Super long-acting polypeptide for NPR-B targeting and application thereof in skeletal system diseases

Abstract

The invention relates to the technical field of biological medicines, in particular to an NPR-B targeted ultra-long-acting polypeptide and application thereof in skeletal system diseases. The ultra-long-acting polypeptide has a parent peptide represented by the following amino acid sequence: R1-Gly-Leu-Ser-Lys (R2)-Gly-[Cys-Ph-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-X1-Ser-Gly-Leu-Gly-Cys]-R3 (disulfide bond ring formation), and the half-life period of the prepared ultra-long-acting polypeptide in a rat body can be greatly prolonged; the C-type natriuretic peptide overcomes the defect that the endogenous NPR-B agonist C-type natriuretic peptide cannot be prepared into a medicine due to too short half-life period, has the characteristics of better biological activity, safety and stability, high synthesis yield, low cost and the like, and can be applied to preparation of medicines for treating skeletal system diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an ultra-long-acting polypeptide for NPR-B targeting and application thereof in skeletal system diseases. Background Art

[0002] The natriuretic peptide family is a group of peptides with similar structures but different origins, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). CNP consists of 22 amino acids and was originally isolated from pig brain. It was later found that the peptide was originally produced in humans by the natriuretic peptide precursor C (Nppc) gene in the form of a single-chain prepropolypeptide containing 126 amino acids. The source CNP is obtained after removing the signal peptide, and further cleavage by the endoprotease furin produces a 53-amino acid active peptide (CNP-53), which is secreted and cleaved to produce a 22-amino acid mature peptide (CNP-22). Both CNP-53 and CNP-22 can bind to the natriuretic peptide receptor B (NPR-B) and induce the production of cyclic monoguanosine phosphate (cGMP) in a dose-dependent manner. CNP-22 is mainly present in body fluids and is considered to be the mature and more biologically active form. CNP is a regulator that acts on the NPR-B receptor of chondrocytes to promote bone growth. 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 can promote the proliferation and differentiation of chondrocytes in the proliferation zone and hypertrophic zone of the growth plate, resulting in bone growth. Therefore, increasing or decreasing the content of any component in this pathway may cause abnormal bone growth. In humans, activating mutations of FGFR-3 are the main cause of hereditary dwarfism. Mice with activated FGFR-3 serve as a model of achondroplasia (the most common form of skeletal dysplasia), and overexpression of CNP rescues these animals from being short. Therefore, CNP and functional variants of CNP are potential therapeutic agents for treating various skeletal dysplasias. However, the plasma half-life of CNP in humans is only 2.6 min, and continuous administration is necessary to make the CNP concentration in plasma higher than its intrinsic concentration (about 5 pM). Vosoglitide, a drug approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of achondroplasia, is a 39-amino acid C-type natriuretic peptide analog that extends the half-life to 20 minutes. However, like most polypeptide compounds, vosoglitide can be taken orally and is easily degraded in the body, with a short half-life. A half-life of only 20 minutes cannot meet clinical needs, and subcutaneous injections are still required once a day until the epiphysis of children is closed. In addition, vosoglitide is expensive, which brings a great mental and financial burden to patients and their guardians. Therefore, how to develop a polypeptide compound that has a longer half-life in the body and still retains similar or improved activity and targets NPR-B is a technical problem that needs to be solved urgently. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention proposes an ultra-long-acting polypeptide for targeting NPR-B and its application in skeletal system diseases. The present invention prepares an ultra-long-acting polypeptide targeting NPR-B, which greatly prolongs its half-life in the body while retaining the activity of the polypeptide to activate NPR-B, thereby extending the dosing interval and reducing the cost of medication. The ultra-long-acting polypeptide can be used to prepare drugs for treating skeletal system diseases.

[0004] The present invention is achieved through the following technical solutions:

[0005] An ultra-long-acting peptide for NPR-B targeting, having a parent peptide represented by the following amino acid sequence:

[0006] R1-Gly-Leu-Ser-Lys(R2)-Gly-Cys 8 -Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-X1-Ser-Gly-Leu-Gly-Cys 24 -R3.

[0007] Further, the R1=-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 absent;

[0008] R2=-Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H,-PEG2-PEG2-γ-Glu-

[0009] CO(CH2) 18 CO2H, or absent;

[0010] R3=(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H)Lys, or absent;

[0011] X1 = Met, Leu or Gly.

[0012] Furthermore, a disulfide bond is formed between the cysteine ​​at position 8 and the cysteine ​​at position 24 in the amino acid sequence.

[0013] The present invention also provides a method for preparing the ultra-long-acting polypeptide for NPR-B targeting, which is manually synthesized according to the Fmoc / t-Bu strategy, comprising the following steps: sequentially connecting amino acids using the Fmoc / t-Bu solid phase synthesis strategy to obtain a polypeptide resin with amino acid protecting groups; removing the protecting groups; and refining and purifying.

[0014] Furthermore, the amino acid connection sequence of the polypeptide resin is: Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-X1-Ser-Gly-Leu-Gly-Cys, X1=Met, Leu or Gly.

[0015] Furthermore, the method for preparing the ultra-long-acting polypeptide may further include a step of grafting side chains onto the polypeptide resin.

[0016] Furthermore, the side chain is R1, R2 or R3.

[0017] Furthermore, the position of the grafted side chain is the first amino acid glycine, the fourth amino acid lysine or the 22nd amino acid cysteine ​​in the amino acid connection sequence of the polypeptide resin.

[0018] Furthermore, the resin is 2-chlorotrityl chloride (2-Cl CTC Resin) resin.

[0019] Furthermore, the method of sequentially connecting amino acids using the Fmoc / t-Bu solid phase synthesis strategy also includes assembling an Fmoc-Cys(Trt)-CTC Resin carrier.

[0020] Furthermore, the preparation of the polypeptide resin further comprises the steps of coupling the amino acid with 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) as coupling agents, using N,N-dimethylformamide (DMF) as solvent, and removing the Fmoc protecting group with 20% piperidine / N,N-dimethylformamide (DMF) solution.

[0021] Furthermore, the amino acid protecting group is one or more of pbf, Boc, Fmoc, tBu, OtBu and Trt.

[0022] Furthermore, the deprotection group comprises a step of using a cleavage solution to carry out the deprotection group step.

[0023] Furthermore, the composition of the lysate includes trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (DODT), m-cresol, and H2O in a volume ratio of 92.5:2.5:2.5:2.5.

[0024] The present invention also provides the use of the ultra-long-acting polypeptide for NPR-B targeting in the preparation of a drug for treating skeletal system diseases.

[0025] Furthermore, the skeletal system disorder is at least one of achondroplasia, hypochondroplasia, osteochondrosis, osteoarthritis, articular cartilage injury (such as ankle cartilage injury), dwarfism, and short stature, preferably achondroplasia.

[0026] Furthermore, the drug includes the above-mentioned ultra-long-acting polypeptide.

[0027] Furthermore, the drug may also include pharmaceutically acceptable carriers and / or excipients.

[0028] Furthermore, the drug is suitable for administration including but not limited to oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, and nasal administration.

[0029] Furthermore, the drug can be prepared into a suitable dosage form according to the adopted administration method.

[0030] Furthermore, the dosage form of the drug includes but is not limited to tablets, capsules, soft capsules, pellets, granules, pills, oral liquids, ointments and patches for skin surfaces, aerosols, nasal sprays, injections, and infusions.

[0031] Furthermore, the drug can be prepared into a solution or lyophilized powder for parenteral administration. Before use, an appropriate solvent or other pharmaceutically acceptable carrier can be added to reconstitute the powder. The liquid formula is generally a buffer solution or an isotonic solution.

[0032] Furthermore, the dosage of the drug is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition, and may depend on the specific disease being treated and other factors, including age, weight, severity of symptoms, route of administration, dosage form, frequency of treatment, and whether other drugs are taken during treatment.

[0033] In a specific embodiment of the present invention, it relates to the following ultra-long-acting polypeptide for NPR-B targeting, having the following amino acid sequence:

[0034] <Compound 1, SEQ ID NO: 1>:

[0035] Gly-Leu-Ser-Lys-Gly-[Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys] (disulfide bond ring);

[0036] <Compound 2, SEQ ID NO:2>:

[0037] Gly-Leu-Ser-Lys-Gly-[Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Gly-Ser-Gly-Leu-Gly-Cys] (disulfide bond cyclization);

[0038] <Compound 3, SEQ ID NO:3>:

[0039] Gly-Leu-Ser-Lys-Gly-[Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys] (disulfide bond cyclization);

[0040] <Compound 4, SEQ ID NO:4>:

[0041] 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] (disulfide bond cyclization);

[0042] <Compound 5, SEQ ID NO:5>:

[0043] CO2HCO(CH2) 18 -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] (disulfide bond cyclization);

[0044] <Compound 6, SEQ ID NO:6>:

[0045] Gly-Leu-Ser-Lys-Gly-[Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys]-(PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H)Lys (disulfide bond cyclization);

[0046] <Compound 7, SEQ ID NO: 7>:

[0047] CO2H CO(CH2) 18 -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] (disulfide bond cyclization);

[0048] <Compound 8, SEQ ID NO: 8>:

[0049] 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] (disulfide bond ring);

[0050] <Compound 9, SEQ ID NO: 9>:

[0051] 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] (disulfide bond ring);

[0052] <Compound 10, SEQ ID NO: 10>:

[0053] CO2HCO(CH2) 18 -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] (disulfide bond cyclization).

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] (1) The ultra-long-acting polypeptide for targeting NPR-B of the present invention can activate cGMP signals in NIH3T3 cells in vitro and significantly prolong the body length and tail length of mice in vivo, thereby improving cartilage development in mice and achieving an improvement effect on skeletal system diseases;

[0056] (2) The ultra-long-acting polypeptide for targeting NPR-B of the present invention has good biological activity and a greatly extended half-life. Taking CNP22 as an example, the half-life of the modified ultra-long-acting polypeptide compound is 300 times that of CNP22 and more than 30 times that of vosoritide, an existing drug for treating achondroplasia. The significant extension of the half-life can reduce the frequency of clinical medication, improve the compliance of clinical patients, and reduce the economic burden on patients;

[0057] (3) The ultra-long-acting polypeptide for NPR-B targeting of the present invention shows stability in the pharmacokinetic experiment of the drug, has good stability, is easy to scale up production, and has low cost;

[0058] (4) Compared with small molecule compounds, the ultra-long-acting polypeptide for NPR-B targeting of the present invention has lower toxicity, a larger safety window, and a smaller dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0060] Figure 1 is the chemical structural formula of compound 1-10 of the present invention;

[0061] Figure 2 The effects of compounds 1-10 of the present invention on the viability of NIH3T3 cells;

[0062] Figure 3 This is a curve diagram of cGMP content produced by NIH3T3 cells stimulated by compounds 1-5 of the present invention;

[0063] Figure 4 This is a curve diagram of cGMP content produced by NIH3T3 cells stimulated by compounds 6 to 10 of the present invention;

[0064] Figure 5 The semi-logarithmic graph of the blood concentration-time curve of compound 6 of the present invention in rats;

[0065] Figure 6 The semi-logarithmic graph of the blood concentration-time curve of compound 7 of the present invention in rats;

[0066] Figure 7 The effects of the compounds 1-10 of the present invention on the body length of mice after administration for 5 weeks;

[0067] Figure 8 The figure shows the effects of compounds 1-10 of the present invention on the tail length of mice after administration for 5 weeks. DETAILED DESCRIPTION

[0068] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0069] Example 1: Preparation of ultra-long-acting polypeptides for NPR-B targeting:

[0070] Materials: Resins were purchased from Xi'an Lanxiao, all protected amino acids were purchased from Chengdu Zhengyuan Company, and coupling agents were purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were analytically pure, and solvents were purchased from Shanghai Titan Company. Centrifuge was purchased from Lu Xiangyi. 5.0 μm reverse phase C 18 A preparative column (46 mm × 250 mm) was used to purify the peptide. The high performance liquid chromatograph was a product of Thermo Fisher Scientific. Mass spectrometry was performed using a Waters mass spectrometer.

[0071] The synthesis method of the polypeptide compound of this embodiment is described by taking polypeptide compound 3 as an example:

[0072] Step 1: Synthesize peptide resin:

[0073] According to the Fmoc / t-Bu strategy, the synthesis scale was 0.5 mmoL, and the following peptide resins were synthesized:

[0074] 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;

[0075] (1) Weigh 1.00 g of 2-chlorotrityl chloride resin (2-Cl CTC Resin, loading 0.50 mmol / g, Xi'an Lanxiao), add it to the reaction column, add 15 mL of N,N-dimethylformamide (DMF) to swell for 30 min, weigh 1.76 g (3 eq) of Fmoc-Cys(Trt)-OH, 0.52 g (4 eq) of N,N-diisopropylethylamine (DIEA), dissolve them in 20 mL of DMF for later use, remove the DMF in the reaction column, wash the resin twice with DMF, add the above-weighed materials to the reaction column, add an appropriate amount of DMF, stir evenly with nitrogen, react for 3 h, and after the reaction is completed, remove the reaction solution, wash three times with DMF, add methanol / DIEA (1:2, v / v) to block for 0.5 h, remove the blocking solution, and wash three times with DMF to obtain Fmoc-Cys(Trt)-CTC Resin;

[0076] (2) Using Fmoc-Cys(Trt)-CTC Resin as a carrier, 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbodiimide (DIC) as coupling agents, and DMF as a solvent, the Fmoc group was removed with a 20% piperidine / DMF solution (twice for 5 min + 7 min). During the coupling process, the coupling effect was monitored with ninhydrin. If the test result was negative, the reaction was complete. If it was positive, it was necessary to re-do the reaction (the amount of Fmoc-protected amino acid, HOBt, and DIC was doubled during the re-dosing, and the reaction time was 1 h. If it was still If it is positive, acetic anhydride / pyridine (7:6, v / v) is added to block for 1 h), and the material is manually added. The condensation reaction is carried out 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, Fmoc-Asp( OtBu)-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, Boc-Gly-OH; the above amino acid feed is equivalent to the synthesis scale of 3eq, and the reaction time is 2h, in each condensation reaction, the amount of Fmoc-protected amino acid, HOBt, and DIC added was equivalent to 3 times the synthetic scale, and 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 was obtained; after all the couplings were completed, the mixture was washed with DMF 3 times, with dichloromethane (DCM) 3 times, with methanol (MeOH) 3 times, and dried under vacuum to obtain 3.4g of dry polypeptide resin.

[0077] Step 2: Removal of full protection of peptide (deprotection group):

[0078] According to the amount of 10mL lysate / g peptide resin, add the dried 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 Lysis solution (lysate: TFA, DODT, m-cresol, H2O in a volume ratio of 92.5:2.5:2.5:2.5) was added to resin, the temperature was raised to room temperature (25°C), the cleavage reaction was carried out for 2 hours, the filtrate was collected by filtration, the resin was washed three times with a small amount of lysate, the filtrate was combined and concentrated under reduced pressure to 1 / 4 of the original volume, and poured into 0°C methyl tert-butyl ether at a rate of 5 mL / min under stirring at 60 rpm, and allowed to stand for 3 hours. After complete precipitation, the supernatant was removed, the precipitate was washed three times with methyl tert-butyl ether, centrifuged at 8000 rpm for 15 minutes, and the solid was vacuum dried 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 weight of 1.45 g.

[0079] Step 3: Refining and purifying peptide compounds:

[0080] The polypeptide compound obtained in step 2 was dissolved in a solution of acetonitrile (ACN):H2O=1:2 (v / v) and then filtered through a 5.0 μm reverse phase C 18 Preparative HPLC purification was performed on a 46 mm × 250 mm column filled with 45% ACN / H2O (containing 1% trifluoroacetic acid) as the starting point, with a gradient (increasing the proportion of ACN at a rate of 0.33% / min), a flow rate of 10 mL / min, and the column was eluted for 60 min, and the fractions containing the polypeptide were collected to obtain a sample with an HPLC purity greater than 90%. Acetic acid / iodine solution was added dropwise to this fraction for cyclization reaction. The reaction was monitored by the liquid phase. The cyclization was completed in 0.5 h. The cyclization reaction solution was quenched with citric acid and purified by HPLC again, starting with 31% ACN / 20mM NaH2PO4 aqueous solution adjusted to pH 6.5 with 1 mol / L NaOH solution, with a gradient (increasing the proportion of ACN at a rate of 0.33% / min), a flow rate of 10 mL / min, and the column was eluted for 60 min, and the fractions containing the polypeptide were collected and freeze-dried to obtain 220 mg of refined peptide with a purity greater than 98.56% and a total yield of 16.4%.

[0081] Step 4: Product confirmation:

[0082] The separated product peptide was identified by LC-MS and confirmed to be the target peptide compound 3 (SEQ ID: 3), [M+H] + Calculated value: 2197.6, [M+2H] + The actual measured value is 1099.72.

[0083] Example 2: Taking compound 9 as an example, the steps of grafting side chains onto the polypeptide resin in this example are described:

[0084] According to the method of Example 1, 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, and the resin was washed thoroughly with N,N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N,N-dimethylformamide (DMF) three times each;

[0085] FmocNH-PEG2-OH (CAS#: 872679-70-4, Quanta BioDesign) was added, which was equivalent to 5 times the synthesis scale. 1-Hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) were used as coupling agents in amounts of 1.2 and 1.3 times the amount of amino acid feed, respectively. After shaking for 2 hours, 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 was obtained by filtration. Resin, then wash the resin three times with N, N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N, N-dimethylformamide (DMF);

[0086] 20% piperidine / N,N-dimethylformamide (DMF) solution was used to remove the Fmoc group (twice for 5 min + 7 min), and FmocNH-PEG2-OH was added, which was equivalent to 5 times the synthesis scale. 1-Hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) were used as coupling agents, and the dosages were 1.2 times and 1.3 times of the amino acid feed, respectively. After shaking for 2 hours, Boc-Gly-Leu-Ser(tBu)-Lys(Fmoc-PEG2- 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)-CTCResin, and then the resin was washed thoroughly with N,N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N,N-dimethylformamide (DMF) three times each;

[0087] The Fmoc group was removed by 20% piperidine / N,N-dimethylformamide (DMF) solution (twice for 5 min + 7 min), and Fmoc-Glu-OtBu was added, which was equivalent to 5 times the synthesis scale. 1-Hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) were used as coupling agents, and the dosages were 1.2 times and 1.3 times of the amino acid feed amount, respectively. After shaking for 2 hours, 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 was obtained by filtration. Resin, then wash the resin three times with N, N-dimethylformamide (DMF), dichloromethane (DCM), dichloromethane (DCM), and N, N-dimethylformamide (DMF);

[0088] 20% piperidine / N,N-dimethylformamide (DMF) solution was used to remove the Fmoc group (twice for 5 min + 7 min), and mono-tert-butyl eicosanoate was added, which was equivalent to 5 times the synthesis scale. 1-Hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) were used as coupling agents, and the dosages were 1.2 times and 1.3 times of the amino acid feed, respectively. After shaking for 2 hours, Boc-Gly-Leu-Ser(tBu)-Lys(PEG2-PEG2-γ-Glu-CO(CH2) 18 The resin was then washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), and then dried in vacuo.

[0089] Then, the polypeptide was fully deprotected, purified and the product was confirmed according to the method of Example 1, and it was determined to be the target polypeptide compound 9 (SEQ ID: 9).

[0090] Based on the synthesis steps described in Example 1-2, the compound 1-10 of the present invention was synthesized. The chemical structure of the compound 1-10 is as follows Figure 1 shown.

[0091] Example 3: CCK8 experiment was used to investigate the effect of candidate compounds on the proliferation of mouse embryonic fibroblasts NIH3T3, and to investigate whether the polypeptide compounds were toxic to NIH3T3 cells. Mouse embryonic fibroblasts NIH3T3 were from the American Type Culture Collection (ATCC). The NIH3T3 cell line used DMEM basal medium (Gibco), and 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100U / mL + streptomycin 100g / mL) were added to the culture medium. The cells were cultured in a humidified environment at 37°C with 5% CO2, and the cell morphology was normal and the growth state was good. The NIH3T3 cell suspension was inoculated into a 96-well plate (5000 cells / well), and the 96-well plate was placed in an incubator and cultured for 24h. After the cells adhered to the wall, the old culture medium was discarded, and new culture medium was added as a control group or compound 1-10 (10mM), and the culture was continued for 48h. 10 μL of CCK8 solution (Biyuntian) was added to each well, and the 96-well plate was incubated in an incubator for 2 hours. The absorbance was measured at 450 nm using an ELISA reader. Figure 2 shown.

[0092] Figure 2The results showed that compounds 1-10 did not affect the proliferation of NIH3T3 cells and no toxic effects were observed, indicating that the ultra-long-acting polypeptide for NPR-B targeting of the present invention had no NIH3T3 cell toxicity.

[0093] Example 4: Candidate compounds activate NIH3T3 cell NPR-B and promote cGMP production. In this example, mouse embryonic fibroblasts NIH3T3 were selected as experimental subjects, and the agonist activity of candidate compounds on NPR-B was evaluated by detecting the production of cyclic guanosine monophosphate (cGMP).

[0094] 1) Cell culture: Mouse embryonic fibroblasts NIH3T3 were obtained from the American Type Culture Collection (ATCC). The NIH3T3 cell line used DMEM basal medium (Gibco), and 10% fetal bovine serum (Gibco) and 1% double antibody (penicillin 100U / mL + streptomycin 100g / mL) were added to the medium. The cells were cultured at 37°C in a humidified environment containing 5% CO2. The cell morphology was normal and the growth state was good.

[0095] 2) cGMP concentration detection: After digestion and centrifugation of NIH3T3 cells (800rpm, 3min), wash once with DMEM medium, discard the supernatant after centrifugation (800rpm, 3min), add 1mL of DMEM medium containing 0.5mM phosphodiesterase inhibitor (IBMX) and resuspend. Count the cells using a cell counting plate, and then dilute the cell density to 5 million cells / mL. Compounds 1-10 and CNP22 of different concentrations (1nM, 10nM, 100nM, 1μM, 10μM, 100μM) were incubated with the cells for 30min (37°C). Finally, cGMP HTRF kits (CISBIO) were used to detect the cGMP concentration. The test results are as follows: Figure 3 and Figure 4 shown.

[0096] Figure 3 The cGMP content curve of NIH3T3 cells stimulated by compounds 1-5 is shown. Figure 4 The graph shows the cGMP content produced by NIH3T3 cells stimulated by compounds 6-10. Figure 3 and Figure 4 The results show that compounds 1-10 of the present invention can activate NIH3T3 cells and promote the production of cGMP, activate cGMP signals in NIH3T3 cells, and the agonist activity of compounds 1-10 is close to that of CNP22.

[0097] Example 5: Pharmacokinetic study of compound 6 and compound 7 in rats:

[0098] In this example, SD rats were used as experimental subjects to evaluate the pharmacokinetic properties of compound 6 and compound 7. Compound 6 or compound 7 was administered subcutaneously to SD rats weighing about 180 g at a dose of 0.5 mg / kg. The injection solution was prepared with PBS buffer and the injection volume was 200 μL / rat. 0.3 mL of blood was collected from the tail vein before and after administration 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, respectively, and placed in a heparinized centrifuge tube and centrifuged to separate the plasma. 100 μL of the supernatant of the sample after centrifugation was drawn into a new centrifuge tube. Then 100 μL of acetonitrile solution containing 0.1% TFA was added to precipitate the impurities. The acetonitrile-treated plasma sample was vortexed for 30 s, 14000 rpm / min, and centrifuged for 20 min. Finally, 30 μL of the supernatant was aspirated and sterilized using a 0.22 μm sterilizing filter, taking care to avoid aspirating the precipitate at the bottom, and then the drug concentration was determined by HPLC.

[0099] Figure 5 The blood concentration-time curve of compound 6 is shown. Figure 6 The blood concentration-time curve of compound 7 was shown. The results showed that the half-lives of compound 6 and compound 7 were 14.20h and 11.52h, respectively. This result shows that compared with CNP22, the half-lives of compound 6 and compound 7 were significantly extended by more than 300 times.

[0100] Example 6: Pharmacodynamic evaluation of candidate compounds in mice:

[0101] In the present embodiment, 3-week-old wild C57BL / 6J male mice were used as research subjects. Mice were randomly divided into 11 groups, 6 in each group, and subcutaneously injected with compound 1-10 or PBS buffer, respectively, at a dosage of 70nmol / kg, and an injection volume of 200 μL / mouse, administered once every 3 days, and the experiment lasted for 35 days (5 weeks). The mice were weighed every other day, and the nose-anus length (the distance from the nose tip of the mouse to the anus) and tail length (the distance from the anus of the mouse to the tip of the tail) of the mice were measured weekly. On the last day of the experiment, the mice were anesthetized with sodium pentobarbital, and the nose-anus length and tail length of the mice were measured, and photos were taken, and then the mice were killed, and the tibia, femur, humerus, and ulna of the left and right sides of the mice were taken, and the length was measured with a caliper.

[0102] Figure 7 The effect of compound 1-10 administration for 5 weeks on the body length of mice was shown. Figure 8 The effect of compound 1-10 administration for 5 weeks on the tail length of mice is shown. Among them, *P<0.05, **P<0.01, ***P<0.001****P<0.0001. Figure 7 and Figure 8It can be seen that after 5 weeks of administration, compounds 1-10 can promote the growth of mouse nose-anus length and mouse tail length to varying degrees. This shows that the ultra-long-acting polypeptide for NPR-B targeting of the present invention can significantly prolong the body length and tail length of mice, improve the development of mouse cartilage, and can be used to prepare drugs for treating skeletal system diseases.

Claims

1. An ultra-long-acting polypeptide for NPR-B targeting, characterized in that: The parent peptide has the following amino acid sequence representation: R1-Gly-Leu-Ser-Lys(R2)-Gly-[Cys 8 -Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-X1-Ser-Gly-Leu-Gly-Cys 24 ]-R3, disulfide bond forming a ring; described R1=-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 does not exist; R2=-Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2) 18 CO2H, -PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H, or absent; R3=(-PEG2-PEG2-γ-Glu-CO(CH2) 18 CO2H)Lys, or absent; X1 = Met, Leu or Gly.

2. The method for preparing an ultra-long-acting polypeptide for NPR-B targeting according to claim 1, characterized in that: The manual synthesis according to the Fmoc / t-Bu strategy includes the following steps: using the Fmoc / t-Bu solid phase synthesis strategy to sequentially connect amino acids to obtain a polypeptide resin with amino acid protecting groups; removing the protecting groups; and refining and purification.

3. The method for preparing an ultra-long-acting polypeptide for NPR-B targeting according to claim 2, characterized in that: The resin is 2-chlorotrityl chloride 2-Cl CTC Resin.

4. The method for preparing an ultra-long-acting polypeptide for NPR-B targeting according to claim 3, characterized in that: The method of sequentially connecting amino acids using the Fmoc / t-Bu solid phase synthesis strategy also includes assembling an Fmoc-Cys(Trt)-CTC Resin carrier.

5. The method for preparing an ultra-long-acting polypeptide for NPR-B targeting according to claim 4, characterized in that: The deprotection group comprises a step of using a cleavage solution to carry out the deprotection group step; the composition of the cleavage solution comprises trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethyl mercaptan), m-cresol, and H2O, and the volume ratio is 92.5:2.5:2.5:2.

5.

6. The use of the ultra-long-acting polypeptide for targeting NPR-B according to claim 1 in the preparation of a drug for treating skeletal system diseases, characterized in that: The skeletal system disorder is at least one of achondroplasia, hypochondrodysplasia, osteochondrosis, osteoarthritis, articular cartilage injury, dwarfism, and short stature; the drug includes the ultra-long-acting polypeptide and may further include a pharmaceutically acceptable carrier and / or excipient.

7. The use of the ultra-long-acting polypeptide for NPR-B targeting according to claim 6, characterized in that: The drug is suitable for administration including but not limited to oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, and nasal administration.

8. The use of the ultra-long-acting polypeptide for NPR-B targeting according to claim 7, characterized in that: The drug can be prepared into dosage forms including but not limited to tablets, capsules, soft capsules, pellets, granules, pills, oral liquids, ointments and patches for skin surfaces, aerosols, nasal sprays, injections, and infusions according to the administration method adopted.

9. The use of the ultra-long-acting polypeptide for NPR-B targeting according to claim 8, characterized in that: The dosage of the drug is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition, and can be determined according to the specific disease being treated and other factors, including age, body weight, severity of symptoms, route of administration, dosage form of the drug, frequency of treatment, and whether other drugs are taken during treatment.

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