CB2 agonist peptide for promoting bone formation and application thereof

Abstract

The invention relates to the field of polypeptide drugs, in particular to a CB2 agonist peptide for promoting bone formation and application thereof. The agonist peptide comprises 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; the CB2-P3 is as shown in SEQ ID NO: 3; and CB2-P4: is as shown in SEQ ID NO: 4. The agonist peptide up-regulates the content of alkaline phosphatase and promotes ALP expression, and has potential clinical applicability and research value in osteoporosis treatment.

Description

Technical Field

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

[0002] Cannabinoid receptors are a class of seven-transmembrane receptors that are widely distributed in the human body. They belong to the G protein-coupled receptor (GPCR) family and are receptor proteins on the cell membrane. They are responsible for receiving and transmitting specific signal molecules and play an important role in regulating a variety of physiological processes and disease development, especially cannabinoid-related signal transduction. In the human body, there are two main types of cannabinoid receptors: type I cannabinoid receptors and type II cannabinoid receptors. Type I cannabinoid receptors (CB1) are mainly distributed in the central nervous system (brain and spinal cord), especially in the cortical area, amygdala, hippocampus and basal ganglia of the brain, and bind to cannabinoids (such as Δ9-tetrahydrocannabinol, THC) and endogenous cannabinoids (2-arachidonic acid compounds, such as 2-AG and endorphin compounds, such as enkephalin) produced in the body; while type II cannabinoid receptors (CB2) are 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 under development.

[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 more expressed in the bone remodeling phase of the bone cycle. In vitro studies of CB2 have shown that CB2 signaling stimulates osteoblast proliferation, differentiation and enhances osteogenic activity. CB2 can upregulate the expression of osteogenic factors such as RUNX2, bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP) and osteocalcin (OCN), thereby promoting osteoblast proliferation and differentiation. Studies have found that CB2 can reduce the number of osteoclasts while promoting osteoblast proliferation. CB2 activation can stimulate bone formation and inhibit bone resorption, so stimulating CB2 can prevent osteoporosis and prevent fractures. Ofek et al. found that young C57BL / 6 mice with CB2 knockout (CB2 − / − / C57BL) show normal peak bone mass, followed by increased age-related bone loss later in life, similar to human postmenopausal osteoporosis. 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 accelerated bone remodeling in CB2 knockout mice in different mouse strains also occurred. Stimulating CB2 signaling with a selective CB2 agonist in ovariectomized (OVX) mice prevented OVX-induced bone loss, and CB2 agonists led to an increase in bone formation rate. It is worth mentioning that CB2 knockout rats did not have any other changes in any organ except bone tissue, further emphasizing the importance of CB2 in the skeleton.

[0004] CB2 receptors are key targets for the development of anti-osteoporosis drugs that aim to improve human bone regeneration. The present invention aims to develop a class of CB2 agonist polypeptide molecules that promote bone formation based on the CB2 target, and achieve targeted distribution of bones through coupling technology, thereby providing a new strategy for the treatment of osteoporosis. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions: The present invention provides a CB2 agonist peptide or a pharmaceutically acceptable salt thereof, wherein the agonist peptide or a pharmaceutically acceptable salt thereof comprises any 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.

[0006] In a preferred embodiment of the present invention, the present invention provides a CB2 agonist peptide or a pharmaceutically acceptable salt thereof, wherein the agonist peptide or a pharmaceutically acceptable salt thereof consists of any 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.

[0007] 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.

[0008] In another preferred embodiment of the present invention, the present invention also provides an expression vector containing the polynucleotide.

[0009] In another preferred embodiment of the present invention, the present invention also provides a host cell containing the expression vector.

[0010] In another preferred embodiment of the present invention, the present invention further provides a pharmaceutical composition, which comprises the CB2 agonist peptide or a pharmaceutically acceptable salt thereof described in the present invention.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0012] In another additional preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or its pharmaceutically acceptable salt and pharmaceutical composition thereof in the treatment of osteoporosis.

[0013] In another additional preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or its pharmaceutically acceptable salt and pharmaceutical composition thereof in promoting osteogenic differentiation.

[0014] In another additional preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or its pharmaceutically acceptable salt and pharmaceutical composition thereof in the preparation of a drug for treating osteoporosis.

[0015] In a preferred embodiment of the present invention, the present invention provides the use of the CB2 agonist peptide or its pharmaceutically acceptable salt and pharmaceutical composition thereof in the preparation of a product promoting osteogenic differentiation.

[0016] The present invention develops a class of CB2 agonist polypeptide molecules with bone-forming promoting properties based on the CB2 target, and couples a bone-targeted polypeptide through coupling technology. The polypeptide upregulates the alkaline phosphatase content and promotes ALP expression, and has potential clinical applicability and research value in the treatment of osteoporosis.

[0017] 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

[0018] Figure 1 This is the result of CCK-8 detection of CB2 agonist peptide toxicity.

[0019] Figure 2These are the results of ALP staining after 14 days of induction, A: no induction solution added; 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.

[0020] Figure 3 This is the expression of osteogenesis-related genes after 14 days of induction. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1 Synthesis of CB2 agonist polypeptide compounds

[0023] 1. Experimental Materials The materials and reagents used in the present invention are purchased from commercial products. The protected amino acids used in the entire 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, and Boc-Tyr(tBu)-OH.

[0024] 2. Taking CB2-P1 as an example, the synthesis and preparation method of the agonist peptide in the present invention is described (the preparation of other agonist peptides in the present invention only requires replacing the synthesis sequence of amino acid raw materials) 1) Resin pretreatment: Weigh 26.3 g of Rink Amide AM Resin into a reactor, add 200 mL of DMF, and stir to swell for 35 min under nitrogen. When swelling is complete, remove the solvent by filtration, and wash the resin with DMF 5 times, 200 mL each time; 2) Prepare amino acid solution: weigh 11.5 g Fmoc-Lys(Boc)-OH and 4.04 g HOBt, dissolve them in 150 mL DMF, then add 3.78 g DIC and mix well. 3) Deprotection: Add 200 mL of 20% piperidine / DMF solvent to the reactor, stir and react for 40 minutes with nitrogen. After the reaction is completed, wash the resin with DMF 5 times, 200 mL each time. After washing, take the resin and test it with ninhydrin reagent, and the resin is positive; 4) Coupling reaction: Add the prepared amino acid solution to the reactor, control the temperature at 25-35℃, and stir the reaction under nitrogen. Monitor the progress of the reaction with ninhydrin reagent until the resin shows negative, indicating that the reaction is complete. After the reaction is completed, wash the resin with DMF 5 times, 200 mL each time. Repeat the above steps and couple the corresponding protected amino acids in sequence according to the CB2-P1 peptide until the peptide main chain is synthesized; 5) Peptide resin cleavage: Prepare a cleavage solution according to TFA / TIS / DTT / H2O=90 / 2.5 / 2.5 / 5, then add the dried peptide resin to the cleavage solution and stir to react for 3 hours. When the reaction is complete, filter, add the filtrate to 5 volumes of ice methyl tert-butyl ether for precipitation, filter, wash the filter cake with methyl tert-butyl ether 5 times, and vacuum dry to obtain a crude product; 6) Purification: The crude product was purified by reverse phase preparative chromatography to obtain a refined product of CB2-P1 with a purity of not less than 90%, and the compound was analyzed by analytical HPLC and LC-MS.

[0025] 3. Preparation results After the preparation was completed, the molecular weight test results of each agonist peptide compound were shown in Table 1, and the results showed that the prepared compounds were all correct.

[0026] Table 1 List of synthetic agonist peptide compounds and molecular weight

[0027] Example 2 Toxicity test of peptide compounds

[0028] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0029] 2. Experimental methods and results The toxicity test of peptide compounds is carried out as follows: hBMSCs in the logarithmic growth phase were plated at 5000 cells / mL in a 96-well plate, and peptide compounds were added to the 96-well plate at different concentrations, 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 indicated that CB2-PX (X=1 / 2 / 3) had no toxicity to cells.

[0030] Example 3 Ability of peptide compounds to promote bone formation

[0031] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0032] 2. Experimental methods and results The following is an osteogenic activity test, as follows: hBMSC cells were used for osteogenic induction. The cells were seeded in 12-well plates and divided into a normal culture group (without induction solution), an induction group (with induction solution), and an experimental group (with induction solution plus peptides). The induction solution was DMEM / F12 medium containing β-glycerophosphate sodium and vitamin C. After 14 days of induction, ALP staining was performed. The results are shown in Figure 2 As shown, compared with the induced group, the alkaline phosphatase levels in the experimental groups were all increased, indicating that CB2-PX (X=1 / 2 / 3 / 4) has the ability to promote osteogenic differentiation.

[0033] Example 4 Activation of osteogenic genes by peptide compounds

[0034] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0035] 2. Experimental methods and results The osteogenic gene expression test is performed as follows: hBMSC cells were used for osteogenic induction. The cells were seeded in 12-well plates and divided into a normal culture group (without induction solution), an induction group (with induction solution), and an experimental group (with induction solution plus peptides). The induction solution was DMEM / F12 medium containing sodium β-glycerophosphate and vitamin C. After 14 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 induced group, the ALP expression in the experimental group was significantly increased, which was consistent with the ALP staining results, indicating that CB2-PX (X=1 / 2 / 3 / 4) has the ability to promote osteogenic differentiation.

[0036] 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 CB2 agonist peptide or a pharmaceutically acceptable salt thereof, wherein the agonist peptide or a pharmaceutically acceptable salt thereof comprises any 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 CB2 agonist peptide or a pharmaceutically acceptable salt thereof, wherein the agonist peptide or a pharmaceutically acceptable salt thereof consists of any 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.

3. A polynucleotide encoding the agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2. An expression vector comprising the polynucleotide according to claim 3.

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

6. A pharmaceutical composition comprising the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier or excipient.

7. The CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and / or the pharmaceutical composition according to claim 6, characterized in that: Used to treat osteoporosis.

8. The CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and / or the pharmaceutical composition according to claim 6, characterized in that: Used to promote osteogenic differentiation.

9. Use of the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and / or the pharmaceutical composition according to claim 6 in the preparation of a drug for treating osteoporosis.

10. Use of the CB2 agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and / or the pharmaceutical composition according to claim 6 in the preparation of a product for promoting osteogenic differentiation.

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