Cyclic peptide and application thereof in medicine for preventing and treating lung fibrosis

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to preparation and application of cyclic peptide. The structure of the cyclic peptide CYP9 is Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2, and the first lysine and the seventh aspartic acid are subjected to cyclization through an amido bond. The biological activity of the cyclopeptide CYP9 is researched, an in-vitro cell model and an in-vivo animal model for pulmonary fibrosis are constructed to evaluate the in-vitro anti-fibrosis activity and the in-vivo treatment effect of the cyclopeptide CYP9, and the cytotoxicity, the serum stability and the pharmacokinetics of the cyclopeptide CYP9 are researched. Results show that compared with a parent peptide YD, the cyclopeptide CYP9 obtained by the invention has better activity indexes and stability in all aspects, and can be used for preparing medicines for preventing pulmonary fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to a cyclic peptide CYP9 and its use in medicines for preventing and treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a pathological change that occurs in the terminal stage of various lung diseases. It is characterized by the proliferation of myofibroblasts and the deposition of extracellular matrix (ECM), which causes damage to the alveolar structure. It is progressive and irreversible, and ultimately leads to respiratory failure or even death in patients. Epidemiological data show that the incidence and prevalence of pulmonary fibrosis are increasing year by year worldwide, and the median survival of patients is only 2-5 years. Currently, there are only two drugs approved for the treatment of pulmonary fibrosis: pirfenidone and nintedanib. Both have unsatisfactory efficacy and can only slow the progression of the disease in some patients, but cannot reverse or cure pulmonary fibrosis. They also have the disadvantages of large side effects and high dosage. Therefore, the development of new and effective drugs to treat pulmonary fibrosis is of great significance in clinical practice.

[0003] Pulmonary fibrosis results from the combined effects of multiple factors, with oxidative stress and inflammation being two key mechanisms. Oxidative stress leads to the accumulation of oxidative species such as reactive oxygen species (ROS), which damage alveolar epithelial and endothelial cells. Inflammation releases cytokines to repair damaged tissue and restore homeostasis. However, repeated and severe damage disrupts the immune system's repair function. Various pro-inflammatory and pro-fibrotic mediators induce fibroblast differentiation into myofibroblasts, leading to the continuous accumulation of ECM, ultimately leading to pulmonary fibrosis and even organ failure. Therefore, therapeutic strategies targeting oxidative stress and inflammation are effective approaches to slow the progression of pulmonary fibrosis.

[0004] YD (APKGVQGPNG) is a natural active peptide purified from Bacillus amyloliquefaciens CBS in 2017 by Jin Cheol Yoo et al. It exerts antioxidant and anti-inflammatory activities in vitro by inhibiting ROS production, increasing antioxidant enzyme expression, activating the Nrf2 / HO-1 pathway, regulating HO-1-related inflammatory processes, and inhibiting inflammatory mediators and the NF-κB signaling pathway. Existing studies using YD for the treatment of liver fibrosis have shown that it can effectively improve liver fibrosis, but YD suffers from issues such as a short half-life, poor bioavailability, and potential for improved efficacy. Summary of the Invention

[0005] The present invention addresses the shortcomings of polypeptide YD and provides a structurally optimized cyclic peptide CYP9.

[0006] Another object of the present invention is to provide a method for preparing the polypeptide.

[0007] Another object of the present invention is to provide use of the polypeptide in preparing a drug for preventing or treating pulmonary fibrosis.

[0008] The structure of the cyclic peptide CYP9 according to a specific embodiment of the present invention is obtained based on the parent peptide YD through the following structural optimization strategy: The amino-terminal alanine and proline and the carboxyl-terminal glycine in the YD amino acid sequence are removed to shorten the peptide chain while retaining the core active sequence.

[0009] The sixth amino acid in the YD sequence is replaced with a D-type amino acid, and the C-terminus of the residual sequence is amidated to enhance the peptide's resistance to protease degradation, improve drug stability, and extend its half-life in the body, which helps to improve the durability and efficacy of the anti-fibrosis drug prepared.

[0010] The first lysine and the last aspartic acid of the YD residue sequence are connected by an amide bond to form a cyclic peptide structure, which further improves the stability and biological activity of the peptide and helps the peptide enter the cell to better exert its effect. Its amino acid sequence is as follows: Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2, where The terminal amino group of lysine at position 1 and the side chain carboxyl group of aspartic acid at position 7 form a ring through an amide bond.

[0011] According to a specific embodiment of the present invention, the cyclic peptide CYP9 has the following structure: .

[0012] The present invention also provides a linear peptide, the sequence of which is: Lys-Gly-Val-dGlu-Gly-Pro-Asn-CONH2.

[0013] According to a specific embodiment of the present invention, a method for preparing the polypeptide is provided, comprising the following steps: Step 1: Using the Fmoc solid-phase synthesis method, the linear peptide resin corresponding to the peptide is synthesized from the carboxyl end to the amino end, and the amino acid used to connect Asp is Fmoc-Asp(OAll)-OH; Step 2: Cyclization: Before removing the last Fmoc protecting group, use DCM containing 20 times the amount of PhSiH3 and 0.1 times the amount of Pd(PPh3)4 to remove the Oall protecting group. To remove the last Fmoc protecting group, use DCM containing 10 times the amount of PyBOP, 10 times the amount of HOBT and 20 times the amount of DIEA dissolved in NMP, mix well, and react overnight to complete the cyclization; Step 3: Prepare a cleavage solution with a volume ratio of TFA:Tis:H2O of 95:2.5:2.5 to cleave the resin from step 1 and remove the remaining protecting groups of the peptide chain. Collect the cleavage solution containing the crude CYP9 peptide chain; Step 4: Purify the cleavage solution obtained in step 3 to obtain the cyclic peptide CYP9; Preferably, in step 1, the resin used in the Fmoc solid phase synthesis method is MBHA resin.

[0014] The present invention provides the use of the polypeptide in preparing a drug for preventing or treating pulmonary fibrosis, specifically comprising: The cyclic peptide CYP9 of the present invention has an ameliorative effect on pulmonary fibrosis and can be used to directly or indirectly treat conditions characterized by pulmonary fibrosis. Pulmonary fibrosis includes idiopathic pulmonary fibrosis and pulmonary fibrosis caused by various factors, including occupational exposure, unhealthy lifestyle habits, trauma, radioactive element damage, adverse drug reactions, and infection by pathogenic microorganisms.

[0015] The present invention also provides a drug or pharmaceutical composition for treating pulmonary fibrosis, comprising the cyclic peptide CYP9 as a primary ingredient and further comprising a pharmaceutically acceptable carrier or excipient. The carrier may be one that reduces drug degradation and loss, and reduces side effects, such as micelles, microemulsions, and gels. Excipients may be materials added to prepare the drug into a suitable dosage form, such as buffers and lyophilization excipients. Liquid formulations typically include buffers, isotonic solutions, and aqueous solutions. Specifically, the pharmaceutical composition comprises the cyclic peptide CYP9 as the active ingredient, and is supported by a pharmaceutically acceptable carrier and / or excipient.

[0016] The pharmaceutical combination of the present invention is suitable for various administration methods, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. Depending on the administration method adopted, the pharmaceutical combination of the polypeptide of the present invention can be supported in various suitable dosage forms, which contain at least one effective amount of the polypeptide of the present invention and at least one pharmaceutically acceptable pharmaceutical carrier.

[0017] Examples of suitable dosage forms include tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin application, aerosols, nasal sprays, and sterile solutions for injection. Pharmaceutical compositions containing the polypeptides of the present invention can be prepared as solutions or lyophilized powders for parenteral administration. The powders can be reconstituted by adding an appropriate solvent or other carrier prior to use. Liquid formulations are typically PBS buffer, isotonic saline solutions, and aqueous solutions.

[0018] The dosage of the polypeptide of the present invention in the pharmaceutical composition can vary within a wide range, and those skilled in the art can easily determine it based on objective factors such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, etc.

[0019] Beneficial effects of the present invention: The present invention provides a cyclic peptide CYP9, and studies are conducted on the biological activity of the cyclic peptide CYP9, including constructing an in vitro cell model of pulmonary fibrosis and an in vivo animal model to evaluate the in vitro anti-fibrotic activity and in vivo therapeutic effect of the cyclic peptide CYP9, and also including studying the cytotoxicity, serum stability, and pharmacokinetics of the cyclic peptide CYP9. The results show that the cyclic peptide CYP9 screened out by chemical modification in the present invention has better biological activity against pulmonary fibrosis diseases. Compared with the parent peptide YD and the control drug, the dosage is significantly reduced and the stability is improved; compared with small molecule compounds, the analogues have low toxicity and a large safety window; compared with large molecule proteins, the preparation cost is more economical and the scalability is higher. The cyclic peptide CYP9 of the present invention can be used to prepare drugs for preventing and treating pulmonary fibrosis diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The effect of peptide YD on the survival rate of mouse embryonic fibroblasts (NIH-3T3).

[0022] Figure 2 Western blot analysis shows the effects of peptide YD on the expression of α-SMA, MMP2, Collagen I and Fibronectin in the NIH-3T3 cell model and its corresponding statistical graphs.

[0023] Figure 3 The mRNA expression levels of α-SMA, MMP2, Collagen I and Fibronectin in NIH-3T3 cell model were detected by qPCR.

[0024] Figure 4 The effect of peptide YD on the survival rate of human lung adenocarcinoma epithelial cells (A549).

[0025] Figure 5 The results of Western blot detection of the effect of parent peptide YD on the expression of α-SMA, MMP2, Collagen I and Fibronectin in the A549 cell model and the corresponding statistical graphs.

[0026] Figure 6The mRNA expression levels of α-SMA, MMP2, Collagen I and Fibronectin in A549 cell model were detected by qPCR.

[0027] Figure 7 The results of Western blot detection of the effect of YD linear analogues on the expression of Collagen Ⅰ in the NIH-3T3 cell model and the corresponding statistical graph.

[0028] Figure 8 Western blot analysis shows the effects of YD-P9 on the expression of α-SMA, Collagen Ⅰ and Fibronectin in the NIH-3T3 cell model and the corresponding statistical graphs.

[0029] Figure 9 Western blot analysis of the effects of parent peptide YD, YD-P9, and the positive control drug pirfenidone (PFD) on the expression of α-SMA, Collagen I, and Fibronectin in mouse lungs and their corresponding statistical graphs.

[0030] Figure 10 The results of Western blot detection of the effect of cyclic peptide CYP9 on the expression of α-SMA, Collagen Ⅰ and Fibronectin in the NIH-3T3 cell model and the corresponding statistical graphs.

[0031] Figure 11 The mRNA expression levels of Collagen I and Fibronectin in the NIH-3T3 cell model were detected by qPCR using cyclic peptide CYP9.

[0032] Figure 12 The results of Western blot detection of the effects of cyclic peptide CYP9 and parent peptide YD on the expression of α-SMA, Collagen I, Fibronectin, E-cadherin and Vimentin in mouse lungs and their corresponding statistical graphs.

[0033] Figure 13 The results of qPCR detection of the effects of cyclic peptide CYP9 and parent peptide YD on the expression of Collagen I, Fibronectin, E-cadherin and Vimentin in mouse lungs.

[0034] Figure 14 These are the results of H&E and immunohistochemical staining of lung tissue pathological sections of mice in each group 21 days after administration of cyclic peptide CYP9.

[0035] Figure 15In vitro serum stability results of parent peptide YD and linear analogs.

[0036] Figure 16 These are the in vitro serum stability results of the cyclic peptide CYP9.

[0037] Figure 17 These are the in vivo half-life results of the parent peptides YD, YD-P9 and cyclic peptide CYP9. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0039] The abbreviations used in the present invention have the following specific meanings: Ala (abbreviated A) is alanine, Pro (abbreviated P) is proline, Lys (abbreviated K) is lysine, Gly (abbreviated G) is glycine, Val (abbreviated V) is valine, Gln (abbreviated Q) is glutamine, Asn (abbreviated N) is asparagine, dGlu (abbreviated e) is D-glutamic acid, and Asp (abbreviated D) is aspartic acid.

[0040] Materials and methods The experimental conditions and methods described in the following examples are conventional conditions and methods unless otherwise specified, and the reagents and instruments described are all commercially available.

[0041] Among them, Western blot results were analyzed by grayscale analysis of protein expression using EvolutionCapt software, and statistics and analysis were performed using GraphPadPrism 8.0 software. Data were expressed as mean ± standard deviation (Mean ± SD), and significant differences were analyzed by one-way analysis of variance and Tukey test. Compared with the control group, **P < 0.01, *P < 0.05; compared with the model group, ## P < 0.01, # P<0.05.

[0042] Example 1 Design and Synthesis of YD Analogs Step 1: The linear peptide was synthesized using the Fmoc solid-phase synthesis method from the carboxyl end to the amino end. The analog sequence with an amide bond at the carboxyl end was synthesized using MBHA resin, and the analog sequence with a carboxylic acid at the carboxyl end was synthesized using dichlororesin. The amino acid sequence of the cyclic peptide CYP9 is shown below: Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2.

[0043] The synthesis specifically comprises the following steps: (1) Resin activation: Weigh the resin and add appropriate amount of DCM to swell on a shaker for 30 min. After drying, add DMF to wash three times, each time for 3 min. (2) Indene test: Add ninhydrin: pyridine: phenol = 1:2:1 indene test reagent to a test tube, dip a small amount of resin into it, and boil it in a boiling water bath for 3 minutes. If the indene test result is yellow, it indicates that the resin is normal; (3) Resin deprotection: Add DMF solution containing 3% redistilled piperidine to remove the protecting group, remove the residual reagent, add DMF to wash, each time for 3 minutes, repeat 4 times, and remove the residual reagent; (4) Indene test: Add ninhydrin: pyridine: phenol = 1:2:1 indene test reagent to a test tube, take a small amount of resin into the test tube, and boil it in a boiling water bath for 3 minutes. If the indene test result is blue-purple, it proves that the protecting group has been removed; (5) Amino acid condensation reaction: weigh 3 times the amount of the amino acid to be condensed and HOBT in a beaker, dissolve in a small amount of DMF, add 6 times excess DIEA to fully dissolve, and finally add 3 times the amount of HBTU and immediately pour into the resin, stir for 1 h, drain the solvent, wash with DMF for 3 min, and repeat 3 times; (6) Indene test: If the indene test result is yellow, it indicates that the condensation is successful; (7) Repeat steps (3) (4) (5) (6) in the order of the amino acid sequence in the compound until all the amino acids in the compound to be synthesized are condensed. Note that when synthesizing CYP9, use Fmoc-Asp(OAll)-OH when connecting the amino acid Asp.

[0044] Step 2: Cyclization: Before removing the last Fmoc protecting group, rinse the resin with DCM for 3 minutes, repeat three times. Add DCM containing 20 times the amount of PhSiH3 and 0.1 times the amount of Pd(PPh3)4 to the resin and react for 1 hour 30 minutes to remove the Oall protecting group. Rinse the resin with DCM for 3 minutes, repeat three times; then rinse the resin with DMF for 3 minutes, repeat three times; finally, remove the last Fmoc protecting group.

[0045] After draining the solvent, wash the resin with DCM for 3 minutes, repeat three times; wash the resin with DMF for 3 minutes, repeat three times; wash the resin with NMP, pour NMP containing 10 times the amount of PyBOP, 10 times the amount of HOBT, and 20 times the amount of DIEA into the resin, mix well, and react overnight to perform cyclization. Drain the solvent, wash with DCM for 3 minutes, repeat three times; wash with DMF for 3 minutes, repeat three times; Step 3: Cleavage of the peptide chain: Wash the resin with DCM for 3 min, repeat twice; then wash with methanol for 3 min; then with DCM for 3 min; and finally with methanol for 3 min, repeat twice. Drain the resin until it is powdery.

[0046] Prepare a cutting solution with TFA:Tis:H2O = 95:2.5:2.5 (V / V / V), add it to the resin and cut for 3 h, collect the cutting solution; use a rotary evaporator to evaporate the cutting solution, then precipitate it with pre-cooled ice ether, add deionized water to extract, collect the aqueous phase with a separatory funnel and divide it into a 50 mL beaker, freeze it in a -80℃ refrigerator overnight, and then freeze-dry it to obtain the crude peptide.

[0047] Step 4: Preparation and purification of peptides: (1) Weigh approximately 40 mg of crude peptide and dissolve it in deionized water. After complete dissolution, remove insoluble matter from the peptide solution using a 0.45 μm filter. Add 0.1% TFA to the elution solvent (acetonitrile and deionized water). (2) HPLC was performed using a C18 reverse preparative column flushed with 100% acetonitrile until the spectrum was stable, equilibrated with an initial concentration of 30% acetonitrile, and injected after setting a flow gradient; (3) After injection, the absorption peak at 220 nm was detected, the main peak was collected, sealed with plastic wrap, pierced with vent holes, and frozen in a -80°C refrigerator overnight and then freeze-dried to obtain the polypeptide CYP9, the structure of which is as follows: .

[0048] (4) After lyophilization, a small amount of the compound was dissolved and eluted on a C18 reverse-phase analytical column with 5%-95% acetonitrile / deionized water for 30 min. The purity of the chromatogram was statistically analyzed by integrating the peak area at 220 nm, and the purity was >95%. The isolated product was characterized and identified by mass spectrometry, confirming that the measured m / z value of the protonated molecular ion peak was 682.35 and the theoretical m / z was 681.34, confirming that the synthesized and purified product was the target product.

[0049] According to the above method, the structure-optimized polypeptide of the parent peptide YD was prepared by Fmoc solid phase synthesis method. The results are shown in Table 1.

[0050] Table 1 Cyclic peptide CYP9 synthesized in Example 1, theoretical m / z, measured m / z and purity

[0051] The compounds in Table 2 were prepared by similar synthetic methods to those in the above examples: Table 2 Structure, theoretical m / z, measured m / z and purity of the compounds synthesized with reference to Example 1

[0052] Example 2: Cytotoxicity evaluation of the mother peptide YD on NIH-3T3 cells The NIH-3T3 cell line was selected to study and observe the cytotoxicity of peptide YD on NIH-3T3 cells.

[0053] NIH-3T3 cells were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS + 1% double antibody at 37°C, 5% CO2 for 24 h. The test substances were added at concentrations of 0, 12.5, 25, 50, 100, and 200 μM. After incubation for 24 h, 10 μL of CCK-8 solution was added and incubated for 2 h. The absorbance at a wavelength of 450 nm was detected by a microplate reader.

[0054] Figure 1 The results showed that within the concentration range of 0-200 μM, the cell survival rate remained at the same level as that of the control group after YD acted on the cells, with no significant difference, indicating that it had no significant toxicity to NIH-3T3 cells.

[0055] Example 3 Detection of the activity of mother peptide YD on NIH-3T3 cell fibrosis in vitro The NIH-3T3 cell line was used in the experiment to study the effect of polypeptide YD on the expression of α-smooth muscle actin (α-SMA), type I collagen (Collagen I), fibronectin and matrix metalloproteinase (MMP2) in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0056] NIH-3T3 cells were seeded in 6-well plates using DMEM (Gibco) medium supplemented with 10% FBS and 1% double-antibody and cultured at 37°C, 5% CO2 for 24 h. The culture medium was then replaced with serum-free medium for 12 h. The cells were then treated with 5 ng / mL TGF-β1 and different doses (200, 100, 50, 25, and 12.5 μM) of peptide YD for 24 h, after which total protein and RNA were extracted. The protein expression and relative expression levels of α-SMA, Collagen I, Fibronectin, and MMP2 were analyzed by Western blot. The mRNA expression levels of α-SMA, Collagen I, Fibronectin, and MMP2 were detected by qPCR.

[0057] Control group (labeled as Control): TGF-β1 and peptide YD were not added to the culture medium; TGF-β1-induced group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; YD-administered group: 5 ng / mL TGF-β1 and different doses of peptide YD were added to the culture medium.

[0058] Figure 2 A shows the expression of Collagen I protein detected by Western blot after TGF-β1 and different doses of peptide YD acted together on NIH-3T3 cells, and the relative expression level of the protein was obtained by analysis. Figure 2 A shows that the minimum effective concentration of YD in the NIH-3T3 cell model is 25 μM.

[0059] Figure 2 B is the protein expression results of α-SMA, Collagen I, Fibronectin and MMP2 detected by Western blot after the treatment of NIH-3T3 cells with TGF-β1, 25 μM peptide YD and TGF-β1, 50 μM peptide YD, as well as the relative expression levels of the proteins obtained by analysis. Figure 2 B shows that TGF-β1 stimulates NIH-3T3 cells to express a large amount of α-SMA, CollagenI, Fibronectin and MMP2 proteins, and YD can inhibit the expression of fibrosis-related proteins at concentrations of 25 and 50 μM.

[0060] Figure 3After the NIH-3T3 cells were treated with TGF-β1 and 25 μM peptide YD, and TGF-β1 and 50 μM peptide YD, the mRNA expression levels of α-SMA, Collagen I, Fibronectin and MMP2 were detected by qPCR. Figure 3 It can be seen that TGF-β1 stimulated a significant increase in the mRNA levels of α-SMA, Collagen I, Fibronectin and MMP2 in NIH-3T3 cells, and YD at concentrations of 25 and 50 μM could effectively inhibit the increase in the mRNA levels of these genes.

[0061] Depend on Figure 2 and Figure 3 It can be seen that polypeptide YD can effectively inhibit the high expression of fibrotic proteins and genes in NIH-3T3 cells induced by TGF-β1.

[0062] Example 4 Investigating the cytotoxicity of the parent peptide YD to A549 cells A549 cell line was selected to investigate the cytotoxicity of peptide YD on A549 cells. A549 cells were seeded into 96-well plates and cultured in 1640 (Gibco) medium containing 10% FBS and 1% double-antibody at 37°C and 5% CO2 for 24 hours. Peptide YD was then added at concentrations of 0, 12.5, 25, 50, 100, and 200 μM. After incubation for 24 hours, 10 μL of CCK-8 solution was added and the cells were incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0063] Figure 4 is the survival rate of A549 cells. The results showed that within the concentration range of 0-200 μM, after the peptide YD acted on the cells, the cell survival rate remained at basically the same level with no significant difference, indicating that the peptide YD had no significant toxicity to A549 cells.

[0064] Example 5 Investigation of the activity of the mother peptide YD on the fibrosis of A549 cells in vitro A549 cell line was selected in the experiment to study and observe the effect of polypeptide YD on the expression of α-SMA, Collagen I, Fibronectin and MMP2 in A549 cells induced by TGF-β1.

[0065] A549 cells were seeded in 6-well plates using 1640 medium containing 10% FBS and 1% double-antibody and cultured at 37°C, 5% CO2 for 24 h. The medium was then replaced with serum-free medium for 12 h. The cells were then treated with 5 ng / mL TGF-β1 and different doses (200, 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 μM) of peptide YD for 24 h. Total protein and RNA were extracted from the cells. The protein expression and relative expression levels of α-SMA, Collagen I, Fibronectin, and MMP2 were detected by Western blot. The mRNA expression levels of α-SMA, Collagen I, Fibronectin, and MMP2 were detected by qPCR.

[0066] Control group (labeled as Control): TGF-β1 and peptide YD were not added to the culture medium; Induction group (labeled with TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; YD-administered group: 5 ng / mL TGF-β1 and different doses of peptide YD were added to the culture medium.

[0067] Figure 5 A is the expression of Collagen I protein detected by Western blot after TGF-β1 and different doses of peptide YD acted together in A549 cells, and the relative expression level of the protein was obtained by analysis. Figure 5 A shows that the minimum effective concentration of YD in the A549 cell model is 12.5 μM.

[0068] Figure 5 B is the protein expression results of α-SMA, Collagen I, Fibronectin and MMP2 detected by Western blot after the treatment of A549 cells with TGF-β1, 12.5 μM peptide YD and TGF-β1, 25 μM peptide YD, as well as the relative expression levels of the proteins obtained by analysis. Figure 5 B shows that TGF-β1 stimulates A549 cells to express a large amount of α-SMA, Collagen I, Fibronectin and MMP2 proteins, and YD can improve the abnormal expression of fibrosis-related proteins at concentrations of 12.5 and 25 μM.

[0069] Figure 6After the A549 cells were treated with TGF-β1 and 12.5 μM peptide YD and TGF-β1 and 25 μM peptide YD, the mRNA expression levels of α-SMA, Collagen I, Fibronectin and MMP2 were detected by qPCR. Figure 6 It can be seen that TGF-β1 stimulated the mRNA levels of α-SMA, Collagen I, Fibronectin and MMP2 in A549 cells to increase significantly, and YD at concentrations of 12.5 and 25 μM could effectively improve the abnormal expression of these gene mRNA levels.

[0070] Depend on Figure 5 and Figure 6 It can be seen that YD can improve the abnormal expression of fibrotic proteins and genes induced by TGF-β1 in A549 cells.

[0071] Example 6 Investigating the activity of YD linear analogs on NIH-3T3 cell fibrosis in vitro NIH-3T3 cell line was used in the experiment to study the effect of YD linear analogs on the expression of Collagen I protein in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0072] NIH-3T3 cells were seeded in 6-well plates using DMEM (Gibco) medium containing 10% FBS and 1% double-antibody and cultured at 37°C, 5% CO2 for 24 h. After changing to serum-free medium and culturing for 12 h, 5 ng / mL TGF-β1 and 25 μM YD linear analogue were added to the cells for 24 h, and then total cell protein was extracted. Western blot was used to detect the expression of Collagen I protein and the relative expression levels of the proteins were analyzed.

[0073] Control group (labeled as Control): TGF-β1 and YD analogs were not added to the culture medium; Induction group (labeled with TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; Drug-treated group: 5 ng / mL TGF-β1 and 25 μM YD linear analog were added to the culture medium.

[0074] Figure 7 After TGF-β1 and YD linear analogs acted together on NIH-3T3 cells, the expression of Collagen I protein was detected by Western blot and the relative expression level of the protein was analyzed.

[0075] Depend on Figure 7It can be seen that YD linear analogs can inhibit the abnormally high expression of Collagen I in the NIH-3T3 cell model at a concentration of 25 μM, especially YD-P9, which exhibits better Collagen I inhibitory activity than the parent peptide YD. This shows that the amino-terminal alanine and proline and the carboxyl-terminal glycine in the amino acid sequence of the parent peptide YD are its key inactive amino acids. Retaining only the core active sequence does not affect its activity. In addition, C-terminal amide bond protection and amino acid substitution at the 6th enzymatic site enhance the anti-fibrosis activity of the analogs in vitro.

[0076] Example 7 Investigating the activity of YD-P9 on fibrosis of NIH-3T3 cells in vitro NIH-3T3 cell line was used in the experiment to study the effect of YD-P9 on the expression of Collagen I protein in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0077] NIH-3T3 cells were seeded in 6-well plates using DMEM (Gibco) medium containing 10% FBS and 1% double-antibody and cultured at 37°C, 5% CO2 for 24 h. After changing to serum-free medium for 12 h, the cells were treated with 5 ng / mL TGF-β1 and different doses (25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.20 μM) of YD-P9 for 24 h. Total cell protein was extracted, and the expression of α-SMA, Collagen I, and Fibronectin proteins was detected by Western blot, as well as the relative expression levels of the proteins.

[0078] Control group (labeled as Control): TGF-β1 and YD-P9 were not added to the culture medium; Induction group (labeled with TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; YD-P9 administration group: 5 ng / mL TGF-β1 and different doses of peptide YD-P9 were added to the culture medium.

[0079] Figure 8 A shows the expression of Collagen I protein detected by Western blot after TGF-β1 and different doses of YD-P9 acted together on NIH-3T3 cells, and the relative expression level of the protein was obtained by analysis. Figure 8 A shows that YD-P9 can inhibit the abnormal expression of Collagen I in the NIH-3T3 cell model at a concentration of 1.56 μM.

[0080] Figure 8B is the protein expression results of α-SMA, Collagen I and Fibronectin detected by Western blot after NIH-3T3 cells were treated with TGF-β1 and 1.56 or 3.12 μM peptide YD-P9, as well as the relative expression levels of the proteins obtained by analysis. Figure 8 As shown in Figure 2, TGF-β1 stimulates NIH-3T3 cells to express a large amount of α-SMA, Collagen I, and Fibronectin proteins. YD-P9 can inhibit the expression of fibrosis-related proteins at concentrations of 3.12 and 1.56 μM. In combination with Example 3, the effective in vitro concentration of the structurally optimized YD analog YD-P9 is lower than that of the parent peptide YD.

[0081] Example 8: Therapeutic effects of parent peptides YD, YD-P9, and the positive control drug pirfenidone (PFD) on pulmonary fibrosis in mice (1) Test substances: peptides YD, YD-P9 and PFD.

[0082] (2) Forty male C57BL / 6J mice (6 weeks old, weighing approximately 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into five groups, with eight mice in each group.

[0083] Groups: sham operation group (Saline), model group (bleomycin (BLM)), PFD positive control group (100 mg / kg), drug-treated groups: YD (0.2 mg / kg), YD-P9 (0.2 mg / kg).

[0084] (3) Establishment of pulmonary fibrosis model: After anesthetizing mice with 1% sodium pentobarbital solution, The mice were placed supine on a surgical board. After disinfection of the neck skin, an approximately 1.5 cm incision was made. The neck muscles and fascia were then bluntly dissected to expose the trachea. BLM (3.5 mg / kg) was injected into the lungs via an insulin needle. The wound was closed and covered with a sterile dressing. The surgical board was rotated upright to ensure even distribution of the drug in the lungs. Mice in the sham-operated group were injected with an equal volume of normal saline. All other procedures were the same. Mice were housed normally after surgery.

[0085] (4) Administration. From the date of modeling, mice in the sham-operated and model groups were intraperitoneally injected with 100 μL of PBS daily. Mice in the drug-treated group were intraperitoneally injected with 100 μL of peptide YD or YD-P9 solution (in PBS) daily. Mice in the positive control group were intragastrically administered with 100 mg / kg PFD daily. Samples were collected after 21 consecutive days of administration.

[0086] Figure 9The expression of α-SMA, Collagen I and Fibronectin proteins in the lung tissue of each group of mice 21 days after administration and the statistical graph of the relative expression levels obtained by analysis show that in BLM model mice, the levels of fibrosis-related proteins in the lungs were significantly reduced after treatment with the test substance, indicating that the test substance has a good effect in improving pulmonary fibrosis.

[0087] YD and YD-P9 demonstrated improvements in pulmonary fibrosis in mouse models at a concentration of 0.2 mg / kg, a lower dose than the positive control drug PFD (100 mg / kg), with comparable therapeutic efficacy. However, the therapeutic efficacy of YD-P9 was not significantly different from that of YD. According to Example 7, the effective dose of YD-P9 in vitro was lower than that of the parent peptide YD, but in Example 8, its in vivo activity was not enhanced.

[0088] Example 9 Investigating the activity of cyclic peptide CYP9 on fibrosis of NIH-3T3 cells in vitro The NIH-3T3 cell line was used in the experiment to study the effect of cyclic peptide CYP9 on the expression of α-SMA, Collagen I and Fibronectin proteins in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0089] NIH-3T3 cells were seeded in 6-well plates using DMEM (Gibco) medium supplemented with 10% FBS and 1% double-antibody and cultured at 37°C, 5% CO2 for 24 h. After 12 h of culture, the cells were treated with serum-free medium (5 ng / mL TGF-β1) and different doses (1.56, 0.78, 0.39, 0.2, 0.1, 0.05, and 0.025 μM) of cyclic peptide CYP9 for 24 h. Total protein and RNA were extracted from the cells. The protein expression of α-SMA, Collagen I, and Fibronectin and the relative expression levels of the proteins were analyzed by Western blot. The mRNA expression levels of Collagen I and Fibronectin were detected by qPCR.

[0090] Control group (labeled as Control): TGF-β1 and cyclic peptide CYP9 were not added to the culture medium; Induction group (labeled with TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium; CYP9 administration group: 5 ng / mL TGF-β1 and different doses of cyclic peptide CYP9 were added to the culture medium.

[0091] Figure 10A shows the expression of Collagen I protein detected by Western blot after TGF-β1 and different doses of cyclic peptide CYP9 acted together on NIH-3T3 cells, and the relative expression level of the protein was obtained by analysis. Figure 10 A shows that CYP9 can inhibit the abnormally high expression of Collagen I in the NIH-3T3 cell model at a concentration of 25 nM.

[0092] Figure 10 B is the protein expression results of α-SMA, Collagen I and Fibronectin detected by Western blot after TGF-β1 and 25 or 50 nM cyclic peptide CYP9 acted on NIH-3T3 cells, as well as the relative expression levels of the proteins obtained by analysis. Figure 10 B shows that TGF-β1 stimulates NIH-3T3 cells to express a large amount of α-SMA, Collagen I and Fibronectin proteins, and CYP9 can inhibit the expression of fibrosis-related proteins at concentrations of 25 and 50 nM.

[0093] Figure 11 After NIH-3T3 cells were treated with TGF-β1 and 25 or 50 nM cyclic peptide CYP9, the mRNA expression levels of Collagen I and Fibronectin were detected by qPCR. Figure 11 It can be seen that TGF-β1 stimulated a significant increase in the mRNA levels of Collagen I and Fibronectin in NIH-3T3 cells, and CYP9 at concentrations of 25 and 50 nM could effectively inhibit the increase in the mRNA levels of these genes.

[0094] Depend on Figure 10 and Figure 11 As shown, the cyclic peptide CYP9 can effectively inhibit the high expression of fibrotic proteins and genes induced by TGF-β1 in NIH-3T3 cells at concentrations of 25 and 50 nM, and the effective concentration is much lower than that of the parent peptide YD and the linear peptide YD-P9 with similar sequences. This shows that the in vitro activity of the linear peptide YD-P9 is further enhanced after the head-to-tail cyclization.

[0095] Example 10 Investigating the therapeutic effects of cyclic peptide CYP9 and parent peptide YD on improving pulmonary fibrosis in mice (1) Test substances: parent peptide YD and cyclic peptide CYP9.

[0096] (2) Forty male C57BL / 6J mice (6 weeks old, weighing approximately 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into five groups, with eight mice in each group.

[0097] Groups: sham operation group (Saline), model group (BLM), drug-treated groups: YD (0.2 mg / kg), cyclic peptide CYP9 (0.2 mg / kg), cyclic peptide CYP9 (0.02 mg / kg).

[0098] (3) The pulmonary fibrosis model was established in the same manner as in Example 8.

[0099] (4) In the drug administration group, the mice in the sham operation group and the model group were intraperitoneally injected with 100 μL of PBS once a day. The mice in the YD administration group were intraperitoneally injected with 100 μL of peptide YD solution (dissolved in PBS) at a dose of 0.2 mg / kg once a day. The mice in the CYP9 administration group were intraperitoneally injected with 100 μL of CYP9 solution (dissolved in PBS) at a dose of 0.2 or 0.02 mg / kg once a day. The samples were collected after 21 consecutive days of drug administration.

[0100] Figure 12 The expression of α-SMA, Collagen I, Fibronectin, E-cadherin and Vimentin proteins in the lung tissues of each group of mice 21 days after administration and the statistical graph of the relative expression levels obtained by analysis show that in BLM model mice, after treatment with the test substance, the levels of fibrosis-related proteins such as α-SMA, Collagen I, Fibronectin and Vimentin in the lungs were significantly reduced, and the level of epithelial cell marker E-cadherin protein was significantly increased, indicating that the test substance has a good effect in improving pulmonary fibrosis.

[0101] Figure 13 The results show the gene expression of Collagen I, Fibronectin, E-cadherin and Vimentin in the lung tissue of each group of mice 21 days after administration. It can be seen that in BLM model mice, after treatment with the test substance, the gene levels of fibrosis-related proteins such as Collagen I, Fibronectin and Vimentin in the lungs were significantly reduced, and the gene level of epithelial cell marker E-cadherin was significantly increased, indicating that the test substance has a good effect in improving lung fibrosis.

[0102] From the above results, it can be seen that the cyclic peptide CYP9 can inhibit and treat BLM-induced pulmonary fibrosis, and its therapeutic effect is better than that of the parent peptide YD. In addition, the effective dose of CYP9 is significantly reduced, and it still has a good therapeutic effect at 0.02 mg / kg.

[0103] Figure 14 These are the results of H&E and immunohistochemical staining of lung tissue pathological sections of mice in each group 21 days after administration in Example 10.

[0104] The method used is: H&E staining: Paraffin sections were oven-baked at 60°C for 1 hour. Dewaxing and hydration: Xylene I (20 min) → Xylene II (20 min) → Absolute Ethanol I (2 min) → Absolute Ethanol II (2 min) → 95% Ethanol I (2 min) → 95% Ethanol II (2 min) → 80% Ethanol (2 min) → Rinse twice with warm water (2 min each). Staining: Hematoxylin staining (3 min) → Rinse twice with tap water (2 min each) → Differentiation with 0.5% hydrochloric acid and alcohol (2 s) → Rinse thoroughly with tap water → Bluing with dilute ammonia solution (5-10 s) → Rinse thoroughly with tap water → Stain with 5% eosin (5 s). Dehydration and clearing: 95% ethanol I (15 s) → 95% ethanol II (15 s) → anhydrous ethanol I (15 s) → anhydrous ethanol II (15 s) → air-dry → clearing in xylene twice (2 min each) → after air-drying, seal the slides with neutral gum.

[0105] Immunohistochemistry: Paraffin sections were oven-baked at 60°C for 1 hour. Dewaxing and hydration: Xylene I (20 min) → Xylene II (20 min) → Absolute Ethanol I (2 min) → Absolute Ethanol II (2 min) → 95% Ethanol I (2 min) → 95% Ethanol II (2 min) → 80% Ethanol (2 min) → Rinse twice with warm water (2 min each). Staining: Hematoxylin staining (3 min) → Rinse twice with tap water (2 min each). Antigen retrieval → Endogenous Peroxidase Blocker (10 min) → Primary Antibody (4°C overnight) → Reaction Enhancement Solution (20 min) → Enhanced American Standard Goat Anti-Rabbit IgG Polymer (20 min) → DAB Development → Hematoxylin Counterstaining → Wash thoroughly with tap water. Dehydration and clearing: 95% ethanol I (15 s) → 95% ethanol II (15 s) → anhydrous ethanol I (15 s) → anhydrous ethanol II (15 s) → air-dry → clearing in xylene twice (2 min each) → after air-drying, seal the slides with neutral gum.

[0106] H&E staining results showed that compared with the sham-operated group, the BLM group had abnormal lung tissue structure, with significant alveolar septal thickening and alveolar stenosis, and inflammatory cell infiltration was observed in the lung tissue. Cyclic peptide CYP9 and parent peptide YD treatment reduced structural damage in the lung tissue of mice and significantly reduced inflammatory infiltration, demonstrating a significant therapeutic and ameliorative effect. Figure 14 The immunohistochemical staining results showed that α-SMA, Collagen I and Fibronectin were expressed in large quantities in the lungs of the BLM group. After 21 days of treatment, the expression level decreased significantly, indicating that the lung fibrosis of mice was improved by treatment, and the therapeutic effect of cyclic peptide CYP9 was better than that of the parent peptide YD.

[0107] Example 11 Comparison of serum stability of parent peptide YD, YD linear analogs and cyclic peptide CYP9 Test substances: YD, YD linear analogs and CYP9.

[0108] Blood was collected from anesthetized C57BL / 6J mice, allowed to stand at room temperature for 30 min, and centrifuged at 5000 rpm / min for 30 min. The upper serum was aspirated, and a 10 mM test substance solution was thoroughly mixed with the serum at a ratio of 1:4. The mixture was then incubated at 37°C and timed. 70 μL of the sample was taken at different time points, and an equal amount of ice-cold acetonitrile was quickly added to precipitate the protein. After thorough vortexing, the sample was centrifuged at 12000 rpm at 4°C for 30 min. The supernatant was collected and filtered. The solution was eluted using a C18 reverse analytical column with a gradient of 5%-95% acetonitrile / deionized water for 30 min. The absorption peak at 220 nm was detected, and the degradation rate of the test substance at different time points was determined by integrating the peak area of ​​the test substance and comparing it with the corresponding peak area of ​​the test substance at 0 min.

[0109] Figure 15 This is the content curve of the parent peptide YD and its linear analogs in serum. The half-life of the parent peptide YD in serum is only 21.31 min. The half-life of YD-P5 after sequence truncation becomes even shorter, but the stability is improved after terminal protection, especially after D-glutamic acid is replaced at the enzymatic hydrolysis site of YD, YD-P9, with an in vitro half-life of 7.975 h, which is nearly 23 times that of YD.

[0110] Figure 16 Figure 3 is the content curve of cyclic peptide CYP9 in serum. The half-life of cyclic peptide CYP9 in serum is 70.17 h. Compared with the linear analogue YD-P9, the in vitro stability of CYP9 is increased by nearly 10 times.

[0111] Example 12 Comparison of the pharmacokinetics of parent peptide YD, YD linear analogs and cyclic peptide CYP9 Test substances: YD, YD linear analogs and CYP9.

[0112] C57BL / 6J mice were injected with 2.5 mg / kg of the test substance via the tail vein. Blood was collected from the eyeballs at different time points. The centrifuge tube was pre-rinsed with 200 μL of 2 mg / kg sodium heparin and centrifuged at 15,000 rpm at 4°C for 10 min. 200 μL of the upper plasma was collected and vortexed with 800 μL of ice-cold methanol. The tube was centrifuged at 15,000 rpm at 4°C for 30 min. 700 μL of the supernatant was concentrated and the precipitate was resuspended in 200 μL of 5% acetonitrile solution. The tube was centrifuged at 15,000 rpm at 4°C for 5 min. The supernatant was aspirated for LC-MS analysis.

[0113] Figure 17This is the drug-drug curve of the test substance in vivo. YD was completely undetectable in the plasma at 20 minutes. The concentrations of the linear analogues YD-P9 and cyclic peptide CYP9 in the body were much higher than those of YD. The half-life of cyclic peptide CYP9 was 0.21 hours, which was nearly doubled compared with 0.11 hours of YD-P9. There was also more drug residue in the plasma at 1 hour, indicating that the action time and concentration of cyclic peptide CYP9 in the body were significantly improved compared with YD and the linear analogue YD-P9.

[0114] In combination with Examples 7, 8, 9 and 10, the structural optimization of the YD linear analogue improved its stability and in vitro activity, but the in vivo activity was not significantly improved. The cyclization gave CYP9 higher stability and membrane permeability, prolonged the in vivo half-life, and may help to improve the durability and efficacy of the anti-fibrosis drug prepared.

[0115] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A cyclic peptide CYP9, characterized in that Its structure is as follows: Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2, where The terminal amino group of lysine at position 1 and the side chain carboxyl group of aspartic acid at position 7 form a ring through an amide bond.

2. A linear peptide YD-P9, characterized in that The sequence of the linear peptide is Lys-Gly-Val-dGlu-Gly-Pro-Asn-CONH2.

3. The cyclic peptide CYP9 according to claim 1, characterized in that Its structure is as follows: 。 4. Use of the cyclic peptide CYP9 according to claim 1 in the preparation of a drug for preventing or treating pulmonary fibrosis.

5. Use of the linear peptide YD-P9 according to claim 2 in the preparation of a medicament for preventing or treating pulmonary fibrosis.

6. A drug for preventing or treating pulmonary fibrosis, characterized in that: The drug uses the cyclic peptide CYP9 described in claim 1 as a main component.

7. A drug for preventing or treating pulmonary fibrosis, characterized in that: The drug contains the linear peptide YD-P9 according to claim 2 as a main component.

8. The drug for preventing or treating pulmonary fibrosis according to claims 6 to 7, characterized in that: The medicine further includes a pharmaceutically acceptable carrier or excipient.

Images