开始下降。甘氨酸(G)为无关氨基酸的代表。
结果如图2B所示，DSSDS单侧延长1个无关氨基酸时对其活性无影响，单侧延长2个无关氨基酸时对其活性有较小影响，延长3个以上无关氨基酸时活性显著下降。双侧延长各1个无关氨基酸时对其活性无影响，双侧各延长2个以上无关氨基酸时活性开始减弱。甘氨酸(G)为无关氨基酸的代表。
3、掺入非天然D型氨基酸的多肽活性检测
3.1合成掺入非天然D型氨基酸的多肽
按照常规方法进行，非天然D型氨基酸分布如表1所示。
表1非天然D型氨基酸分布
编号 非天然D型氨基酸分布 非天然D型氨基酸分布
P1 ESSES DSSDS
P2 ESSES D DSSDS D
P3 ESSE DS DSSD DS
P4 ESS DES DSS DDS
P5 ES DSES DS DSDS
P6 DESSES DDSSDS
3.2细胞实验
培养成骨细胞系MC-3T3E1细胞，使用终浓度为1μg/ml的不同长度的多肽孵育24小时后，QPCR检测ALP基因表达水平。
3.3结果
结果如图3A所示，ESSES C端的S替换为非天然D型氨基酸后活性有所增强，而其他位置的氨基酸替换会导致活性下降。
结果如图3B所示，DSSDS C端的S替换为非天然D型氨基酸后活性有所增强，而其他位置的氨基酸替换会导致活性下降。
4、多肽修饰后的变体活性检测
4.1合成修饰后的变体
按照常规方法进行，多肽修饰如表2所示。
表2不同修饰的多肽
Figure PCTCN2020132556-appb-000001
4.2细胞实验
培养成骨细胞系MC-3T3E1细胞，使用终浓度为1μg/ml的不同长度的多肽孵育24小时后，QPCR检测ALP基因表达水平。
4.3结果
结果如图4A所示，甲基化、豆蔻化、PEG修饰、氟元素修饰、生物素修饰、FAM荧光标记、二硫键成环的ESSES都有活性。
结果如图4B所示，甲基化、豆蔻化、PEG修饰、氟元素修饰、生物素修饰、FAM荧光标记、二硫键成环的DSSDS都有活性。
5、多肽偶联物的活性检测
5.1合成多肽偶联物
按照常规方法进行，多肽偶联物如表3所示。
表3多肽偶联物
编号 修饰方式 修饰方式
PC1 未修饰的ESSES 未修饰的DSSDS
PC2 N-偶联阿伦磷酸钠 N-偶联阿伦磷酸钠
PC3 C-偶联阿伦磷酸钠 C-偶联阿伦磷酸钠
PC4 N-偶联京尼平苷酸 N-偶联京尼平苷酸
PC5 C-偶联京尼平苷酸 C-偶联京尼平苷酸
PC6 N-偶联RGD多肽 N-偶联RGD多肽
PC7 C-偶联RGD多肽 C-偶联RGD多肽
5.2细胞实验
培养成骨细胞系MC-3T3E1细胞，使用终浓度为2μg/ml的多肽偶联物孵育24小时后，QPCR检测ALP基因表达水平。
5.3结果
结果如图5A所示，以阿伦磷酸钠为代表ESSES偶联双膦酸盐后具有活性。以京尼平苷酸为代表ESSES偶联中药单体后具有活性。以RGD为代表ESSES偶联寡肽后具有活性。
结果如图5B所示，以阿伦磷酸钠为代表DSSDS偶联双膦酸盐后具有活性。以京尼平苷酸为代表DSSDS偶联中药单体后具有活性。以RGD为代表DSSDS偶联寡肽后具有活性。
6、多肽缓释剂型的活性检测
6.1合成微球缓释剂型
步骤：将PLGA搅拌溶解后，滴加入致孔溶液，并超声乳化形成乳化液； 其中PLGA的LA与GA的摩尔比例为50:50；然后将乳化液逐滴加入到搅拌的外水相中，并加入预设体积的去离子水，改变速率持续搅拌，直至溶剂完全挥发。其中外水相为水溶性表面活性剂加入去离子水中，然后将有机溶剂完全挥发后的溶液进行离心，并用去离子水清洗，除去上清液，从而获得PLGA微球。将含有多肽的NaOH溶液加入到所述PLGA微球中混合均匀，并放到摇床继续反应，将反应后的PLGA微球用去离子水反复离心洗涤，冷冻干燥后备用。
6.2细胞实验
培养成骨细胞系MC-3T3E1细胞，使用终浓度为2μg/ml的微球孵育1-7天后，QPCR检测ALP基因表达水平。
6.3结果
结果如图6A所示，以PLGA 50:50微球为代表，ESSES缓释剂型存在显著的生物学活性。
结果如图6B所示，以PLGA 50:50微球为代表，DSSDS缓释剂型存在显著的生物学活性。
实施例3多肽B5P对小鼠骨形成的影响
1、材料
野生型小鼠来源：全部小鼠均为SPF级，购自北京维通利华(Charles River)实验动物技术有限公司。
2、多肽B5P溶液配制：称取1.000克氢氧化钠于烧杯中，加少量生理盐水溶解，然后倒入1000毫升容量瓶里，分3次洗烧杯，将溶液全部倒入容量瓶里，最后用生理盐水稀释至刻度线。摇匀后即得到1000mg/mL的多肽存贮母液。使用时按所需浓度等比例稀释。
3、药物处理
将小鼠随机分为两组，对照组和实验组，对照组小鼠每周3次注射生理盐水，实验组小鼠每周3次注射20mg/kg多肽B5P；一个月后收集股骨样本。
4、Micro-CT扫描
将收集的股骨样本进行Micro-CT扫描，分析骨体积分数(BV/TV)；骨小梁数(TB.N)；骨小梁分离度(TB.Sp)；骨材料密度(BMD)。
4.1步骤
(1)将上述股骨样本置于4％多聚甲醛固定24小时后，转移至0.5％多聚甲醛溶液中防止结晶体在骨组织形成影响样本扫描。
(2)将样本放置于14mm管径的Micro-CT专用扫描管中，样本水平摆放，且横轴垂直于Micro-CT扫描轴，将扫描管放置于Micro-CT扫描机箱样本盘中。
(3)扫描程序相关参数如下：扫描电压为70kVp，扫描能量功率为14W，扫描电流为200μA，曝光时间为300ms，扫描BH 1200mg HA/cc，扫描精度为10μm，扫描滤光器(Filter)为0.5mm AI Filter。后期图像数据采用Mimics 13.0软件重建并分析。
(4)股骨数据重建时，骨小梁数据分析选取自股骨生长板下起始1mm，皮质骨内层的全部骨小梁；皮质骨数据分析选取生长板下5mm-6mm处皮质骨。
4.2结果
Micro-CT扫描结果如图7所示，应用多肽B5P后小鼠骨小梁密度显著升高，骨小梁厚度显著增加，皮质骨厚度增加。小鼠股骨骨体积分数(BV/TV)和骨小梁数目(TB.N)明显升高，骨小梁分离度(TB.Sp)降低，说明小鼠股骨骨密度升高，骨小梁分布更密集；骨材料密度(BMD)指标升高，表明小鼠股骨矿化程度增强。小鼠皮质骨骨体积分数(皮质骨的BV/TV)增加，骨小梁厚度显著增加，皮质骨厚度增加说明应用多肽B5P后小鼠骨量明显增加。
5、H&E染色
5.1步骤
1)切片：以常规方式制作组织的石蜡切片；
2)烤片：切片放置65℃烘箱30min；
3)脱蜡水化：依次对组织切片进行脱蜡处理，二甲苯I 30min，二甲苯II 30min。然后进行切片水化处理，无水乙醇I 5min，无水乙醇II 5min，90％酒精5min，75％酒精5min，蒸馏水5min。
4)染色：苏木素5min水洗，PBS浸泡5min，使细胞核返蓝。伊红染色2min，水洗。
5)脱水透明：梯度乙醇进行组织脱水，80％酒精、90％酒精、95％酒精分别10s后，室温条件下晾干。无水乙醇I 5min，无水乙醇II 5min，室温晾干。浸入二甲苯中5min透明后，中性树脂封片。
6)观察：正置显微镜下观察拍照。
5.2结果
将上述股骨样本进行H&E染色，结果显示小鼠注射多肽B5P后，生长板下骨小梁更致密(图8)。
6、小鼠双荧光标记实验
6.1步骤
(1)将上述股骨样本(二甲酚橙与钙黄绿素腹腔注射剂量为80mg/kg，间隔时间为2周)进行硬组织切片，切片厚度约为15μm。
(2)DAPI染色：硬组织切片滴加1：1000稀释的DAPI染液，染色5min。在PBS中浸泡冲洗3次，每次时间5min。使用抗荧光淬灭封片剂封片。倒置荧光显微镜下观察红绿双色荧光标记，并统计骨沉积率。
6.2结果
结果如图9所示，红色标记与绿色标记相隔2周，间距为新骨沉积。与对照组相比，实验组股骨骨沉积量明显增加。分析结果表明小鼠注射多肽B5P后，骨矿化沉积率(MAR)显著升高。
7、Von Kossa染色
7.1步骤
(1)硝酸银染液染色：配置0.2％硝酸银溶液，滴加于股骨硬组织切片上，覆盖股骨组织，强光照射15min，流水冲洗切片2-3s。
(2)硫代硫酸钠孵育：配置5％硫代硫酸钠溶液，滴加于切片上，覆盖股骨组织，5s，流水冲洗切片2-3s。
(3)甲基绿衬染：甲基绿染液衬染5min，流水冲洗5s，冲去浮色，65℃烘箱烘干30min。
(4)封片与观察：切片滴加二甲苯透明5min后，中性树脂封片。在体式显微镜下观察并拍照。
7.2结果
骨组织中的钙离子与银离子置换呈黑色，表示矿化骨的密度与骨矿化水平。Von Kossa染色结果如图10所示，多肽B5P注射组生长板下骨小梁密度增加，定量分析显示实验组吸光度升高，说明骨矿化程度显著增强。
8、骨矿化相关指标Dmp-1检测
8.1免疫组化染色
(1)切片：股骨组织脱钙后，常规石蜡组织切片；
(2)烤片：切片放65℃烘箱30min；
(3)脱蜡水化：依次对组织切片进行脱蜡处理，二甲苯I 30min，二甲苯II30min。然后进行切片水化处理，无水乙醇I 5min，无水乙醇II 5min，90％酒精5min，75％酒精5min。蒸馏水5min。浸入PBS中冲洗三次，每次时长3min。
(4)抗原修复：根据抗体的要求，对组织抗原进行相应的修复。
(5)内源性过氧化物酶灭活：切片上滴加适量的过氧化酶阻断溶液，室温下孵育10min(用以阻断内源性过氧化物酶的活性)。滴加PBS冲洗三次，每次5min。
(6)组织封闭：切片上滴加适量的正常非免疫动物血清，室温下孵育10min。
(7)孵育一抗：甩去组织上的血清，在组织上滴加适量的一抗，在室温下孵育60min或4℃过夜。滴加PBS 3min冲洗三次。
(8)孵育二抗：切片滴加适量生物素标记的二抗，室温下孵育10min，用PBS冲洗三次，每次3min。
(9)除去PBS，每张切片加1滴或50μl链霉素抗生物素标记的第二抗体(试剂D)，室温下孵育10min，用PBS冲洗三次，每次3min。
(10)DAB显色：滴加DAB显色液(现配现用)，显微镜下观察3-10min。待显色结束后，自来水冲洗，终止显色。
(11)细胞核复染：甲基绿染液复染，PBS返蓝。
(12)脱水透明：梯度乙醇进行组织脱水，80％酒精、90％酒精、95％酒精分别10s后，室温条件下晾干。无水乙醇I 5min，无水乙醇II 5min室温晾干。在二甲苯中透明5min后。进行封片和观察。
8.2结果
结果图11所示，B5P组免疫组化显色程度升高。半定量结果显示多肽B5P注射组相比于对照组吸光度升高，说明Dmp-1表达水平升高。
实施例3多肽B5P对骨质疏松症小鼠骨量的影响
1、骨质疏松症小鼠模型构建
(1)选用12周龄雌性WT小鼠18只，分为3组(对照组，OVX+多肽B5P组，OVX+PTH组)。
(2)35mg/kg剂量戊巴比妥钠麻醉小鼠。
(3)小鼠下腹部备皮，碘伏消毒，75％酒精擦去碘伏，于肋骨下1cm腹部中线偏左0.5cm做一个0.5cm长度切口。
(4)剪开筋膜与肌肉，钝性拓宽手术视野。在下腹部找到白色脂肪组织牵拉出体外，延子宫上行找到输卵管与卵巢。
(5)结扎输卵管后剪下卵巢与输卵管，双侧切除后，将子宫与脂肪归置入腹。
(6)分层缝合皮肤与肌肉。
(7)骨质疏松症模型小鼠自建模后三个月，模型趋于稳定后，通过尾静脉注射多肽B5P，总计注射4周，每周3次，剂量为20mg/kg；PTH给药方式为皮下给药，剂量为0.1μg/kg，注射4周，每周3次。
2、Micro-CT扫描
步骤同实施例1。
结果如图12所示，与对照组相比，多肽B5P组与PTH组小鼠骨量明显增加，骨体积分数(BV/TV)，骨小梁数目(TB.N)，骨材料密度(BMD)，骨小梁厚度(TB.Th)等指标均显著升高。皮质骨重建与数据分析表明，与对照组相比，B5P组与PTH组小鼠皮质骨厚度显著增加，皮质骨骨体积分数增加(图13)。说明多肽B5P能够增加骨质疏松症小鼠皮质骨骨量。
3、荧光双标实验
步骤同实施例1。
结果如图14所示，B5P组与PTH组，骨沉积量较对照组小鼠显著升高，骨矿化沉积率(MAR)明显升高，说明B5P治疗后小鼠骨形成功能升高。
4、H&E染色
步骤同实施例1。
结果如图15所示，与对照组相比，B5P组骨小梁密度显著增加。另外，PTH组骨小梁密度也显著增加。
5、VonKossa染色
步骤同实施例1。
结果如16所示，与对照组相比，小鼠骨量在多肽B5P与PTH作用下显著增加，且B5P组与PTH组小鼠骨矿化程度明显升高。
实施例4多肽B5P对骨折愈合的影响
1、构建小鼠骨折模型
按照文献[Glycosylation of dentin matrix protein 1 is critical for fracture healing via promoting chondrogenesis.Frontiers of Medicine.2019 Oct；13(5):575-589]中描述的方法构建小鼠骨折模型。建模1周后，小鼠每周3次注射20mg/kg多肽B5P，两周后取材评价小鼠骨折断端骨痂形成情况与骨痂密度。
2、荧光双标实验
步骤同实施例1。
结果如图17所示，实验组近骨折断端皮质骨骨沉积量较对照组显著增加。分析结果显示实验组骨矿化沉积速率较对照组显著升高。
3、H&E染色
步骤同实施例1。
结果如图18所示，B5P组与对照组相比骨折部位连接更紧密，骨痂密度显著增加。
4、Micro-CT扫描
步骤基本同实施例1。不同点在于：骨折样本数据重建时，骨痂展示部位为骨折断端上下各500μm，数据分析选取自相应部位皮质骨外的骨痂部分。
结果如图19所示，骨折模型小鼠注射B5P组骨折部位愈合加快。骨痂部位重建结果显示，多肽B5P注射后骨痂较对照组致密，B5P组骨痂密度显著高于对照组。
5、骨矿化相关指标Dmp-1免疫组化染色
步骤同实施例1。
对小鼠骨折样本进行脱钙后，通过石蜡切片与成骨相关指标免疫组化染色评价骨折部位对愈合情况。Dmp-1免疫组化染色结果如图20所示，多肽B5P组骨折部位Dmp-1表达情况显著升高。
实施例5多肽B5P对种植体骨结合的影响
1、构建小鼠股骨钛钉植入模型
(1)制备直径1mm，2mm长钛钉，头尾两端尖锐部分用砂纸挫平。将钛钉浸泡于75％酒精30min消毒后，在PBS中清洗。
(2)选用8周龄雌性WT小鼠12只，分为2组(对照组，钛钉+B5P组)。35mg/kg剂量戊巴比妥钠麻醉小鼠。
(3)小鼠右侧腿部备皮，碘伏消毒，75％酒精擦去碘伏，自髌骨外侧至股 骨头外侧做一切口。
(4)延股骨外侧肌肉边缘钝性分离肌肉，暴露股骨中段。将剥离子插入股骨下方，支撑股骨中段。
(5)慢速手机安装1mm钻头，钻开股骨中段外侧皮质骨。
(6)安放钛钉，妥善固位后分层缝合肌肉与皮肤。
(7)小鼠于术后第二周开始注射药物，总计注射3周，每周3次，剂量为20mg/kg，于术后第四周取样。
2、Micro-CT扫描
步骤基本同实施例1。不同点在于：钛钉数据重建时，钛钉周围骨量数据分析选取自钛钉内径1mm，外径1.1mm圆内骨组织。钛钉图片展示时，骨阈值为212-535，钛钉阈值为535-1000。
结果如图21所示，与对照组相比，B5P组小鼠骨量明显增加，骨体积分数(BV/TV)，骨小梁厚度(TB.Th)等指标均显著升高，说明多肽B5P治疗后钛钉表面骨密度与厚度增加。
3、VanGieson染色
步骤：按照常规方法进行VanGieson染色。
结果如图22所示，B5P组钛钉表面骨组织表面积显著高于对照组，虚线标注为钛钉表面骨所在位置。分析骨表面积/钛钉表面积可见钛钉表面骨量显著增加。
4、荧光双标实验
步骤同实施例1。
结果如图23所示，左侧为钛钉，钛钉表面覆盖骨组织，钙黄绿素与二甲酚橙标记相隔一周，多肽B5P注射后钛钉表面骨沉积速率明显升高。
5、Von Kossa染色
步骤同实施例1。
结果显示多肽B5P组钛钉表面骨结合部位较对照组多，吸光度分析结果表 明钛钉周围骨矿化程度显著高于对照组(图24)。
实施例6多肽B5P对老年骨质疏松猕猴骨量的影响
22岁左右的老年雌性猕猴购自广西省南宁市灵康赛诺科生物科技有限公司，按照20mg/kg每周2次静脉注射多肽12周后，评价猴的股骨和腰椎骨量，探讨应用多肽B5P治疗老年骨质疏松猴的可行性。
1、Micro-CT扫描
步骤同实施例1。
结果如图25所示，与对照组无关多肽注射相比，多肽B5P组猕猴的骨体积分数(BV/TV)，骨小梁数目(TB.N)，骨小梁厚度(TB.th)指标均升高。上述结果表明，与对照组无关多肽注射相比，多肽B5P组猕猴的松质骨含量增加。
2、Q-CT扫描
步骤：按照常规方法进行Q-CT扫描
结果如图26所示，与对照组无关多肽注射相比，多肽B5P组猕猴髋部的骨密度在注射12周后升高5.9％。与对照组无关多肽注射相比，多肽B5P组猕猴第二到第四腰椎的骨密度在注射12周后升高6.1％。
3、H&E染色
步骤同实施例1。
结果如图27所示，与对照组无关多肽注射相比，多肽B5P组猕猴第四腰椎的松质骨含量增加。
4、三点弯曲实验
步骤：使用上海企想检测仪器有限公司的WD-T1动物骨骼三点弯曲强度试验机进行超检测。将骨骼标本放在有一定距离的两个支撑点上，在两个支撑点中点上方向标本施加向下的载荷，骨骼标本的三个接触点形成相等的两个力矩时即发生三点弯曲。
结果如图28所示，与对照组无关多肽注射相比，多肽B5P组猕猴股骨的抗 压和抗折能力增强。
实施例7多肽B5P对干细胞成骨向分化的影响
通过在体外培养的骨髓间充质干细胞(BMSCs)中加入多肽B5P并进行矿化诱导，检测成骨相关基因的表达与矿化相关染色，验证多肽B5P在体外的促矿化能力。
1、骨髓间充质干细胞(BMSCs)培养和矿化诱导
步骤：BMSCs细胞融合达80％以上后，弃去原培养基，添加成骨诱导培养基(DMEM中加入10％FBS，5μg/ml胰岛素，0.1μM地塞米松，0.2mM维生素C和10mMβ-甘油磷酸盐)。每隔2天换液1次，分化两周后检测矿化相关基因表达或进行染色分析。
2、细胞RNA抽提
(1)细胞RNA：取上述细胞，将细胞培养液吸走，PBS洗3遍。加入1ml的Trizol试剂，轻轻吹打，将其转移至灭RNA酶的1.5ml EP管中。
(2)加入0.2ml的氯仿，剧烈振荡20s后，冰上静置10min。4℃下以12000rpm，离心，20min。
(3)吸上清至1.5ml离心管中。此处应小心吸取，以免碰触下层蛋白层。
(4)加入等体积的预冷异丙醇，轻轻颠倒5次，冰上静置10min，在4℃下以12000rpm离心，20min。
(5)小心弃去上清后，加入1ml预冷后的75％乙醇(以DEPC水配置)，洗涤RNA沉淀。在4℃下离心(8000rpm)，20min。
(6)小心弃上清，RNA沉淀空气干燥10min。加入20ul DEPC水溶解。
3、实时荧光定量核酸扩增检测系统(qPCR)
(1)RT-qPCR反应体系(20ul)(表4)：
表4 qPCR反应体系
Figure PCTCN2020132556-appb-000002
(2)注意事项：每个标本及每个基因均设3副孔，混匀后上样。
RT-qPCR程序：
95℃ 10s，1循环；
95℃ 5s，60℃ 30s，38个循环。
引物序列如表5所示。
表5 RT-qPCR相关基因引物序列
Figure PCTCN2020132556-appb-000003
Figure PCTCN2020132556-appb-000004
(3)结果
在矿化诱导的BMSC中分别加入0.5μg/ml或1μg/ml多肽B5P，7天后提取RNA进行RT-qPCR检测。结果如图29所示，成骨后期标志物Alpl，Ocn与成骨分化前期标志物Runx2，Osx基因表达水平均显著升高。
4、矿化染色
4.1 ALP染色
(1)配置染液：A液(磷酸盐染色液)0.3μl，B液体(偶联重氮盐)0.6μl，DD H 2O 900μl。
(2)BMSCs矿化诱导7天后，每孔滴加4％多聚甲醛500μl，4℃冰箱中固定30min。
(3)弃去固定液，PBS溶液洗涤3次。
(4)滴加上述染液，37℃杂交炉孵育1小时。
(5)小心吸去染液，DD H 2O清洗浮色。
(6)白光板上拍照。
4.2茜素红染色
(1)BMSCs矿化诱导21天后，每孔滴加4％多聚甲醛500μl，4℃冰箱中固定30分钟。
(2)弃去固定液，PBS溶液洗涤3次。
(3)滴加500μl成品茜素红染液，染色15min。
(4)小心吸去染液，DD H2O清洗浮色。
(5)镜下观察结晶生成情况，并在白光板上拍照。
4.3 Von kossa染色
(1)BMSCs矿化诱导21天后，每孔滴加4％多聚甲醛500μl，4℃冰箱中固定30分钟。
(2)弃去固定液，PBS溶液洗涤3次。
(3)滴加0.1％硝酸银染液，强光照射下染色10min。
(4)小心吸去染液，DD H2O清洗浮色。
(5)镜下观察黑色矿化结节生成情况，并在白光板上拍照。
4.4结果
通过培养小鼠BMSC，加入0.5μg/ml或1μg/ml多肽B5P，并进行矿化诱导。矿化诱导后第7天进行ALP染色，观察BMSC成骨向分化效果。矿化诱导后第21天，进行茜素红与Von kossa染色观察BMSC矿化效果。结果如图30所示，BMSCs矿化诱导7天后ALP染色增强，BMSCs矿化结节形成显著增加。提示多肽B5P能够促进BMSC成骨向分化。
5、信号通路研究
5.1 Western blot
提取分别加入0.5μg/ml或1μg/ml B5P诱导14天后的BMSC的蛋白质，通过Western blot验证细胞重要信号转导通路FAK，AKT，ERK等的变化。结果如图31A所示，FAK-AKT信号通路显著改变，磷酸化FAK与AKT表达升高。
5.2抑制整合素α5
进一步研究FAK-AKT通路的重要受体整合素受体，通过抑制FAK-AKT通路的整合素受体(整合素α5)研究其对B5P促成骨作用的影响
5.2.1干扰实验
步骤：细胞生长至占瓶底90％时消化，按每孔20万个细胞接种于6孔培养板中,待细胞生长至占板底40％时，按照Lipofectamine 3000说明书分组进行转染，对照组转染NC siRNA序列为CGGCAAGCTGACCCTGAAGT(SEQ ID NO.11)，实验组转染整合素α5siRNA序列为GGACCAGGAAGCTATTTCT(SEQ ID NO.12)。转染48小时后收获细胞进行检测。
5.2.2染色
ALP染色(图31B)和茜素红染色(图31C)结果显示，抑制整合素α5后B5P促成骨作用被阻断。提示B5P可能通过激活整合素α5，进而激活FAK-AKT信号通路促进干细胞成骨向分化。
实施例8多肽B5P的生物安全性评价
通过红细胞凝集，血清生化指标评价与重要脏器的病理切片分析等方法探讨多肽B5P的生物安全性。
1、红细胞凝集试验
1.1步骤
(1)取野生型小鼠血浆0.5ml，在EP管中室温静置一小时以上(或37℃水浴1小时，4℃冰箱静置2小时或者过夜)以1800rpm离心10分钟。取下层沉淀，加入1ml生理盐水稀释。吸取10μl稀释液，加入1ml生理盐水(共稀释1000倍)。以1800rpm离心10分钟。
(2)取沉淀，加入1ml红细胞储存液(阿氏液)，充分混匀，以1800rpm离心5min,弃上清。加入生理盐水稀释成1％红细胞悬液。
(3)取96孔板，加入上述红细胞悬液50μl。
(4)试剂加入情况如表6所示。
表6 HA加样表
Figure PCTCN2020132556-appb-000005
(3)正置显微镜下观察红细胞凝集情况，拍照并作记号。
1.2结果
结果如图32所示，在不同浓度在0、1、10、1000、1000ng/ml均不导致明显的红细胞凝集。代表性图片显示加入10mg/ml多肽B5P，红细胞散在分布， 不发生明显细胞凝集或溶血现象。
2、ELISA检测
通过ELISA检测小鼠注射多肽B5P后，血清中BUN(血尿素氮)，CK(肌酸激酶)，ALT(谷丙转氨酶)，ALS(醛固酮)水平，评估小分子多肽对生化指标的影响。
2.1步骤
2.1.1小鼠血清采集
(1)小鼠眼眶后静脉丛采血：
取内径为1.0-1.5mm的玻璃毛细管，临用前折断成2～2.5cm长的毛细管段，浸入1％肝素溶液中，干燥后用。取血时左手抓住鼠两耳之间的颈背部皮肤以固定头部，轻轻向下压迫颈部两侧，引起头部静脉血液回流困难使眼眶静脉丛充血，右手持毛细管由内眦部插入结膜，再轻轻向眼底部方向推进，轻轻旋转毛细管以划破静脉丛，让血液顺毛细管流出，接收入事先准备的容器中。采血后纱布轻压眼部止血。
(2)经上述的方法取得血液后，在EP管中室温静置一小时以上(或37℃水浴1小时，4℃冰箱静置2小时或者过夜)以3000rpm离心10分钟，上清即为血清。可在-80℃保存。
2.1.2、酶联免疫吸附剂测定(ELISA)
(1)平衡：取出CK(肌酸激酶)，BUN(血尿素氮)，ALS(醛固酮)ALT(谷丙转氨酶)，ELISA kit室温平衡20min。
(2)稀释洗涤液：dd H 2O稀释20X洗涤液至1X。
(3)血清预备：使用不含热源和内毒素的试管(在后续实验操作过程中需要避免任何细胞刺激)收集血液后，室温下3000rpm离心10分钟后，小心吸取，将血清和红细胞分离。
(4)加样：取出所需数量的孔板，放在96孔底板上，板上标记加样顺序。在空白和标准品孔中，加入20μl Matrix solution，标准品分别加入0、0.025、0.05、0.1、0.2、0.4ng/ml标准品各20μl。样品孔中加入对照组与多肽B5P组小鼠稀释 后血清各10μl，再加入样本稀释液40μl，空白孔不加。
(5)手动洗板：沿着孔壁缓缓加入洗板液，静置1min后，弃去洗板液，在吸水纸上轻拍。重复5次。加入不同样本时及时更换枪头，避免污染。
自动洗板：在每个反应孔中加入350μl洗涤液，程序为浸泡1min，洗板5次。
(6)除空白孔外，在标准品反应孔与样本反应孔中加入辣根过氧化物酶(HRP)标记的检测抗体100μl，封板膜封住反应孔后，在37℃水浴锅或恒温箱孵育60min。
(7)洗板：上述方式洗板5次。
(8)显色：在每个反应孔中加入底物A，B各50μl，37℃避光孵育15min。
(9)测量：每孔加入终止液50μl，10min后，酶标仪450nm波长读取吸光度。制作标准曲线后，用相应的公式计算样品中各蛋白含量。
2.2结果
结果如图33所示，野生型小鼠注射200mg/kg多肽B5P 1月后，BUN，CK，ALT，ALS指标均无显著差异。
3、H&E染色
通过注射200mg/kg多肽B5P，对重要脏器进行H&E染色进行病理学评价，探讨多肽B5P对内脏器官是否具有慢性毒理作用。对心，肺，肝，脾，肾等组织石蜡切片，并进行H&E染色。结果如图34所示，注射多肽B5P一个月后，与对照组相比，重要脏器(心、肺、肝、脾、肾)未产生肿瘤或炎性浸润等明显病理性改变。
本实施例的结果表明，在观察期一个月内未发现有明显的细胞毒性与组织器官病理改变，说明多肽B5P具有较好的生物安全性。
实施例9多肽B5P对大鼠妊娠结局的影响
1、步骤
将SPF级Wistar大鼠雌雄配对后筛选80只孕鼠，分为溶剂对照组及低，中，高剂量4组，各20只。统一静脉注射多肽，每周2次，持续2周。于妊娠第18.5天处死大鼠，解剖后立即数子宫着床点数以及活胎数死胎数，计量子宫重量，胎盘重量，卵巢HE切片后计量黄体数等。
2、结果
结果如表7所示，未发现低，中，高剂量注射的多肽B5P对大鼠妊娠结局存在影响。
表7多肽B5P对大鼠妊娠结局影响的统计表
Figure PCTCN2020132556-appb-000006
实施例10多肽B5P对老年猕猴血清生化指标的影响
1、步骤
将20-23岁的老年雌性猕猴随机分为2组。对照组注射无关多肽，实验组20mg/kg每周2次静脉注射多肽B5P。注射后观察2小时内的动物行为并录像。注射4小时后抽取2ml血液，离心分离血清后自动生化分析仪测试CK，HDLC等 8项生化指标的含量。
2、结果
22岁左右的老年雌性猕猴，20mg/kg每周2次静脉注射多肽12周，结果如图35所示，多肽B5P在注射后未导致血清生化指标的改变。
上述实施例仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (10)

  1. 一种多肽，其特征在于，所述多肽序列由丝氨酸，和谷氨酸或天冬氨酸两种氨基酸组成，所述多肽含有2-50个氨基酸，所述丝氨酸，和谷氨酸或天冬氨酸的个数比为(0.25～2)：1；
    优选地，所述多肽具有如下通式：(XSSXS)n，其中，X为谷氨酸或天冬氨酸，S为丝氨酸，n为自然数；
    更优选地，n为1、2、3、4；
    最优选地，n为1。
  2. 一种多肽衍生物，其特征在于，所述多肽衍生物包括权利要求1所述的多肽的修饰产物、权利要求1所述的多肽经一个或多个氨基酸添加和/或替换后获得的变体，权利要求1所述的多肽与其他物质的偶联物；
    优选地，所述修饰包括甲基化修饰、豆蔻酰化修饰、PEG修饰、氟元素修饰、生物素修饰、荧光标记修饰、环化修饰、羧化修饰、脂肪酸修饰、乙酰化修饰、磷酸化修饰、糖基化修饰、酰胺化修饰或其他已知的多肽修饰；
    优选地，所述其他物质包括蛋白、多肽、药物；更优选地，所述蛋白包括血清白蛋白；所述多肽包括Fc区、信号肽、多肽标记；所述药物包括双膦酸盐类药物、环烯醚萜化合物；最优选地，所述双膦酸盐类药物包括阿仑膦酸、伊班膦酸盐、唑来膦酸；环烯醚萜化合物包括京尼平尼酸、京尼平-龙胆双糖苷、京尼平苷和京尼平苷酸；
    优选地，所述氨基酸添加和/或替换包括在多肽序列的氨基端、羧基端或内部任意位置进行添加和/或替换氨基酸。
  3. 根据权利要求2所述的多肽衍生物，其特征在于，所述变体选自以下组：
    所述变体是在权利要求1所述的多肽的氨基端或羧基端添加1至3个无关氨基酸获得的；优选地，无关氨基酸包括甘氨酸；
    所述变体是在权利要求1所述的多肽的氨基端和羧基端同时添加1至2个无关氨基酸获得的；优选地，无关氨基酸包括甘氨酸；
    所述变体是在权利要求1所述的多肽的羧基端用D型氨基酸的非天然氨基酸替换天然氨基酸获得的；优选地，所述变体是在权利要求1所述的多肽的羧基端用丝氨酸的非天然D型氨基酸替换天然氨基酸获得的。
  4. 权利要求1所述的多肽或权利要求2或3所述的多肽衍生物的可药用盐或酯。
  5. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1所述的多肽或权利要求2或3所述的多肽衍生物；优选地，所述药物组合物还包括药学上可接受的载体；更优选地，所述药学上可接受的载体包括稀释剂、填充剂、赋形剂、粘合剂、润湿剂、崩解剂、泡腾剂、表面活性剂、吸收促进剂、润滑剂、吸附载体、缓释微球、埋植剂、微球、脂质体、微乳、水凝胶、纳米粒、蛋白酶抑制剂、生物黏附剂；最优选地，所述缓释微球是PLGA缓释微球。
  6. 编码权利要求1所述的多肽或编码权利要求2或3所述的多肽衍生物的核酸分子或包含其的载体。
  7. 包含权利要求6所述的核酸分子或包含其的载体的宿主细胞。
  8. 一种方法，其特征在于，所述方法包括以下任一项所述的方法：
    1)一种促进成骨细胞或能分化成成骨细胞的细胞的分化、增殖、成熟或钙化的方法，所述方法包括施用权利要求1所述的多肽、权利要求2或3所述的多肽衍生物或权利要求5所述的药物组合物；
    2)一种体外扩增干细胞的方法，所述方法包括将权利要求1所述的多肽、权利要求2或3所述的多肽衍生物或权利要求5所述的药物组合物与干细胞接触；
    优选地，所述能分化成成骨细胞的细胞包括成骨细胞前体细胞、间充质干细胞、基质细胞、成肌细胞；
    3)一种预防或治疗骨疾病的方法，所述方法包括给有需要的受试者施用权利要求1所述的多肽、权利要求2或3所述的多肽衍生物或权利要求5所述的药物组合物；
    4)一种在有需要的受试者中增强骨形成的方法，所述方法包括给有需要的受试者施用权利要求1所述的多肽、权利要求2或3所述的多肽衍生物或权利要求5所述的药物组合物；
    5)一种在有需要的受试者中诱导骨沉积的方法，所述方法包括给有需要的受试者施用权利要求1所述的多肽、权利要求2或3所述的多肽衍生物或权利要求5所述的药物组合物；
    6)一种在有需要的受试者中诱导骨成熟的方法，所述方法包括给有需要的受试者施用权利要求1所述的多肽、权利要求2或3所述的多肽衍生物或权利要求5所述的药物组合物。
  9. 权利要求1所述的多肽的用途，其特征在于，所述用途包括以下任一项所述的用途：
    1)在制备权利要求2或3所述的多肽衍生物中的用途；
    2)在制备权利要求5所述的药物组合物中的用途；
    3)在制备促进骨形成的药物中的用途；
    4)在制备促进成骨细胞分化、增殖、成熟或钙化的药物中的用途；
    5)在制备促进干细胞成骨向分化的药物中的用途；
    5)在制备预防或治疗骨疾病的药物中的用途；
    优选地，所述骨疾病包括骨质疏松症、佝偻病、骨软化症、成骨不全、大理石骨病、纤维异常增生、佩吉特病、慢性甲状旁腺功能亢进症、甲状腺功能亢进症、类风湿性关节炎、Gorham-Stout病、McCune-Albright综合征、各种癌症的溶骨性转移或多发性骨髓瘤、骨量丢失、全身骨骼脆弱、关节退变、非愈合性骨折、由糖尿病引起的骨科和牙科问题、植入性牙周炎、对骨移植物/植入物/骨替代性材料的不良反应、牙周病、骨骼老化、骨折、骨缺损、骨移植、植骨、骨癌、关节置换、关节修复、融合、小关节修复、骨质退变、牙种植体和 修复、骨髓缺损、肢端肥大症患者的骨病、囊性纤维化相关骨病、无动力性骨病、与慢性肾病相关的肾性骨营养不良、与胱氨酸病相关的骨病和与高草酸尿相关的骨病；优选地，所述骨质疏松症包括绝经后骨质疏松症、男性和女性的老年性骨质疏松症、糖皮质激素诱导的骨质疏松症、制动性骨质疏松症、失重引起的骨质疏松症、移植后骨质疏松症、迁移性骨质疏松症、特发性骨质疏松症、青少年骨质疏松症。
  10. 权利要求2或3所述的多肽衍生物的用途，其特征在于，所述用途包括以下任一项所述的用途：
    1)在制备权利要求5所述的药物组合物中的用途；
    2)在制备促进骨形成的药物中的用途；
    3)在制备促进成骨细胞分化、增殖、成熟或钙化的药物中的用途；
    4)在制备促进干细胞成骨向分化的药物中的用途；
    5)在制备预防或治疗骨疾病的药物中的用途；
    优选地，所述骨疾病包括骨质疏松症、佝偻病、骨软化症、成骨不全、大理石骨病、纤维异常增生、佩吉特病、慢性甲状旁腺功能亢进症、甲状腺功能亢进症、类风湿性关节炎、Gorham-Stout病、McCune-Albright综合征、各种癌症的溶骨性转移或多发性骨髓瘤、骨量丢失、全身骨骼脆弱、关节退变、非愈合性骨折、由糖尿病引起的骨科和牙科问题、植入性牙周炎、对骨移植物/植入物/骨替代性材料的不良反应、牙周病、骨骼老化、骨折、骨缺损、骨移植、植骨、骨癌、关节置换、关节修复、融合、关节修复、骨质退变、牙种植体和修复、骨髓缺损、肢端肥大症患者的骨病、囊性纤维化相关骨病、无动力性骨病、与慢性肾病相关的肾性骨营养不良、与胱氨酸病相关的骨病和与高草酸尿相关的骨病；优选地，所述骨质疏松症包括绝经后骨质疏松症、男性和女性的老年性骨质疏松症、糖皮质激素诱导的骨质疏松症、制动性骨质疏松症、失重引起的骨质疏松症、移植后骨质疏松症、迁移性骨质疏松症、特发性骨质疏松症、青少年骨质疏松症。
PCT/CN2020/132556 2020-11-29 2020-11-29 一种促骨形成多肽及其应用 WO2022110092A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/132556 WO2022110092A1 (zh) 2020-11-29 2020-11-29 一种促骨形成多肽及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/132556 WO2022110092A1 (zh) 2020-11-29 2020-11-29 一种促骨形成多肽及其应用

Publications (1)

Publication Number Publication Date
WO2022110092A1 true WO2022110092A1 (zh) 2022-06-02

Family

ID=81753859

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/132556 WO2022110092A1 (zh) 2020-11-29 2020-11-29 一种促骨形成多肽及其应用

Country Status (1)

Country Link
WO (1) WO2022110092A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103665142A (zh) * 2013-11-20 2014-03-26 北京博恩康生物科技有限公司 一种诱导成骨短肽及其制备方法和用途
CN103880928A (zh) * 2014-03-28 2014-06-25 甘少磊 一种诱导成骨短肽及其制备方法和用途
US20200230286A1 (en) * 2017-05-03 2020-07-23 The University Of Akron Post-3d printing functionalization of polymer scaffolds for enhanced bioactivity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103665142A (zh) * 2013-11-20 2014-03-26 北京博恩康生物科技有限公司 一种诱导成骨短肽及其制备方法和用途
CN103880928A (zh) * 2014-03-28 2014-06-25 甘少磊 一种诱导成骨短肽及其制备方法和用途
US20200230286A1 (en) * 2017-05-03 2020-07-23 The University Of Akron Post-3d printing functionalization of polymer scaffolds for enhanced bioactivity

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
AMSO ZAID, CORNISH JILLIAN, BRIMBLE MARGARET A: "Short Anabolic Peptides for Bone Growth ", MEDICINAL RESEARCH REVIEWS, MEDICAL RESEARCH REVIEWS, vol. 36, no. 4, 1 July 2016 (2016-07-01), US , pages 579 - 640, XP055935687, ISSN: 0198-6325, DOI: 10.1002/med.21388 *
EMILY M LINDLEY; FERNANDO A GUERRA; JACK T KRAUSER; SERGIO M MATOS; EVALINA L BURGER; VIKAS V PATEL: "Small peptide (P-15) bone substitute efficacy in a rabbit cancellous bone model", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B: APPLIED BIOMATERIALS, vol. 94B, no. 2, 31 August 2010 (2010-08-31), pages 463 - 468, XP0055671191, ISSN: 1552-4973, DOI: 10.1002/jbm.b.31676 *
FRANCISCO GOMAR ; RAFAEL OROZCO ; JOSE LUIS VILLAR ; FEDERICO ARRIZABALAGA: "P-15 small peptide bone graft substitute in the treatment of non-unions and delayed union. A pilot clinical trial", INTERNATIONAL ORTHOPAEDICS, vol. 31, no. 1, 8 June 2006 (2006-06-08), Berlin, DE , pages 93 - 99, XP019489820, ISSN: 1432-5195 *
KANIE, K.: "Screening of Osteogenic- Enhancing Short Peptides from BMPs for Biomimetic Material Applications", MATERIIALS, vol. 9, no. 9, 1 January 2016 (2016-01-01), pages 1 - 17, XP055704446, DOI: 10.3390/ma9090730 *
QIU KAI , CHEN XIN , LI TIANQUAN , WAN CHANGXIU: "The Advance of Bioactive Peptide RGD in the Research of Bone Regeneration", JOURNAL OF BIOMEDICAL ENGINEERING, vol. 20, no. 3, 25 September 2003 (2003-09-25), pages 546 - 549, XP055935693 *
SHOHEI KASUGAI,RYUICHI FUJISAWA,YOSHIHIRO WAKI,KEN-ICHI MIYAMOTO,KEIICHI OHYA: "Selective drug delivery system to bone: small peptide (Asp)6 conjugation", JOURNAL OF BONE AND MINERAL RESEARCH, vol. 15, no. 5, 1 May 2000 (2000-05-01), US , pages 936 - 943, XP008008188, ISSN: 0884-0431, DOI: 10.1359/jbmr.2000.15.5.936 *
WANG CUNYANG, LIU YAN, FAN YUBO, LI XIAOMING: "The use of bioactive peptides to modify materials for bone tissue repair", REGENERATIVE BIOMATERIALS, vol. 4, no. 3, 16 April 2017 (2017-04-16), pages 191 - 206, XP055935678, ISSN: 2056-3418, DOI: 10.1093/rb/rbx011 *
XU ZIXING, CHEN JIANTING, XU WEIHONG, ZHU XIA, WANG CHANGSHENG, LUO HONGBIN, LI GUISHUANG, CHEN RONGSHENG: "Osteogenic Effect of Peptides Anchored Aminated Tissue Engineered Bone for Repairing Femoral Defect in Rats", CHINESE JOURNAL OF REPARATIVE AND RECONSTRUCTIVE SURGERY, vol. 27, no. 5, 23 April 2013 (2013-04-23), pages 520 - 528, XP055935685, ISSN: 1008-1892, DOI: 10.7507/1008-1892.20130118 *

Similar Documents

Publication Publication Date Title
CN112457371B (zh) 一种促骨形成多肽及其应用
Mayahara et al. In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats
EP2083846B1 (en) N-terminal fgf variants having increased receptor selectivity and uses thereof
RU2557654C2 (ru) Терапевтические агенты для снижения уровней паратиреоидного гормона
AU2008353306B2 (en) An synthetic peptide containing bone forming peptide 1(BFP 1) for stimulating osteoblast differentiation, and pharmaceutical composition comprising the synthetic peptide
US7754683B2 (en) Bioactive peptide of bone morphogenetic protein-2
JP4786047B2 (ja) ペプチド誘導体
Gulseren et al. Dentin phosphoprotein mimetic peptide nanofibers promote biomineralization
KR20050010778A (ko) 결합조직 자극 펩티드
JP2009502782A (ja) 硬組織形成を促進するためのタンパク質製剤
CN109789188A (zh) 具有低峰-谷比的pth化合物
CA2269655C (en) Peptide compositions with growth factor-like activity
CN113388005B (zh) 增强成骨细胞活性的多肽及其在治疗骨科疾病中的应用
WO2012158169A1 (en) Methods and compositions for tissue repair
US9238055B2 (en) Peptide bone forming peptide 4 for promoting osteogenesis or vascularization and use thereof
WO2006041205A1 (ja) 血管形成促進剤
JP2002540216A (ja) アドレノメデュリン又はアドレノメデュリンアゴニストを用いた骨障害の治療
WO2022110092A1 (zh) 一种促骨形成多肽及其应用
CN110225920B (zh) 具有细胞渗透性和骨组织再生能力的双功能新颖肽及其用途
US6352973B1 (en) Bone stimulating factor
KR19990022716A (ko) 골 자극 인자
US20140256635A1 (en) Anti-angiogenic molecules, nanostructures and uses thereof
WO2021187478A1 (ja) 自己組織化ペプチドを含む組成物
US8066980B2 (en) Osteogenic synthetic peptides, pharmaceutical compositions comprising the same, and medium containing the same
WO2023272658A1 (zh) 增强成骨细胞活性的多肽及其在治疗骨科疾病中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20962982

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20962982

Country of ref document: EP

Kind code of ref document: A1