A bone formation-promoting G5-like peptide or a pharmaceutically acceptable salt thereof

Abstract

The present invention belongs to the field of polypeptide drugs, and particularly relates to a G5-like peptide for promoting bone formation or a pharmaceutically acceptable salt thereof. The like peptide or its pharmaceutically acceptable salt comprises the amino acid sequence shown below or consists of the amino acid sequence shown below: 1) G5-1: SEQ ID NO: 1; 2) G5-2: SEQ ID NO: 2; 3) G5-3: SEQ ID NO: 3; or 4) G5-4: SEQ ID NO: 4. The G5-like peptide or its pharmaceutically acceptable salt significantly up-regulates the expression of alkaline phosphatase, significantly promotes the ability of osteogenic differentiation, has potential clinical applicability and research value in the treatment of osteoporosis, and is non-toxic to other tissues, safe and reliable.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drugs, and particularly relates to a G5-like peptide for promoting bone formation or a pharmaceutically acceptable salt thereof. Background Art

[0002] Bone regeneration is a complex process involving a combination of many biological factors. Growth factors are related to bone repair / regeneration and are bioactive peptides or protein hormones that affect immune function and the proliferation, chemotaxis, and differentiation of epithelial cells, osteocytes, and connective tissue cells. These hormones bind to specific cell surface tyrosine kinase receptors that are present on various target cells, including osteoblasts, osteocytes, and fibroblasts.

[0003] Among the growth factors related to bone repair / regeneration, an important growth factor called osteogenic growth peptide (OGP) has been widely studied. OGP is a polypeptide composed of 14 amino acid residues and is a promoting factor with a systemic response to bone marrow injury, existing in the serum of mammals at micromolar concentrations. It has been found that the C-terminal OGP(10 - 14) pentapeptide (hereinafter simply referred to as G5 peptide) is the smallest amino acid sequence that retains all OGP-like activities. The literature shows that OGP / G5 plays an important role in bone repair / regeneration, mainly in stimulating the proliferation, differentiation, alkaline phosphatase activity, and matrix mineralization of osteoblast lineage cells. In addition, OGP regulates the expression of transforming growth factor (TGF), insulin-like growth factor (IGF), and basic fibroblast growth factor (bFGF), thereby increasing bone formation and trabecular bone density in vivo. OGP can stimulate the activity of osteoblasts and promote the growth of osteoblasts and fibroblasts. G5 has important value for the research and treatment of osteoporosis.

[0004] The use of small molecule polypeptides for disease treatment has become a new trend. In this study, G5-like peptides were developed for osteoporosis research and osteogenic promotion, providing new drug options for the treatment of osteoporosis. Summary of the Invention

[0005] The present invention provides a G5-like peptide for promoting bone formation or a pharmaceutically acceptable salt thereof, and the like peptide or its pharmaceutically acceptable salt includes the following amino acid sequence or is composed of the following amino acid sequence:

[0006] 1) SEQ ID NO: 1;

[0007] 2) SEQ ID NO: 2;

[0008] 3) SEQ ID NO: 3; or

[0009] 4) SEQ ID NO: 4.

[0010] Preferably, the G5-like peptide is also conjugated with an osteogenesis-promoting functional domain.

[0011] Preferably, the amino acid sequence of the osteogenesis-promoting functional domain is as shown in SEQ ID NO: 5.

[0012] Preferably, the G5-like peptide is directly conjugated with the osteogenesis-promoting functional domain through a peptide bond.

[0013] Preferably, the G5-like peptide or its pharmaceutically acceptable salt comprises the amino acid sequence shown below or consists of the amino acid sequence shown below:

[0014] G5-1: SEQ ID NO: 6;

[0015] G5-2: SEQ ID NO: 7;

[0016] G5-3: SEQ ID NO: 8; or

[0017] G5-4: SEQ ID NO: 9.

[0018] In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition, which comprises the bifunctional polypeptide or its pharmaceutically acceptable salt of the present invention.

[0019] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the pharmaceutical composition is used for promoting osteogenic differentiation or treating osteoporosis.

[0021] In another preferred embodiment of the present invention, the present invention provides the use of the bifunctional polypeptide or its pharmaceutically acceptable salt and its pharmaceutical composition of the present invention in the preparation of a drug for treating osteoporosis and / or promoting osteogenic differentiation.

[0022] Preferably, the treatment of osteoporosis is achieved by promoting bone differentiation.

[0023] The present invention has developed a G5-like peptide, which is directly fused with SESSE that promotes bone formation to obtain a fusion polypeptide, which significantly up-regulates the expression of alkaline phosphatase, significantly promotes the ability of osteogenic differentiation, has potential clinical applicability and research value in the treatment of osteoporosis, and is non-toxic to other tissues, safe and reliable.

[0024] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the drawings to fully understand the purpose, features and effects of the present invention. Brief Description of the Drawings

[0025] Figure 1 It is the toxicity result diagram of CCK-8 detection of G5-X (X = 1 / 2 / 3 / 4).

[0026] Figure 2 It is the ALP staining result diagram of hBMSC cells induced by compound G5-X (X = 1 / 2 / 3 / 4) after 14 days of induction, where A: without induction solution; B: with induction solution; C: induction solution + SESSE; D: induction solution + G5-1; E: induction solution + G5-2; F: induction solution + G5-3; G: induction solution + G5-4.

[0027] Figure 3 It is the expression of osteogenesis-related genes in hBMSC cells induced by compound G5-X (X = 1 / 2 / 3 / 4) after 14 days. Specific implementation mode

[0028] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] Example 1 Synthesis of kinins

[0030] 1. Experimental materials

[0031] The materials and reagents used in the present invention are all purchased from commercial products. Taking G5-1 as an example, the synthesis preparation method of kinins in the present invention is described (for the preparation of other kinins in the present invention, only the synthesis sequence of amino acid raw materials needs to be replaced).

[0032] 2. Weigh 15 mmol equivalents of resin and put it into the reactor, add DCM (dichloromethane) to swell for half an hour, then pump out DCM, add the first amino acid (15 mmol) in the sequence, DIEA (diisopropylethylamine), an appropriate amount of DMF (dimethylformamide), DCM solution (the appropriate amount means an amount that can make the resin agitate sufficiently), and bubble with nitrogen for 60 min. Then add about 75 mmol equivalents of methanol, react for half an hour, pump out the reaction solution, and wash it with DMF and DCM.

[0033] 3. Add an appropriate amount of 20% piperidine / DMF to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash it, and detect it with a detection reagent.

[0034] 4. Add the second amino acid in the sequence (also 45 mmol), 45 mmol of HBTU (benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), and DIEA to the reactor. Bubble nitrogen through the reaction mixture for half an hour, then remove the liquid, wash it with DMF and DCM, and detect it with ninhydrin.

[0035] 5. Add the amino acids in the sequence successively in the manner of Steps 3 and 4. Remove the liquid, wash it with DMF, and detect it with the detection reagent.

[0036] 6. After drying the resin with nitrogen, remove it from the reaction column, weigh it, pour it into a flask, and then add a certain amount of 95% TFA (trifluoroacetic acid) cleavage solution to the flask. Shake and react for 2 h to cleave the polypeptide from the resin support and remove the side-chain protecting groups of the amino acids.

[0037] 7. Filter off the resin to obtain the filtrate. Then add a large amount of ether to the filtrate to precipitate the crude product, and then centrifuge and wash to obtain the crude product of this sequence.

[0038] 8. Analysis, purification, and mass spectrometry detection: Use an ESI (electrospray ionization) ion source mass spectrometer to detect the correctness of the molecular weight of this sequence, and use high-performance liquid chromatography to purify the crude product to the required purity.

[0039] 9. Collect the purified target polypeptide solution and put it into a freeze dryer for concentration and freeze-drying into a white powder.

[0040] 10. Preparation results

[0041] After the preparation, the molecular weight detection results of each agonist peptide compound are shown in Table 1. The results show that the prepared compounds are all correct.

[0042] Table 1 List of synthetic agonist peptide compounds and molecular weights

[0043]

[0044] Example 2 Toxicity test of peptide compounds

[0045] 1. Experimental materials

[0046] All materials and reagents used in this evaluation were purchased from commercial products.

[0047] 2. Experimental methods and results

[0048] Taking the compound G5-X (X = 1 / 2 / 3 / 4) as an example, the toxicity test of the peptide compound is carried out as follows:

[0049] hBMSCs in the logarithmic growth phase were seeded into 96-well plates at a density of 5000 cells / mL. Peptide compounds were added to the 96-well plates at different concentrations, with 3 replicates for each concentration. After incubation in a carbon dioxide incubator for 24 hours, the medium was discarded and CCK-8 reagent was added, and the detection was carried out at a wavelength of 450 nm. The results are as Figure 1 shown. Compared with the group without polypeptide, there was no statistical difference among different concentration groups, indicating that the addition of G5-like peptide was non-toxic to cells.

[0050] Example 3 Osteogenic induction of peptide compounds on hBMSC cells and ALP staining

[0051] 1. Experimental materials

[0052] All materials and reagents used in this evaluation were purchased from commercial products.

[0053] 2. Experimental methods and results

[0054] Taking compound G5-X (X = 1 / 2 / 3 / 4) as an example, the osteoclast induction test was carried out as follows:

[0055] hBMSC cells were used for osteogenic induction. The cells were seeded into 12-well plates and divided into a normal culture group (without induction medium), an induction group (with induction medium), and an experimental group (with induction medium plus peptide). The induction medium was DMEM / F12 medium containing β-glycerophosphate and vitamin C. After 14 days of induction, ALP staining was performed. The results are as Figure 2 shown. Compared with the induction group, the alkaline phosphatase content in the experimental group increased, indicating that the addition of G5-X (X = 1 / 2 / 3 / 4) promoted the ability of osteogenic differentiation.

[0056] Example 4 Effect of peptide compounds on the expression of osteogenesis-related genes

[0057] 1. Experimental materials

[0058] All materials and reagents used in this evaluation were purchased from commercial products.

[0059] 2. Experimental methods and results

[0060] Taking compound G5-X (X = 1 / 2 / 3 / 4) as an example, the osteoclast gene expression test was carried out as follows:

[0061] hBMSC cells were used for osteogenic induction. The cells were seeded into 12-well plates and divided into a normal culture group (without induction medium), an induction group (with induction medium), and an experimental group (with induction medium plus peptide). The induction medium was DMEM / F12 medium containing β-glycerophosphate and vitamin C. After 14 days of induction, the total RNA of the cells was collected for qPCR. The results are as Figure 3As shown, compared with the induction group, the expression of ALPL in the experimental group was significantly increased, indicating that the addition of G5-X (X = 1 / 2 / 3 / 4) promoted the ability of osteogenic differentiation.

[0062] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A G5-like peptide for promoting bone formation or a pharmaceutically acceptable salt thereof, wherein the G5-like peptide consists of the amino acid sequence shown as follows: G5-1: SEQ ID NO: 6; G5-2: SEQ ID NO: 7; G5-3: SEQ ID NO: 8; or G5-4: SEQ ID NO:

9.

2. A pharmaceutical composition, which comprises the G5-like peptide or a pharmaceutically acceptable salt thereof according to claim 1.

3. The pharmaceutical composition according to claim 2, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition according to claim 2, wherein The pharmaceutical composition is used for promoting osteogenic differentiation.

5. The pharmaceutical composition according to claim 2, wherein The pharmaceutical composition is used for treating osteoporosis.

6. Use of the G5-like peptide or a pharmaceutically acceptable salt thereof according to claim 1 and / or the pharmaceutical composition according to any one of claims 2-5 in the preparation of a medicament for treating osteoporosis.

7. The application according to claim 6, characterized in that, The treatment of osteoporosis is achieved by promoting bone differentiation.

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