A CB2 agonist peptide for promoting bone formation and its applications

Abstract

The present invention relates to the field of polypeptide drugs, and particularly relates to a CB2 agonist peptide for promoting bone formation and its application. The agonist peptide comprises an amino acid sequence shown in any of the following: CB2-P1: as shown in SEQ ID NO: 1; CB2-P2: as shown in SEQ ID NO: 2; CB2-P3: as shown in SEQ ID NO: 3; CB2-P4: as shown in SEQ ID NO: 4. The agonist peptide up-regulates the content of alkaline phosphatase, promotes the expression of ALP, and has potential clinical applicability and research value in the treatment of osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drugs, and particularly relates to a CB2 agonist peptide for promoting bone formation and its application. Background Art

[0002] Cannabinoid receptors are a class of seven-transmembrane receptors widely distributed in the human body. They belong to the G protein-coupled receptor (GPCR) family and are receptor proteins on the cell membrane, responsible for receiving and transmitting specific signaling molecules, and play an important role in regulating various physiological processes and disease development, especially signal transduction related to cannabinoids. In the human body, there are mainly two types of cannabinoid receptors, type I cannabinoid receptor and type II cannabinoid receptor: type I cannabinoid receptor (Cannabinoid Receptor Type 1, CB1) is mainly distributed in the central nervous system (brain and spinal cord), especially in areas such as the cerebral cortex, amygdala, hippocampus, and basal ganglia of the brain, and binds to cannabinoid substances (such as Δ9-tetrahydrocannabinol, abbreviated as THC) and endogenous cannabinoids produced in the body (2-arachidonoyl glycerol, such as 2-AG, and endorphin-like compounds, such as enkephalin); while type II cannabinoid receptor (Cannabinoid Receptor Type 2, CB2) is mainly distributed in the immune system and bone marrow, such as white blood cells, macrophages, and lymphocytes. By regulating CB2, the activity and function of the immune system can be affected, and a variety of immune-related drugs targeting CB2 are being developed.

[0003] In recent years, a large number of studies have found that in osteocytes, CB2 plays an important role in maintaining the balance between bone resorption and bone formation. CB2 is expressed more in the bone remodeling stage of the bone cycle. In vitro studies of CB2 have shown that CB2 signal transduction stimulates the proliferation, differentiation, and enhanced osteogenic activity of osteoblasts. CB2 can up-regulate the expression of osteogenic factors such as RUNX2, bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), and osteocalcin (OCN), thereby promoting the proliferation and differentiation of osteoblasts. Some studies have found that while promoting the proliferation of osteoblasts, CB2 can also reduce the number of osteoclasts. Activation of CB2 can stimulate bone formation and inhibit bone resorption. Therefore, activating CB2 can prevent osteoporosis and fractures. Ofek et al. found that young C57BL / 6 mice with CB2 knocked out (CB2 − / − / C57BL) shows normal peak bone mass, followed by an increase in age-related bone loss in later life, similar to postmenopausal osteoporosis in humans. This age-related bone loss phenotype suggests that CB2 polymorphisms are associated with osteoporosis and bone strength. Coincidentally, Karsak et al. found that the same result of bone loss due to accelerated bone remodeling occurred in knockout CB2 mice in different mouse strains. Stimulating CB2 signaling with a selective CB2 agonist in ovariectomized (OVX) mice prevents OVX-induced bone loss, and the CB2 agonist leads to an increase in bone formation rate. It is worth mentioning that CB2 knockout rats show no other changes in any organ except bone tissue, further emphasizing the importance of CB2 in the skeleton.

[0004] The CB2 receptor is a key target for the development of future anti-osteoporosis drugs aimed at enhancing the bone regeneration ability in humans. The present invention aims to develop a class of CB2 agonist polypeptide molecules with osteogenic promotion based on the CB2 target, and achieve targeted distribution to bone through coupling technology, thereby providing a new strategy for the treatment of osteoporosis. Summary of the Invention

[0005] To solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The present invention provides a CB2 agonist peptide or a pharmaceutically acceptable salt thereof, and the agonist peptide or the pharmaceutically acceptable salt thereof comprises an amino acid sequence shown in any of the following:

[0007] CB2-P1: as shown in SEQ ID NO: 1;

[0008] CB2-P2: as shown in SEQ ID NO: 2;

[0009] CB2-P3: as shown in SEQ ID NO: 3;

[0010] CB2-P4: as shown in SEQ ID NO: 4.

[0011] In a preferred embodiment of the present invention, the present invention provides a CB2 agonist peptide or a pharmaceutically acceptable salt thereof, and the agonist peptide or the pharmaceutically acceptable salt thereof is composed of an amino acid sequence shown in any of the following:

[0012] CB2-P1: as shown in SEQ ID NO: 1;

[0013] CB2-P2: as shown in SEQ ID NO: 2;

[0014] CB2-P3: as shown in SEQ ID NO: 3;

[0015] CB2-P4: as shown in SEQ ID NO: 4.

[0016] In a preferred embodiment of the present invention, the present invention also provides a polynucleotide encoding the CB2 agonist peptide or a pharmaceutically acceptable salt thereof.

[0017] In another preferred embodiment of the present invention, the present invention also provides an expression vector containing the polynucleotide.

[0018] In yet another preferred embodiment of the present invention, the present invention also provides a host cell containing the expression vector.

[0019] In still another preferred embodiment of the present invention, the present invention also provides a pharmaceutical composition, which comprises the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to the present invention.

[0020] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0021] In an additional preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to the present invention and its pharmaceutical composition in the treatment of osteoporosis.

[0022] In an additional further preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to the present invention and its pharmaceutical composition in promoting osteogenic differentiation.

[0023] In an additional still another preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to the present invention and its pharmaceutical composition in the preparation of a medicament for treating osteoporosis.

[0024] In a preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to the present invention and its pharmaceutical composition in the preparation of a product for promoting osteogenic differentiation.

[0025] Based on the CB2 target, the present invention develops a class of CB2 agonist polypeptide molecules with osteogenic promotion, and couples a polypeptide with bone targeting through a coupling technology. The polypeptide up-regulates the content of alkaline phosphatase, promotes the expression of ALP, and has potential clinical applicability and research value in the treatment of osteoporosis.

[0026] 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

[0027] Figure 1 It is a graph showing the results of detecting the toxicity of the CB2 agonist polypeptide by CCK-8.

[0028] Figure 2 It is the ALP staining result diagram after 14 days of induction. A: Without induction solution; B: With induction solution; C: Induction solution + SESSE; D: Induction solution + CB2-P1; E: Induction solution + CB2-P2; F: Induction solution + CB2-P3; G: Induction solution + CB2-P4.

[0029] Figure 3 It is the expression of osteogenesis-related genes after 14 days of induction. Specific implementation mode

[0030] 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. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0031] Example 1 Synthesis of CB2 agonist polypeptide compound

[0032] 1. Experimental materials

[0033] The materials and reagents used in the present invention are all purchased from commercial products. The protected amino acids used in the whole synthesis process are as follows: Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-His-OH, Fmo-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-O, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(iLEde)-OH, Boc-Tyr(tBu)-OH.

[0034] 2. Taking CB2-P1 as an example, illustrate the synthesis preparation method of the agonist peptide in the present invention (for the preparation of other agonist peptides in the present invention, only the synthesis sequence of amino acid raw materials needs to be replaced)

[0035] 1) Resin pretreatment: Weigh 26.3 g of Rink Amide AM Resin and add it to the reaction kettle. Add 200 mL of DMF, and stir and swell it for 35 min while passing nitrogen. When the swelling is complete, filter off the solvent by suction, and wash the resin 5 times with DMF, 200 mL each time;

[0036] 2) Preparation of amino acid solution: Weigh 11.5 g of Fmoc-Lys(Boc)-OH and 4.04 g of HOBt, dissolve them in 150 mL of DMF, then add 3.78 g of DIC and mix well;

[0037] 3) Deprotection: Add 200 mL of 20% piperidine / DMF solvent to the reaction kettle, pass nitrogen and stir for 40 min. After the reaction is completed, wash the resin with DMF 5 times, 200 mL each time. After the washing is completed, take the resin and detect it with ninhydrin reagent. The resin shows a positive result;

[0038] 4) Coupling reaction: Add the prepared amino acid solution to the reaction kettle, control the temperature at 25 - 35 °C, pass nitrogen and stir for reaction. Monitor the reaction process with ninhydrin reagent during the reaction. When the resin shows a negative result, it indicates that the reaction is completed. After the reaction is completed, wash the resin with DMF 5 times, 200 mL each time. Repeat the above operations, and couple the corresponding protected amino acids in sequence according to the CB2-P1 peptide sequence until the peptide backbone synthesis is completed;

[0039] 5) Peptide resin cleavage: Prepare the cleavage solution according to TFA / TIS / DTT / H2O = 90 / 2.5 / 2.5 / 5, then add the dried peptide resin mentioned above to the cleavage solution and stir for reaction for 3 h. When the reaction reaches the time, filter, add the filtrate to 5 times the volume of ice methyl tert-butyl ether for precipitation, filter by suction, wash the filter cake with methyl tert-butyl ether slurry 5 times, and obtain the crude product after vacuum drying;

[0040] 6) Purification and refinement: Refine and purify the obtained crude product through a reverse-phase preparative chromatography system to obtain a CB2-P1 refined product with a purity of not less than 90%, and analyze the compound by analytical HPLC and LC-MS.

[0041] 3. Preparation results

[0042] After the preparation is completed, 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.

[0043] Table 1 List of synthetic agonist peptide compounds and molecular weights

[0044]

[0045] Example 2 Toxicity test of peptide compounds

[0046] 1. Experimental materials

[0047] All materials and reagents used in this evaluation are purchased from commercial products.

[0048] 2. Experimental methods and results

[0049] The following is the toxicity test of peptide compounds, specifically as follows:

[0050] hBMSCs in the logarithmic growth phase were seeded into a 96-well plate at a density of 5000 cells / mL. Peptide compounds were added to the 96-well plate at different concentrations, with three replicates for each concentration. After incubation in a carbon dioxide incubator for 24 hours, the culture 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. There was no statistical difference between different concentration groups compared with the group without adding polypeptide, indicating that CB2-PX (X = 1 / 2 / 3) has no toxicity to cells.

[0051] Example 3 Ability of peptide compounds to promote bone formation

[0052] 1. Experimental materials

[0053] All materials and reagents used in this evaluation were purchased from commercial products.

[0054] 2. Experimental methods and results

[0055] The following osteogenic activity test was carried out as follows:

[0056] hBMSC cells were used for osteogenic induction. The cells were seeded into a 12-well plate 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 induction for 14 days, 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 CB2-PX (X = 1 / 2 / 3 / 4) has the ability to promote osteogenic differentiation.

[0057] Example 4 Activation effect of peptide compounds on osteogenic genes

[0058] 1. Experimental materials

[0059] All materials and reagents used in this evaluation were purchased from commercial products.

[0060] 2. Experimental methods and results

[0061] The following osteogenic gene expression test was carried out as follows:

[0062] hBMSC cells were used for osteogenic induction. The cells were seeded into a 12-well plate 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 induction for 14 days, total RNA of the cells was collected for qPCR. The results are as Figure 3As shown, compared with the induction group, the expression of ALP in the experimental group was significantly increased, which was consistent with the results of ALP staining, indicating that CB2-PX (X = 1 / 2 / 3 / 4) has the ability to promote osteogenic differentiation.

[0063] 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 CB2 agonist peptide, which is composed of any one of the following amino acid sequences: CB2-P1: as shown in SEQ ID NO: 1; CB2-P2: as shown in SEQ ID NO: 2; CB2-P3: as shown in SEQ ID NO: 3; CB2-P4: as shown in SEQ ID NO:

4.

2. A polynucleotide encoding the agonist peptide according to claim 1.

3. An expression vector containing the polynucleotide according to claim 2.

4. A host cell containing the polynucleotide according to claim 2 and / or the expression vector according to claim 3.

5. A pharmaceutical composition comprising the CB2 agonist peptide according to claim 1 and a pharmaceutically acceptable carrier or excipient.

6. The CB2 agonist peptide according to claim 1 or the pharmaceutical composition according to claim 5, characterized in that, For the treatment of osteoporosis.

7. The CB2 agonist peptide according to claim 1 or the pharmaceutical composition according to claim 5, characterized in that, For promoting osteogenic differentiation.

8. Use of the CB2 agonist peptide according to claim 1 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for the treatment of osteoporosis.

Images