Bone remodeling modulating polypeptides and uses

Abstract

The application provides bone reconstruction regulating polypeptides and application. On the basis of the first experiment of the inventors, bone reconstruction regulating polypeptides are designed and newly synthesized by using solid-phase polypeptide synthesis method through sequence alignment, structure analysis, physicochemical property and function prediction. The polypeptides have the characteristics of short length, simple synthesis, low cost, low cytotoxicity, high biological stability, moderate half-life, good bone targeting, and strong bone resorption regulating ability. The differentiation and function of osteoclasts and osteoblasts can be controlled by adjusting the concentration of the synthesized bone reconstruction regulating polypeptides, the ordered regulation of bone resorption and bone formation is realized, and the polypeptides have a broad potential application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bone remodeling regulation technology, specifically relating to bone remodeling regulatory peptides and their applications. Background Technology

[0002] The development and therapeutic targets of current anti-osteoporosis drugs are almost entirely focused on bone remodeling (the dynamic balance between bone formation and bone resorption), i.e., how to inhibit bone resorption or promote bone formation. Clinically, anti-osteoporosis drugs are also divided into anti-resorption drugs, such as bisphosphonates and calcitonin, and bone-forming drugs, such as teriparatide, based on these two points. However, the former inhibits bone resorption while also inhibiting bone formation, significantly increasing the incidence of adverse reactions with long-term use; while the latter promotes bone formation while also promoting bone resorption, potentially increasing the incidence of fractures after two years of use. To date, there is no anti-resorption drug that inhibits bone resorption without inhibiting bone formation, nor is there any bone-forming drug that promotes bone formation without promoting bone resorption! This greatly limits the effectiveness and scope of application of these drugs in clinical practice. Currently, bone remodeling modulating drugs mainly include bisphosphonates, calcitonin, selective estrogen receptor modulators, parathyroid hormone analogs, and RANKL inhibitors. Among these, only parathyroid hormone analogs are bone remodeling modulating peptide drugs, derived from the hormone parathyroid hormone itself, which has bone remodeling modulating effects. Currently, there are no non-hormonal peptide drugs available domestically or internationally. Summary of the Invention

[0003] Bone tissue is composed of bone matrix and cells, with 90% of the organic component of the bone matrix being type I collagen. Although collagen is widely used in bone tissue engineering materials, cell culture, medical aesthetics, and even daily life, it has also faced considerable skepticism. The reasons are mainly twofold: First, because type I collagen consists of triple-helix collagen fiber bundles with a large molecular weight, it has long been thought to only play a supporting, protective, or interstitial role in anchoring cytokines. Second, type I collagen is a heterotrimeric protein composed of two α1 chains and one α2 chain, with a complex peptide chain structure, making it difficult to identify the core sequence that binds to its receptor. Therefore, its activity cannot be explained from a molecular mechanism perspective, leading to skepticism about the application of collagen products in health and medical fields, and the scarcity of drugs developed specifically targeting collagen. Therefore, identifying and designing core type I collagen peptides that bind to receptors, and revealing the mechanism by which type I collagen regulates bone remodeling, will not only establish and further refine new mechanisms of bone metabolism regulation but also provide new evidence for the application of collagen and its peptides.

[0004] Furthermore, in the treatment of osteoporotic metabolic bone diseases, achieving specific bone targeting of drugs can significantly increase therapeutic efficacy. While bisphosphonates can specifically target bone formation and resorption surfaces by binding to hydroxyapatite (HA) on the bone surface, long-term or high-dose use may lead to osteonecrosis. Oligopeptides, such as glutamate (Glu) and aspartic acid (Asp), not only possess the characteristics of being enzymatically degradable, not forming colloids with metals, and having no long-term health risks, but they can also sensitively sense changes in the crystallinity of HA on the bone resorption surface, thereby achieving targeting of the bone resorption surface. However, the uncertainty of bone targeting efficacy often increases due to differences in the number of linked oligopeptides and the structure of the linked polypeptides.

[0005] The inventors synthesized novel bone remodeling regulatory peptides using a solid-phase peptide synthesis method through sequence alignment, structural analysis, physicochemical property verification experiments, and functional validation. These peptides consist of nine amino acids and are simple and inexpensive to synthesize. Furthermore, the bone remodeling regulatory peptides of this invention exhibit low cytotoxicity, high biological stability, and a moderate half-life, demonstrating broad potential applications. Experiments have confirmed that the synthesized bone remodeling regulatory peptides can specifically regulate the differentiation and function of osteoclasts and osteoblasts, achieving orderly regulation of bone resorption and bone formation. Based on this, we designed and synthesized bone tissue-targeting bone remodeling regulatory peptides using aspartic acid (ASP)8, and verified their specific bone tissue targeting ability in vivo, showing promise for development into new clinical drugs for osteoporosis, osteosclerosis, and other metabolic bone diseases.

[0006] The purpose of this invention is to provide a bone remodeling regulatory polypeptide and its applications. These polypeptides are characterized by short length, simple synthesis, low cost, low cytotoxicity, high biological stability, moderate half-life, good bone targeting, and broad potential application prospects. Furthermore, the bone remodeling regulatory polypeptide synthesized in this invention can specifically regulate the differentiation and function of osteoclasts and osteoblasts, thereby achieving orderly regulation of bone resorption and bone formation.

[0007] The bone remodeling regulatory polypeptide of the present invention has a core sequence as described in any one or more of the following:

[0008] Gly-Xaa-Pro-Gly-Xaa-Xaa-Gly-Xaa-Xaa, see SEQ ID NO.1,

[0009] Furthermore, the second amino acid Xaa is Ala, Pro, or Gly; the fifth amino acid Xaa is Pro or Ala; the sixth amino acid Xaa is Ala, Gln, Thr, Ser, or a hydroxylated Pro; the eighth amino acid Xaa is Phe, Ser, Asp, or Tyr; the ninth amino acid Xaa is Ala, Gln, Pro, Arg, or a hydroxylated Pro; and the third amino acid Pro is hydroxylated. The hydroxylated Pro is hydroxyproline (Hyp).

[0010] Furthermore, the present invention preferably includes one or more of the above-mentioned core sequences, specifically as follows:

[0011] A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln, see SEQ ID NO.2

[0012] A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln, see SEQ ID NO.3,

[0013] A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala, see SEQ ID NO.4,

[0014] A1-5-3: Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro, see SEQ ID NO.5,

[0015] OSC pep : Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala, see SEQ ID NO. 6.

[0016] Furthermore, one or more of the above core sequences are preferred, as follows:

[0017] A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln,

[0018] A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln,

[0019] A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala,

[0020] A1-5-3: Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro.

[0021] The bone remodeling regulatory polypeptides include a series of polypeptides that can play a role in regulating bone remodeling, obtained by modifying the core sequence through N-terminus or C-terminus, elongating the amino acid sequence, substituting or replacing amino acids, cyclizing or chiralizing them.

[0022] The aforementioned bone remodeling regulation refers to controlling the differentiation and function of osteoclasts and osteoblasts by regulating the concentration of synthesized bone remodeling regulatory peptides, thereby achieving orderly regulation of bone resorption and bone formation.

[0023] The bone remodeling regulatory polypeptide has at least two aspartic acid residues, preferably eight consecutive aspartic acid residues, linked to the amino terminus of its amino acid sequence.

[0024] The application of the bone remodeling regulatory polypeptide described above in the preparation of preparations that promote bone resorption and inhibit bone formation, or preparations that inhibit bone resorption and promote bone formation.

[0025] When the above-mentioned bone remodeling regulatory peptides are

[0026] A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln,

[0027] A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln,

[0028] A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala,

[0029] A1-5-3:Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro,

[0030] When any one or more of the above are present, the concentration of bone remodeling-regulating peptide in the preparation for promoting bone resorption and inhibiting bone formation is <500 μg / mL, preferably <200 μg / mL, and the concentration of bone remodeling-regulating peptide in the preparation for inhibiting bone resorption and promoting bone formation is ≥500 μg / mL, preferably 500-1000 μg / mL.

[0031] When the above-mentioned bone remodeling regulatory peptide is OSC pep When Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala is used, the concentration of bone remodeling-regulating peptide in the preparation that promotes bone resorption and inhibits bone formation is <8000 μg / mL, preferably 1000-5000 μg / mL, and the concentration of bone remodeling-regulating peptide in the preparation that inhibits bone resorption and promotes bone formation is ≥8000 μg / mL, preferably 8000-10000 μg / mL.

[0032] The application of the bone remodeling regulatory polypeptide described in this invention in the preparation of agents for the treatment of metabolic bone diseases, including osteoporosis and osteosclerosis.

[0033] Furthermore, when the aforementioned bone remodeling regulatory peptide is

[0034] A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln,

[0035] A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln,

[0036] A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala,

[0037] A1-5-3:Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro,

[0038] When any one or more of the following are present, the concentration of bone remodeling-regulating peptide in the preparation for treating osteoporosis is ≥500 μg / mL, preferably 500-1000 μg / mL; the concentration of bone remodeling-regulating peptide in the preparation for treating osteosclerosis is <500 μg / mL, preferably <200 μg / mL.

[0039] When the above-mentioned bone remodeling regulatory peptide is OSC pep When Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala is used to treat osteoporosis, the concentration of bone remodeling-regulating peptides in the formulation is ≥8000 μg / mL, preferably 8000-10000 μg / mL; and the concentration of bone remodeling-regulating peptides in the formulation for treating osteosclerosis is <8000 μg / mL, preferably 1000-5000 μg / mL.

[0040] The collagen receptor OSCAR (osteoclast-associated receptor) can be expressed in monocytes and osteoclasts, providing a significant breakthrough for research on the mechanism of collagen. However, type I collagen is a heterotrimer with a complex peptide chain structure, and its interaction with OSCAR remains unclear. Our research on the protein structure of mouse OSCAR (mOSCAR) revealed five highly conserved amino acid sites that can bind to collagen molecules. Therefore, we hypothesize that type I collagen can bind to OSCAR and participate in bone remodeling. Furthermore, our experiments showed that high levels of OSCAR are also expressed on osteoblasts and osteocytes involved in bone formation. We further hypothesize that type I collagen can bind to OSCAR not only to regulate bone resorption but also to regulate bone formation.

[0041] Therefore, through sequence alignment, structural analysis, physicochemical property verification, and functional validation, we innovatively designed and synthesized four peptides derived from type I collagen and one peptide derived from type II and III collagen, each containing a core sequence of nine amino acids, using a solid-phase peptide synthesis method. These five peptides were collectively termed bone remodeling regulatory peptides. The newly synthesized five bone remodeling regulatory peptides were found to possess low cytotoxicity, high biological stability, and appropriate half-lives. Subsequently, we constructed an in vitro osteoclast differentiation model using primary mouse bone marrow mononuclear cells (BMMs) under RANKL and M-CSF induction conditions. Simultaneously, we constructed a bone resorption model using bovine bone slices and BMMs under RANKL and M-CSF induction conditions. We observed the formation of bone resorption lacunae using WGA staining. The results showed that low concentrations (<500 μg / mL, even below 200 μg / mL had good effects) of type I collagen bone remodeling regulatory peptides (A1-1 / A1-2-1 / A1-4-1 / A1-5-3) promoted osteoclast differentiation, while high concentrations (≥500 μg / mL, 500-1000 μg / mL had significant effects) of type I collagen bone remodeling regulatory peptides inhibited osteoclast differentiation and bone resorption. Bone remodeling regulatory peptides derived from type II and III collagen (OSC) showed a different effect. pep While exhibiting the same concentration-specific regulatory effect on bone resorption, its effective low concentrations (<8000 μg / mL) and 1000-5000 μg / mL show significant effects, while its effective high concentrations (≥8000 μg / mL, preferably 8000-10000 μg / mL) demonstrate higher bioactivity, indicating that type I collagen bone remodeling regulatory peptides possess higher biological activity. Osteogenesis was induced in primary pre-osteoblasts derived from neonatal mouse skulls under β-glycerophosphate and vitamin C conditions to construct a bone formation model. Osteoblast differentiation was observed using alkaline phosphatase (ALP) staining. The results showed that low concentrations (<500 μg / mL, preferably <200 μg / mL) of type I collagen bone remodeling regulatory peptides inhibited osteoblast differentiation, while high concentrations (≥500 μg / mL, preferably 500-1000 μg / mL) promoted osteoblast differentiation. When the bone remodeling regulatory peptide is OSC... pep At concentrations <8000 μg / mL, preferably 1000-5000 μg / mL, osteoblast differentiation was inhibited, while concentrations ≥8000 μg / mL, preferably 8000-10000 μg / mL, promoted osteoblast differentiation. Therefore, we conclude that bone remodeling regulatory peptides can specifically regulate bone remodeling at concentrations.

[0042] Having first experimentally clarified the role of bone remodeling regulatory peptides in bone formation / resorption, we will further explore the feasibility of their clinical application. If bone remodeling regulatory peptides can specifically target bone tissue, the therapeutic effect can be significantly increased. Among existing osteoporosis treatments, FDA-approved bisphosphonates can specifically target the bone formation and resorption surfaces by binding to hydroxyapatite on the bone surface; however, long-term or high-dose use may lead to osteonecrosis. Oligopeptides such as glutamate (Glu) and aspartic acid (Asp) have the advantages of being enzymatically degradable, not forming colloids with metals, and having no long-term health risks. Different numbers of Asp linkages result in different bone targeting capabilities; studies have found that as the number of Asp linkages increases, the binding ability to hydroxyapatite strengthens, with 8, 11, and 14 Asp linkages showing the best bone tissue targeting capabilities. Simultaneously, Asp exists in two conformations: left-handed (L-Asp) and right-handed (D-Asp). Studies have found that (D-Asp)n is more stable than (L-Asp)n. Therefore, (D-Asp)8 was used to link the five segments of the bone remodeling regulatory peptide of this invention, and it was labeled with the red fluorescent excitation group Cy5. The red fluorescent Cy5 label has stronger solubility, stability, and fluorescence intensity than the conventional FITC green fluorescent label. In vivo experimental results showed that, compared with physiological saline and the Cy5 control group, the Cy5-labeled (D-Asp)8-linked bone remodeling regulatory peptide of this invention was found to fluoresce only in the femur, tibia, vertebrae, skull, and alveolar bone 1-7 days after tail vein injection in mice, exhibiting good bone tissue specificity. Excitingly, the bone tissue-targeting bone remodeling regulatory peptide we designed and synthesized has a half-life of 7 days in bone, which is much longer than the bone half-life of conventional (Asp)8-loaded drugs.

[0043] Building upon our initial experimental findings, this invention synthesizes five novel bone remodeling regulatory peptides using a solid-phase peptide synthesis method through sequence alignment, structural analysis, physicochemical property verification, and functional validation. These peptides consist of nine core amino acids, each only one-fifth the length of triple-helix peptides derived from type II and III collagen, making their synthesis simple and inexpensive. Furthermore, the bone remodeling regulatory peptides of this invention exhibit low cytotoxicity, high biological stability, moderate half-life, and strong bone resorption regulation capabilities, demonstrating broad potential applications. In vitro experiments have confirmed that the synthesized bone remodeling regulatory peptides can specifically regulate the differentiation and function of osteoclasts and osteoblasts, achieving orderly regulation of bone resorption and bone formation. Based on this, we designed and synthesized bone tissue-targeting bone remodeling regulatory peptides using (ASP)8, and in vivo validation of their specific bone tissue targeting ability suggests their potential development into novel clinical drugs for treating osteoporosis and other metabolic bone diseases. Attached Figure Description

[0044] Figure 1(A) Positive TRAP staining indicates that the extracted primary osteoclast precursor cells can differentiate into osteoclasts; (B) Immunofluorescence and (C) Western blot results show that both osteoclast precursor cells and osteoclasts express the collagen receptor OSCAR; (D) Positive ALP staining on day 14 of osteogenic induction of primary pre-osteoblasts indicates that the extracted primary pre-osteoblasts can differentiate into osteoblasts; (E) Immunohistochemical results on days 0, 7, 14, 21, and 35 of osteogenic induction of primary pre-osteoblasts show that both primary pre-osteoblasts and osteoblasts at different differentiation stages express the collagen receptor OSCAR; (F) Western blot results show that both primary pre-osteoblasts and the MLO-Y4 osteoblast line express the collagen receptor OSCAR, ***P<0.001.

[0045] Figure 2 (A) Multiple sequence alignment results of human and (B) mouse Col1a1 and Col1a2 showed that there is a sequence on type I collagen that may bind to the collagen receptor OSCAR (boxed part); (C) Prediction of the three-dimensional structure of mouse OSCAR protein showed that the D2 domain (gray background) of mouse OSCAR protein is highly conserved at sites 135, 145, 169, 177, and 182 and can bind to collagen-derived peptides.

[0046] Figure 3 (A) The hydroxylation level of amino acids in protein sequences that may bind to the collagen receptor OSCAR in mouse type I collagen; (B) Four stable sequences were screened based on isoelectric point, number of positive and negative charge residues, number of atoms, half-life, instability coefficient, average hydrophilicity and aliphatic amino acid index (grey part).

[0047] Figure 4 Synthetic type II collagen peptides (OSC) pep The mass spectrometry and chromatographic analysis results of the synthesized peptides and four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) showed that the purity of the synthesized peptides was >98%.

[0048] Figure 5 The results of the solid-state combination experiment showed that the synthesized type II collagen polypeptide (OSC) pep ) and 4 type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) can bind to OSCAR. *P<0.05.

[0049] Figure 6(A) TRAP staining and WGA staining results (B) showed that four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) could promote osteoclast differentiation and bone resorption at low concentrations (<500 μg / mL, preferably <200 μg / mL) and inhibit osteoclast differentiation and bone resorption at high concentrations (≥500 μg / mL, preferably 500-1000 μg / mL); type II collagen peptides (OSC) pep (C) ALP staining results showed that: <500μg / mL, preferably <200μg / mL, four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) could inhibit osteoblast differentiation and bone formation; ≥500μg / mL, preferably 500-1000μg / mL, four type I collagen peptides could promote osteoblast differentiation and bone formation; <8000μg / mL, preferably 1000-5000μg / mL, type II collagen peptides (OSC) could promote osteoblast differentiation and bone formation. pep It can inhibit osteoblast differentiation and bone formation, ≥8000 μg / mL, preferably 8000-10000 μg / mL type II collagen peptide (OSC). pep (D) It can promote osteoblast differentiation and bone formation; (D) The results of the cytotoxicity test showed that collagen peptides at all concentrations had no cytotoxic effects. * P<0.05, ** P<0.01.

[0050] Figure 7 Mass spectrometry and chromatographic analysis results of synthesized Cy5-labeled (Asp) 8-I type collagen peptide (A1-1) showed that the purity of the synthesized peptide was >98%.

[0051] Figure 8 (A) Fluorescence imaging results of various tissues and femur of mice injected with Cy5-labeled (Asp)8-bone remodeling regulatory peptide via tail vein showed that Cy5 fluorescence was expressed only in the femur, tibia, vertebrae, skull, and alveolar bone, and no expression was observed in other tissues; (B) Confocal microscopy observation of femoral hard tissue sections showed that the Cy5-labeled (Asp)8-bone remodeling regulatory peptide injection group expressed red fluorescence, while the Cy5 control group did not express it; (C) Blood half-life measurement results of fluorescence imaging showed that the half-life of Cy5 and Cy5-labeled (Asp)8-bone remodeling regulatory peptide in blood was 1.5 hours; (D) Bone half-life measurement results of fluorescence imaging showed that the half-life of Cy5-labeled (Asp)8-bone remodeling regulatory peptide in bone was more than 7 days.

[0052] For ease of description, all amino acids in the attached diagrams are replaced with their corresponding known uppercase letters, with hydroxylated proline (Pro) replaced by the letter O. Detailed implementation method:

[0053] The following examples are intended to further illustrate the present invention, but not to limit it.

[0054] Example 1

[0055] We determined that osteoclasts and osteoblasts express the collagen receptor OSCAR.

[0056] 1) Bone marrow cells were aseptically isolated from the bilateral femurs and tibias of wild-type female mice. The bone marrow cavity was repeatedly washed with culture medium to obtain bone marrow cells. After culturing in α-MEM complete medium containing M-CSF (30 ng / mL) for 24 hours, non-adherent cells were aspirated, centrifuged, and resuspended. These cells were then cultured again in α-MEM complete medium containing M-CSF (30 ng / mL) for 3 days to obtain adherent primary mouse bone marrow mononuclear macrophages (osteoclast precursor cells). M-CSF (50 ng / mL) and RANKL (100 ng / mL) were used to induce differentiation into osteoclasts, which were then identified by TRAP staining. Figure 1 A), via OSCAR immunofluorescence ( Figure 1 B) and Western blot experiment ( Figure 1 C) Identify osteoclast precursor cells and osteoclasts that express the collagen receptor OSCAR;

[0057] 2) Wild-type mouse skulls were aseptically isolated, and cells were separated by collagenase digestion. Nucleated cells were obtained by density gradient centrifugation and administered with fetal bovine serum containing 10 mM β-glycerophosphate sodium, 50 μM vitamin C, and 10... -7 Osteogenic induction was performed using M dexamethasone culture medium, and the results were identified by ALP staining. Figure 1 D), OSCAR immunohistochemistry was performed on days 0, 7, 14, 21, and 35 of osteogenic induction. Figure 1 E) and Western blot experiments ( Figure 1 F) Identify that pre-osteoblasts and the MLO-Y4 osteoblast line express the collagen receptor OSCAR.

[0058] Example 2

[0059] For human beings ( Figure 2 A) and mice ( Figure 2 B) Perform multiple sequence alignment on the α1 and α2 subunits of type I collagen to identify sequences on type I collagen that may bind to the collagen receptor OSCAR. Figure 2In boxes A and B, the upper sequence in each group represents Col1a1, and the lower sequence represents Col1a2. Furthermore, by predicting the protein structure of mouse OSCAR (mOSCAR), we found that it possesses five highly conserved amino acid sites that can bind to collagen molecules. Figure 2 C).

[0060] Example 3

[0061] Based on physicochemical properties and functional predictions (isoelectric point, molecular weight, number of positive and negative charges, half-life, instability index, average hydrophilicity, and fatty amino acid index), four stable sequences derived from type I collagen and one sequence derived from type II and III collagen were screened. The selected sequences derived from type I collagen and type II and III collagen were synthesized into peptides using a solid-phase peptide synthesis method (i.e., five bone remodeling regulatory peptides were synthesized).

[0062] 1) By analyzing the hydroxylation levels of amino acids in protein sequences of mouse type I collagen that may bind to the collagen receptor OSCAR (… Figure 3 A), and analysis of the isoelectric point, number of positive and negative charge residues, number of atoms, half-life, instability coefficient, average hydrophilicity and aliphatic amino acid index of the above sequences ( Figure 3 B), through comprehensive analysis of the above parameters, four sequences with stable performance were selected. Figure 3 The gray parts (B) are named A1-1, A1-2-1, A1-4-1, and A1-5-3 respectively.

[0063] 2) The four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) and one type II / III collagen peptide (OSC) that can bind to the collagen receptor OSCAR were synthesized and purified using a solid-phase peptide synthesis method. pep ).

[0064] The five sequences are as follows:

[0065] A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln,

[0066] A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln,

[0067] A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala,

[0068] A1-5-3:Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro,

[0069] OSC pep :Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala

[0070] The third amino acid Pro in each of the above sequences is hydroxylated, and the sixth amino acid Pro in A1-2-1 is hydroxylated.

[0071] Using Wang Resin resin, Fmoc-amino acid-OH and DIEA were added and mixed with DIEA to complete the ligation of the first amino acid. The Fmoc protecting group was eluted with 20% piperidine DMF, and the second Fmoc-amino acid-OH, HBTU, and DIEA were added and the mixture was shaken to complete the ligation of the second amino acid. Subsequent amino acids were ligated sequentially using the same method. After eluting the Fmoc protecting group, the peptide was cleaved from the resin to obtain the crude peptide. The target peptide was purified by high-performance liquid chromatography (HPLC), and after lyophilization, the purity was determined by LC-MS to be >98%. Figure 4 ).

[0072] Example 4

[0073] The synthesized type II collagen polypeptide (OSC) was verified through solid-state bonding experiments. pep ) and four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) can bind to the collagen receptor OSCAR.

[0074] 1) The extracellular region of mOSCAR was amplified by RT-PCR, and the expression vector of the His tag fusion protein was inserted. The constructed vector was then transfected into HEK293T cells for expression. The mOSCAR-His fusion protein was purified by ion exchange chromatography and molecular sieve chromatography.

[0075] 2) Collagen peptides were dissolved in 10 mM acetic acid and coated onto 96-well microtiter plates overnight. The plates were blocked with phosphate buffer containing bovine serum albumin at room temperature for 1 hour. Then, mOSCAR-His fusion protein was added and incubated at 37°C for 3 hours. The plates were then incubated with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (which specifically reacts with His) at room temperature for 1 hour. The binding of mOSCAR-His fusion protein was detected by o-phenylenediamine hydrochloride. The reaction was terminated with 3 M H₂SO₄, and the absorbance at 492 nm was measured to confirm the type II collagen peptide (OSCAR-His fusion protein). pep ) and four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) can bind to mOSCAR. Figure 5 ).

[0076] Example 5

[0077] After verifying that the five bone remodeling regulatory peptides have similar physicochemical properties and the ability to bind to the mouse collagen receptor OSCAR, we conducted this part of the experimental verification. In vitro cell experiments verified that the five synthesized collagen peptides can specifically regulate the differentiation and function of osteoclasts and osteoblasts at specific concentrations, and that they do not exhibit cytotoxic effects.

[0078] 1) Primary bone remodeling modulators (BMMs) were cultured in vitro according to the method described in Part 1 above. Cytotoxicity assays were performed using A1-1 / A1-4-1 collagen peptides as an example. Different concentrations (0, 1, 10, 100, 200, 500, 800, 1000, 1500 μg / mL) of collagen peptides were used for intervention for 24 and 48 hours. The cytotoxic effect of the bone remodeling modulators was determined using a CCK-8 assay kit. It was confirmed that the above concentrations of bone remodeling modulators had no cytotoxic effect on the cells. Figure 6 D).

[0079] 2) Primary bone marrow mesenchymal stem cells (BMMs) were treated with five collagen peptides at different concentrations, and their differentiation into osteoclasts was induced by M-CSF and RANKL. The effect of different concentrations of collagen peptides on osteoclast differentiation was verified by TRAP staining, confirming that the bone remodeling regulatory peptides could promote osteoclast differentiation at low concentrations and inhibit it at high concentrations. Figure 6 A).

[0080] 3) A peptide-primary BMMs-bone slice culture system was constructed using the aforementioned bone remodeling regulatory peptides at different concentrations. Simultaneously, M-CSF and RANKL were used to induce differentiation into osteoclasts. Wheat germ agglutinin (WGA) staining was used to verify the effect of collagen peptides on osteoclast bone resorption, confirming that both bone remodeling regulatory peptides could promote osteoclast bone resorption at low concentrations and inhibit it at high concentrations. Figure 6 B).

[0081] TRAP and WGA staining results showed that four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) could promote osteoclast differentiation and bone resorption at low concentrations (<500 μg / mL, preferably <200 μg / mL) and inhibit osteoclast differentiation and bone resorption at high concentrations (≥500 μg / mL, preferably 500-1000 μg / mL); type II collagen peptides (OSC) pep It can promote osteoclast differentiation and bone resorption at low concentrations (<8000μg / mL, preferably 1000-5000μg / mL) and inhibit osteoclast differentiation and bone resorption at high concentrations (≥8000μg / mL, preferably 8000-10000μg / mL).

[0082] 4) Primary preosteoblasts were cultured in vitro according to the method described in Part 1 above, and treated with five collagen peptides at different concentrations, while simultaneously receiving 10 mM β-glycerophosphate sodium, 50 μM vitamin C, and 10 -7Osteogenic induction was performed using dexamethasone, and the effect of collagen peptides on osteoblast differentiation was verified by ALP staining. The results confirmed that bone remodeling regulatory peptides could inhibit osteoblast differentiation and promote bone formation at low concentrations and at high concentrations. Figure 6 C). ALP staining results showed that: <500 μg / mL, preferably <200 μg / mL, four type I collagen peptides (A1-1, A1-2-1, A1-4-1, A1-5-3) could inhibit osteoblast differentiation and bone formation; ≥500 μg / mL, preferably 500-1000 μg / mL, four type I collagen peptides could promote osteoblast differentiation and bone formation; <8000 μg / mL, preferably 1000-5000 μg / mL, type II collagen peptides (OSC) pep It can inhibit osteoblast differentiation and bone formation, ≥8000 μg / mL, preferably 8000-10000 μg / mL type II collagen peptide (OSC). pep It can promote osteoblast differentiation and bone formation.

[0083] Example 6

[0084] Cy5-labeled bone tissue-targeting bone remodeling regulatory peptides were designed and synthesized. In vivo animal experiments were conducted to determine the bone tissue targeting of the synthesized peptides.

[0085] 1) Five Cy5 fluorescently labeled bone tissue-targeting bone remodeling regulatory peptides were synthesized and purified using a solid-phase peptide synthesis method.

[0086] Using Wang Resin resin, the first amino acid was ligated by adding Fmoc-amino acid-OH and DIEA and shaking. The Fmoc protecting group was eluted with 20% piperidine DMF, and the second Fmoc-amino acid-OH, HBTU, and DIEA were added and shaken to ligate the second amino acid. Subsequent amino acids were ligated sequentially using the same method. After eluting the Fmoc protecting group, Cy5.0 NHS fluorescence was added, and the mixture was reacted in anhydrous acetonitrile solution in the dark for 30 minutes. The peptide was cleaved from the resin to obtain the crude peptide. The target peptide was purified by high-performance liquid chromatography (HPLC), and after lyophilization, the purity was determined by LC-MS to be >98%. The mass spectrometry and chromatographic analysis results of the Cy5-labeled (Asp) 8-I type collagen peptide (taking A1-1 as an example) are shown below. Figure 7 .

[0087] 2) After verifying that the five bone remodeling regulatory peptides have similar physicochemical properties and the ability to bind to the mouse collagen receptor OSCAR, we will use A1-4-1 as a representative for this part of the experiment. In vivo animal experiments confirmed that the bone tissue-targeting bone remodeling regulatory peptide can specifically target bone tissue, and its half-life was determined.

[0088] The synthesized Cy5-labeled (Asp)8-bone remodeling regulatory peptide was injected into mice via the tail vein. Fluorescence imaging of tissues (brain, heart, lung, liver, spleen, subcutaneous fat) and bone tissues was performed at 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, 4 d, and 7 d after injection. The results showed that, compared with the saline and Cy5 control groups, the Cy5-(Asp)8-bone remodeling regulatory peptide group was expressed only in the femur, tibia, vertebrae, skull, and alveolar bone at 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, 4 d, and 7 d after injection; no expression was observed in other tissues and organs (results at 2 h and 96 h are shown in [details omitted]). Figure 8 A); simultaneously, hard tissue sections of the femur were taken, and the expression of Cy5 fluorescence in bone was observed under a confocal microscope. The results showed that the Cy5-labeled (Asp)8-bone remodeling regulatory peptide injection group expressed red fluorescence, while the Cy5 control group did not express it. Figure 8 B) indicates that the Cy5-labeled (Asp)8-bone remodeling regulatory peptide can specifically target bone tissue. The synthesized Cy5-labeled (Asp)8-bone remodeling regulatory peptide was injected into rats via the tail vein. Blood samples were collected from the tail vein at 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, 4 d, and 7 d after injection. Serum was collected after centrifugation, and fluorescence quantification was performed using a fluorescence imaging system. The results showed that the half-life of the Cy5-labeled (Asp)8-bone remodeling regulatory peptide in the blood was approximately 1.5 hours. Figure 8 C); Fluorescence quantification of bone tissue at various time points was performed using a fluorescence imaging system. The results showed that the half-life of Cy5-labeled (Asp)8-bone remodeling regulatory peptide in bone exceeded 7 days. Figure 8 D).

[0089] The bone remodeling regulatory peptide designed and synthesized in this invention has a concentration-specific regulatory effect on the differentiation and function of osteoclasts and osteoblasts. By using the (Asp)8 linkage method, the bone remodeling regulatory peptide can specifically target bone tissue, and it is expected to be developed into a new clinical drug for treating osteoporosis and other metabolic bone diseases. sequence list <110> Xiangya No. 2 Hospital of Central South University <120> Bone remodeling regulatory peptides and their applications <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Gly Xaa Pro Gly Xaa Xaa Gly Xaa Xaa 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (3)..(3) <223> The third amino acid Pro is modified by hydroxylation. <400> 2 Gly Ala Pro Gly Pro Gln Gly Phe Gln 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (3)..(3) <223> The third amino acid Pro is modified by hydroxylation. <220> <221> SITE <222> (6)..(6) <223> The sixth amino acid, Pro, is modified by hydroxylation. <400> 3 Gly Ala Pro Gly Ala Pro Gly Ser Gln 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (3)..(3) <223> The third amino acid Pro is modified by hydroxylation. <400> 4 Gly Pro Pro Gly Pro Ala Gly Phe Ala 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (3)..(3) <223> The third amino acid Pro is modified by hydroxylation. <400> 5 Gly Pro Pro Gly Ala Thr Gly Phe Pro 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (3)..(3) <223> The third amino acid Pro is modified by hydroxylation. <400> 6 Gly Ala Pro Gly Pro Ala Gly Phe Ala 1 5

Claims

1. The application of bone remodeling regulatory peptides in the preparation of agents that inhibit bone resorption and promote bone formation, characterized in that, When bone remodeling regulatory peptides are A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln, A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln, A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala, A1-5-3: Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro, The bone resorption inhibitory preparation contains a bone remodeling-regulating polypeptide concentration ≥500 μg / mL; When the bone remodeling regulating peptide is OSCpep: Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala, the concentration of the regulating peptide in the bone resorption inhibitory preparation is ≥8000 μg / mL; The third amino acid Pro in each of the above sequences is hydroxylated, and the sixth amino acid Pro in A1-2-1 is hydroxylated.

2. The application of the bone remodeling regulatory polypeptide according to claim 1, characterized in that, Eight consecutive aspartic acid residues are attached to the amino terminus of the amino acid sequence.

3. The application of the bone remodeling regulatory polypeptide according to claim 1 or 2, characterized in that, When bone remodeling regulatory peptides are A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln, A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln, A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala, A1-5-3: When any one or more of Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro are selected, The bone resorption inhibitory preparation contains a bone remodeling-regulating polypeptide concentration of 500-1000 μg / mL; When the bone remodeling regulatory peptide is OSCpep: Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala, the concentration of the bone remodeling regulatory peptide in the bone resorption inhibitor is 8000-10000 μg / mL.

4. A preparation for treating metabolic bone diseases including osteoporosis and osteosclerosis, characterized in that, Including bone remodeling regulatory peptides, when bone remodeling regulatory peptides are A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln, A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln, A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala, A1-5-3: Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro, The concentration of bone remodeling regulatory peptides in the formulation is ≥500 μg / mL; When the bone remodeling regulatory peptide is OSCpep: Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala, the concentration of the bone remodeling regulatory peptide in the formulation is ≥8000 μg / mL. The third amino acid Pro in each of the above sequences is hydroxylated, and the sixth amino acid Pro in A1-2-1 is hydroxylated. The formulation described herein controls the differentiation and function of osteoclasts and osteoblasts by regulating the concentration of bone remodeling-regulating peptides. Different concentrations have diametrically opposite effects, thereby inhibiting bone resorption or promoting osteogenic function to achieve the purpose of treating osteoporosis.

5. The preparation for treating metabolic bone diseases including osteoporosis and osteosclerosis according to claim 4, characterized in that, The bone remodeling regulatory polypeptide has eight consecutive aspartic acid residues linked to the amino terminus of its amino acid sequence.

6. The preparation for treating metabolic bone diseases, including osteoporosis and osteosclerosis, according to claim 4 or 5, is characterized in that, When bone remodeling regulatory peptides are A1-1: Gly-Ala-Pro-Gly-Pro-Gln-Gly-Phe-Gln, A1-2-1: Gly-Ala-Pro-Gly-Ala-Pro-Gly-Ser-Gln, A1-4-1: Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala, A1-5-3: Any one or more of Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe-Pro The bone remodeling regulatory peptide concentration in the formulation is 500-1000 μg / mL; When the bone remodeling regulatory peptide is OSCpep: Gly-Ala-Pro-Gly-Pro-Ala-Gly-Phe-Ala, the concentration of the bone remodeling regulatory peptide in the formulation is 8000-10000 μg / mL.

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