A pth long-acting polypeptide compound and preparation and application thereof

Abstract

This invention relates to the field of biomedicine, disclosing a long-acting pTH polypeptide compound and its preparation and application. The polypeptide has an octadecanoic acid or eicosanoic acid and its derivatives modified by at least one amino acid residue at the 13th, 26th, and 27th amino acid residues of the pTH (1-34) parent peptide sequence, and is covalently bound to the polypeptide backbone via an AEEA-AEEA-γ-Glu linker. This invention significantly enhances the binding ability of the polypeptide to serum albumin and prolongs its half-life by utilizing fatty acid side chain modification. The long-acting pTH polypeptide compound of this invention exhibits significant osteogenic activity, promoting the proliferation of MC3T3-E1 cells and generating osteogenic markers and calcium nodules under osteogenic induction conditions, with a 2-6 fold increase in binding ability to serum albumin. This invention also provides a solid-phase synthesis method for this polypeptide compound and a pharmaceutical composition containing it, which can be used to treat osteoporosis and hypoparathyroidism, effectively solving the compliance problem of existing drugs requiring daily injections.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a long-acting pTH polypeptide compound, its preparation, and its application in the treatment of osteoporosis and hypoparathyroidism. Background Technology

[0002] Osteoporosis (OP) is a systemic bone metabolic disease characterized by decreased bone mass and deterioration of bone microstructure. Developing long-acting therapeutic drugs is a key challenge in current clinical research. Teriparatide (pTH 1-34), currently the only approved bone formation promoter, requires daily subcutaneous injection due to its short half-life (1 hour), leading to poor patient compliance.

[0003] Hypoparathyroidism, characterized by low levels of parathyroid hormone (pTH), is a rare disorder that is either congenital or more commonly acquired after neck surgery. pTH is secreted by four parathyroid glands located next to the thyroid gland in the neck and controls calcium homeostasis, vitamin D-dependent calcium absorption, renal calcium reabsorption, and renal phosphate clearance. pTH stimulates the release of calcium from bones and enhances calcium absorption in the intestines. Decreased pTH levels lead to hypocalcemia and include symptoms such as neuromuscular hypersensitivity, including paresthesia, muscle twitching, laryngospasm, tetany, and seizures, and can be fatal if left untreated. The current standard of care is high-dose calcium and active vitamin D; however, many patients have reported an increased incidence of depression, as well as an increased risk of infections and kidney complications such as calcification and renal insufficiency, despite treatment causing fluctuations in calcium levels.

[0004] pTH is an 84-amino acid-long polypeptide containing a 34-amino acid-long N-terminal bioactive domain, and has been found to be an effective pTH receptor agonist. pTH (1-34) activates the intracellular cAMP / PKA and PLC / PKC dual signaling pathways by specifically binding to the extracellular domain of the pTH1R receptor, thereby regulating osteoblast differentiation and bone formation. Its binding mode and the division of labor of the receptor functional regions are as follows: (1) N-terminal core region (positions 1-14): mediates the conformational activation of the receptor. Ser¹ and Val² bind to the β-sheet region of the extracellular domain (ECD) of the pTH1R receptor through hydrogen bonds, triggering conformational rearrangement of the transmembrane domain (TMD) of the receptor. (2) Intermediate helical region (positions 15-26): stabilizes the receptor-ligand complex. The α-helix structure binds to the hydrophobic pocket of the ECD through hydrophobic interactions, enhancing the binding affinity. (3) C-terminal extension region (positions 27-34): determines the specific recognition of the receptor. Trp 23 Arg 28It forms charge complementarity with the TMD region of pTH1R, ensuring the specificity of signal transduction.

[0005] Currently, recombinant pTH is administered via daily subcutaneous injection. However, pTH is unstable in vitro and has a short half-life in vivo, leading to fluctuations in pTH levels and potentially causing nausea and vomiting. US7550434, US7144861, US6770623, WO2006 / 129995, and WO2013 / 108235 disclose compositions for stabilizing pTH in vitro, and WO2011143406 discloses recombinant pTH analogs with enhanced pharmacokinetics and pharmacodynamics, comprising a modified pTH fragment of up to 36 amino acids. However, there is a pressing need in the art for long-acting pTH biologics that improve control over serum calcium levels and reduce side effects to minimize the need for daily subcutaneous injections.

[0006] Recombinant proteins and peptides used in pharmaceuticals often suffer from increased serum clearance. The factors leading to the removal of administered proteins from circulation consist of two components: renal filtration and proteolysis. Typically, proteins with a molecular weight greater than 70 kDa cannot be cleared by glomerular filtration because they are too large to be filtered; however, smaller molecular weight proteins can be filtered by the glomerulus and are found in the urine. One method to increase the effective molecular weight of proteins and produce products with reduced immunogenicity is to encapsulate the proteins in polyethylene glycol (PEG). PEG is believed to slow renal clearance by providing an increased hydrodynamic volume in PEGylated proteins (Maxfield et al., Polymer, 16:505-509 (1975)). However, PEGylation of proteins leads to a decrease in affinity for their receptors, thereby reducing biological activity. An alternative approach to improving the PK and PD of protein biologics is disclosed in WO2009 / 013461. Compared to growth hormone, human growth hormone fused with the extracellular domain of the human growth hormone receptor (GHR) increased PK and PD, with PK increasing by approximately 200-fold. The effects of GHR on non-growth hormone peptides are disclosed in the currently unpublished PCT / GB2016 / 053218, where leptin and granulocyte colony-stimulating factor (GSCF) exhibit increased PK and PD upon fusion with GHR.

[0007] Fatty acid side chain modification enhances drug binding to serum albumin through hydrophobic interactions. Ruan S et al. designed a series of pTH (1-34) analogs containing fatty acids, deoxycholic acid, and biotin groups. Using solid-liquid phase synthesis technology, they replaced any two Lys residues in the peptide chain with Arg, with the remaining Lys residues serving as modification sites, synthesizing fatty acid side chain analogs with different linker units. In vitro bioactivity and albumin affinity assays were used to screen for analogs containing AEEA-γGlu-C. 18 AEEA-AEEA-Pro-C 18 Side-chain analogs were developed as candidate therapeutic agents. In vitro functional evaluation showed that the modified analogs retained the complete pTH1R receptor activation capacity, while significantly enhancing albumin-binding affinity. This study reveals the significant impact of fatty acid side chains and spacer arm structure on drug performance, providing important theoretical basis for the development of once-weekly pTH analogs.

[0008] Peptide drugs generally suffer from low oral bioavailability, therefore they are mostly administered in injectable form. Currently, commercially available parathyroid hormone fragment pTH(1-34) injections are primarily in acetate form, mainly administered subcutaneously. Their pharmacokinetics exhibit a typical "rapid absorption-rapid elimination" pattern: the drug diffuses into the bloodstream through the capillary endothelial space, reaching peak concentration in approximately 30 minutes; the half-life is approximately 1 hour (shortened to 5 minutes with intravenous injection). Although subcutaneous injection achieves a bioavailability as high as 95%, approximately 90% of the drug is rapidly cleared by glomerular filtration, resulting in a short duration of blood drug concentration, requiring patients to inject 20 μg daily. This high-frequency administration leads to significant medication adherence problems, and long-term injections may cause lipoatrophy at the injection site, limiting its clinical application to some extent. Therefore, prolonging the drug's half-life has become a primary clinical challenge. While existing technologies such as polyethylene glycol modification can prolong the half-life, they often result in a decrease in biological activity. Fatty acid side-chain modification, by enhancing binding to serum albumin and reducing glomerular filtration rate and enzymatic hydrolysis rate, is considered an ideal strategy for achieving long-acting fatty acids. However, how to achieve effective long-acting fatty acids while maintaining their biological activity remains a key technical bottleneck that urgently needs to be overcome. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a long-acting pTH polypeptide compound and its application in the treatment of osteoporosis and hypoparathyroidism.

[0010] This invention is based on pTH (1-34) crystal structure analysis. Lys at positions 13, 26, and 27 are located on the molecular surface, far from the acceptor binding interface. Modification at these sites has minimal impact on activity, and the ε-amino group of Lys readily forms an amide bond, providing a possibility for long-lasting modification. Fatty acid side chain C 18 Moderate hydrophobicity, balancing albumin binding and solubility, C 20 This results in stronger hydrophobicity, enhancing the interaction with albumin. Based on this, the present invention aims to improve the interaction between pTH(1-34) and Lys... 13 / 26 / 27 Introducing C 18 / C 20 The fatty acid side chain is designed for long-lasting effects.

[0011] The present invention includes: Compound Synthesis and Structural Identification: Solid-phase synthesis (SPPS) was employed, with protecting amino acids progressively coupled to a solid support from the C-terminus to the N-terminus based on the peptide sequence. After synthesis, the peptide resin was cleaved and deprotected, followed by separation and purification using reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain high-purity pTH(1-34) analogs. Structural Characterization: Mass spectrometry was used to characterize the structure, determining its molecular weight and peptide sequence. Primary mass spectrometry (MS) was used to determine the molecular weight of the analog to verify its consistency with theoretical values; secondary mass spectrometry (MS / MS) was used to analyze the amino acid sequence of the analog to ensure the accuracy of the synthesis.

[0012] In vitro activity assay: Using osteoblast cell lines, such as MC3T3-E1 cells, experiments such as CCK-8 assay, enzyme-linked immunosorbent assay, and alizarin red staining were conducted to detect the effects of the analog on osteoblast proliferation, differentiation, and mineralization, thereby determining the bioactivity of the analog.

[0013] Long-term efficacy assessment: Surface plasmon resonance (SPR) technology was used to dynamically monitor the binding kinetic parameters (KD value) of the analog to human serum albumin (HSA). By quantifying the interaction strength with the natural protein transport system, the effect of structural modification on prolonging the drug's half-life was verified.

[0014] The first aspect of this invention provides a pTH long-acting polypeptide compound, wherein the parent peptide sequence of the polypeptide is: H-Ser 1 -Val 2 -Ser 3 -Glu 4 -Ile 5 -Gln 6 -Leu 7 -Met 8 -His 9 -Asn 10 -Leu 11-Gly 12 -Lys 13 -His 14 -Leu 15 -Asn 16 -Ser 17 -Met 18 -Glu 19 -Arg 20 -Val 21 -Glu 22 -Trp 23 -Leu 24 -Arg 25 -Lys 26 -Lys 27 -Leu 28 -Gln 29 -Asp 30 -Val 31 -His 32 -Asn 33 -Phe 34 -OH, and an aliphatic chain modifying group is attached to at least one amino acid residue selected from the 13th, 26th, and 27th positions; the aliphatic chain modifying group is covalently linked to the polypeptide backbone via a linker unit.

[0015] Furthermore, the polypeptide compounds screened through simulation design in this invention have a parent peptide represented by one of the following amino acid sequences: pTH-1:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic acid)-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-2:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys(AEEA-AEEA-γ-Glu-Eicosanoic acid)-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-3:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-G lu-Trp-Leu-Arg-Lys(AEEA-AEEA-γ-Glu-Octadecanedioicacid)-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-4:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Va l-Glu-Trp-Leu-Arg-Lys(AEEA-AEEA-γ-Glu-Eicosanoicacid)-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-5: Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic acid)-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-6:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Va l-Glu-Trp-Leu-Arg-Lys-Lys(AEEA-AEEA-γ-Glu-Eicosanoic acid)-Leu-Gln-Asp-Val-His-Asn-Phe-OH.

[0016] Furthermore, the fatty chain is selected from octadecanoic acid, eicosanoic acid and their derivatives.

[0017] Furthermore, the connecting unit is AEEA-AEEA-γ-Glu.

[0018] The second aspect of this invention provides a method for preparing the aforementioned polypeptide compound, which employs a solid-phase synthesis method. Based on the polypeptide sequence, protecting amino acids are progressively coupled to a solid-phase support from the C-terminus to the N-terminus. After synthesis, a polypeptide resin is obtained. The polypeptide resin is then cleaved, deprotected, and purified to obtain the aforementioned polypeptide compound.

[0019] A third aspect of the present invention provides a pharmaceutical composition comprising any of the polypeptide compounds described above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0020] The fourth aspect of the present invention provides the use of any of the above-described polypeptide compounds or pharmaceutical compositions in the preparation of a medicament for treating osteoporosis, thereby solving the problem of long-term compliance among osteoporosis patients and the problem of prolonged dosing frequency.

[0021] The fifth aspect of the present invention provides the use of any of the above-described polypeptide compounds or pharmaceutical compositions in the preparation of a medicament for treating hypoparathyroidism, which can be used as an alternative treatment for hypoparathyroidism.

[0022] The present invention has the following advantages over the prior art: 1. This invention utilizes fatty acid side chain modification to modify the Lys groups at positions 13 / 26 / 27 of the pTH(1-34) polypeptide, designing a series of analog compounds pTH-1 to pTH-6, mainly involving C18 and C20 fatty acid side chain modifications. Finally, these analogs were screened for osteogenic bioactivity in vitro. MC3T3-E1 cells highly expressing the pTH1R receptor were selected, and the polypeptide compounds were applied to these cells. The proliferative effect of the compounds on cell proliferation was evaluated. Under osteogenic induction conditions, the levels of biomarkers Col-1, OPN, and OCN in the early, middle, and late stages were measured, and the formation of calcium nodules was detected in the late stage of induction. Experimental results showed that pTH-2 exhibited osteogenic activity similar to pTH(1-34).

[0023] 2. The polypeptide compound designed and synthesized in this invention has hydrophobic fatty acid side chains, which can enhance the binding ability of the polypeptide drug to serum albumin through hydrophobic interactions. This binding process is reversible, which to some extent increases steric hindrance, shields the action sites of hydrolytic enzymes, and reduces the cleavage efficiency of trypsin; it also reduces the glomerular filtration rate; and it reversibly binds to serum albumin to form a drug-protein complex with a molecular weight >60kDa, avoiding transmembrane transport and reducing the drug's renal excretion, hepatic metabolism, and hemodynamic effects, thus prolonging the drug's half-life while retaining its biological activity. The binding of the analog to serum albumin was dynamically monitored using surface plasmon resonance (SPR) technology. The experimental results show that compared with the prototype peptide, the affinity of the analog to serum albumin is improved. Among them, the least effective analog pTH-5 showed an affinity increase of about 2 times, while the most effective pTH-3 showed an affinity increase of more than 6 times, making it the most promising long-acting candidate molecule for development.

[0024] 3. The polypeptide compound of this invention exhibits significant osteogenic activity, promoting the proliferation of MC3T3-E1 cells and generating osteogenic markers and calcium nodules under osteogenic induction conditions. Furthermore, its binding affinity to serum albumin is increased by 2-6 times. It holds promise as an effective candidate drug for the clinical treatment of hypothyroidism and osteoporosis.

[0025] 4. The polypeptide compounds of this invention provide a new research direction for hypothyroidism and osteoporosis. Attached Figure Description

[0026] Figure 1 The HPLC analysis chromatogram of pTH-1 is shown. Figure 2 This is a mass spectrometry analysis result of pTH-1. Figure 3 This is a pTH-1 secondary mass spectrometry detection and analysis chromatogram; Figure 4 The HPLC analysis chromatogram of pTH-2 is shown. Figure 5 This is a mass spectrometry analysis result of pTH-2. Figure 6 This is a pTH-2 secondary mass spectrometry detection and analysis chromatogram; Figure 7 The HPLC analysis chromatogram of pTH-3 is shown. Figure 8 This is a mass spectrometry analysis result of pTH-3. Figure 9 This is a pTH-3 secondary mass spectrometry detection and analysis chromatogram; Figure 10 The HPLC analysis chromatogram of pTH-4 is shown. Figure 11 This is a mass spectrometry analysis result of pTH-4. Figure 12 This is a pTH-4 secondary mass spectrometry detection and analysis chromatogram; Figure 13 The HPLC analysis chromatogram of pTH-5 is shown. Figure 14 This is a mass spectrometry analysis result of pTH-5. Figure 15 This is a pTH-5 secondary mass spectrometry detection and analysis chromatogram; Figure 16 The HPLC analysis chromatogram of pTH-6 is shown. Figure 17 This is a mass spectrometry analysis result of pTH-6. Figure 18 This is a pTH-6 secondary mass spectrometry detection and analysis chromatogram; Figure 19The results of cell viability assays after 24 h of treatment with different concentrations of pTH (1-34) and analogues (n=3); Figure 20 Cell viability assay results after treatment with different concentrations of pTH (1-34) and analogues for 48 h (n=3); Figure 21 Cell viability assay results after 72 h of treatment with different concentrations of pTH (1-34) and analogues (n=3); Figure 22 The Col-1 content in cell supernatant of pTH (1-34) and its analogues at a concentration of 10 nM (n=3). Figure 23 The OPN content in the cell supernatant of pTH (1-34) and its analogues at a concentration of 10 nM (n=3). Figure 24 The OCN content in the cell supernatant of pTH (1-34) and its analogues at a concentration of 10 nM (n=3). Figure 25 Alizarin Red staining results of cells induced to osteogenicity by pTH(1-34) and its analogues at a concentration of 10 nM for 21 days. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments.

[0028] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the experimental materials and reagents used in the following examples were obtained commercially.

[0031] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, all reagents or instruments used are commercially available.

[0032] The abbreviations and their meanings in this invention are as follows:

[0033] Example 1: Preparation of pTH-1 peptide resin 1. Amino acid resin swelling: Weigh 4.0 g Fmoc-Phe-Wang Resin (sub=0.377mmol / g) onto weighing paper, add it to the top of the column, add 40 mL DMF, stir with nitrogen gas, and allow it to swell completely for 1 h. Then remove the DMF, add 40 mL DMF and wash twice, 2 min each time.

[0034] 2. Deprotection: Add 40 mL of DBLK solution, purge with nitrogen and stir, repeat twice, the first time for 6 min and the second time for 8 min.

[0035] 3. Washing after deprotection: Add 40 mL of DMF for washing after deprotection, wash a total of 6 times, 2 min each time.

[0036] 4. Ninhydrin detection: After the 5th deprotection wash, dip a small amount of resin into a test tube with a glass rod, add a small amount of anhydrous ethanol solution containing 20% ​​ninhydrin, heat at 115 °C for 5 min, and the resin changes from colorless and transparent to blue, proving that the Fmoc protecting group has been removed.

[0037] 5. Amino acid coupling: Weigh 2.685 g of Fmoc-Asn(Trt)-OH and 0.669 g of HOBt into a beaker, add 20 mL of DMF and stir to dissolve. Then add 1.5 mL of DIC, react in an ice-water bath for 5 min, and then add to the reaction column. Add another 20 mL of DMF and stir under nitrogen for 2.5 h. After the reaction is complete, perform a ninhydrin test. The reaction is complete when the resin is colorless and transparent. After removing the solvent with a water pump, add 40 mL of DMF for washing, repeating twice, 2 min each time.

[0038] 6. Peptide chain synthesis: Repeat steps 2 to 5 to sequentially couple all amino acids according to the pTH (1-34) sequence.

[0039] 7. Peptide resin shrinkage and drying: Wash four times with 40 mL DMF, 2 min each time, then wash twice each with dichloromethane and methanol, 2 min each time. Finally, drain the resin using a water pump and weigh it. A total of 15.574 g of peptide resin was obtained in this experiment, with a yield of 99.6%.

[0040] Example 2: Preparation of pTH-1 polypeptide compound 1. Preparation of lysis buffer: Mix TFA, Anisole, Thioanisole, 3-MPa and TIS in an Erlenmeyer flask in a ratio of 90:2.5:2.5:2.5:2.5. Stir gently with a glass rod until well mixed, and store at -20°C for later use.

[0041] 2. Peptide resin lysis: Add the shrunken and dried peptide resin to a round-bottom flask equipped with a magnetic stir bar, add 80 mL of lysis buffer (the volume of the lysis buffer is about 5-8 mL / g), and then rinse the inner wall of the round-bottom flask with a small amount of lysis buffer to completely immerse the peptide resin in the lysis buffer. Slowly turn on the magnetic stir bar to allow the reaction to proceed. React at room temperature for 3 h. After the lysis reaction is complete, filter the lysis buffer through a sintered glass funnel and collect the filtrate into an Erlenmeyer flask. Rinse the round-bottom flask and resin twice with a small amount of lysis buffer and combine the rinses with the filtrate.

[0042] 3. Crude peptide precipitation: Take 200 mL of pre-cooled isopropyl ether and add it to four centrifuge cups (the total volume of isopropyl ether is approximately 8-10 times the volume of the lysis reaction solution). Divide the filtrate into four portions and slowly add them to the isopropyl ether separately, stirring constantly. After all the filtrate has been added, rinse the Erlenmeyer flasks with a small amount of isopropyl ether and add the rinsed flasks to the centrifuge cups to ensure complete transfer of the filtrate. Stir thoroughly with a glass rod to ensure the filtrate and isopropyl ether are evenly mixed.

[0043] 4. Centrifugal Drying: Centrifuge at 3500 r / min for 5 min. After centrifugation, discard the supernatant and add 200 mL of isopropyl ether. Stir with a glass rod to ensure the filter cake is fully dispersed in the isopropyl ether. Wash away any residual lysis buffer. Centrifuge again under the same conditions, repeating 3 times. After the third centrifugation, discard the supernatant and purge the precipitate with nitrogen to accelerate the evaporation of isopropyl ether from the filter cake surface. Seal the centrifuge cup with sealing film and make evenly punctured holes. Place it in a desiccator and vacuum dry with a water pump for 1 h. Then, continue drying with an oil pump. After complete drying, weigh the crude peptide to obtain 5.793 g of pTH(1-34) crude peptide, with a yield of 93.74% and a purity of 56.30%. Purify the crude peptide by HPLC to obtain 2.12 g of refined peptide with a purity of 98.87%, with a total yield of 36.61%.

[0044] The preparation method of pTH-2~6 polypeptide compounds is completely consistent with that in Examples 1 and 2.

[0045] The HPLC, mass spectrometry, and secondary mass spectrometry analyses of the pure pTH-1 polypeptide compound are shown in the following figures. Figure 1 , Figure 2 and Figure 3 As shown.

[0046] The HPLC, mass spectrometry, and secondary mass spectrometry analyses of the pure pTH-2 polypeptide compound are shown in the following figures. Figure 4 , Figure 5 and Figure 6 As shown.

[0047] The HPLC, mass spectrometry, and secondary mass spectrometry analyses of the pure pTH-3 polypeptide compound are shown in the following figures. Figure 7, Figure 8 and Figure 9 As shown.

[0048] The HPLC, mass spectrometry, and secondary mass spectrometry analyses of the pure pTH-4 polypeptide compound are shown in the following figures. Figure 10 , Figure 11 and Figure 12 As shown.

[0049] The HPLC, mass spectrometry, and secondary mass spectrometry analyses of the pure pTH-5 polypeptide compound are shown in the following figures. Figure 13 , Figure 14 and Figure 15 As shown.

[0050] The HPLC, mass spectrometry, and secondary mass spectrometry analyses of the pure pTH-6 polypeptide compound are shown in the following figures. Figure 16 , Figure 17 and Figure 18 As shown.

[0051] The results of synthesizing the pTH-1-pTH-6 compound in the above embodiments are shown in the table below.

[0052] Synthesis results of pTH peptide analogues

[0053] Example 3: Evaluation of MC3T3-E1 cell proliferation (1) Cell seeding: Take MC3T3-E1 cells in the logarithmic growth phase, discard the original culture medium, and wash three times with 2 mL PBS. Digest the cells according to the cell passage method, and collect the cell pellet by centrifugation. Add 3 mL of complete culture medium to resuspend the cells. Count the number of cells using a cell counting chamber, and then dilute the cell suspension to 8 × 10⁻⁶ with culture medium. 4 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well plate to make the number of cells per well approximately 8000. Incubate the cells in a cell culture incubator at 37°C and 5% CO2 for 24 h to allow the cells to adhere.

[0054] (2) pTH (1-34) and analogue treatment: Discard the original culture medium in the 96-well plate. Add 100 μl of sample-containing culture medium (concentration gradient: 2.5, 5, 10, 20, 40 nM) to each well of the positive control group and experimental group. Add 100 μl of culture medium to the blank control group. Set up 5 parallel wells for each sample. After adding PBS to the outer ring of the 96-well plate, place it in an incubator for culture. Cell proliferation was detected after 24 and 48 h.

[0055] (3) Add CCK-8 reagent: After culturing for the set time, remove the 96-well plate from the incubator. Discard the original culture medium, add 100 μl of culture medium containing 10% CCK-8 to each well, and then put the 96-well plate back into the incubator for 1.5 h.

[0056] (4) Measurement of absorbance: After incubation, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0057] The cell proliferation results at 24, 48, and 72 hours are shown in the table below: pTH-1~6 cell proliferation results

[0058] Cell viability assays (n=3) after treatment with different concentrations of pTH (1-34) and analogues for 24 h, 48 h, and 72 h are shown below. Figure 19 , 20 As shown in Figure 21. The results showed that after 24 h of treatment with pTH(1-34) and its analogues, the proliferation regulation of MC3T3-E1 cells exhibited a partially concentration-dependent characteristic. Among them, pTH(1-34) showed a dose-dependent proliferative effect in the concentration range of 2.5-10 nM, while the high concentration group of 20-40 nM had almost no proliferative activity. At the same time point, cell activity was slightly inhibited in all concentration groups of pTH-1; pTH-2 showed a proliferative trend at concentrations of 2.5-10 nM, and the cell proliferation-promoting activity was the best at 10 nM, with cell activity at 110%, while cell activity was inhibited at concentrations of 20 and 40 nM; all concentration groups of pTH-3-6 had no significant proliferative effect, and cell activity was inhibited to some extent at a concentration of 40 nM.

[0059] After 48 h of treatment with pTH(1-34) and its analogues, the effects of pTH(1-34) and pTH-2 on cells at each concentration group were the same as at 24 h: low concentration groups (2.5-10 nM) showed a proliferative effect, while high concentration groups (20-40 nM) showed an inhibitory effect. pTH-1 showed a more pronounced inhibitory effect on cell viability. For pTH-3-5, unlike at 24 h, only the 5 nM concentration showed a proliferative effect, while other concentrations showed no proliferative or inhibitory effects. pTH-6 showed neither proliferative nor inhibitory effects at any concentration.

[0060] After 72 h of treatment with pTH(1-34) and its analogues, the proliferative activity of pTH(1-34) and pTH-2 was further enhanced overall, but the general trend was the same as that at 24 and 48 h, with no significant changes. The other analogues did not show significant proliferative effects.

[0061] Example 4: Osteogenesis Marker Detection 1. Col-1 detection (1) Preparation of standard curves: The standard was added to the corresponding wells in sequence according to the concentration gradient of 0, 39, 78, 156, 312, 625, 1250 and 2500 pg / mL. Three replicates were set for each concentration, with 100 μL in each well.

[0062] (2) Sample addition and incubation: For the treatment group, the supernatant of cells cultured for osteogenic induction for 5, 7 and 10 days was centrifuged and 100 μL was added to each well. For the control group, only 100 μL of complete culture medium was added. For the basal induction group, only 100 μL of osteogenic induction culture medium was added. The plates were sealed and incubated in a 37℃ incubator for 1 h.

[0063] (3) Add detection antibody: Discard the liquid in the microplate, invert the microplate onto filter paper and gently pat it dry. Add 100 μl of working solution A to each well, seal the plate and incubate it in a 37°C incubator for 1 h.

[0064] (4) Washing: After incubation, discard the liquid, add 350 μL of washing solution to each well, let stand for 1 min and then discard the washing solution. Repeat washing 3 times.

[0065] (5) Add detection antibody: Add 100 μL of working solution B to each well. After sealing, incubate at 37°C for 30 min.

[0066] (6) Wash again: Repeat step (4) 5 times.

[0067] (7) Add substrate solution: Add 90 μL of TMB substrate solution to each well and incubate the microplate in a 37°C incubator for 15 min in the dark.

[0068] (8) Add stop solution: After incubation, add 50 μL of stop solution to each well to terminate the reaction.

[0069] (9) Measure absorbance and analyze results: Immediately after the reaction is terminated, measure absorbance at a wavelength of 450 nm. Plot a standard curve with the concentration of the standard as the x-axis and the corresponding absorbance value as the y-axis. Use linear regression to obtain the standard curve equation. Substitute the absorbance value of the sample to be tested into the standard curve equation to calculate the concentration of osteocalcin in the sample.

[0070] 2. OPN detection The operation method is the same as in 5.4.3.1 Col-1 detection.

[0071] 3. OCN detection The operation method is the same as that for Col-1 testing in 5.4.3.1, except that the testing time is 15, 17, or 21 days.

[0072] 4. Staining of calcium nodules (1) Cell fixation: After osteogenic induction for 21 days, the culture medium was discarded. The cells were washed three times with D-PBS for 2 min each time. 2 mL of 4% paraformaldehyde was added to each well and the cells were fixed at room temperature for 30 min.

[0073] (2) Washing: Discard the fixative and rinse the cells twice with D-PBS for 2 min each time.

[0074] (3) Staining: Add 2 mL of alizarin red staining solution to each well and stain at room temperature for 10 min.

[0075] (4) Washing: Repeat step (2) 3 times.

[0076] (5) Microscopic observation: Observe the staining of calcium nodules under an inverted optical microscope and take pictures for record.

[0077] The results of osteogenic marker testing are shown in the table below:

[0078] The results of detecting pTH (1-34) and its analogue cell supernatant Col-1 content (n=3), OPN content (n=3), and OCN content (n=3) at a concentration of 10 nM are as follows: Figure 22 , 23 As shown in Figure 24. The results showed that at days 5, 7, and 10 of osteogenic induction, the secretion of the early osteogenic marker Col-1 in each control group was lower than that in the induction group. In the induction group, the secretion of Col-1 increased with the extension of induction time from day 5 to day 7, reaching a peak on day 7, and then decreased from day 7 to day 10. This indicates that there is a tendency for osteogenic differentiation under this concentration and culture medium system. Among them, pTH(1-34) and pTH-2 can significantly activate the osteogenic differentiation ability of MC3T3-E1. On day 7, the peak Col-1 secretion levels were 3357 pg / mL and 3601 pg / mL, respectively, which were 5.9% and 13.7% higher than those in the basal induction group. Compared with the basal induction group, pTH-1 showed lower Col-1 expression levels at all time points. pTH-3 and pTH-4 showed a slight increase in Col-1 secretion on day 5, but decreased on days 7 and 10. pTH-5 and pTH-6 showed lower Col-1 secretion levels on days 5, 7, and 10 than the basal induction group, and pTH-6 showed a significantly lower Col-1 secretion level on day 10 than the control group.

[0079] Significant differences were observed in OPN secretion levels among the groups at different mid-term osteogenic induction time points. The basal OPN secretion level in the control group remained low, indicating that osteogenic activity was not activated under control conditions. OPN expression in the basal induction group gradually increased over time, validating the effectiveness of the induction system. pTH(1-34) at a concentration of 10 nM significantly promoted OPN secretion, with its expression reaching a peak on day 10, 71.4% higher than the basal induction group (p<0.001), and showing a time-dependent increase. On day 5 of osteogenic induction, there were no significant differences in OPN secretion among the induction groups, but all were higher than the control group. On day 7, OPN secretion significantly increased in the pTH(1-34), pTH-2, pTH-4, pTH-5, and pTH-6 groups, but pTH-3 showed inhibition, with OPN secretion lower than the control group. On day 10, OPN secretion decreased in all groups, but OPN expression in pTH-5 was significantly higher than that in pTH(1-34).

[0080] The levels of OCN, a late osteogenic marker, in pTH (1-34) and its analogues showed a time-dependent increase at days 13, 15, and 17 of osteogenic induction. Compared with the control group, the OCN secretion levels in each induction group were significantly increased, with the pTH (1-34) group showing the highest OCN secretion at all time points, reaching 876 pg / mL at day 17. At day 13, the secretion levels in the pTH-2 and pTH-3 groups were comparable to those in the pTH (1-34) group, but decreased at day 17. The OCN secretion levels in all other groups were lower than those in the pTH (1-34) group at all time points.

[0081] Alizarin Red staining results after 21 days of osteogenic induction with pTH(1-34) and its analogues at a concentration of 10 nM. The control group was induced using complete culture medium. Figure 25 As shown, no red calcium nodules were generated after alizarin red staining. Each induction group was induced using osteogenic induction medium, and red calcium nodules were generated to varying degrees after staining. Among them, the number and size of red calcium nodules in the pTH(1-34) and pTH-2 groups were higher than those in the other groups, showing a significant effect in promoting osteogenic differentiation.

[0082] Example 5: Long-term testing SPR was used to characterize pTH(1-34) and its analogues for extended use. By detecting their binding to HSA, key parameters such as binding affinity and dissociation rate were analyzed to evaluate their extended use.

[0083] 1. Solution preparation: (1) Activation buffer ① Preparation of 0.4 M EDC solution: Weigh 1.534 g of EDC hydrochloride, add 10 mL of ultrapure water and stir to dissolve. Adjust the pH to 4.5-5.0 with hydrochloric acid and set aside. ② Preparation of 0.1 M NHS solution: Weigh 0.2302 g NHS, add 10 mL of ultrapure water and stir to dissolve.

[0084] The solution is obtained by mixing 0.4 M EDC and 0.1 M NHS solution at a ratio of 1:1.

[0085] (2) Blocking reagent: Weigh 0.9754 g of ethanolamine hydrochloride, add 8 mL of ultrapure water and stir to dissolve. Adjust the pH to 8.5 with sodium hydroxide and bring the volume to 10 mL with ultrapure water.

[0086] (3) Fixation reagent: Weigh 1.361 mg of sodium acetate trihydrate, add 8 mL of ultrapure water and stir to dissolve, adjust the pH to 4.5 with acetic acid, and make up to 10 mL with ultrapure water.

[0087] (4) Ligand buffer: Weigh 2.383 mg HEPES, 8.766 mg NaCl and 0.8767 mg EDTA, add 8 mL of ultrapure water, stir to dissolve, add 50 μL of Tween 20, stir evenly, adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide, and make up to 10 mL with ultrapure water.

[0088] (5) Analytical buffer: Measure 0.1 mL of DMSO and 10 mL of PBS solution, mix well, add 0.5 μl of Tween 20, and adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide.

[0089] (6) Regeneration buffer: Weigh 0.7507 mg glycine, add about 8 mL of ultrapure water and stir to dissolve, adjust the pH to 2.0 with hydrochloric acid, and make up to 10 mL with ultrapure water.

[0090] 2. Experimental Procedure: (1) Chip installation and system equilibration: Open the SPR instrument software and install the sensor chip into the flow cell of the instrument according to the instrument operation manual. Rinse the system with analysis buffer at a flow rate of 10 μL / min until the baseline is stable to ensure that the system reaches equilibrium.

[0091] (2) Chip surface activation: Inject activation buffer into the flow cell at a flow rate of 10 μL / min to activate the carboxyl groups on the chip surface for 420 s.

[0092] (3) HSA immobilization: Human serum albumin was diluted to 20 μg / mL in immobilization buffer and injected into the sample channel at a flow rate of 10 μL / min. The immobilization process of HSA was observed by monitoring the changes in the SPR signal until the signal reached a stable value. The immobilization time depends on the HSA concentration and binding status, and is generally 10-30 min. Subsequently, blocking reagent was injected to block unreacted active groups at a flow rate of 10 μL / min for 420 s. The flow cell was rinsed with analytical buffer to allow the SPR signal to reach a stable baseline again.

[0093] (4) Peptide binding: pTH(1-34) and analogues were diluted to eight concentrations (0.78-50 μM) with the same analytical buffer. They were injected into the sample channel at a flow rate of 20 μL / min and bound for 100 s.

[0094] (5) Peptide dissociation: Inject analytical buffer to flush the flow cell at 10 μL / min for 180 s. This allows the bound peptide to dissociate from HSA, and the signal change curve during the dissociation phase is observed. Repeat 8 cycles according to the ascending order of analyte concentration.

[0095] (6) Chip regeneration process: Inject the regeneration buffer into the flow cell at a flow rate of 10 μL / min to remove the peptides bound to the chip surface and restore the chip surface to its initial state. Then rinse the flow cell again with the analysis buffer to stabilize the baseline.

[0096] (7) Data Acquisition and Processing: The SPR instrument records sensor images in real time. The sensor images are processed using the instrument's accompanying data analysis software, and the blank control is subtracted to obtain the net binding curve. Based on the net binding curve, a 1:1 binding model is used to fit the data of the binding and dissociation stages, and kinetic parameters such as the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) are calculated to evaluate the binding affinity between the peptide and HSA.

[0097] Based on SPR real-time kinetic analysis technology, the interaction between pTH(1-34) and its analogues and HSA was investigated. Experimental data were acquired and analyzed using Biacore 8K Manager software. The binding rate constant (Ka), dissociation rate constant (Kb), and dissociation equilibrium constant (KD) were obtained by fitting a kinetic model. The specific values ​​are shown in the table below. The results show that: pTH (1-34) and similar SPR kinetic fitting parameters

[0098] SPR kinetic analysis showed that fatty acid side chain modification significantly enhanced the affinity of the peptide for HSA. Based on dissociation equilibrium constant (KD) analysis, the affinity of each analog to HSA, from highest to lowest, was: pTH-3 > pTH-2 > pTH-4 > pTH-1 > pTH-6 > pTH-5 > pTH (1-34). Compared to the prototype pTH (1-34) (KD = 5.52 × 10⁻⁶), the affinity was significantly higher. -5 M), the binding affinity of each analogue to HSA was significantly enhanced: pTH-3 (KD=6.61×10 -6 M) increased by approximately 8.4 times, pTH-2 (KD=7.17×10) -6 M) increased by approximately 7.7 times, pTH-4 (KD=8.60×10 -6 M) increased by approximately 6.4 times, pTH-1 (KD=1.31×10) -5 M) increased by approximately 4.2 times, pTH-6 (KD=1.46×10) -5 M) increased by approximately 3.8 times, pTH-5 (KD=2.29×10) -5 The affinity (M) increased by approximately 2.4 times. This significant increase in affinity suggests that the in vivo half-life of the analogues may be correspondingly extended.

[0099] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A pTH long-acting polypeptide compound, characterized in that, The parent peptide sequence of the polypeptide is: H-Ser 1 -Val 2 -Ser 3 -Glu 4 -Ile 5 -Gln 6 -Leu 7 -Met 8 -His 9 -Asn 10 -Leu 11 -Gly 12 -Lys 13 -His 14 -Leu 15 -Asn 16 -Ser 17 -Met 18 -Glu 19 -Arg 20 -Val 21 -Glu 22 -Trp 23 -Leu 24 -Arg 25 -Lys 26 -Lys 27 -Leu 28 -Gln 29 -Asp 30 -Val 31 -His 32 -Asn 33 -Phe 34 -OH, and an aliphatic chain modifying group is attached to at least one amino acid residue selected from the 13th, 26th, and 27th positions; the aliphatic chain modifying group is covalently linked to the polypeptide backbone via a linker unit.

2. The polypeptide compound according to claim 1, characterized in that, If you want to make a difference, you will be able to see the results. pTH-1:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic acid)-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-2:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys(AEEA-AEEA-γ-Glu-Eicosanoic acid)-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-3:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-G lu-Trp-Leu-Arg-Lys(AEEA-AEEA-γ-Glu-Octadecanedioicacid)-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-4:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys(AEEA-AEEA-Eico-Eico acid)-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-5:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-G lu-Trp-Leu-Arg-Lys-Lys(AEEA-AEEA-γ-Glu-Octadecanedioicacid)-Leu-Gln-Asp-Val-His-Asn-Phe-OH; pTH-6:Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Va l-Glu-Trp-Leu-Arg-Lys-Lys(AEEA-AEEA-γ-Glu-Eicosanoic acid)-Leu-Gln-Asp-Val-His-Asn-Phe-OH.

3. The polypeptide compound according to claim 1 or 2, characterized in that, The aliphatic chain modifying groups are selected from octadecanoic acid, eicosanoic acid and their derivatives.

4. The polypeptide compound according to claim 1 or 2, characterized in that, The connecting unit is AEEA-AEEA-γ-Glu.

5. A method for preparing a polypeptide compound according to any one of claims 1-4, characterized in that, A solid-phase synthesis method was used, based on the polypeptide sequence, to progressively couple the protecting amino acids from the C-terminus to the N-terminus onto a solid-phase support. After synthesis, a polypeptide resin was obtained. The polypeptide resin was then cleaved, deprotected, and purified to obtain the polypeptide compound.

6. A pharmaceutical composition, characterized in that, Includes the polypeptide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4, and a pharmaceutically acceptable carrier.

7. Use of the polypeptide compound according to any one of claims 1-4 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating osteoporosis.

8. The use of the polypeptide compound according to any one of claims 1-4 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating hypoparathyroidism.

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