Glycopeptide molecule

Abstract

The invention relates to the field of biological medicine, and provides a glycopeptide molecule, the glycopeptide molecule is a glycopeptide molecule or a glycopeptide assembly which is constructed by combining oligosaccharide fragments with oligopeptide fragments and has an osteogenesis promoting function, and the glycopeptide molecule has the effects of being safer, small in side effect and capable of effectively preventing and treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the preparation and application of a glycopeptide molecule. Background Art

[0002] Osteoporosis (OP) is a systemic skeletal disease characterized by low bone mass and destruction of bone tissue microstructure, resulting from an imbalance in bone homeostasis. Clinical patients mainly experience pain, fractures, and spinal deformities. With the increasing aging of the world's population, osteoporosis has become a public health issue of global concern. According to statistics, the number of people suffering from osteoporosis in my country exceeds 70 million, and osteoporotic fractures occur approximately 2.6 million times each year. The market for osteoporosis drugs has broad prospects. Currently, the commonly used anti-osteoporosis drugs mainly include the following: (1) Vitamin D, calcium, etc., which are the first-line drugs for preventing osteoporosis; (2) Anti-bone resorption drugs: such as bisphosphonates, estrogen, selective estrogen receptor agonists (represented by raloxifene) and calcitonin, etc. These drugs can inhibit osteoclast bone resorption and slow down the bone loss process; (3) Pro-osteoblast drugs: such as parathyroid hormone, teriparatide, oxides, these drugs can promote osteoblast bone formation; (4) Other drugs: such as statins, denosumab, etc. Although drugs including bisphosphonates or parathyroid hormone derivatives can be used to treat osteoporosis, they also cause many side effects, such as jaw necrosis and renal impairment. Therefore, it is urgent to find new strategies and new drugs for the treatment of osteoporosis.

[0003] In addition to cell dysfunction, such as abnormal osteoblast matrix secretion and abnormal bone metabolism caused by enhanced bone absorption capacity of osteoblasts, abnormal extracellular matrix content and structure can also lead to imbalance of bone homeostasis. Extracellular matrix components mainly include collagen, glycosaminoglycans, minerals and some trace elements. Chondroitin sulfate (CS), as an important component of glycosaminoglycans, is composed of D-glucuronic acid and N-acetylgalactosamine disaccharide repeating units, which can regulate the bone matrix microenvironment and maintain bone homeostasis. In the chemical structure of chondroitin sulfate, the sulfate group is modified by replacing one or more OH groups on the disaccharide units N-acetylgalactosamine C4, C6 and D-glucuronic acid C2, C3. These four modifications can produce 16 isomers of repeated disaccharide units, thereby producing a rich variety of structures. The diversity of the structure allows it to interact with many protein molecules. Therefore, in addition to its structural role, chondroitin sulfate also has important biological functions such as inflammation regulation, cell proliferation, cell differentiation, cell migration, tissue morphogenesis, organogenesis, infection and wound repair. Chondroitin sulfate has been widely studied in cartilage, such as the treatment of osteoarthritis, but its contribution to hard bone, especially in osteoporosis, has been little studied.

[0004] At present, the main obstacle to the drug development of chondroitin sulfate is its complex chemical structure and its source; commercial chondroitin sulfate mainly relies on animal tissue extraction (such as cattle, pigs and sharks). Although this traditional method has been used for many years, it still has many inherent defects, such as contamination by other glycosaminoglycans, structural heterogeneity, long-term breeding, easy degradation, etc. In order to overcome the above problems, the present invention designs and prepares a series of glycopeptide molecules with clear structures to replace natural chondroitin sulfate for the treatment of osteoporosis. Summary of the invention

[0005] The present invention has discovered a glycopeptide molecule, which is a glycopeptide molecule (glycopeptide assembly) constructed by combining an oligosaccharide fragment with an oligopeptide fragment to have the function of promoting osteogenesis. The glycopeptide molecule is safer, has fewer side effects, and can effectively prevent and treat osteoporosis. The present invention is completed based on this.

[0006] In a first aspect, the present invention provides a glycopeptide molecule, the structure of which is shown in Formula I. The glycopeptide molecule comprises an oligosaccharide fragment, an oligopeptide fragment, and a linker connecting the oligosaccharide fragment and the oligopeptide fragment.

[0007]

[0008] wherein ((I)), n is an integer of 1-6, and R is H or SO3H;

[0009] The oligopeptide fragment (oligopeptide) is an amino acid, a dipeptide, a trimeptide or a tetrapeptide protected by N-fluorenylmethoxycarbonyl (Fomc) composed of amino acids;

[0010] Furthermore, the amino acids are selected from the group consisting of: leucine (Leu), isoleucine (Ile), alanine (Ala), valine (Val), proline (Pro), phenylalanine (Phe), tryptophan (Trp) and / or methionine (Met).

[0011] The oligosaccharide fragment is a linear polysaccharide chain composed of D-glucuronic acid and N-acetylgalactosamine residue units, and its structure is shown in Formula III;

[0012]

[0013] In the above formula (II), the number of R being SO3H is 1 to (4n+1), wherein n is selected from an integer of 1 to 6.

[0014] The linker is selected from (a) an azide group and an alkynyl ring through an addition reaction to obtain a linker triazole heterocycle; (b) an azide group and a cyclooctyne through a cycloaddition reaction to obtain a product; (c) a conjugated diene and a substituted olefin through a Diels-Alder reaction to generate a linker substituted cyclohexene, such as a furan group and a maleic anhydride group through a cycloaddition reaction to obtain a six-membered cycloaddition product; (d) a hydrazine group and a hydroxyl group react to obtain a linker hydrazine bond or a hydrazine bond; (e) a thiol group and an alkene, an alkyne, a hydrogen halide, an isocyanate, and an epoxide react to obtain a linker. The structure of the linker is shown in formula (III);

[0015]

[0016] More preferably, the molecular structure of the glycopeptide of the present invention is as shown in IV:

[0017] Chondroitin sulfate (CS)-linker-oligopeptide, (IV)

[0018] The chondroitin sulfate is extracted from animal tissue or chemically synthesized.

[0019] In a second aspect, the present invention provides a glycopeptide composition, comprising the glycopeptide molecule of the present invention and a pharmaceutically and / or health-care acceptable carrier, diluent, excipient and / or adjuvant.

[0020] Furthermore, the glycopeptide composition of the present invention can be prepared as medicine, health care product or food.

[0021] In a third aspect, the present invention provides a combination preparation, which comprises a first active ingredient and / or a second active ingredient, wherein the first active ingredient is a glycopeptide molecule of the present invention, and the second active ingredient is a drug or active agent that can be used to treat or prevent osteoporosis and related diseases.

[0022] The second active ingredient is exemplarily selected from calcium, magnesium, estrogen, bisphosphonates, calcitonin, vitamin K1, vitamin K2, vitamin K4, vitamin K5, vitamin C, vitamin A, vitamin D2, vitamin D3, vitamin B1, vitamin B2, vitamin B6 or other trace vitamins, vitamin-like compounds folic acid or pantothenic acid, etc.

[0023] Furthermore, the dosage forms of the medicine, health product, food or combined preparation of the present invention include tablets, sugar-coated tablets, film-coated tablets, tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, electuary preparations, pills, powders, ointments, pills, suspensions, powders or solutions.

[0024] In a fourth aspect, the present invention provides a method for preparing a glycopeptide molecule, the preparation method comprising the following steps:

[0025] S01 Oligosaccharide enzymatic hydrolysis and separation: Chondroitin sulfate is hydrolyzed by enzyme to obtain chondroitin sulfate oligosaccharide fragments;

[0026] Synthesis of oligosaccharide azide S02: introducing an azide group into the oligosaccharide fragment obtained by hydrolysis and separation to obtain oligosaccharide azide CSn-N3;

[0027] Synthesis of S03 glycopeptide: CSn-N3 and oligopeptide were self-assembled using the "Click" reaction to obtain a new type of glycopeptide molecule.

[0028] Furthermore, in the method for preparing the glycopeptide molecule, the prepared glycopeptide molecule includes sulfation on the sugar chain molecule in chondroitin sulfate, wherein the number of sulfate groups is 1 to (4n+1), and n is selected from an integer of 1 to 6.

[0029] Furthermore, the chondroitin sulfate includes but is not limited to naturally extracted chondroitin sulfate and chemically synthesized chondroitin sulfate.

[0030] In a fifth aspect, the glycopeptide molecules, glycopeptide compositions and / or combined preparations of the present invention are used in the preparation of drugs for preventing and / or treating osteoporosis and related diseases.

[0031] Furthermore, the use comprises administering an effective amount of a glycopeptide molecule, a glycopeptide composition and / or a combined preparation to a subject (patient).

[0032] Furthermore, the osteoporosis includes primary osteoporosis and secondary osteoporosis, wherein the primary osteoporosis includes postmenopausal osteoporosis and senile osteoporosis.

[0033] Furthermore, the secondary osteoporosis includes osteoporosis secondary to endocrine diseases, rheumatic diseases, gastrointestinal diseases, and drug treatment.

[0034] Furthermore, the secondary osteoporosis includes osteoporosis caused by glucocorticoids, primary hyperparathyroidism, hyperthyroidism, primary biliary cirrhosis, hypogonadism, diabetes, hypertension, atherosclerosis, chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, osteoarthritis, gonadal hormone therapy, antiepileptic drug therapy, and chemotherapy drug therapy.

[0035] Beneficial Effects

[0036] The present invention adopts an enzymatic chemistry method to combine oligosaccharide fragments with oligopeptides to construct a glycopeptide assembly with osteogenic function. Compared with the traditional chemical method, it has the advantages of simple steps and high efficiency. Compared with natural chondroitin sulfate, the glycopeptide molecules prepared by the present invention have a clear glycopeptide small molecule structure, which solves the safety problem of animal-derived chondroitin sulfate. At the same time, the glycopeptide molecules can effectively promote the expression of osteogenesis-related genes and bone formation in osteoporotic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Synthesis flow chart of glycopeptide molecule Peptide-CSn

[0038] Figure 2 ESI-MS spectrum of CS tetrasaccharide (CS4)

[0039] Figure 3 ESI-MS spectrum of CS hexasaccharide (CS6)

[0040] Figure 4 ESI-MS spectrum of Fomc-Y-CS4

[0041] Figure 5 ESI-MS spectrum of FF-CS4

[0042] Figure 6 ESI-MS spectrum of IFF-CS4

[0043] Figure 7 ESI-MS image of EFFF-CS4

[0044] Figure 8 ESI-MS spectrum of Fomc-Y-CS6

[0045] Fig. 9 ESI-MS spectrum of FF-CS6

[0046] Fig.10 ESI-MS spectrum of IFF-CS6

[0047] Fig.11 ESI-MS spectrum of EFFF-CS6

[0048] Fig.12 (a) Particle size, (b) zeta potential and (c) TEM image of Fomc-Y-CS4 in water

[0049] Fig.13 Flow chart of the formation of nanomicelles by Fomc-Y-CS4

[0050] Fig.14 The effect of natural CS and glycopeptide molecules on promoting the expression of alkaline phosphatase in osteoblasts

[0051] Fig.15 Comparison of the effects of Fomc-Y-CS4, oligosaccharides and natural CS on promoting osteogenic gene expression

[0052] Fig.16 Effect of tetrasaccharide glycopeptide Fomc-Y-CS4 on promoting osteogenic gene expression

[0053] Fig.17 Effects of tetrasaccharide glycopeptides FF-CS4, IFF-CS4, and EFFF-CS4 on promoting osteogenic gene expression

[0054] Fig.18 The effect of hexasaccharide glycopeptides Fomc-Y-CS6, FF-CS6, IFF-CS6 and EFFF-CS6 on promoting osteogenic gene expression Fig.19 Effects of natural CS and glycopeptide molecules on bone mass changes in vivo. (a) Micro-CT three-dimensional reconstruction images. (bf) Quantitative statistical analysis of trabeculae. BV / TV (bone volume fraction), Tb.N (trabecular number), Tb.Th (trabecular thickness), Tb.Sp (trabecular separation), SMI (structural model index). *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0055] Oligopeptide: A peptide chain containing several to a dozen amino acid residues is collectively referred to as an oligopeptide, and its oligopeptide fragment (oligopeptide) is an amino acid, a dipeptide, a tripeptide or a tetrapeptide protected by an N-fluorenylmethoxycarbonyl (Fomc) composed of amino acids; the amino acids constituting the oligopeptide include a combination of leucine (Leu), isoleucine (Ile), alanine (Ala), valine (Val), proline (Pro), phenylalanine (Phe), tryptophan (Trp) and / or methionine (Met); wherein the combination of amino acids includes a combination of identical amino acids and / or different amino acids.

[0056] Oligosaccharide: a carbohydrate substance formed by 2 to 20 monosaccharides connected by glycosidic bonds. In the present invention, the oligosaccharide is a linear polysaccharide chain composed of D-glucuronic acid and N-acetylgalactosamine residue units. In the present invention, the oligosaccharide is derived from natural or chemically synthesized chondroitin sulfate.

[0057] As described herein, "self-assembly" is a process in which each component spontaneously forms a highly ordered structure across multiple scales under the drive of various non-covalent interactions such as hydrophobic interactions, π-π interactions, hydrogen bonds, electrostatic interactions, van der Waals forces, etc. In the present invention, glycopeptides are a class of small molecules in which sugar units are covalently linked to peptide segments, have a chemical composition similar to that of natural glycoprotein molecules, and can simulate the structure and function of natural glycoproteins to a certain extent. On the one hand, the peptide segments as the main chain have a secondary structure similar to that of natural proteins, and it is possible to assemble to obtain a higher-order structure or aggregated structure similar to that of natural proteins. On the other hand, the sugar units present in the side groups or ends of glycopeptides have diverse structures, which give glycopeptides many biologically active functions. Therefore, using glycopeptides as self-assembly primitives, glycopeptide assemblies with novel molecular structures, controllable self-assembly behaviors and rich functions can be constructed. The oligosaccharides obtained in the "glycopeptide molecules" described in the present invention can provide osteogenic sugar fragments with clear structures. The hydrophobic and π-π interactions of the hydrophobic peptide segments give the glycopeptide the ability to self-assemble. The glycopeptide molecules assemble into nano-micelles in water. The oligosaccharide fragments in the outer layer are looser than the random coils of chondroitin sulfate polysaccharides, which can effectively improve the bioavailability of glycopeptide nanoparticles and thus improve their osteogenic ability. In the present invention, the glycopeptide assembly and the glycopeptide molecule can be interchangeable.

[0058] As used herein, the term "glycopeptide composition" is prepared by mixing the Peptide-CSn of the present invention having the desired purity and a pharmaceutically and / or health-care acceptable carrier or excipient, wherein the carrier is in the form of a lyophilized preparation or an aqueous solution. The pharmaceutically and / or health-care acceptable carrier is generally non-toxic to the recipient at the dosage and concentration used, and is formulated for ingestion in liquid form, solid form, aerosol form or other forms. In certain aspects, the glycopeptide composition provided in the present invention can be used to treat or prevent osteoporosis and its related conditions.

[0059] Micelles: nano-sized core-shell micelles formed by self-assembly of amphiphilic block copolymers having hydrophilic and hydrophobic groups in water. In the present invention, glycopeptide molecules containing chondroitin sulfate oligosaccharides are self-assembled to form micelles with hydrophobic polypeptide as the core and sugar chains as the shell.

[0060] "CS-A" described in this article: Chondroitin sulfate (CS) is a type of glycosaminoglycan covalently linked to proteins to form proteoglycans. It is composed of repeating units of D-glucuronic acid and N-acetylgalactosamine disaccharide, and is connected to the serine residue of the core protein through a sugar-like linking region; although the main chain structure of the polysaccharide is not complex, it is highly heterogeneous in terms of the degree of sulfation, the distribution of sulfate groups and two different isomeric uronic acids in the chain. According to the differences in the degree and position of sulfate group substitution, it can be divided into multiple isomers such as A, B, C, D, and E; the "CS-A" described in this article is one of the isomers.

[0061] Glycopeptide compositions may generally contain, for example, pharmaceutically acceptable salts, buffers, preservatives and / or compatible carriers. As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to mammals, including humans.

[0062] The term "pharmaceutically acceptable" refers to compositions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications, commensurate with a reasonable benefit / risk ratio.

[0063] The term "combination preparation" refers to a combination preparation in the present invention that may also contain more than one active compound required for the specific disease to be treated, preferably those with complementary activities and no side effects, such as antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, etc. In the present invention, glycopeptide molecules, glycopeptide compositions and combination preparations can be prepared as tablets, sugar-coated tablets, film-coated tablets, tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, powders, pastes, pills, suspensions, powders, solutions, etc. In one embodiment, nanoparticle preparations are preferred.

[0064] The term "effective amount" refers to an amount effective for treatment or prevention that can be administered to an individual in a single dose or as part of a series of doses; that is, an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0065] The "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers that are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. In the composition, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, or a buffer. In addition, auxiliary substances may also be present in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc.

[0066] The term "subject" refers to any subject, particularly a mammalian subject, for which diagnosis, prognosis, immunization or treatment is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals such as bears, sports animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows; primates such as apes, monkeys, gorillas and chimpanzees; canines such as dogs and wolves; felines such as cats, lions and tigers; equines such as horses, donkeys and zebras; food animals such as cattle, pigs and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs, etc. In this embodiment, the subject is a mouse.

[0067] The terms "treat" and "prevent" refer to obtaining a desired pharmacological and / or physiological effect. The effect may be complete or partial prevention of a disease or its symptoms, and / or partial or complete cure of a disease and / or its symptoms, and include ① preventing the disease from occurring in a subject, who may have a predisposition to the disease but has not yet been diagnosed as having the disease; ② inhibiting the disease, i.e., blocking its formation; and ③ alleviating the disease and / or its symptoms, i.e., causing the disease and / or its symptoms to regress.

[0068] Osteoporosis is a bone disease that increases the risk of fractures. In osteoporosis, bone mineral density (BMD) decreases, bone microarchitecture is destroyed, and the amount and diversity of non-collagenous proteins in the bone change. The disease category of osteoporosis includes two major categories: primary and secondary. Primary osteoporosis is further divided into three types: postmenopausal osteoporosis, senile osteoporosis, and idiopathic osteoporosis (including juvenile type). Secondary osteoporosis includes osteoporosis caused by endocrine system diseases, connective tissue diseases, chronic kidney diseases, gastrointestinal diseases and nutritional diseases, blood system diseases, neuromuscular system diseases, long-term immobilization, post-organ transplantation, and long-term use of glucocorticoids, immunosuppressants or anticancer drugs.

[0069] The short peptide Fomc-Y described herein is N-fluorenylmethoxycarbonyl L-tyrosine, FF is phenylalanine-phenylalanine, IFF is isoleucine-phenylalanine-phenylalanine, and EFFF is glutamic acid-phenylalanine-phenylalanine-phenylalanine.

[0070] The administration methods of the present invention include but are not limited to enteral administration and parenteral administration.

[0071] Example 1 Glycopeptide molecules and drug preparation thereof

[0072] 1.1 Preparation of CS oligosaccharides

[0073] (1) Enzymatic hydrolysis and separation of CS tetrasaccharide (CS4) and CS hexasaccharide (CS6)

[0074] The natural CS was added to a methanol solution containing acetyl chloride and reacted for 7 days. The above precipitate was dissolved in a NaOH solution and reacted overnight, and then concentrated by rotary evaporation to obtain desulfated CS. The desulfated CS was dissolved in a NaOAc / AcOH buffer solution (PH=5) and hyaluronidase was added. The CS tetrasaccharide and hexasaccharide mixture was obtained by enzymolysis at 37°C for 7 days. After the product was filtered, concentrated and precipitated, it was separated and purified by ion exchange resin to obtain CS4 and CS6, respectively.

[0075] (2) Enzymatic hydrolysis and separation of CS disaccharide (CS2), CS octasaccharide (CS8), CS decasaccharide (CS10), and CS dodecasaccharide (CS12);

[0076] His-CHSase ABC I (chondroitin sulfate lyase ABC I) was added to the natural CS solution, the reaction system was enzymatically hydrolyzed at 37°C for 10 hours, and the enzymatic hydrolyzate was separated and purified using a gel chromatography column to obtain CS2, CS8, CS10, and CS12.

[0077] 1.2 Synthesis of oligosaccharide azides

[0078] Dissolve CS4, NaN3 and 2-chloro-1.3-dimethylimidazolium chloride in deionized water and react overnight. The reaction solution is concentrated, desalted and freeze-dried to obtain oligosaccharide azide CS4-N3. The same method is used to synthesize other oligosaccharide azides (CSn-N3).

[0079] 1.3 Synthesis of Peptide-CSn (n = 2, 3) (Peptide = Fomc-Y, FF, IFF, EFFF)

[0080] (1) Preparation of Peptide-CS4

[0081] CS4-N3, Fomc-Y (N-fluorenylmethoxycarbonyl L-tyrosine), sodium ascorbate, and CuSO4 were added to a DMF / H2O mixed solution and reacted overnight. The reaction solution was concentrated under reduced pressure, dialyzed for desalination, and then separated by HPLC to obtain the unsulfated glycopeptide molecule product. The above product and the sulfation reagent were dissolved in DMF and reacted overnight; the above reaction solution was concentrated under reduced pressure, dialyzed and freeze-dried to obtain Fomc-Y-CS4. Glycopeptide molecules FF-CS4, IFF-CS4, and EFFF-CS4 were prepared using the same steps (FF (phenylalanine-phenylalanine), IFF (isoleucine-phenylalanine-phenylalanine), EFFF (glutamic acid-phenylalanine-phenylalanine-phenylalanine)).

[0082] (2) Peptide-CS6 was obtained by the same preparation method as above

[0083] 1.4 Synthesis of Peptide-CSn (n = 1, 4, 5, 6)

[0084] Using the same "click" method as in step 1.3, CSn-N3 and short peptides Fomc-Y, FF, IFF, EFFF were dissolved in a DMF / H2O mixed solution, and then sodium ascorbate and CuSO4 were added to react overnight. The reaction solution was dialyzed and HPLC separated to obtain the final products Peptide-CS2, Peptide-CS8, Peptide-CS10, and Peptide-CS12.

[0085] 1.5 Preparation of glycopeptide nanomedicines

[0086] A glycopeptide molecule aqueous solution is prepared, and nano-micelles are obtained after standing and assembling at room temperature.

[0087] 1.6 Glycopeptide molecular structures are shown in Tables 1 and 2 below

[0088] Table 1 Molecular structures of Fomc-Y and FF glycopeptides

[0089]

[0090]

[0091] Table 2 Schematic diagram of the molecular structure of IFF and EFFF glycopeptides

[0092]

[0093] 1.7 Experimental Results

[0094] (1) Hyaluronidase was used to hydrolyze the desulfated CS to obtain chondroitin sulfate fragments and obtain glycosidic bonds of natural configuration; the efficient aqueous "Click" reaction was used to combine with the oligopeptide fragments through a linker to construct a new type of glycopeptide molecule. Among them, the glycopeptide molecules Peptide-CSn of CS tetrasaccharide (CS4), CS hexasaccharide (CS6), CS disaccharide (CS2), CS octasaccharide (CS8), CS decasaccharide (CS10), and CS dodecasaccharide (CS12) were synthesized (see Figure 1 ); In the reaction, the linker is an azide group and an alkynyl ring, which undergo an addition reaction to obtain a triazole heterocycle.

[0095] (2) ESI-MS spectra of CS4 and CS6 (see Figure 2 ), m / z(CS4)=775.23, m / z(CS6)=1154.34 (see Figure 3 ).

[0096] (3) ESI-MS spectrum of the glycopeptide molecule Fomc-Y-CS-4 (see Figure 4), m / z=1320, contains one SO3H, m / z=699, contains two SO3H.

[0097] (4) ESI-MS spectra of glycopeptide molecules FF-CS4, IFF-CS4, and EFFF-CS4 (see Figure 5-7 ),in Figure 5 The m / z of FF-CS4 in the sample is 1410, which contains two SO3H; Figure 6 The m / z of IFF-CS4 is 1442, containing one SO3H; Figure 7 The m / z of EFFF-CS4 in the sample is 1670, which contains two SO3H;

[0098] (5) ESI-MS spectra of glycopeptide molecules Fomc-Y-CS6, FF-CS6, IFF-CS6 and EFFF-CS6 (see

[0099] Figures 8 to 11 ), Figure 8 Fomc-Y-CS6 has m / z = 849, containing one SO3H, and m / z = 889, containing two SO3H;

[0100] Fig. 9 The m / z of FF-CS6 in the sample is 623, which contains three SO3H; Fig.10 In IFF-CS6, m / z = 675, containing three SO3H; Fig.11 The m / z of EFFF-CS6 is 1984, which contains one SO3H.

[0101] (6) Particle size, zeta potential and TEM image of Fomc-Y-CS4 in water (see Fig.12 ) and nanomicelle formation flow chart (see Fig.13 ).

[0102] Example 2 Cell Osteogenesis Induction

[0103] 2.1 Osteogenic induction medium configuration:

[0104] (1) Preparation of dexamethasone storage solution: Weigh 3.925 mg of dexamethasone and dissolve it in 10 ml of anhydrous ethanol. Filter through a 0.22 μm filter. The concentration of dexamethasone is now 1 mM. Dispense into 10 sterile EP tubes and store at 4°C in the dark.

[0105] (2) Preparation of Vc (ascorbic acid) storage solution: Weigh 17.612 mg of Vc (ascorbic acid) and dissolve it in 10 ml of PBS. Filter it through a 0.22 μm filter to a concentration of 10 mM. Dispense it into 10 sterile EP tubes and store it at 4°C.

[0106] (3) Preparation of β-glycerophosphate sodium storage solution: Weigh 3.0611 g of β-glycerophosphate sodium and dissolve it in 9.1 ml of PBS. Filter it through a 0.22 μm filter to a concentration of 1 M. Dispense it into 10 sterile EP tubes and store it at 4°C.

[0107] (4) Prepare 100 ml of osteogenic induction working solution: 10 μl 1 mM dexamethasone + 500 μl 10 mM Vc (ascorbic acid) + 1000 μl 1 M β-glycerophosphate sodium + 1000 μl double antibody + 10 ml FBS, and finally adjust the volume to 100 ml with α-MEM medium.

[0108] (5) According to the experimental design, add the prepared sample solution into the osteogenic induction medium.

[0109] 2.2 When osteoblasts and bone marrow mesenchymal cells at generation P2 covered about 90% of the bottom of the dish, wash once with PBS, digest the cells, centrifuge, and count. 4 The cells were seeded in 6-well plates at a density of 2×10 4 The cells were seeded at a density of 10 cells / well in 12-well plates.

[0110] 2.3 Wait for the cells to adhere to the wall on the second day, discard the original culture medium, and prepare the sample to the required working solution concentration in the 6-well plate with osteogenic induction medium, and finally adjust the volume of osteogenic induction medium to 1500μl per well. Prepare the sample to the required working solution concentration in the 12-well plate with osteogenic induction medium, and finally adjust the volume of osteogenic induction medium to 1000μl per well. After the addition of the drug is completed, it is recorded as the first day. In the following two days, add the sample solution in the same way and replace the osteogenic induction culture medium.

[0111] 2.4 After 7 days of osteogenic induction, alkaline phosphatase staining and cell RNA extraction were performed.

[0112] Example 3 Promoting the expression of osteoblast alkaline phosphatase

[0113] Primary and subcultured osteoblasts, P3 cells were plated in 12-well plates. A control group (mineralization induction medium + PBS, Ctrl group), a natural CS group (mineralization induction medium + different concentrations of natural CS preparations, CS group), and a glycopeptide molecule group (mineralization induction medium + different concentrations of glycopeptide molecule preparations) were set up. The concentration gradient was set to 10ug / ml, 20ug / ml, 50ug / ml, and 100ug / ml. After 7 days of osteogenic induction, alkaline phosphatase staining was performed. The glycopeptide used in the glycopeptide molecule was selected from Fomc-Y-CS4.

[0114] The experimental results showed that both the full-length CS and the glycopeptide molecule Fomc-Y-CS4 can promote the osteogenic differentiation of preosteoblasts and the expression of cell alkaline phosphatase. As the drug concentration increases, the osteogenic function becomes more obvious (see Fig.14 ).

[0115] Example 4 Comparison of the effects of glycopeptide molecules, CS oligosaccharides and natural CS on promoting osteogenic gene expression

[0116] Primary and subcultured osteoblasts, P3 cells were plated in 6-well plates. A control group (Ctrl, i.e., PBS group), natural CS, CS disaccharide (CS2), CS tetrasaccharide (CS4), CS hexasaccharide (CS6), and glycopeptide molecule (Fomc-Y-CS4) groups were set up, wherein CS disaccharide (CS2), CS tetrasaccharide (CS4), CS hexasaccharide (CS6), and glycopeptide molecule (Fomc-Y-CS4) were from the glycopeptide molecules prepared in Example 1 and Example 2. After 7 days of osteogenic induction, cell RNA was extracted to detect the effects of the control group and the experimental group on osteogenic genes.

[0117] The experimental results showed that CS polysaccharide, CS oligosaccharide and glycopeptide molecule (Fomc-Y-CS4) groups can promote the expression of osteogenic gene Ocn, and the osteogenic differentiation ability of glycopeptide molecule is better than that of CS polysaccharide and CS oligosaccharide (see Fig.15 ).

[0118] Example 5 Effect of Glycopeptide Molecules on Promoting Osteogenic Gene Expression

[0119] Primary and subcultured osteoblasts, P3 cells were plated in 6-well plates. A control group, a natural CS group, and a glycopeptide molecule (Fomc-Y-CS4) group were set up. The sample concentration was set to 50ug / ml and 100ug / ml, and cell RNA was extracted after 7 days of osteogenic induction to detect the effects of each group on osteogenic genes. The glycopeptide molecule prepared in Example 1 was used as the glycopeptide molecule.

[0120] The experimental results showed that both the natural CS group and the glycopeptide molecule group Fomc-Y-CS4 could promote the expression of osteogenic genes Ocn, Bsp, Opg, and Alp, proving that chondroitin sulfate oligosaccharides have the function of full-length chondroitin sulfate and can promote the expression of osteoblast-related genes (see Fig.16 ).

[0121] In addition, the osteogenic effects of tetrasaccharide glycopeptide molecules FF-CS4, IFF-CS4, EFFF-CS4 and hexasaccharide glycopeptide molecules Fomc-Y-CS6, FF-CS6, IFF-CS6 and EFFF-CS6 were also verified (the concentration of glycopeptide molecules was set to 100ug / ml). The results showed that the osteogenic marker genes Alp, Col1, and Ocn of osteoblasts treated with these seven glycopeptide molecules were significantly upregulated, confirming that glycopeptide molecules can promote osteogenic differentiation and the expression of osteogenic genes (see Fig.17 and Fig.18 ).

[0122] Example 6 Effect of glycopeptide molecules on the changes of bone mass in vivo after osteoporosis model construction

[0123] Twenty-four 8-week-old ICR female mice were randomly divided into 4 groups, sham operation group (SHAM group), saline group (CON group), CS group and glycopeptide group, 6 mice in each group. The abdominal cavity of mice in the SHAM group was opened under surgical conditions, and the ovaries were not removed. A certain amount of saline was injected into the tail vein 2 months after surgery; bilateral ovaries were removed by surgery in the CON group, and an equal volume of saline was injected into the tail vein 2 months after surgery; bilateral ovaries were removed by surgery in the CS group and glycopeptide group, and 100 mg / kg (100 mg drug per kg of mouse body weight) of CS and glycopeptide molecules were injected into the tail vein 2 months after surgery. The injection was repeated three times a week for 2 consecutive months. After sampling and perfusion fixation, the femur samples of mice were taken for Micro-CT scanning and analysis, and the changes in bone mass in the control group and the experimental group were analyzed. The glycopeptide used in the glycopeptide molecule was selected from Fomc-Y-CS4.

[0124] The three-dimensional reconstruction data showed that the bone mass of the femoral metaphysis increased; the results of quantitative analysis of trabeculae showed that compared with the SHAM group, the BV / TV of the CON group decreased, the separation of trabeculae increased, the structural pattern index (SMI) increased, and the trabecular structure changed from plate-like to rod-like. The number of trabeculae and the average thickness of trabeculae increased, and the separation of trabeculae decreased in the CS group and the glycopeptide group compared with the CON group, indicating that the CS group and the glycopeptide group can promote the increase of bone mass in osteoporotic mice (see Fig.19 ).

Claims

1. A glycopeptide molecule, as shown in formula I, the glycopeptide molecule comprises an oligosaccharide fragment, an oligopeptide fragment, and a linker connecting the oligosaccharide fragment and the oligopeptide fragment, the oligosaccharide fragment is a linear polysaccharide chain composed of D-glucuronic acid and N-acetylgalactosamine residue units, and the structure is shown in formula (II); the oligopeptide fragment (oligopeptide) is an amino acid, a dipeptide, a tripeptide or a tetrapeptide protected by an N-fluorenylmethoxycarbonyl (Fomc) composed of amino acids; the linker is any one of (III); In the formulae (I) and (II), n is an integer of 1-6, R is H or SO3H, and the number of R being SO3H is 1 to (4n+1).

2. The glycopeptide molecule according to claim 1, wherein the amino acids of the oligopeptide fragment in the glycopeptide molecule are selected from a combination of leucine (Leu), isoleucine (Ile), alanine (Ala), valine (Val), proline (Pro), phenylalanine (Phe), tryptophan (Trp) and / or methionine (Met).

3. The glycopeptide molecule according to claim 1, wherein the structure of the glycopeptide molecule is as shown in Formula IV, Chondroitin sulfate (CS)-linker-oligopeptide (IV); in, The chondroitin sulfate is extracted from animal tissue or chemically synthesized.

4. A glycopeptide composition, comprising the glycopeptide molecule according to any one of claims 1 to 3 and a pharmaceutically and / or health-care acceptable carrier, diluent, excipient and / or adjuvant.

5. A combined preparation, comprising a first active ingredient and / or a second active ingredient, wherein the first active ingredient is the glycopeptide molecule according to any one of claims 1 to 3, and the second active ingredient is a drug or active agent that can be used to treat or prevent osteoporosis and related diseases.

6. The combined preparation according to claim 5, characterized in that The second active ingredient is selected from calcium, magnesium, estrogen, bisphosphonates, calcitonin, vitamin K1, vitamin K2, vitamin K4 vitamin K5, vitamin C, vitamin A, vitamin D2, vitamin D3, vitamin B1, vitamin B2, vitamin B6 or other trace vitamins or vitamin-like compounds folic acid or pantothenic acid.

7. A method for preparing a glycopeptide molecule, the method comprising the following steps: S01 Oligosaccharide enzymatic hydrolysis and separation: Chondroitin sulfate was hydrolyzed by enzyme to obtain chondroitin sulfate oligosaccharide fragments; Synthesis of oligosaccharide azide S02: introducing an azide group into the oligosaccharide fragment obtained by hydrolysis and separation to obtain oligosaccharide azide CSn-N3; Synthesis of S03 glycopeptide: CSn-N3 and oligopeptide were reacted by "Click" reaction to obtain a new type of glycopeptide molecule.

8. The method for preparing the glycopeptide molecule according to claim 7, characterized in that In the preparation method of the glycopeptide molecule, the prepared glycopeptide molecule includes sulfation on the oligosaccharide fragment molecule in chondroitin sulfate, wherein the number of sulfate groups is 1 to (4n+1), and n is selected from an integer of 1 to 6.

9. Use of the glycopeptide molecule, glycopeptide composition or combined preparation according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating osteoporosis and related diseases.

10. The use according to claim 9, characterized in that The osteoporosis includes primary osteoporosis and secondary osteoporosis.

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