Super long-acting polypeptide for NPR-B targeting and application thereof in skeletal system diseases
By designing NPR-B-targeted ultra-long-acting peptides, the problem of short half-life of CNP analogs has been solved, achieving long-acting therapeutic effects for skeletal system diseases and significantly improving treatment adherence.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2026-03-24
AI Technical Summary
Existing CNP analogs, such as vosolidaretin, have a short half-life in the human body, requiring frequent administration, which leads to economic and psychological burdens on patients. They are also expensive and cannot meet the long-term treatment needs of conditions such as skeletal dysplasia.
An ultra-long-acting NPR-B-targeting peptide was designed. By introducing disulfide bonds and specific side chain modifications (such as PEG2-γ-Glu-CO(CH2)18CO2H) into the peptide, its in vivo half-life was extended and its activation activity against NPR-B was maintained.
The peptide compound has a significantly extended half-life, reaching 300 times that of CNP22, reducing the dosing frequency to once a month, lowering medication costs, improving patient compliance, and exhibiting good biological activity and low toxicity.
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Abstract
Description
[0001] This application is a divisional application of the Chinese patent application No. 202411747622.0, filed on December 02, 2024, with the title of "A long-acting polypeptide for NPR-B targeting and its application in skeletal system disorders". TECHNICAL FIELD
[0002] The present application relates to the technical field of biological medicine, in particular to a long-acting polypeptide for NPR-B targeting and its application in skeletal system disorders. BACKGROUND
[0003] The natriuretic peptide family is a group of structurally similar but different peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). CNP is composed of 22 amino acids, which is initially isolated from pig brain, and then found to be initially produced in the human body by the natriuretic peptide precursor C (Nppc) gene containing a 126-amino acid single-chain pro-polypeptide form. After removing the signal peptide, the source CNP is obtained, and through further cleavage by endoprotease furin, an active peptide of 53 amino acids (C-type Natriuretic Peptide-53, CNP-53) is produced, which is secreted and enzymatically cleaved to produce a mature peptide of 22 amino acids (C-type Natriuretic Peptide-22, CNP-22). CNP-53 and CNP-22 can both bind to natriuretic peptide receptor B (NPR-B) and induce the production of cyclic guanosine monophosphate (cGMP) in a dose-dependent manner. CNP-22 mainly exists in body fluids and is considered to be a mature and more bioactive form.
[0004] CNP is a bone growth promoting regulator acting on the NPR-B receptor of chondrocytes. Downstream signaling of CNP / NPR-B inhibits the Fibroblast Growth Factor Receptor 3 (FGFR-3) pathway at the level of Mitogen-Activated Protein Kinase (MAPK). Inhibition at MAPK promotes proliferation and differentiation of chondrocytes in the proliferative and hypertrophic zones of the growth plate, leading to bone growth. Therefore, increasing or decreasing the amount of any component in this pathway can cause abnormal bone growth. In humans, activating mutations of FGFR-3 are the main cause of genetic dwarfism. Mice with activated FGFR-3 serve as a model for achondroplasia (the most common form of skeletal dysplasia), and overexpression of CNP saves these animals from short stature. Therefore, CNP and functional variants of CNP are potential therapeutic agents for treating various skeletal dysplasias.
[0005] However, the plasma half-life of CNP in humans is only 2.6 min, and in order to keep the CNP concentration in the plasma higher than its inherent concentration (about 5 pM), continuous administration is necessary. The drug vosoritide approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of achondroplasia is a C-type natriuretic peptide analogue containing 39 amino acids, which prolongs the half-life to 20 min. However, like most polypeptide compounds, vosoritide can be taken orally and is easily degraded in the body, and the problem of short half-life. The half-life of only 20 min cannot meet the clinical needs, and still requires once-daily subcutaneous injection, which continues until the epiphyseal closure of the child patient. In addition, vosoritide is expensive, which brings great mental and economic burden to the patient and the patient's guardian.
[0006] Therefore, how to develop a polypeptide compound targeting NPR-B with a longer half-life in the body and still retaining similar or improved activity is a technical problem that needs to be solved. SUMMARY
[0007] In view of the deficiencies of the prior art, the present application provides a super-long-acting polypeptide for NPR-B targeting and its application in skeletal system disorders. The super-long-acting polypeptide greatly prolongs its half-life in the body while retaining the activity of activating NPR-B, thereby prolonging the administration interval and reducing the cost of medication. The super-long-acting polypeptide can be applied to the preparation of a drug for treating diseases in the skeletal system.
[0008] The present application is realized by the following technical solutions: In one aspect, the present application provides a polypeptide compound for NPR-B targeting, which has the following structure: R1-Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-X1-Ser-Gly-Leu-Gly-Cys-R3, wherein a disulfide bond is formed between the two Cys (cysteine); R1is selected from -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, or is absent; R2is selected from -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, or is absent; R3is selected from -Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H), or is absent; X1is selected from -Met, Leu or Gly; R1, R2, R3are not simultaneously absent.
[0009] Preferably, the polypeptide compound is selected from any one or more of: (1) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 1), wherein a disulfide bond is formed between the two Cys; (2) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gly-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 2), wherein a disulfide bond is formed between the two Cys; (3) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 3), wherein a disulfide bond is formed between the two Cys; (4) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (5) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (6) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R3, R3 is -Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H), in which disulfide bonds are formed between the two Cys; (7) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (8) Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R2 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (9) Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R2 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (10) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R1 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between two Cys.
[0010] Preferably, X1 is Leu.
[0011] Preferably, R1 is selected from -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2). 18 CO2H,-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H or -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H.
[0012] Preferably, R2 is absent.
[0013] Preferably, R3 is absent.
[0014] More preferably, the polypeptide compound is selected from: (1) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R3, R3 is -Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H), in which disulfide bonds are formed between the two Cys; (2) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between two Cys.
[0015] On the other hand, the present invention also provides the use of the aforementioned polypeptide compound in the preparation of a medicament for treating skeletal system diseases.
[0016] Furthermore, the skeletal system disorder is at least one of achondroplasia, chondrodysplasia, osteochondrosis, osteoarthritis, articular cartilage damage (e.g., ankle cartilage damage), dwarfism, and short stature, preferably achondroplasia.
[0017] Furthermore, the drug may also include pharmaceutically acceptable carriers and / or excipients.
[0018] Furthermore, the drug is suitable for administration via methods including but not limited to oral, transdermal, intravenous, intramuscular, local, and nasal administration.
[0019] Furthermore, the drug can be formulated into a suitable dosage form depending on the route of administration used.
[0020] Furthermore, the dosage forms of the drug include, but are not limited to, tablets, capsules, soft capsules, drop pills, granules, pills, oral liquids, ointments and patches for skin application, aerosols, nasal sprays, injections, and infusions.
[0021] Furthermore, the drug can be formulated into a solution or lyophilized powder for parenteral administration. Before use, the powder can be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier. The liquid formulation is generally a buffer solution or an isotonic solution.
[0022] Furthermore, the dosage of the drug is an amount that can alleviate or delay the progression of the disease, degenerative or damaging condition, and may be determined depending on the specific disease being treated and other factors, including age, weight, severity of symptoms, route of administration, drug formulation, frequency of treatment, and whether other drugs are being used concurrently during treatment.
[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The peptide compound for NPR-B targeting of the present invention can achieve ultra-long-lasting effect while also efficiently activating cGMP signal in NIH3T3 cells in vitro, significantly prolonging body length and tail length in mice in vivo, thereby improving cartilage development in mice and achieving the effect of improving skeletal system diseases. (2) The peptide compound for NPR-B targeting of the present invention has good biological activity and a significantly extended half-life. Taking CNP22 as an example, the half-life of the modified peptide compound is 300 times that of CNP22 and more than 30 times that of vosolidaretin, the only existing treatment for achondroplasia. Vosolidaretin requires daily subcutaneous injection and long-term use, usually continuing until the bone matures (growth plate closes), with treatment courses ranging from several years to decades, which imposes a huge drug burden on patients and society. The significant extension of the half-life can reduce the dosing frequency from once a day to once a month, which can improve patient compliance, reduce the economic burden on patients, and address a major clinical need. (3) The peptide compound for NPR-B targeting of the present invention shows stability in the pharmacokinetics of the drug, has good stability, is easy to scale up for production, and has low cost; (4) The peptide compounds for NPR-B targeting of the present invention have lower toxicity, a larger safety window, and require smaller dosages compared with small molecule compounds. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 The chemical structural formulas of polypeptide compounds 1-10 are shown.
[0026] Figure 2 The effects of peptide compounds 1-10 on the viability of NIH3T3 cells were shown.
[0027] Figure 3The graph shows the cGMP content produced by NIH3T3 cells stimulated by peptide compounds 1-5.
[0028] Figure 4 The graph shows the cGMP content produced by NIH3T3 cells stimulated by peptide compound 6-10.
[0029] Figure 5 The semi-logarithmic plot of rat blood concentration-time curves for polypeptide compound 6 is shown.
[0030] Figure 6 The semi-logarithmic plot of rat blood concentration-time curves for polypeptide compound 7 is shown.
[0031] Figure 7 The effect of administration of peptide compounds 1-10 for 5 weeks on the nasal-anal length in mice was shown.
[0032] Figure 8 The effect of administration of peptide compounds 1-10 for 5 weeks on tail length in mice was shown. Detailed Implementation
[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described in detail below through specific embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation schemes obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0034] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such further embodiments obtained through combinations. (I) Definitions and Explanations
[0035] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. It should be understood that this invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this invention are all widely used terms and routine procedures in their respective fields. It should also be understood that the terminology used in this invention is for describing specific embodiments only and is not intended to be limiting.
[0036] In this invention, the terms "comprising," "including," "having," and "containing," and any variations thereof, are intended to cover non-exclusive inclusion. The terms are intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but do not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof.
[0037] In this invention, the term "γGlu" or "γ-Glu" refers to the γ-glutamyl group, a chemical group derived from glutamic acid, characterized by the γ-carboxyl group of glutamic acid participating in the formation of peptide bonds or other chemical bonds.
[0038] In this invention, the term "PEG2" refers to 2-(2-(2-aminoethoxy)ethoxy)acetic acid, CAS No. 134978-97-5.
[0039] The polypeptide compound provided by the present invention has two cysteine residues (Cys), and the thiol groups (-SH) of the two Cys form disulfide bonds (-SS-) under oxidative conditions, thereby forming a cyclic structure (disulfide bond cyclication) between the two Cys in the polypeptide.
[0040] In one specific embodiment, the polypeptide compound is selected from any one or more of the following polypeptide compounds 1-10: The polypeptide compound 1 has the amino acid sequence shown in SEQ ID NO: 1, Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein a disulfide bond is formed between the two Cys; it has the structure shown in formula (a); Equation (a) The polypeptide compound 2 has the amino acid sequence shown in SEQ ID NO: 2, Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gly-Ser-Gly-Leu-Gly-Cys, wherein a disulfide bond is formed between the two Cys; it has the structure shown in formula (b); Formula (b) The polypeptide compound 3 has the amino acid sequence shown in SEQ ID NO: 3: Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, wherein a disulfide bond is formed between the two Cys; it has the structure shown in formula (c). Formula (c) The structure of polypeptide compound 4 is R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, where R1 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2). 18 CO2H; also expressed as CO2H(CH2) 18 CO-γ-Glu-Gly-Ser-Gly-Ser-Gly-Ser-Gly-Gly-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys; and a disulfide bond is formed between the two Cys; it has the structure shown in formula (d); Formula (d) The structure of the polypeptide compound 5 is R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, where R1 is -PEG2-PEG2-γ-Glu-CO(CH2). 18 CO2H; also expressed as CO2H(CH2) 18 CO-γ-Glu-PEG2-PEG2-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys; and a disulfide bond is formed between the two Cys; it has the structure shown in formula (e); Equation (e) The structure of polypeptide compound 6 is Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R3, where R3 is -Lys(-PEG2-PEG2-γ-Glu-CO(CH2)). 18 CO2H); also expressed as Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H); and a disulfide bond is formed between the two Cys; it has the structure shown in formula (f); Equation (f) The structure of the polypeptide compound 7 is R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, where R1 is -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2). 18 CO2H; also expressed as CO2H(CH2) 18 CO-γ-Glu-PEG2-PEG2-Tyr-Lys-Gly-Ala-Asn-Lys-Lys-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein a disulfide bond is formed between two Cys; it has the structure shown in formula (g); Formula (g) The structure of the polypeptide compound 8 is Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, where R2 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2). 18CO2H; also expressed as Gly-Leu-Ser-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, wherein a disulfide bond is formed between two Cys; it has the structure shown in formula (h); Formula (h) The structure of the polypeptide compound 9 is Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, where R2 is -PEG2-PEG2-γ-Glu-CO(CH2). 18 CO2H; also expressed as Gly-Leu-Ser-Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys; and a disulfide bond is formed between the two Cys; it has the structure shown in formula (i); Formula (i) The structure of the polypeptide compound 10 is R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, where R1 is -PEG2-PEG2-γ-Glu-CO(CH2). 18 CO2H; also expressed as CO2H(CH2) 18 CO-γ-Glu-PEG2-PEG2-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys; and a disulfide bond is formed between the two Cys; it has the structure shown in formula (j).
[0041] Equation (j) (II) Detailed Technical Solution
[0042] For the purpose of clarity and concise description, features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of the invention may include some embodiments having a combination of all or some of the features described.
[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.
[0044] The resin used in this invention was purchased from Xi'an Lanxiao, all protected amino acids were purchased from Chengdu Zhengyuan Company, and the coupling agent was purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were of analytical grade, and the solvent was purchased from Shanghai Titan Company. The centrifuge was purchased from Lu Xiangyi. A 5.0 μm reverse-phase C2000 micrometer was used. 18 A preparative column (46 mm × 250 mm) was used to purify the peptides. The high-performance liquid chromatograph (HPLC) was a Thermo Fisher Scientific product. Mass spectrometry analysis was performed using a Waters mass spectrometer. Example 1: Preparation of polypeptide compound 3
[0045] The method for synthesizing polypeptide compounds of the present invention is illustrated using polypeptide compound 3 as an example.
[0046] The polypeptide compound 3 has the amino acid sequence described in SEQ ID NO: 3, wherein a disulfide bond is formed between the two Cys. The amino acid sequence shown in SEQ ID NO: 3 is Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys. Step 1: Synthesis of polypeptide resin:
[0047] The following peptide resins were synthesized manually using the Fmoc / t-Bu strategy at a scale of 0.5 mmol / L: Boc-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTCResin; (1) Weigh 1.00g of 2-chlorotriphenylmethyl chloride resin (2-Cl CTC Resin, loading 0.50mmol / g, Xi'an Lanxiao), add it to the reaction column, add 15mL of N,N-dimethylformamide (DMF) to swell for 30min, weigh Fmoc-Cys(Trt)-OH: 1.76g (3eq), N,N-diisopropylethylamine (DIEA): 0.52g (4eq), dissolve in 20mL of DMF and set aside, remove the DMF from the reaction column, wash the resin thoroughly with DMF twice, add the weighed materials to the reaction column, add an appropriate amount of DMF, stir evenly with nitrogen, react for 3h, the reaction is finished, remove the reaction liquid, wash with DMF 3 times, add methanol / DIEA (1:2, v / v) to block for 0.5h, remove the blocking liquid, wash with DMF 3 times, to obtain Fmoc-Cys(Trt)-CTC Resin; (2) Using Fmoc-Cys(Trt)-CTC Resin as the carrier, 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbodiimide (DIC) as coupling agents, and DMF as the solvent, the Fmoc group was removed by a 20% Piperidine / DMF solution (twice, 5 min + 7 min). The coupling effect was monitored with ninhydrin hydrate during the coupling process. If the test was negative, the reaction was complete; if it was positive, it needed to be repeated once (when repeating, the amount of Fmoc-protected amino acid, HOBt, and DIC should be doubled, and the reaction time should be 1 h; if it still does not improve, the reaction should be repeated). If positive, acetic anhydride / pyridine (7:6, v / v) is added for blocking for 1 hour. Then, the mixture is manually fed, and condensation reactions are performed sequentially from the C-terminus to the N-terminus to connect Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Asp(O The amino acids are: tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, and Boc-Gly-OH; the above amino acid feed amounts to a synthesis scale of 3 eq, with a reaction time of 2... In each condensation reaction, the amounts of Fmoc protecting amino acids, HOBt, and DIC were three times the synthetic scale, yielding Boc-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC Resin. After all coupling was completed, the resin was washed three times with DMF, three times with dichloromethane (DCM), and three times with methanol (MeOH). The resin was then dried under vacuum to obtain 3.4 g of dried polypeptide resin. Step 2: Removal of polypeptide full protection (deprotection group):
[0048] Add the following to the dried peptide resin at a rate of 10 mL lysis buffer / g peptide resin: Boc-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC The resin was lysed with a lysis buffer (prepared by mixing TFA, DODT, m-cresol, and H2O in a volume ratio of 92.5:2.5:2.5:2.5), and the mixture was heated to room temperature (25°C) for 2 hours. The lysate was collected by filtration, and the resin was washed three times with a small amount of the lysis buffer. The filtrates were combined and concentrated under reduced pressure to 1 / 4 of the original volume. The solution was then poured into 0°C methyl tert-butyl ether at a stirring speed of 5 mL / min under 60 rpm. The mixture was allowed to stand for 3 hours until precipitation was complete. The supernatant was removed, and the precipitate was washed three times with methyl tert-butyl ether. The precipitate was centrifuged at 8000 rpm for 15 minutes and dried under vacuum to obtain Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, with a crude product weight of 1.45 g. Step 3: Purification of polypeptide compound:
[0049] The polypeptide compound obtained in step 2 was dissolved in an acetonitrile (ACN):H2O solution of 1:2 (v / v), and passed through a 5.0 μm reverse-phase C2O filter. 18 Preparative HPLC purification was performed on a packed 46 mm × 250 mm column. Starting with 45% ACN / H₂O (containing 1% trifluoroacetic acid), the ACN ratio was increased in a gradient of 0.33% / min at a flow rate of 10 mL / min. The column was eluted for 60 min, and the fraction containing the peptide was collected, yielding a sample with HPLC purity greater than 90%. Acetic acid / iodine solution was added dropwise to this fraction to initiate a cyclization reaction. The reaction was monitored in HPLC, and cyclization was completed in 0.5 h. The cyclization reaction solution was quenched with citric acid and purified again by HPLC. Starting with a 31% ACN / 20 mM NaH₂PO₄ aqueous solution adjusted to pH 6.5 with 1 mol / L NaOH solution, the column was eluted in a gradient of 0.33% / min at a flow rate of 10 mL / min for 60 min. The fraction containing the peptide was collected, freeze-dried, and 220 mg of peptide was obtained with a purity greater than 98.56%, and the overall yield was 16.4%. Step 4: Product confirmation:
[0050] The isolated product polypeptide was identified using liquid chromatography-mass spectrometry (LC-MS) and confirmed to be the target polypeptide compound 3, [M+H]. +The calculated value is 2197.6, [M+2H] + The measured value is 1099.72. Example 2: Preparation of polypeptide compound 9
[0051] Taking polypeptide compound 9 as an example, the step of grafting side chains onto polypeptide resin is explained.
[0052] The structure of peptide compound 9 is: Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, where R2 is -PEG2-PEG2-γ-Glu-CO(CH2). 18 CO2H, in which disulfide bonds are formed between two Cys.
[0053] The specific steps are as follows: Boc-Gly-Leu-Ser(tBu)-Lys-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC Resin was obtained according to the method in Example 1. The resin was thoroughly washed three times each with N,N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N,N-dimethylformamide (DMF). Adding FmocNH-PEG2-OH (CAS#: 872679-70-4, Quanta BioDesign) is equivalent to a 5-fold increase in synthetic scale. Using 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) as coupling agents at 1.2 and 1.3 times the amount of amino acid feed, respectively, after shaking for 2 hours, the mixture was filtered to obtain Boc-Gly-Leu-Ser(tBu)-Lys(Fmoc-PEG2)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC Resin was then used to thoroughly wash the resin three times each with N,N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N,N-dimethylformamide (DMF) in sequence. The Fmoc group was removed using a 20% piperidine / N,N-dimethylformamide (DMF) solution (twice, 5 min + 7 min). Adding FmocNH-PEG2-OH was equivalent to a 5-fold increase in synthetic scale. Using 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) as coupling agents at 1.2 and 1.3 times the amount of amino acid feed, respectively, the mixture was shaken for 2 hours and then filtered to obtain Boc-Gly-Leu-Ser(tBu)-Lys(Fmoc-PEG2- The resin was then thoroughly washed three times each with N,N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N,N-dimethylformamide (DMF) in sequence. The Fmoc group was removed using a 20% piperidine / N,N-dimethylformamide (DMF) solution (twice, 5 min + 7 min). Adding Fmoc-Glu-OtBu equates to a 5-fold increase in synthetic scale. Using 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) as coupling agents at 1.2 and 1.3 times the amount of amino acid feed, respectively, the mixture was shaken for 2 hours and then filtered to obtain Boc-Gly-Leu-Ser(tBu)-Lys(Fmoc-PEG2-PEG2-γ-Glu)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC Resin was then used to thoroughly wash the resin three times each with N,N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N,N-dimethylformamide (DMF) in sequence. The Fmoc group was removed by a 20% piperidine / N,N-dimethylformamide (DMF) solution (twice, 5 min + 7 min). Adding tert-butyl eicosanoate (TBE) was equivalent to a 5-fold increase in synthetic scale. Using 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) as coupling agents at 1.2 and 1.3 times the amount of amino acid feed, respectively, the mixture was shaken for 2 hours and then filtered to obtain Boc-Gly-Leu-Ser(tBu)-Lys(-PEG2-PEG2-γ-Glu-CO(CH2)). 18CO2H)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-CTC Resin, followed by thorough washing of the resin three times each with N,N-dimethylformamide (DMF), dichloromethane (DCM), and dichloromethane (DCM), and then vacuum drying.
[0054] The peptide was then deprotected, purified, and the product was identified as the target peptide compound 9, following the method described in Example 1.
[0055] Based on the synthesis steps described in Examples 1-2, the following polypeptide compounds 1, 2, 4-8, and 10 were synthesized. The chemical structural formulas of polypeptide compounds 1-10 are shown below. Figure 1 As shown.
[0056] <Polypeptide compound 1>: Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein a disulfide bond is formed between the two Cys. <Polypeptide compound 2>: Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gly-Ser-Gly-Leu-Gly-Cys, where a disulfide bond is formed between the two Cys. <Polypeptide compound 4>: R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys. <Peptide Compound 5>: R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, where R1 is -PEG2-PEG2-γ-Glu-CO(CH2). 18 CO2H, in which disulfide bonds are formed between the two Cys. <Peptide Compound 6>: Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R3, where R3 is -Lys(-PEG2-PEG2-γ-Glu-CO(CH2)). 18 CO2H), in which disulfide bonds are formed between the two Cys. <Peptide Compound 7>: R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, where R1 is -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys. <Polypeptide compound 8>: Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R2 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys. <Polypeptide compound 10>: R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R1 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys. Once the prepared product is identified as the target polypeptide compound, it will be used in subsequent experiments. Example 3: Effects of polypeptide compounds 1-10 on the proliferation of mouse embryonic fibroblasts NIH3T3
[0057] The CCK8 assay was used to investigate the effects of candidate peptide compounds on the proliferation of mouse embryonic fibroblasts NIH3T3, and to examine whether the peptide compounds were toxic to NIH3T3 cells. The mouse embryonic fibroblasts NIH3T3 were obtained from the American Type Culture Collection Center (ATCC).
[0058] The NIH3T3 cell line was cultured in DMEM basal medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL. Cells were cultured at 37°C in a humidified environment with 5% CO2, exhibiting normal morphology and good growth. The NIH3T3 cell suspension was seeded into 96-well plates (5000 cells / well) and incubated for 24 hours. After cell attachment, the old medium was discarded, and fresh medium was added as a control group or peptide compound 1-10 (10 mM), and cultured for another 48 hours. 10 μL of CCK8 solution (Beyotime) was added to each well, and the 96-well plates were incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader.
[0059] Experimental results are as follows Figure 2 As shown, the results indicate that peptide compounds 1-10 did not affect the proliferation of NIH3T3 cells and no toxic effects were observed, indicating that the ultra-long-acting peptides for NPR-B targeting in this invention have no NIH3T3 cell toxicity. Example 4: Peptide compound 1-10 activates NPR-B in NIH3T3 cells and promotes cGMP production.
[0060] In this embodiment, mouse embryonic fibroblasts NIH3T3 were selected as the experimental subject. The agonistic activity of candidate peptide compounds on NPR-B was evaluated by detecting the production of cyclic guanosine monophosphate (cGMP).
[0061] 1) Cell Culture: Mouse embryonic fibroblasts NIH3T3 were obtained from the American Type Culture Collection Center (ATCC). The NIH3T3 cell line was cultured in DMEM basal medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin 100 U / mL + streptomycin 100 g / mL. The cells were cultured at 37°C in a humid environment with 5% CO2. The cells showed normal morphology and good growth.
[0062] 2) cGMP concentration detection: NIH3T3 cells were digested and centrifuged (800 rpm, 3 min), washed once with DMEM medium, centrifuged again (800 rpm, 3 min), the supernatant was discarded, and the cells were resuspended in 1 mL of DMEM medium containing 0.5 mM phosphodiesterase inhibitor (IBMX). Cell counting was performed using a cell counting chamber, and the cell density was subsequently diluted to 5 million cells / mL. Different concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM) of peptide compounds 1-10 and CNP22 were incubated with the cells for 30 min (37℃). Finally, the cGMP concentration was detected using cGMP HTRF kits (CISBIO). The detection results are as follows: Figure 3and Figure 4 As shown.
[0063] Figure 3 The graphs showing the cGMP levels produced by NIH3T3 cells stimulated by peptide compounds 1-5 are presented. Figure 4 The graph shows the cGMP content produced by NIH3T3 cells stimulated by peptide compound 6-10. Figure 3 and Figure 4 The results showed that peptide compounds 1-10 of the present invention could activate NIH3T3 cells and promote the production of cGMP, thereby activating cGMP signaling in NIH3T3 cells, and the agonistic activity of peptide compounds 1-10 was close to that of CNP22. Example 5: Pharmacokinetic study of peptide compounds 6 and 7 in rats
[0064] This study used SD rats as experimental subjects to evaluate the pharmacokinetic properties of peptide compounds 6 and 7. SD rats weighing approximately 180g were administered peptide compounds 6 or 7 subcutaneously at a dose of 0.5 mg / kg, with the injection solution prepared in PBS buffer at a volume of 200 μL / rat. Blood samples of 0.3 mL were collected from the tail vein before administration and at 1 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after administration. The plasma was separated by centrifugation in heparinized centrifuge tubes. 100 μL of the supernatant was transferred to a new centrifuge tube. Then, 100 μL of acetonitrile solution containing 0.1% TFA was added to precipitate impurities. The acetonitrile-treated plasma sample was vortexed for 30 s and centrifuged at 14000 rpm for 20 min. Finally, aspirate 30 μL of the supernatant and filter it through a 0.22 μm sterile filter, taking care to avoid aspirating the precipitate at the bottom. Then, perform HPLC to determine the drug concentration.
[0065] Figure 5 The plasma concentration-time curves of peptide compound 6 are shown. Figure 6 The plasma concentration-time curves for peptide compound 7 are presented. The results show that the half-lives of peptide compounds 6 and 7 are 14.20 h and 11.52 h, respectively. These results indicate that the half-lives of peptide compounds 6 and 7 are significantly extended by more than 300 times compared to CNP22. Example 6: Pharmacodynamic evaluation of peptide compounds 1-10 in mice
[0066] This study used 3-week-old wild-type C57BL / 6J male mice as research subjects. The mice were randomly divided into 11 groups of 6 mice each. Each group received a subcutaneous injection of either polypeptide compound 1-10 or PBS buffer at a dose of 70 nmol / kg, with an injection volume of 200 μL per mouse, administered every 3 days for 35 days (5 weeks). Mice were weighed every other day, and the nose-to-anus length (distance from the tip of the nose to the anus) and tail length (distance from the anus to the tip of the tail) were measured weekly. On the last day of the experiment, mice were anesthetized with sodium pentobarbital, and their nose-to-anus length and tail length were measured and photographed. The mice were then euthanized, and the tibia, femur, humerus, and ulna on both sides were harvested and their lengths measured using calipers.
[0067] Figure 7 The effects of peptide compounds 1-10 administered for 5 weeks on the nose-anal length in mice were demonstrated. Figure 8 The effects of peptide compounds 1-10 administered for 5 weeks on tail length in mice were demonstrated. * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. By Figure 7 and Figure 8 It was found that after 5 weeks of administration, peptide compounds 1-10 all promoted the growth of nose-anal length and tail length in mice to varying degrees. This indicates that the ultra-long-acting peptides for NPR-B targeting of this invention can significantly prolong the body and tail length of mice, improve cartilage development, and can be applied to the preparation of drugs for treating skeletal system diseases.
Claims
1. A polypeptide compound for NPR-B targeting, characterized in that, The polypeptide compound has the following structure: R1-Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-X1-Ser-Gly-Leu-Gly-Cys-R3, wherein a disulfide bond is formed between the two Cys. R1 is selected from -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, or is absent; R2 is selected from -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H,-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, or may not be present; R3 is selected from -Lys(PEG2-PEG2-γ-Glu-CO(CH2)). 18 CO2H), or not present; X1 is selected from Met, Leu, or Gly; R1, R2, and R3 do not exist simultaneously.
2. The polypeptide compound according to claim 1, wherein the polypeptide compound is selected from any one or more of the following (1)-(10): (1) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 1), wherein, Disulfide bonds are formed between the two Cys; (2) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gly-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 2), wherein a disulfide bond is formed between the two Cys; (3) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 3), wherein a disulfide bond is formed between the two Cys; (4) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (5) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (6) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R3, R3 is -Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H), in which disulfide bonds are formed between the two Cys; (7) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (8) Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R2 is -Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (9) Gly-Leu-Ser-Lys(R2)-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R2 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; (10) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys, R1 is -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between the two Cys; More preferably, the polypeptide compound is selected from (1) or (2): (1) Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R3, R3 is -Lys(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H), in which disulfide bonds are formed between the two Cys; (2) R1-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, R1 is -Lys-Lys-Asn-Ala-Gly-Lys-Tyr-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, in which disulfide bonds are formed between two Cys.
3. The use of the polypeptide compound of claim 1 or 2 in the preparation of a medicament for treating skeletal system disorders.
4. In the application according to claim 3, the skeletal system disease is at least one of achondroplasia, chondrodysplasia, osteochondrosis, osteoarthritis, articular cartilage damage, dwarfism, and short stature.
5. In the application according to claim 3 or 4, the medicament further comprises a pharmaceutically acceptable carrier and / or excipients.
6. The application according to any one of claims 3-5, wherein the drug is suitable for oral administration, transdermal administration, intravenous administration, intramuscular administration, local administration, and nasal administration.
7. The application according to any one of claims 3-6, wherein the dosage form of the drug includes tablets, capsules, soft capsules, drop pills, granules, pills, oral liquids, ointments and patches for skin application, aerosols, nasal sprays, injections, and infusions.