A bifunctional anti-osteoporosis polypeptide that inhibits bone resorption and promotes bone formation

Abstract

The present invention relates to the field of polypeptide drugs, and particularly relates to a bifunctional anti-osteoporosis polypeptide that inhibits bone resorption and promotes bone formation. The bifunctional polypeptide comprises a bone resorption inhibition functional domain and a bone formation promotion functional domain. The amino acid sequence of the bone resorption inhibition functional domain is as shown in SEQ ID NO: 1. The bone formation promotion functional domain comprises n repeated bone formation promoting active peptides, and the amino acid sequence of the bone formation promoting active peptide is as shown in SEQ ID NO: 2, where n = 1-6. The bifunctional polypeptide inhibits the growth of osteoclasts, down-regulates the expression of Trap and Ctsk, and up-regulates the expression of the bone formation ability biochemical marker BALP, significantly increases bone mass in the OVX mouse model, 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 relates to the field of polypeptide drugs, and particularly 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 imbalances in 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 mass; osteoclasts are responsible for absorbing and decomposing aged or damaged bone mass. These two processes are coordinated with each other to maintain the stability of bone density. However, during the occurrence of osteoporosis, it is often due to the increased activity of osteoclasts or the weakened function of osteoblasts, resulting in the bone resorption rate exceeding the bone formation rate. This imbalance may be caused by various factors, including genetic factors, changes in hormone levels, abnormal regulation of cytokines, and environmental factors, etc.

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

[0004] Targeted RANKL treatment strategies have become a new means of treating osteoporosis. Denosumab is a humanized monoclonal antibody against RANKL and has important applications in the treatment of osteoporosis and skeletal 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 (receptor activator of nuclear factor-κB ligand). Osteoclasts are important cells involved in bone resorption. Denosumab specifically binds to RANKL, preventing the formation and interaction of RANKL with RANK, thereby inhibiting the differentiation and activation of osteoclasts, significantly reducing the bone resorption rate, and effectively increasing bone density and reducing the fracture risk. Denosumab has been applied 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 only treats the symptoms rather than the root cause of osteoporosis. In the field of osteoporosis, polypeptide 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 polypeptide drugs can stimulate the proliferation and differentiation of osteoblasts and enhance the synthesis of bone matrix, thereby promoting bone formation. Based on the RANKL protein target and assisted by an AI algorithm, the present invention has developed a polypeptide molecule with high affinity for RANKL, and coupled another bone formation-promoting polypeptide molecule through a linker to form a bifunctional anti-osteoporosis polypeptide that simultaneously inhibits bone resorption and promotes bone formation. Summary of the Invention

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

[0007] Preferably, the bone resorption inhibition functional domain and the bone formation promotion functional domain are directly connected or connected through a linker peptide.

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

[0009] Preferably, the flexible linker peptide is composed of glycine and serine.

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

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

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

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

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

[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 or a pharmaceutically acceptable salt thereof and a pharmaceutical composition thereof in the preparation of a medicament for treating osteoporosis.

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

[0018] The present invention has developed a polypeptide molecule that can have a high affinity for RANKL. By coupling another polypeptide molecule that promotes bone formation through a linker, a bifunctional anti-osteoporosis polypeptide that simultaneously inhibits bone resorption and promotes bone formation is formed. It inhibits the growth of osteoclasts, down-regulates the expression of Trap and Ctsk, and up-regulates the expression of the biochemical marker BALP of bone formation ability. It significantly increases bone mass in the OVX mouse model, has potential clinical applicability and research value in the treatment of osteoporosis, and is non-toxic to other tissues, safe and reliable.

[0019] The following will further illustrate the concept, specific structure and technical effects of the present invention with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a graph showing the results of detecting the toxicity of the bifunctional polypeptide by CCK-8.

[0021] Figure 2 It is a graph showing the results of TRAP staining of osteoclasts and the quantitative results after 5 days of induction. A: Cell TRAP staining map; B: Statistical chart of the number of osteoclasts (OC); C: Statistical chart of the relative area of osteoclasts (OC); where **(p < 0.01); ****(p< 0.0001).

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

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

[0024] Figure 5 It is the Micro-CT and quantitative result diagram of the femur of OVX mice.

[0025] Figure 6 It is the HE staining result diagram of the heart, liver, spleen, lungs, and kidneys of OVX mice. Detailed implementation manners

[0026] The present invention will be described in detail below with reference to 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 sources unless otherwise specified.

[0027] Example 1 Synthesis of peptide compounds

[0028] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and those skilled in the art can prepare the agonists and pharmaceutically acceptable salts thereof of the present invention in a synthesis 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

[0030] The materials and reagents used in the present invention are all purchased from commercial products.

[0031] 2. Synthesis preparation method of the agonist in the present invention

[0032] 1) Condensation: Add 10 mL of V (piperidine):V (DMF) = 1:4 to the resin Fmoc-Lys(dde)-Wang Resin, react for 20 min, and remove the Fmoc protecting group.

[0033] 2) Add 10 mL of DMF, MeOH, and DCM successively to wash the resin 9 times (3 times each), filter, drain, pick a little resin, and detect it with the kaiser test. The solution is blue. Calculate the feeding amount of Fmoc-Gly-OH according to the loading of Fmoc-Lys(dde)-Wang Resin of 0.3 mmol / g (resin weight 1 g * molecular weight * loading * 2 = 0.18 g), the amount of condensing agent TBTU (321.1 * 2 * 1 * 0.3 = 0.193 g), 0.1 ml of MMN, 10 mL of DMF, at a temperature of about 35 °C, react for 40 minutes, filter off the liquid, add 10 mL of DMF to wash the resin, 6 times, and filter.

[0034] 3) Drain, pick a little resin, and detect it with the kaiser test. If the solution is not blue and the resin is transparent, it indicates that Fmoc-Gly-OH has been condensed onto the resin. Add 10 mL of V (hexahydropyridine):V(DMF) = 1:4 to the resin and react for 20 min to remove the Fmoc protecting group.

[0035] 4) Add 10 mL of DMF, MeOH, and DCM successively to wash the resin 9 times (3 times each), filter, drain, pick a little resin, and detect it with the kaiser test. The solution is blue, and prepare to condense the next amino acid Thr.

[0036] 5) Condense Thr Fmoc-Thr(tBu)-OH, the feeding amount of the amino acid is 0.24 g, the amount of condensing agent TBTU is 0.193 g, 0.1 ml of MMN, 10 mL of DMF, at a temperature of about 35 °C, react for 40 minutes, filter off the liquid, add 10 mL of DMF to wash the resin, 6 times, and filter.

[0037] 6) Drain, pick a little resin, and detect it with the kaiser test. If the solution is not blue and the resin is transparent, it indicates that Fmoc-Thr(tBu)-OH has been condensed onto the resin. Add 10 mL of V (hexahydropyridine):V(DMF) = 1:4 to the resin and react for 20 min to remove the Fmoc protecting group.

[0038] 7) Add 10 mL of DMF, MeOH, and DCM successively to wash the resin 9 times (3 times each), filter, drain, pick a little resin, and detect it with the kaiser test. The solution is blue, and prepare to condense the next amino acid Asp.

[0039] 8) Condense from the C-terminus D to the N-terminus M successively using the raw material Boc-Met-OH, and then remove dde with 1% hydrazine hydrate and connect eicosanoic acid.

[0040] 9) Add DCM to wash the resin, then add ether to wash twice, drain, and dry in vacuum for 2 h.

[0041] 10) Cleavage: Place the obtained peptide-resin in a round-bottom flask, add 20 mL of cleavage reagent (82.5% TFA), and react for 3 h. Then filter and concentrate the filtrate by rotary evaporation.

[0042] 11) Add 10 mL of ice-cold ether to the concentrated solution to precipitate. Centrifuge at 5000 r / m for 5 min, then discard the supernatant, repeat three times, and dry the precipitate in vacuum. 620 mg of crude peptide is obtained.

[0043] 12) Crude product, analytical pure

[0044] Conditions for liquid phase analysis: Chromatographic column: analytical column (250*4.6 mm, Kromasil-C18-5um); mobile

[0045] phase, 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.

[0046] 13) Purify the crude polypeptide using a reverse chromatographic column. Freeze it in the refrigerator, and after it is frozen solid, freeze-dry it into a solid powder to obtain the polypeptide.

[0047] Example 2 Toxicity test of peptide compounds

[0048] 1. Experimental materials

[0049] The materials and reagents used in this evaluation were all purchased from commercial products.

[0050] 2. Experimental methods and results

[0051] The following is the toxicity test of peptide compounds, specifically as follows:

[0052] Seed RAW264.7 in the logarithmic growth phase, inoculate it into a 96-well plate at 5000 cells / mL, add different concentrations of peptide compounds such as 0, 10, 100, etc. to the 96-well plate, make 3 replicates for each concentration, after incubating in a carbon dioxide incubator for 24 hours, discard the culture medium and add CCK-8 reagent, and detect at a wavelength of 450 nm. The results are as Figure 1 shown. Compared with the group without adding polypeptide, there is no statistical difference among different concentration groups, indicating that the bifunctional polypeptide is non-toxic to cells.

[0053] Example 3 Inhibitory effect of peptide compounds on osteoclast formation

[0054] 1. Experimental materials

[0055] The materials and reagents used in this evaluation were all purchased from commercial products.

[0056] 2. Experimental methods and results

[0057] Osteoclast induction test was carried out as follows:

[0058] Osteoclast induction was performed using bone marrow-derived macrophages. The cells were seeded in 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 bifunctional polypeptide). The induction medium was DMEM medium containing 50 ng / mL RANKL and 100 ng / mL C-MFC. After induction for 5 - 7 days, TRAP staining was performed. The results are as Figure 2 shown. Compared with the induction group, the number and area (volume) of osteoclasts in the experimental group were significantly reduced, indicating that the bifunctional polypeptide has the ability to inhibit osteoclast differentiation.

[0059] Example 4 Inhibitory effect of peptide compound on osteoclasts

[0060] 1. Experimental materials

[0061] The materials and reagents used in this evaluation were all purchased from commercial products.

[0062] 2. Experimental methods and results

[0063] The following osteoclast gene expression test was carried out as follows:

[0064] Osteoclast induction was performed using bone marrow-derived macrophages. The cells were seeded in 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 bifunctional polypeptide). The induction medium was DMEM medium containing 50 ng / mL RANKL and 100 ng / mL C-MFC. After induction for 5 - 7 days, total RNA of the cells was collected for qPCR. The results are as Figure 3 shown. Compared with the induction group, the expressions of Trap and Ctsk in the experimental group were significantly decreased, which was consistent with the results of osteoclast TRAP staining and quantification, also indicating that the bifunctional polypeptide has the ability to inhibit osteoclast differentiation.

[0065] Example 5 Activation effect of peptide compound on osteogenic ability of OVX mice

[0066] 1. Experimental materials

[0067] OVX model female mice. The materials and reagents used in this evaluation were all purchased from commercial products.

[0068] 2. Experimental methods and results

[0069] The following is a serum ELISA test, which is specifically as follows:

[0070] C57 mice at 12 weeks of age were subjected to OVX modeling and divided into a Sham group, an OVX group, and an OVX + bifunctional polypeptide group (1 mg / kg). Then, 100 μl of the peptide compound was subcutaneously injected 5 times a week for 6 consecutive weeks. After 6 consecutive weeks of drug administration, mouse serum was collected for ELISA detection. The results are as Figure 4 shown. Compared with the OVX group, the biochemical marker BALP of bone formation ability in the bifunctional polypeptide group (1 mg / kg) was significantly increased, indicating that the bifunctional polypeptide has the ability to promote bone formation.

[0071] Example 6 Testing of the change in bone mass of OVX model mice by the peptide compound

[0072] 1. Experimental materials

[0073] OVX model female mice.

[0074] 2. Experimental methods and results

[0075] Taking compound X as an example, the following is the bone mass change test, which is specifically as follows:

[0076] C57 mice at 12 weeks of age were subjected to OVX modeling and divided into a Sham group, an OVX group, and an OVX + bifunctional polypeptide group (1 mg / kg). Then, 100 μl of the peptide compound was subcutaneously injected 5 times a week for 6 consecutive weeks. After six weeks, the lower limb femurs of the mouse small intestine were taken. Fixed in 4% paraformaldehyde and then subjected to Micro-CT detection. N = 3. The results are as Figure 5 shown. Compared with the OVX group, the bone mass in the bifunctional polypeptide group (1 mg / kg) increased, indicating that the bifunctional polypeptide has the function of treating osteoporosis.

[0077] Example 7 Safety evaluation of mice after using the peptide compound

[0078] 1. Experimental materials

[0079] The materials and reagents used in this evaluation were all purchased from commercial products. OVX model female mice.

[0080] 2. Experimental methods and results

[0081] The following is the safety evaluation of mice, which is specifically as follows:

[0082] C57 mice at 12 weeks of age were subjected to OVX modeling and divided into a Sham group, an OVX group, and an OVX + bifunctional polypeptide group (1 mg / kg). Then, 100 μl of the peptide compound was subcutaneously injected 5 times a week for 6 consecutive weeks. After six weeks, the hearts, livers, spleens, lungs, and kidneys of the mice were taken, fixed in 4% paraformaldehyde, and then subjected to HE staining for detection. N = 3. The results are as Figure 6 shown. Compared with the Sham group, there were no obvious lesions in the tissues of the bifunctional polypeptide group (1 mg / kg), indicating that the bifunctional polypeptide is non-toxic to the heart, liver, spleen, lung, and kidney, and is safe and reliable.

[0083] 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 labor. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should be within the protection scope determined by the claims.

Claims

1. A bifunctional anti-osteoporosis polypeptide for inhibiting bone resorption and promoting bone formation or a pharmaceutically acceptable salt thereof, the polypeptide consisting of a bone resorption inhibitory functional domain, a flexible linker peptide, and a bone formation promoting functional domain, the amino acid sequence of the bone resorption inhibitory functional domain being as shown in SEQ ID NO: 1, and the amino acid sequence of the bone formation promoting functional domain being as shown in SEQ ID NO:

2.

2. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein The flexible linker peptide consists of glycine and serine.

3. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to claim 2, wherein, The amino acid sequence of the flexible linker peptide is GSG.

4. The bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, The amino acid sequence of the bifunctional anti-osteoporosis polypeptide is MPKGSFNYAWVLDGLKAERWTGIDTGKGGSGSESSE.

5. A pharmaceutical composition, the pharmaceutical composition comprising the bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3.

6. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

7. Use of the bifunctional anti-osteoporosis polypeptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 and / or the pharmaceutical composition according to claim 5 or 6 in the preparation of a medicament for treating osteoporosis.

8. The application according to claim 7, wherein The treatment of osteoporosis is achieved by inhibiting bone resorption and / or promoting bone formation.

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