Difunctional anti-osteoporosis polypeptide for inhibiting bone resorption and promoting bone formation

Abstract

The invention relates to the field of polypeptide medicines, in particular to a bifunctional anti-osteoporosis polypeptide for inhibiting bone resorption and promoting bone formation. The bifunctional polypeptide comprises a bone resorption inhibiting functional domain and a bone formation promoting functional domain, the amino acid sequence of the bone resorption inhibiting functional domain is shown as SEQ ID NO: 1, the bone formation promoting functional domain comprises n repeated bone formation promoting active oligopeptides, the amino acid sequence of the bone formation promoting active oligopeptides is shown as SEQ ID NO: 2, and n is equal to 1-6. The bifunctional polypeptide inhibits the growth of osteoclasts, down-regulates the expression of Trap and Ctsk, and up-regulates the expression of a bone formation ability biochemical marker BALP, significantly increases the bone mass in an OVX mouse model, has potential clinical applicability and research value in osteoporosis treatment, and is non-toxic to other tissues, safe and reliable.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drugs, and in particular to a bifunctional anti-osteoporosis polypeptide that inhibits bone resorption and promotes bone formation. Background Art

[0002] The pathogenesis of osteoporosis is extremely complex, involving the imbalance of multiple physiological processes, among which the imbalance of bone metabolism is the core link. The metabolic process of bone tissue consists of two parts: bone formation and bone resorption. Under normal physiological conditions, osteoblasts continuously synthesize new bone matrix and promote the deposition of minerals to form new bone; osteoclasts are responsible for absorbing and decomposing aged or damaged bone. These two processes coordinate with each other to maintain the stability of bone density. However, in the occurrence of osteoporosis, it is often because the activity of osteoclasts increases or the function of osteoblasts weakens, causing the bone resorption rate to exceed the bone formation rate. This imbalance may be caused by a variety of factors, including genetic factors, changes in hormone levels, abnormal regulation of cytokines, and environmental factors.

[0003] The occurrence of osteoporosis is related to a variety of cytokines, among which the osteoclastogenic factor RANKL plays a vital role. RANKL (Receptor Activator of Nuclear Factor κ B Ligand, RANKL) is an important cytokine that belongs to the tumor necrosis factor (TNF) superfamily. It is mainly produced by osteoblasts and some immune cells and plays a key regulatory role in bone metabolism. RANKL activates downstream signaling pathways by binding to its receptor RANK, promoting the generation, maturation and activity of osteoclasts, thereby increasing bone resorption. The RANKL-RANK signaling pathway is a key pathway for osteoclast differentiation and activation. When RANKL binds to RANK, it triggers a series of intracellular signal transduction events, including the activation of transcription factors NF-κB, etc. These events ultimately lead to the differentiation of osteoclast precursor cells into mature osteoclasts and enhance their activity.

[0004] The therapeutic strategy of targeting RANKL has become a new means of treating osteoporosis. Denosumab is a humanized anti-RANKL monoclonal antibody, which has important applications in the treatment of osteoporosis and bone complications caused by cancer. The mechanism of action of Denosumab is to interfere with the formation and function of osteoclasts by targeting and inhibiting RANKL (nuclear factor κB ligand). Osteoclasts are important cells involved in bone resorption. Denosumab specifically binds to RANKL, preventing the formation and interaction of RANKL and RANK, thereby inhibiting the differentiation and activation of osteoclasts, significantly reducing the rate of bone resorption, and effectively increasing bone density and reducing the risk of fractures. Denosumab has been used in the clinical treatment of osteoporosis, showing good clinical effects and providing a new direction for the management of bone metabolic diseases.

[0005] However, although Denosumab is well tolerated by patients, it cannot promote bone formation and can only treat the symptoms of osteoporosis, not the root cause. In the field of osteoporosis, peptide drugs achieve therapeutic effects by regulating the bone remodeling process and balancing the functions of osteoblasts and osteoclasts. Its main mechanisms include promoting bone formation and inhibiting bone resorption. Some peptide drugs can stimulate the proliferation and differentiation of osteoblasts, enhance the synthesis of bone matrix, and thus promote bone formation. Based on the RANKL protein target and with the assistance of AI algorithms, the present invention has developed a peptide molecule that has a high affinity with RANKL, and coupled another peptide molecule that promotes bone formation through a linker, forming a dual-functional anti-osteoporosis peptide that simultaneously inhibits bone resorption and promotes bone formation. Summary of the invention

[0006] Based on the above purpose, this study developed a bifunctional polypeptide molecule for anti-osteoporosis. The present invention provides the bifunctional polypeptide or a pharmaceutically acceptable salt thereof, wherein the bifunctional polypeptide comprises a bone resorption inhibition domain and a bone formation promotion domain, wherein the amino acid sequence of the bone resorption inhibition domain is as shown in SEQ ID NO: 1, and the bone formation promotion domain comprises n repeated short peptides that promote bone formation activity, wherein the amino acid sequence of the short peptides that promote bone formation activity is as shown in SEQ ID NO: 2, where n=1-6.

[0007] Preferably, the bone resorption inhibiting domain is directly connected to the bone formation promoting domain or is connected via a connecting peptide.

[0008] Preferably, the connecting peptide is a flexible connecting peptide.

[0009] Preferably, the flexible connecting peptide consists of glycine and serine.

[0010] Preferably, the amino acid sequence of the flexible connecting peptide is GSG.

[0011] Preferably, the amino acid sequence formula of the bifunctional polypeptide is MPKGSFNYAWVLDGLKAERWTGIDTGKGGSG(SESSE)n, where n=1-6.

[0012] Preferably, the bifunctional polypeptide is synthesized by organic solid phase synthesis.

[0013] Preferably, the transmembrane domain and the glucagon-like peptide functional domain are coupled by chemical synthesis.

[0014] In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition, which comprises the bifunctional polypeptide of the present invention or a pharmaceutically acceptable salt thereof.

[0015] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0016] In another preferred embodiment of the present invention, the present invention provides the use of the bifunctional polypeptide of the present invention or its pharmaceutically acceptable salt and pharmaceutical composition thereof in the preparation of a method for treating osteoporosis.

[0017] Preferably, the treatment of osteoporosis is achieved by inhibiting bone resorption and / or promoting bone formation.

[0018] The present invention develops a polypeptide molecule that has high affinity with RANKL, and couples another polypeptide molecule that promotes bone formation through a linker to form a bifunctional anti-osteoporosis polypeptide that simultaneously inhibits bone resorption and promotes bone formation. The polypeptide inhibits osteoclast growth, downregulates the expression of Trap and Ctsk, and upregulates the expression of BALP, a biochemical marker of bone formation ability, and significantly increases bone mass in an OVX mouse model. The polypeptide has potential clinical applicability and research value in the treatment of osteoporosis, is non-toxic to other tissues, and is safe and reliable.

[0019] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of CCK-8 detection of bifunctional peptide toxicity.

[0021] Figure 2 The following are the results of TRAP staining and quantitative results of osteoclasts after 5 days of induction: A: cell TRAP staining; B: statistical graph of the number of osteoclasts (OC); C: statistical graph of the relative area of ​​osteoclasts (OC); **(p < 0.01); ****(p < 0.0001).

[0022] Figure 3This is the expression of osteoclast-related genes in OVX mice.

[0023] Figure 4 This is the expression of BALP in the serum of OVX mice.

[0024] Figure 5 This is the Micro-CT and quantitative results of the femur of OVX mice.

[0025] Figure 6 This is the HE staining result of the heart, liver, spleen, lung and kidney of OVX mice. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Example 1 Synthesis of peptide compounds

[0028] The reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art, and those skilled in the art can prepare the agonist peptides of the present invention and their pharmaceutically acceptable salts in a synthetic manner not limited to the embodiments of the present invention. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0029] 1. Experimental Materials The materials and reagents used in the present invention were purchased from commercial products.

[0030] 2. Synthetic preparation method of the agonist peptide of the present invention 1) Condensation of Gly: Add 10 mL of V (hexahydropyridine): V (DMF) = 1:4 to the resin Fmoc-Lys(dde)-Wang Resin and react for 20 min to remove the Fmoc protecting group.

[0031] 2) Add 10mL DMF, MeOH and DCM in sequence to wash the resin 9 times (3 times each), filter, drain, pick some resin, and detect with Kaiser test. The solution is blue. According to the loading of Fmoc-Lys(dde)-Wang Resin of 0.3mmol / g, calculate the feeding amount of Fmoc-Gly-OH (resin weight 1g*molecular weight*loading*2=0.18g), the amount of condensation agent TBTU (321.1*2*1*0.3=0.193g), MMN0.1ml, 10mL DMF, temperature about 35 degrees, react for 40 minutes, filter out the liquid, add 10mL DMF to wash the resin 6 times, and filter.

[0032] 3) Drain and pick up some resin, and use Kaiser test to detect. If the solution does not appear blue and the resin is transparent, it means that Fmoc-Gly-OH has been condensed onto the resin. Add 10mL V (hexahydropyridine): V (DMF) = 1:4 to the resin and react for 20 minutes to remove the Fmoc protecting group.

[0033] 4) Add 10mL DMF, MeOH, and DCM in sequence to wash the resin 9 times (3 times each), filter, drain, pick some resin, and test with Kaiser test. The solution is blue, ready to condense the next amino acid Thr.

[0034] 5) Condensation of Thr Fmoc-Thr(tBu)-OH, amino acid feed amount 0.24g, condensation agent TBTU amount 0.193g, MMN 0.1ml, 10mL DMF, temperature around 35 degrees, reaction for 40 minutes, filter out the liquid, add 10mL DMF to wash the resin, 6 times, and filter.

[0035] 6) Drain and pick up some resin, and use Kaiser test to detect. If the solution does not appear blue and the resin is transparent, it means that Fmoc-Thr(tBu)-OH has been condensed onto the resin. Add 10mL V (hexahydropyridine): V (DMF) = 1:4 to the resin and react for 20 minutes to remove the Fmoc protecting group.

[0036] 7) Add 10 mL of DMF, MeOH, and DCM in sequence to wash the resin 9 times (3 times each), filter, drain, pick some resin, and test with Kaiser test. The solution is blue, ready to condense the next amino acid Asp.

[0037] 8) Condense the D at the C-terminus to the M at the N-terminus in sequence, using Boc-Met-OH as the raw material, and then remove the DDE with 1% hydrazine hydrate, and then connect the eicosanoic acid.

[0038] 9) Add DCM to wash the resin, then add ether to wash twice, drain and vacuum dry for 2 h.

[0039] 10) Cleavage: Place the obtained peptide-resin in an eggplant-shaped bottle, add 20 mL of cleavage reagent (82.5% TFA), react for 3 h, then filter and concentrate the filtrate by rotary evaporation.

[0040] 11) Add 10 mL of ice ether to the concentrate to precipitate. Centrifuge at 5000 r / m for 5 min, then discard the supernatant. Repeat three times and vacuum dry the precipitate to obtain 620 mg of crude peptide.

[0041] 12) Crude product analytical grade Liquid phase analysis conditions: Chromatographic column: analytical column (250*4.6mm, Kromasil-C18-5um); mobile Phase A: 0.1% TFA in 100% water; Phase B: 0.1% TFA in 100% acetonitrile, flow rate: 1.0 mL min -1 , detection wavelength 220 nm.

[0042] 13) Purify the crude polypeptide using a reverse phase chromatography column. Freeze it in a refrigerator, and after freezing solid, freeze it in a freeze dryer to form a solid powder to obtain the polypeptide.

[0043] Example 2 Toxicity test of peptide compounds

[0044] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0045] 2. Experimental methods and results The toxicity test of peptide compounds is carried out as follows: RAW264.7 in the logarithmic growth phase was plated at 5000 cells / mL in a 96-well plate, and peptide compounds were added to the 96-well plate at different concentrations of 0, 10, 100, etc., with 3 replicates for each concentration. After incubation in a carbon dioxide incubator for 24 hours, the culture medium was discarded, CCK-8 reagent was added, and detection was performed at a wavelength of 450nm. The results are shown in Figure 1 As shown, there was no statistical difference between the different concentration groups compared with the group without peptide addition, which indicates that the bifunctional peptide has no toxicity to cells.

[0046] Example 3 Inhibitory effect of peptide compounds on osteoclast formation

[0047] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0048] 2. Experimental methods and results Perform osteoclast induction test as follows: Bone marrow-derived macrophages were used for osteoclast induction. The cells were inoculated in a 12-well plate and divided into a normal culture group (without induction solution), an induction group (with induction solution) and an experimental group (with induction solution plus bifunctional peptides). The induction solution was DMEM medium containing 50ng / mL RANKL and 100ng / mL C-MFC. TRAP staining was performed after 5-7 days of induction. The results are shown in Figure 2 As shown, compared with the induced group, the number of osteoclasts and the area (volume) of osteoclasts in the experimental group were significantly reduced, indicating that the bifunctional polypeptide has the ability to inhibit osteoclast differentiation.

[0049] Example 4 Inhibitory effect of peptide compounds on osteoclasts

[0050] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0051] 2. Experimental methods and results The osteoclast gene expression test is performed as follows: Bone marrow-derived macrophages were used for osteoclast induction. The cells were inoculated in a 12-well plate and divided into a normal culture group (without induction solution), an induction group (with induction solution) and an experimental group (with induction solution plus bifunctional peptides). The induction solution was DMEM medium containing 50ng / mL RANKL and 100ng / mL C-MFC. After 5-7 days of induction, the total RNA of the cells was collected for qPCR. The results are shown in Figure 3 As shown, compared with the induction group, the expression of Trap and Ctsk in the experimental group was significantly decreased, which was consistent with the osteoclast TRAP staining and quantitative results, and also indicated that the bifunctional polypeptide had the ability to inhibit osteoclast differentiation.

[0052] Example 5 Activation of osteogenic capacity of OVX mice by peptide compounds

[0053] 1. Experimental Materials OVX model female mice. Materials and reagents used in this evaluation were purchased from commercial products.

[0054] 2. Experimental methods and results The following serum ELISA test is performed as follows: The 12-week-old C57 mice were subjected to OVX modeling and divided into the Sham group, OVX group, and OVX+bifunctional peptide group (1 mg / kg). Then, the peptide compound was subcutaneously injected 100ul / time, five times a week, for 6 consecutive weeks. After 6 consecutive weeks of administration, the mouse serum was collected for ELISA testing. The results are shown in Figure 4 As shown, compared with the OVX group, the biochemical marker of bone formation ability BALP in the bifunctional polypeptide group (1 mg / kg) was significantly increased, indicating that the bifunctional polypeptide has the ability to promote bone formation.

[0055] Example 6 Testing of the Effect of Peptide Compounds on Bone Mass Changes in OVX Model Mice

[0056] 1. Experimental Materials OVX model female mice.

[0057] 2. Experimental methods and results Taking compound X as an example, a bone mass change test is conducted as follows: OVX model was established in 12-week-old C57 mice, which were divided into Sham group, OVX group and OVX+ bifunctional peptide group (1 mg / kg), and then the peptide compound was subcutaneously injected 100ul / time, five times a week, for 6 consecutive weeks. After six weeks, the femur of the mouse small intestine was obtained. It was fixed in 4% paraformaldehyde and then subjected to Micro-CT examination. N=3. The results are shown in Figure 5 As shown, compared with the OVX group, the bone mass in the bifunctional peptide group (1 mg / kg) increased, which indicates that the bifunctional peptide has the function of treating osteoporosis.

[0058] Example 7 Safety evaluation of peptide compounds in mice

[0059] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products. OVX model female mice.

[0060] 2. Experimental methods and results The following is a safety evaluation of mice, as follows: The 12-week-old C57 mice were subjected to OVX modeling and divided into Sham group, OVX group and OVX+ bifunctional peptide group (1 mg / kg), and then subcutaneously injected with 100ul / time of peptide compound, five times a week, for 6 consecutive weeks. After six weeks, the heart, liver, spleen, lung and kidney of the mice were taken, fixed in 4% paraformaldehyde, and then HE staining was performed. N=3. The results are shown in Figure 6 As shown, compared with the Sham group, the bifunctional polypeptide group (1 mg / kg) had no obvious lesions in tissues, which indicates that the bifunctional polypeptide is non-toxic to the heart, liver, spleen, lungs and kidneys and is safe and reliable.

[0061] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians 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 be within the scope of protection determined by the claims.

Claims

1. A bifunctional anti-osteoporosis polypeptide that inhibits bone resorption and promotes bone formation, or a pharmaceutically acceptable salt thereof, wherein the bifunctional polypeptide comprises a bone resorption inhibiting domain and a bone formation promoting domain, the amino acid sequence of the bone resorption inhibiting domain is as shown in SEQ ID NO: 1, the bone formation promoting domain comprises n repeated short peptides that promote bone formation, the amino acid sequence of the short peptides that promote bone formation is as shown in SEQ ID NO: 2, where n=1-6.

2. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The bone resorption inhibiting domain is directly connected to the bone formation promoting domain or is connected via a connecting peptide.

3. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The connecting peptide is a flexible connecting peptide.

4. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The flexible connecting peptide consists of glycine and serine.

5. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The amino acid sequence of the flexible connecting peptide is GSG.

6. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The general formula of the amino acid sequence of the bifunctional anti-osteoporosis polypeptide is MPKGSFNYAWVLDGLKAERWTGIDTGKGGSG (SESSE) n, where n=1-6.

7. A pharmaceutical composition comprising the bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

9. Use of the bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 and / or the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for treating osteoporosis.

10. The use according to claim 9, characterized in that: The treatment of osteoporosis is achieved by inhibiting bone resorption and / or promoting bone formation.

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