A cyclic peptide and its use in the prevention and treatment of pulmonary fibrosis

Abstract

This invention belongs to the field of biochemistry, specifically relating to the preparation and application of a cyclic peptide. The structure of the cyclic peptide CYP9 is Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2, wherein lysine at position 1 and aspartic acid at position 7 are cyclically linked by an amide bond. This invention investigates the biological activity of the cyclic peptide CYP9, including constructing in vitro cell models and in vivo animal models of pulmonary fibrosis to evaluate its in vitro antifibrotic activity and in vivo therapeutic effects, as well as studying its cytotoxicity, serum stability, and pharmacokinetics. The results show that, compared with the parent peptide YD, the cyclic peptide CYP9 obtained in this invention exhibits superior activity and stability in all aspects, and can be used to prepare drugs for the prevention of pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a cyclic peptide CYP9 and its use in drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a pathological change that occurs in the end stage of various lung diseases. It is characterized by myofibroblast proliferation and extracellular matrix (ECM) deposition, leading to damage to alveolar structure. This process is progressive and irreversible, ultimately causing respiratory failure and even death. Epidemiological data shows that the incidence and prevalence of pulmonary fibrosis are increasing globally, with a median survival of only 2-5 years. Currently, only pirfenidone and nintedanib are approved for the treatment of pulmonary fibrosis. Both are not ideally effective, only slowing disease progression in some patients, but not reversing or curing pulmonary fibrosis. Furthermore, they have drawbacks such as significant side effects and high dosage. Therefore, developing novel and effective drugs for the treatment of pulmonary fibrosis is of great importance in clinical practice.

[0003] Pulmonary fibrosis is the result of multiple factors, among which oxidative stress and inflammatory response are two important mechanisms. Oxidative stress leads to the accumulation of oxidative substances such as reactive oxygen species (ROS), damaging alveolar epithelial cells and endothelial cells. Inflammatory responses release cytokines to repair damaged tissues and restore homeostasis. However, repeated and severe damage disrupts the repair function of the immune system, and various pro-inflammatory and pro-fibrotic mediators induce fibroblasts to differentiate into myofibroblasts, resulting in the continuous accumulation of ECM, ultimately leading to pulmonary fibrosis and even organ failure. Therefore, treatment strategies targeting oxidative stress and inflammatory responses are effective ways to slow the progression of pulmonary fibrosis.

[0004] YD (APKGVQGPNG) is a natural bioactive peptide purified from Bacillus amyloliquefaciens CBS by Jin Cheol Yoo et al. in 2017. It exerts its in vitro antioxidant and anti-inflammatory activities by inhibiting ROS generation, 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. Current research using YD to treat liver fibrosis shows that it can effectively improve liver fibrosis; however, YD suffers from a short half-life, poor bioavailability, and requires further improvement in efficacy. Summary of the Invention

[0005] This invention addresses the shortcomings of polypeptide YD by providing a structurally optimized cyclic peptide CYP9.

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

[0007] Another object of the present invention is to provide the use of the said polypeptide in the preparation of medicaments for the prevention or treatment of pulmonary fibrosis.

[0008] 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:

[0009] The amino-terminal alanine and proline, and the carboxyl-terminal glycine in the YD amino acid sequence are removed to shorten the peptide chain length while retaining the core active sequence.

[0010] Replacing the 6th amino acid in the YD sequence with a D-type amino acid and amidating the C-terminus of the residual sequence enhances the peptide's resistance to protease degradation, improves drug stability, and prolongs its half-life in vivo, thus contributing to improved durability and efficacy of the prepared antifibrotic drug.

[0011] The first lysine and the last aspartine of the YD residual sequence are linked by an amide bond to form a cyclic peptide structure, which further improves the peptide's stability and biological activity, and also facilitates the peptide's entry into the cell to exert its function. Its amino acid sequence is as follows:

[0012] Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2, where...

[0013] The terminal amino group of lysine at position 1 and the carboxyl group of aspartic acid side chain at position 7 form a ring through an amide bond.

[0014] The cyclic peptide CYP9 according to a specific embodiment of the present invention has the following structure:

[0015]

[0016] The present invention also provides a linear peptide, the sequence of which is:

[0017] Lys-Gly-Val-dGlu-Gly-Pro-Asn-CONH2.

[0018] According to a specific embodiment of the present invention, a method for preparing the polypeptide is provided, comprising the following steps:

[0019] Step 1: Using the Fmoc solid-phase synthesis method, the linear polypeptide resin corresponding to the polypeptide is synthesized from the carboxyl terminus to the amino terminus. The amino acid used when linking Asp is Fmoc-Asp(OAll)-OH.

[0020] Step 2: Cycloning: 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. After removing the last Fmoc protecting group, use NMP containing 10 times the amount of PyBOP, 10 times the amount of HOBT and 20 times the amount of DIEA, mix well, and react overnight to complete the cyclization.

[0021] Step 3: Prepare the cleavage solution according to a TFA:Tis:H2O volume ratio of 95:2.5:2.5, cleave the resin from Step 1, and remove the remaining protecting groups of the peptide chain. Collect the cleavage solution containing crude CYP9 peptide chains;

[0022] Step 4: Purify the cleavage solution obtained in Step 3 to obtain the cyclic peptide CYP9;

[0023] Preferably, in step 1, the resin used in the Fmoc solid-phase synthesis method is MBHA resin.

[0024] This invention provides the use of the polypeptide in the preparation of drugs for the prevention or treatment of pulmonary fibrosis, specifically including:

[0025] The cyclic peptide CYP9 of this invention has an ameliorative effect on pulmonary fibrosis and can be used to directly or indirectly treat diseases characterized by pulmonary fibrosis. Pulmonary fibrosis includes idiopathic pulmonary fibrosis and pulmonary fibrosis caused by various factors such as occupational exposure, unhealthy lifestyle habits, trauma, radiation damage, adverse drug reactions, and pathogenic microbial infections.

[0026] This invention also provides a medicament or pharmaceutical composition for treating pulmonary fibrosis, wherein the medicament or pharmaceutical composition has cyclic peptide CYP9 as the main component, and further comprises a pharmaceutically acceptable carrier or excipient. The carrier can be a carrier capable of reducing drug degradation and loss, and reducing side effects, such as micelles, microemulsions, gels, etc.; the excipient can be a material added to form a suitable dosage form of the drug, such as a buffer, a lyophilization excipient, etc. Liquid formulations are generally buffer solutions, isotonic solutions, and aqueous solutions. Specifically, a pharmaceutically acceptable carrier and / or excipient are added to support the pharmaceutical composition with cyclic peptide CYP9 as the active ingredient.

[0027] The pharmaceutical combinations of the present invention are suitable for various routes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, and nasal administration. Depending on the route of administration used, the pharmaceutical combinations of the polypeptides of the present invention can support various suitable dosage forms, comprising at least one effective amount of the polypeptide of the present invention and at least one pharmaceutically acceptable pharmaceutical carrier.

[0028] 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 suitable for injection. Pharmaceutical compositions containing the polypeptides of this invention can be formulated as solutions or lyophilized powders for parenteral administration. The powder can be reconstituted by adding a suitable solvent or other suitable carrier before use. Liquid formulations are generally PBS buffer, isotonic saline solution, and aqueous solution.

[0029] The amount 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 some objective factors such as the type of disease, the severity of the disease, the patient's weight, the dosage form, the route of administration, etc.

[0030] The beneficial effects of this invention are:

[0031] This invention provides a cyclic peptide CYP9 and studies its biological activity, including constructing in vitro cell models and in vivo animal models of pulmonary fibrosis to evaluate its in vitro anti-fibrotic activity and in vivo therapeutic effects. The study also includes investigations into the cytotoxicity, serum stability, and pharmacokinetics of CYP9. Results show that the chemically modified and screened cyclic peptide CYP9 of this invention exhibits superior biological activity against pulmonary fibrosis. Compared with the parent peptide YD and the control drug, it requires significantly lower dosage and exhibits improved stability. Compared with small molecule compounds, the analogue is less toxic and has a wider safety window. Compared with large molecule proteins, its preparation cost is more economical and its scalability is higher. The cyclic peptide CYP9 of this invention can be used to prepare drugs for the prevention and treatment of pulmonary fibrosis. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The survival rate of mouse embryonic fibroblasts (NIH-3T3) with the mother peptide YD.

[0034] Figure 2 The results of Western blot analysis of the effects of the parent peptide YD on the expression of α-SMA, MMP2, Collagen I and Fibronectin in the NIH-3T3 cell model and their corresponding statistical graphs are presented.

[0035] Figure 3 The expression levels of α-SMA, MMP2, Collagen I, and Fibronectin mRNA in the NIH-3T3 cell model were detected by qPCR using the parent peptide YD.

[0036] Figure 4 The survival rate of the mother peptide YD on human lung adenocarcinoma epithelial cells (A549) is shown.

[0037] Figure 5The results of Western blot analysis of the effects of the parent peptide YD on the expression of α-SMA, MMP2, Collagen I and Fibronectin in the A549 cell model and their corresponding statistical graphs are shown.

[0038] Figure 6 The mRNA expression levels of the mother peptide YD in the A549 cell model were detected by qPCR for α-SMA, MMP2, Collagen I and Fibronectin.

[0039] Figure 7 The results of Western blot analysis of the effect of YD linear analogues on Collagen I expression in the NIH-3T3 cell model and the corresponding statistical graphs are shown.

[0040] Figure 8 The results of Western blot analysis of 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 are shown.

[0041] Figure 9 The results of Western blot analysis of the effects of the parent peptides YD, YD-P9, and the positive control drug pirfenidone (PFD) on the expression of α-SMA, Collagen I, and Fibronectin in mouse lungs, along with their corresponding statistical graphs.

[0042] Figure 10 The results of Western blot analysis of the effects of the cyclic peptide CYP9 on the expression of α-SMA, Collagen I, and Fibronectin in the NIH-3T3 cell model, and the corresponding statistical graphs.

[0043] Figure 11 The mRNA expression levels of the cyclic peptide CYP9 on Collagen I and Fibronectin in the NIH-3T3 cell model were detected by qPCR.

[0044] Figure 12 The results of Western blot analysis 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.

[0045] Figure 13 The results show the effects of cyclic peptide CYP9 and parent peptide YD on the expression of Collagen I, Fibronectin, E-cadherin and Vimentin in mouse lungs, as detected by qPCR.

[0046] Figure 14 The results of H&E and immunohistochemical staining of lung tissue sections from mice in each group 21 days after administration of cyclic peptide CYP9.

[0047] Figure 15 Results of in vitro serum stability of the parent peptide YD and its linear analogue.

[0048] Figure 16 The results show the in vitro serum stability of the cyclic peptide CYP9.

[0049] Figure 17 The in vivo half-life results are for the parent peptide YD, YD-P9 and cyclic peptide CYP9. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0051] The specific meanings of the abbreviations used in this invention are as follows:

[0052] 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.

[0053] Materials and Methods:

[0054] Unless otherwise specified, the experimental conditions and methods described in the following examples are conventional conditions and methods, and the reagents or instruments mentioned are all commercially available.

[0055] Western blot results were analyzed for protein expression using EvolutionCapt software (grayscale analysis), and statistical analysis was performed using GraphPadPrism 8.0 software. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA and Tukey's test were used for significance analysis. Compared with the control group, **P < 0.01, *P < 0.05; compared with the model group, ## P < 0.01, # P < 0.05.

[0056] Example 1: Design and Synthesis of YD Analogs

[0057] Step 1: The linear peptide was synthesized using the Fmoc solid-phase synthesis method, proceeding from the carboxyl terminus to the amino terminus. Analog sequences with an amide bond at the carboxyl terminus were synthesized using MBHA resin, while analog sequences with a carboxylic acid bond at the carboxyl terminus were synthesized using dichloropolymer resin. The amino acid sequence of the cyclic peptide CYP9 is shown below:

[0058] Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2.

[0059] The synthesis specifically includes the following steps:

[0060] (1) Resin activation: Weigh the resin, add an appropriate amount of DCM, and swell on a shaker for 30 min. After drying, add DMF to wash 3 times, 3 min each time.

[0061] (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 in water for 3 minutes. If the indene test result is yellow, it indicates that the resin is normal.

[0062] (3) Resin deprotection: Add DMF solution containing 3% redistilled piperidine to remove the protecting group, remove the residual reagent, add DMF to wash, 3 min each time, repeat 4 times, and remove the residual reagent.

[0063] (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 in a water bath for 3 min. If the indene test result is blue-purple, it proves that the protecting group has been removed.

[0064] (5) Amino acid condensation reaction: Weigh 3 times the amount of amino acid to be condensed and HOBT in a beaker, dissolve in a small amount of DMF, add 6 times the excess of DIEA to dissolve completely, and finally add 3 times the amount of HBTU and immediately pour into the resin, stir for 1 h, remove the solvent, wash with DMF for 3 min, and repeat 3 times.

[0065] (6) Indene test: If the indene test result is yellow, it indicates that the condensation was successful;

[0066] (7) Repeat steps (3)(4)(5)(6) to condense the amino acids in the compound in sequence until all the amino acids in the compound to be synthesized are condensed. Note that when synthesizing CYP9, Fmoc-Asp(OAll)-OH is used when linking the amino acid Asp.

[0067] Step 2: Cycloning:

[0068] Before removing the last Fmoc protecting group, the resin was cleaned with DCM for 3 min, repeated 3 times. DCM containing 20 times the amount of PhSiH3 and 0.1 times the amount of Pd(PPh3)4 was then poured into the resin, and the reaction was carried out for 1 h 30 min to remove the Oall protecting group. The resin was then cleaned with DCM for 3 min, repeated 3 times; followed by DMF cleaning for 3 min, repeated 3 times; finally, the last Fmoc protecting group was removed.

[0069] After removing the solvent, wash the resin with DCM for 3 min, repeating 3 times; wash the resin with DMF for 3 min, repeating 3 times; wash the resin with NMP, then add NMP containing 10 times the amount of PyBOP, 10 times the amount of HOBT, and 20 times the amount of DIEA to the resin, mix well, and let it react overnight to carry out cyclization. Remove the solvent, wash with DCM for 3 min, repeating 3 times; wash with DMF for 3 min, repeating 3 times.

[0070] Step 3: Cutting the peptide chains: Wash the resin with DCM for 3 min, repeat twice; wash with methanol for 3 min; wash with DCM for 3 min; finally wash with methanol for 3 min, repeat twice. Dry the resin until it becomes powder.

[0071] A cutting solution with a ratio of TFA:Tis:H2O = 95:2.5:2.5 (V / V / V) was prepared and added to the resin for cutting for 3 h. The cutting solution was collected. The cutting solution was evaporated using a rotary evaporator, and then precipitated with pre-cooled ice-cold ether. Deionized water was added for extraction, and the aqueous phase was collected by separatory funnel and dispensed into 50 mL beakers. The beakers were then frozen overnight at -80℃ and freeze-dried to obtain crude peptides.

[0072] Step 4: Preparation and purification of peptides:

[0073] (1) Weigh about 40 mg of crude peptide and dissolve it in deionized water. After it is completely dissolved, remove the insoluble substances in the peptide solution using a 0.45 μm filter. Add 0.1% TFA to the elution solvent (acetonitrile and deionized water).

[0074] (2) High performance liquid chromatography uses a C18 reverse preparative column, washes with 100% acetonitrile until the spectral lines are stable, equilibrates with an initial concentration of 30% acetonitrile, and injects the sample after setting the flow gradient.

[0075] (3) After injection, the absorption peak at 220 nm was detected, the main peak was collected, sealed with plastic wrap, the vent hole was punched, and the sample was stored overnight at -80℃. Then, it was freeze-dried to obtain the peptide CYP9, the structure of which is as follows:

[0076] .

[0077] (4) After lyophilization, a small amount of the compound was dissolved and eluted with 5%-95% acetonitrile / deionized water for 30 min on a C18 reverse-phase analytical column. The purity was statistically determined by integrating the peak area of ​​the 220 nm chromatogram. The purity was >95%. The separated product was characterized and identified by mass spectrometry. The measured m / z value of the protonated molecular ion peak was confirmed to be 682.35, and the theoretical m / z was 681.34. The synthesized and purified product was confirmed to be the target product.

[0078] The structure-optimized peptide of the parent peptide YD was prepared by Fmoc solid-phase synthesis according to the above method, and the results are shown in Table 1.

[0079] Table 1. Cyclic peptide CYP9 synthesized in Example 1, theoretical m / z, measured m / z, and purity.

[0080]

[0081] Following a similar synthetic method to the examples described above, the compounds listed in Table 2 were prepared:

[0082] Table 2 shows the structure, theoretical m / z, measured m / z, and purity of the compounds synthesized according to Example 1.

[0083]

[0084] Example 2: Evaluation of the cytotoxicity of the parent peptide YD as the test substance against NIH-3T3 cells.

[0085] The NIH-3T3 cell line was selected to study and observe the cytotoxicity of peptide YD on NIH-3T3 cells.

[0086] NIH-3T3 cells were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS and 1% penicillin antibody at 37°C and 5% CO2 for 24 h. 0, 12.5, 25, 50, 100, and 200 μM of the test substance were added and incubated for 24 h. After incubation, 10 μL of CCK-8 solution was added and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader.

[0087] Figure 1 The study showed that, within the concentration range of 0-200 μM, the cell survival rate of YD after treatment remained basically the same as that of the control group, with no significant difference, indicating that it had no significant toxicity to NIH-3T3 cells.

[0088] Example 3: Detection of the activity of the parent peptide YD on NIH-3T3 cell in vitro fibrosis.

[0089] The NIH-3T3 cell line was used in the experiment to study the effect of peptide YD on the expression of α-smooth muscle actin (α-SMA), type I collagen (Collagen I), fibronectin and matrix metalloproteinase (MMP2) induced by transforming growth factor TGF-β1 in NIH-3T3 cells.

[0090] NIH-3T3 cells were seeded in 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% penicillin and cultured at 37°C and 5% CO2 for 24 h. After being cultured in serum-free medium for 12 h, 5 ng / mL TGF-β1 and different doses (200, 100, 50, 25 and 12.5 μM) of peptide YD were added and the cells were treated for 24 h. Total protein and RNA were extracted from the cells. Western blot was used to detect the protein expression of α-SMA, Collagen I, Fibronectin and MMP2 and to analyze the relative expression levels of the proteins. qPCR was used to detect the mRNA expression levels of α-SMA, Collagen I, Fibronectin and MMP2.

[0091] Control group (labeled as Control): The culture medium did not contain TGF-β1 and peptide YD;

[0092] TGF-β1 induction group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0093] YD administration group: 5 ng / mL TGF-β1 and different doses of peptide YD were added to the culture medium.

[0094] Figure 2 A represents the results of Western blot analysis of Collagen I protein expression and the relative expression levels of NIH-3T3 cells after treatment with TGF-β1 and different doses of peptide YD. Figure 2 As can be seen from A, the lowest effective concentration of YD in the NIH-3T3 cell model is 25 μM.

[0095] Figure 2 B represents the results of Western blot analysis of α-SMA, Collagen I, Fibronectin, and MMP2 protein expression in NIH-3T3 cells after treatment with TGF-β1, 25 μM peptide YD, and TGF-β1, 50 μM peptide YD in the treatment group, along with the analysis of the relative protein expression levels. Figure 2B indicates that TGF-β1 stimulates NIH-3T3 cells to express large amounts of α-SMA, CollagenI, Fibronectin, and MMP2 proteins, while YD at concentrations of 25 and 50 μM can inhibit the expression of fibrosis-related proteins.

[0096] Figure 3 After treating NIH-3T3 cells with TGF-β1, 25 μM peptide YD, and 50 μM peptide YD in the treatment group, the mRNA expression levels of α-SMA, Collagen I, Fibronectin, and MMP2 were detected by qPCR. Figure 3 It was found that TGF-β1 stimulation significantly increased the mRNA levels of α-SMA, Collagen I, Fibronectin, and MMP2 in NIH-3T3 cells, while YD at concentrations of 25 and 50 μM could effectively inhibit the increase in mRNA levels of these genes.

[0097] 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 induced by TGF-β1 in NIH-3T3 cells.

[0098] Example 4 investigated the cytotoxicity of the parent peptide YD on A549 cells.

[0099] The A549 cell line was selected to study and observe 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% penicillin antibody at 37°C and 5% CO2 for 24 h. Peptide YD at concentrations of 0, 12.5, 25, 50, 100, and 200 μM was added, and the cells were incubated for 24 h. Then, 10 μL of CCK-8 solution was added, and the cells were incubated for another 2 h. The absorbance at 450 nm was measured using a microplate reader.

[0100] Figure 4 The results showed that, within the concentration range of 0-200 μM, the cell survival rate remained basically the same after treatment with peptide YD, with no significant difference, indicating that peptide YD had no significant toxicity to A549 cells.

[0101] Example 5 investigated the activity of the parent peptide YD on A549 cell in vitro fibrosis.

[0102] The A549 cell line was used in the experiment to study and observe the effects of peptide YD on the expression of α-SMA, Collagen I, Fibronectin and MMP2 induced by TGF-β1 in A549 cells.

[0103] A549 cells were seeded in 6-well plates using 1640 medium containing 10% FBS and 1% penicillin and antibiotics. The cells were cultured at 37°C and 5% CO2 for 24 h. After being replaced with serum-free medium for 12 h, 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 were added and the cells were treated for 24 h. Total protein and RNA were extracted from the cells. Western blot was used to detect the protein expression of α-SMA, Collagen I, Fibronectin, and MMP2, and the relative expression levels of these proteins were analyzed. qPCR was used to detect the mRNA expression levels of α-SMA, Collagen I, Fibronectin, and MMP2.

[0104] Control group (labeled Control): The culture medium did not contain TGF-β1 and peptide YD;

[0105] Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0106] YD administration group: 5 ng / mL TGF-β1 and different doses of peptide YD were added to the culture medium.

[0107] Figure 5 A549 cells were treated with TGF-β1 and different doses of peptide YD. Western blot analysis was used to detect Collagen I protein expression and to determine the relative expression levels. Figure 5 As can be seen from A, the lowest effective concentration of YD in the A549 cell model is 12.5 μM.

[0108] Figure 5 B represents the results of Western blot analysis of α-SMA, Collagen I, Fibronectin, and MMP2 protein expression in A549 cells after treatment with TGF-β1 (12.5 μM) and 12.5 μM peptide YD in the treatment group, and TGF-β1 (25 μM) peptide YD in the treatment group, along with the analysis of the relative protein expression levels. Figure 5 B indicates that TGF-β1 stimulates A549 cells to express large amounts of α-SMA, Collagen I, Fibronectin, and MMP2 proteins, and YD at concentrations of 12.5 and 25 μM can improve the abnormal expression of fibrosis-related proteins.

[0109] Figure 6After treating A549 cells with TGF-β1, 12.5 μM peptide YD in the treatment group and TGF-β1, 25 μM peptide YD in the treatment group, the mRNA expression levels of α-SMA, Collagen I, Fibronectin, and MMP2 were detected by qPCR. Figure 6 It was found that TGF-β1 stimulation significantly increased the mRNA levels of α-SMA, Collagen I, Fibronectin, and MMP2 in A549 cells, and YD at concentrations of 12.5 and 25 μM could effectively improve the abnormal expression of these genes' mRNA levels.

[0110] Depend on Figure 5 and Figure 6 It is known that YD can improve the abnormal expression of fibrotic proteins and genes induced by TGF-β1 in A549 cells.

[0111] Example 6 investigated the activity of YD linear analogues against NIH-3T3 cell fibrosis in vitro.

[0112] The experiment used the NIH-3T3 cell line to study the effect of YD linear analogues on the expression of Collagen I protein in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0113] NIH-3T3 cells were seeded in 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% penicillin and cultured at 37°C and 5% CO2 for 24 h. After being cultured in serum-free medium for 12 h, 5 ng / mL TGF-β1 and 25 μM YD linear analog were added and the cells were treated for 24 h. Total cell protein was extracted, and Collagen I protein expression was detected by Western blot and the relative expression level of the obtained protein was analyzed.

[0114] Control group (labeled as Control): No TGF-β1 and YD analogue were added to the culture medium;

[0115] Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0116] Drug administration group: 5 ng / mL TGF-β1 and 25 μM YD linear analog were added to the culture medium.

[0117] Figure 7 After NIH-3T3 cells were treated with TGF-β1 and YD linear analogues, Western blot was used to detect Collagen I protein expression and analyze the relative expression levels of the protein.

[0118] Depend on Figure 7It was found that the linear analogues of YD could inhibit the abnormally high expression of Collagen I in the NIH-3T3 cell model at a concentration of 25 μM, especially YD-P9, which showed better Collagen I inhibitory activity than the parent peptide YD. This suggests that the alanine, proline, and glycine at the N-terminus and C-terminus of the parent peptide YD amino acid sequence are its inactive key amino acids; retaining only the core active sequence does not affect its activity. Furthermore, the protection of the C-terminal amide bond and the amino acid substitution at the 6th enzymatic cleavage site enhance the in vitro antifibrotic activity of the analogues.

[0119] Example 7 investigated the activity of YD-P9 against NIH-3T3 cell fibrosis in vitro.

[0120] The experiment used the NIH-3T3 cell line 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.

[0121] NIH-3T3 cells were seeded in 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% penicillin antibiotics and cultured at 37°C and 5% CO2 for 24 h. After being cultured in serum-free medium for 12 h, 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 were added and the cells were treated for 24 h. Total cell protein was extracted, and the expression results of α-SMA, Collagen I, and Fibronectin proteins were detected by Western blot, and the relative expression levels of the obtained proteins were analyzed.

[0122] Control group (labeled as Control): The culture medium did not contain TGF-β1 and YD-P9;

[0123] Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0124] YD-P9 administration group: 5 ng / mL TGF-β1 and different doses of peptide YD-P9 were added to the culture medium.

[0125] Figure 8 A represents the results of Western blot analysis of Collagen I protein expression and the relative expression levels of NIH-3T3 cells after treatment with TGF-β1 and different doses of YD-P9. Figure 8 As shown in Figure A, YD-P9 at a concentration of 1.56 μM can inhibit the abnormal expression of Collagen I in the NIH-3T3 cell model.

[0126] Figure 8 B represents the protein expression of α-SMA, Collagen I, and Fibronectin in NIH-3T3 cells after treatment with TGF-β1 and 1.56 or 3.12 μM peptide YD-P9, as well as the analysis of their relative expression levels using Western blot. Figure 8 As shown in B, TGF-β1 stimulates NIH-3T3 cells to express large amounts of α-SMA, Collagen I, and Fibronectin proteins, and YD-P9 can inhibit the expression of fibrosis-related proteins at concentrations of 3.12 and 1.56 μM. Combined with Example 3, the in vitro effective concentration of the structurally optimized YD analog YD-P9 is lower than that of the parent peptide YD.

[0127] Example 8: Therapeutic effects of parent peptide YD, YD-P9 and the positive control drug pirfenidone (PFD) on improving pulmonary fibrosis in mice.

[0128] (1) Test substances: polypeptides YD, YD-P9 and PFD.

[0129] (2) Forty male C57BL / 6J mice (6 weeks old, weighing about 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 5 groups of 8 mice each.

[0130] Groups: sham operation group (Saline), model group (bleomycin (BLM)), PFD positive control group (100 mg / kg), and drug administration groups: YD (0.2 mg / kg) and YD-P9 (0.2 mg / kg).

[0131] (3) Establishment of a pulmonary fibrosis model: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution.

[0132] Mice were fixed supine on a surgical board. After disinfection of the neck skin, an incision of approximately 1.5 cm was made in the skin. The neck muscles and fascia were then bluntly dissected to expose the trachea. BLM (3.5 mg / kg) was injected into the lungs via the trachea using an insulin injection needle. The wound was closed and covered with a sterile dressing. The surgical board was then rotated upright to ensure even distribution of the drug in the lungs. Mice in the sham-operated group received an equal volume of saline solution; all other procedures were performed identically. Mice were fed normally after surgery.

[0133] (4) Drug administration. From the date of mouse modeling, mice in the sham-operated group and the model group were injected intraperitoneally with 100 μL of PBS once a day, mice in the drug administration group were injected intraperitoneally with 100 μL of polypeptide YD or YD-P9 solution (dissolved in PBS) once a day, and mice in the positive control group were administered 100 mg / kg PFD once a day by gavage. Samples were collected after 21 days of continuous administration.

[0134] Figure 9The graph shows the expression of α-SMA, Collagen I, and Fibronectin proteins in the lung tissue of mice in each group 21 days after drug administration, and the statistical analysis of their relative expression levels. It can be seen that in BLM model mice, the level of fibrosis-related proteins in the lungs was significantly reduced after treatment with the test substance, indicating that the test substance has a good effect on improving lung fibrosis.

[0135] Both YD and YD-P9 showed ameliorative effects on pulmonary fibrosis in model mice at a concentration of 0.2 mg / kg, a lower dose than the positive control drug PFD (100 mg / kg), and their therapeutic effects were comparable. However, the therapeutic effect 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.

[0136] Example 9 investigated the activity of cyclic peptide CYP9 against NIH-3T3 cell fibrosis in vitro.

[0137] 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 induced by transforming growth factor TGF-β1 in NIH-3T3 cells.

[0138] NIH-3T3 cells were seeded in 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% penicillin antibiotics and cultured at 37°C and 5% CO2 for 24 h. After being cultured in serum-free medium for 12 h, 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 were added and the cells were treated for 24 h. Total protein and RNA were extracted from the cells. Western blot was used to detect the expression of α-SMA, Collagen I, and Fibronectin proteins and to analyze the relative expression levels of the proteins. qPCR was used to detect the mRNA expression levels of Collagen I and Fibronectin.

[0139] Control group (labeled as Control): The culture medium did not contain TGF-β1 and cyclic peptide CYP9;

[0140] Induction group (labeled TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0141] CYP9 administration group: 5 ng / mL TGF-β1 and different doses of cyclic peptide CYP9 were added to the culture medium.

[0142] Figure 10A represents the results of Western blot analysis of Collagen I protein expression and the relative expression levels of NIH-3T3 cells after treatment with TGF-β1 and different doses of the cyclic peptide CYP9. Figure 10 As shown in Figure A, CYP9 at a concentration of 25 nM can inhibit the abnormally high expression of Collagen I in the NIH-3T3 cell model.

[0143] Figure 10 B represents the protein expression results of α-SMA, Collagen I, and Fibronectin after treatment of NIH-3T3 cells with TGF-β1 and 25 or 50 nM cyclic peptide CYP9, and the relative expression levels of these proteins were analyzed by Western blot. Figure 10 B indicates that TGF-β1 stimulates NIH-3T3 cells to express large amounts of α-SMA, Collagen I, and Fibronectin proteins, while CYP9 at concentrations of 25 and 50 nM can inhibit the expression of fibrosis-related proteins.

[0144] Figure 11 After treating NIH-3T3 cells with TGF-β1 and the 25 or 50 nM cyclic peptide CYP9, the mRNA expression levels of Collagen I and Fibronectin were detected by qPCR. Figure 11 It was found that TGF-β1 stimulation significantly increased the mRNA levels of Collagen I and Fibronectin in NIH-3T3 cells, while CYP9 at concentrations of 25 and 50 nM could effectively inhibit the increase in the mRNA levels of these genes.

[0145] Depend on Figure 10 and Figure 11 It was found that the cyclic peptide CYP9 could 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 was much lower than that of the parent peptide YD and the linear peptide YD-P9 with a similar sequence. Therefore, the in vitro activity of the linear peptide YD-P9 was further enhanced after cyclization of its head and tail.

[0146] Example 10 investigated the therapeutic effects of cyclic peptide CYP9 and parent peptide YD on improving pulmonary fibrosis in mice.

[0147] (1) Test substances: parent peptide YD and cyclic peptide CYP9.

[0148] (2) Forty male C57BL / 6J mice (6 weeks old, weighing about 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 5 groups of 8 mice each.

[0149] Groups: sham surgery group (Saline), model group (BLM), and drug administration groups: YD (0.2 mg / kg), cyclic peptide CYP9 (0.2 mg / kg), and cyclic peptide CYP9 (0.02 mg / kg).

[0150] (3) The pulmonary fibrosis model was established in the same way as in Example 8.

[0151] (4) Mice in the treatment group, sham surgery group and model group were injected intraperitoneally with 100 μL of PBS once a day. Mice in the YD treatment group were injected intraperitoneally with 100 μL of polypeptide YD solution (dissolved in PBS) once a day at a dose of 0.2 mg / kg. Mice in the CYP9 treatment group were injected intraperitoneally with 100 μL of CYP9 solution (dissolved in PBS) once a day at a dose of 0.2 or 0.02 mg / kg. The samples were collected after 21 consecutive days of treatment.

[0152] Figure 12 The graph shows the expression levels of α-SMA, Collagen I, Fibronectin, E-cadherin, and Vimentin proteins in the lung tissue of mice in each group 21 days after drug administration, along with the statistical analysis of their relative expression levels. It can be seen 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, while the level of E-cadherin protein, an epithelial cell marker, was significantly increased, indicating that the test substance has a good effect on improving pulmonary fibrosis.

[0153] Figure 13 The gene expression of Collagen I, Fibronectin, E-cadherin, and Vimentin in the lung tissue of mice in each group 21 days after drug administration was shown. 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, while the gene level of the epithelial cell marker E-cadherin was significantly increased, indicating that the test substance has a good effect on improving pulmonary fibrosis.

[0154] The results above show that 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.

[0155] Figure 14 The results of H&E and immunohistochemical staining of lung tissue sections from mice in each group 21 days after administration in Example 10 are shown.

[0156] The method used is as follows:

[0157] H&E staining: Paraffin sections were baked in a 60℃ oven for 1 h. Dewaxing and hydration: Xylene I (20 min) → Xylene II (20 min) → Anhydrous ethanol I (2 min) → Anhydrous 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 time). Staining: Hematoxylin staining (3 min) → Rinse twice with tap water (2 min each time) → Differentiation with 0.5% hydrochloric acid alcohol (2 s) → Rinse thoroughly with tap water → Blue with dilute ammonia water (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 → clear with xylene twice (2 min each time) → after air drying, seal with neutral resin.

[0158] Immunohistochemistry: Paraffin sections were baked in a 60℃ oven for 1 hour. Dewaxing and hydration: Xylene I (20 min) → Xylene II (20 min) → Anhydrous ethanol I (2 min) → Anhydrous 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 time). Staining: Hematoxylin staining (3 min) → Rinse twice with tap water (2 min each time). Antigen retrieval → Endogenous peroxidase inhibitor (10 min) → Primary antibody (4℃ overnight) → Reaction enhancement solution (20 min) → Enhancement of US standard goat anti-rabbit IgG polymer (20 min) → DAB staining → Hematoxylin counterstaining → Rinse 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 → clear with xylene twice (2 min each time) → after air drying, seal with neutral resin.

[0159] H&E staining results showed that, compared with the sham-operated group, the BLM group mice exhibited abnormal lung tissue structure, with significant thickening of alveolar septa and narrowing of alveolar cavities, and inflammatory cell infiltration was observed in the lung tissue. Treatment with cyclic peptide CYP9 and parent peptide YD reduced the degree of structural damage and significantly decreased inflammatory infiltration in the lung tissue of mice, demonstrating significant therapeutic and ameliorative effects. Figure 14 Immunohistochemical staining results showed that α-SMA, Collagen I and Fibronectin were highly expressed in the lungs of the BLM group. After 21 days of treatment, the expression level decreased significantly, indicating that the pulmonary fibrosis of mice was treated and improved. The therapeutic effect of cyclic peptide CYP9 was better than that of parent peptide YD.

[0160] Example 11 compares the serum stability of parent peptide YD, YD linear analogue and cyclic peptide CYP9.

[0161] Test substances: YD, YD linear analogues and CYP9.

[0162] Blood was collected from C57BL / 6J mice after anesthesia. The mixture was allowed to stand at room temperature for 30 min, then centrifuged at 5000 rpm for 30 min. The supernatant serum was collected, and a 10 mM test solution was thoroughly mixed with the serum at a ratio of 1:4. The mixture was then incubated at 37°C and timed. At different time points, 70 μL samples were taken, and an equal volume of ice-cold acetonitrile was quickly added to precipitate the protein. After thorough vortexing, the mixture was centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was collected, filtered, and eluted with a gradient of 5%-95% acetonitrile / deionized water for 30 min using a C18 reverse-phase analytical column. The absorption peak at 220 nm was detected. 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.

[0163] Figure 15 The content curves of the parent peptide YD and its linear analogues in serum are shown. The half-life of parent peptide YD in serum is only 21.31 min. The half-life of YD-P5 after sequence truncation is even shorter, but the stability is improved after end protection. In particular, YD-P9, after replacing the D-glutamate at the enzymatic hydrolysis site of YD, has an in vitro half-life of 7.975 h, which is nearly 23 times that of YD.

[0164] Figure 16 The curve shows the concentration of cyclic peptide CYP9 in serum. The half-life of cyclic peptide CYP9 in serum is 70.17 h. Compared with the linear analog YD-P9, the in vitro stability of CYP9 is nearly 10 times improved.

[0165] Example 12 compares the pharmacokinetics of parent peptide YD, linear YD analogues, and cyclic peptide CYP9.

[0166] Test substances: YD, YD linear analogues and CYP9.

[0167] C57BL / 6J mice were injected with 2.5 mg / kg of the test substance via the tail vein. Blood samples were collected from the eyeballs at different time points. Centrifuge tubes were pre-washed with 200 μL of 2 mg / kg heparin sodium and centrifuged at 15,000 rpm for 10 min at 4°C. 200 μL of the supernatant plasma was collected and mixed with 800 μL of ice-cold methanol by vortexing. The mixture was centrifuged at 15,000 rpm for 30 min at 4°C. 700 μL of the supernatant was collected, concentrated, and the precipitate was resuspended in 200 μL of 5% acetonitrile solution. The mixture was centrifuged at 15,000 rpm for 5 min at 4°C. The supernatant was then collected for LC-MS analysis.

[0168] Figure 17 The in vivo pharmacokinetic curves show that YD was completely undetectable in plasma at 20 min. The concentrations of the linear analog YD-P9 and cyclic peptide CYP9 in vivo were much higher than those of YD. The half-life of cyclic peptide CYP9 was 0.21 h, which was nearly double that of YD-P9 (0.11 h). There was also more drug residue in plasma at 1 h, indicating that the duration of action and concentration of cyclic peptide CYP9 in vivo were significantly improved compared to YD and the linear analog YD-P9.

[0169] In conjunction with Examples 7, 8, 9 and 10, structural optimization of the YD linear analogue improved its stability and in vitro activity, but did not significantly enhance its in vivo activity. Cyclization endowed CYP9 with higher stability and membrane permeability, and prolonged its in vivo half-life, which may help improve the durability and efficacy of the prepared antifibrotic drug.

[0170] 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 variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A cyclic peptide CYP9, characterized in that, The structure is as follows: Lys-Gly-Val-dGlu-Gly-Pro-Asp-CONH2, wherein, The first lysine terminal amino group and the seventh aspartic acid side chain carboxyl group are cyclized by 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, The 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 drug for preventing or treating pulmonary fibrosis.

6. A medicine for preventing or treating a pulmonary fibrosis disease, characterized by, The drug takes the cyclic peptide CYP9 according to claim 1 as the main component.

7. A medicine for preventing or treating a pulmonary fibrosis disease, characterized by, The drug takes the linear peptide YD-P9 according to claim 2 as the main component.

8. The medicament for preventing or treating pulmonary fibrosis disease according to claim 6 or 7, characterized by, The drug further comprises a pharmaceutically acceptable carrier or excipient.

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