DR8 cyclic peptide analogue and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a DR8 cyclic peptide analogue and application thereof. The structure of the DR8 cyclopeptide analogue is R1-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2, in the structural formula, R1 is a chain alkyl group with 1 to 30 carbon atoms, and a fourth amino acid and an eighth amino acid are subjected to cyclization through the chain alkyl group. The biological activity of the DR8 cyclic peptide analogue is researched, and an in-vitro cell model and an in-vivo animal model of renal fibrosis are constructed to evaluate indexes such as in-vitro anti-fibrosis activity, inhibition effect and in-vivo treatment effect of the DR8 cyclic peptide analogue. And the cytotoxicity, serum stability and pharmacokinetics of the DR8 cyclopeptide analogue are researched. Results show that compared with the polypeptide DR3penA, the DR8 cyclic peptide analogue obtained by the invention has better activity indexes and stability in all aspects, and can be used for preparing medicines for preventing renal fibrosis diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a DR8 cyclic peptide analog and application thereof. Background Art

[0002] Renal fibrosis is an important driver and pathological manifestation of the development of chronic kidney disease. The main manifestation of renal fibrosis is the deposition of extracellular matrix in the renal interstitium, which leads to renal scarring and decreased or even loss of renal function, and eventually develops into end-stage renal disease. However, its treatment methods are limited and the mortality rate is high. Although the underlying mechanisms of renal fibrosis have been comprehensively studied, there is no approved treatment for clinical use. Therefore, the development of new and effective drugs to treat renal fibrosis is of great significance in clinical practice.

[0003] DR8 (DHNNPQIR) is an antioxidant peptide extracted from rapeseed protein, which can remove reactive oxygen species in cells and reduce the damage of free radicals to the body. Existing technology uses DR8 to treat pulmonary fibrosis and renal fibrosis. The results show that DR8 can effectively improve pulmonary and renal fibrosis. The activity and stability of DR3penA, an analogue obtained by designing and modifying DR8, are significantly improved. However, DR3penA still has problems such as short half-life in vivo, low bioavailability, and the efficacy needs to be improved. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a DR8 cyclic peptide analog.

[0005] Another object of the present invention is to provide a method for preparing the DR8 cyclic peptide analog.

[0006] Another object of the present invention is to provide the application of the DR8 cyclic peptide analog.

[0007] According to a specific embodiment of the present invention, a DR8 cyclic peptide analog has the following general structure:

[0008] R1-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2, where

[0009] R1 is a C1-C30 alkyl group,

[0010] The 4th amino acid and the 8th amino acid are cyclized via a chain alkyl group.

[0011] The DR8 cyclic peptide analogue of the present invention overcomes the problems of poor stability and low bioavailability of traditional polypeptides by modifying the N-terminal chain alkyl acid of DR8 and the cyclization strategy of the 4th amino acid and the 8th amino acid, while showing significant anti-fibrosis activity, excellent serum stability and low cost advantages.

[0012] Preferably, R1 is a C6-C16 alkyl group, or R1 is a C10-C12 alkyl group, or R1 is a C12-C18 alkyl group.

[0013] Preferably, the amino acid at position 4 and the amino acid at position 8 are cyclized by a substituted or unsubstituted C4-C12 chain alkyl group, or the amino acid at position 4 and the amino acid at position 8 are cyclized by a substituted or unsubstituted C4-C10 chain alkyl group, or the amino acid at position 4 and the amino acid at position 8 are cyclized by a substituted or unsubstituted C4-C8 chain alkyl group.

[0014] Preferably, the cysteine ​​at position 4 and the cysteine ​​at position 8 are cyclized via a butane chain, a hexane chain or an octane chain.

[0015] "Alkanyl" in the sense of the present invention is preferably the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, cyclopentyl, n-hexyl, neohexyl, n-heptyl, n-neutetyl, n-nonyl, n-decyl, etc.

[0016] In the present invention, the term "substituted or unsubstituted" means that the group is substituted or unsubstituted with one or more substituents selected from hydrogen, deuterium, a halogen atom, a hydroxyl group, a nitrile group, a nitro group, an amino group, an amidine group, a hydrazine group, a hydrazone group, a carboxyl group or a carboxylate thereof, a sulfonic acid group or a sulfonate thereof, a phosphoric acid group or a phosphate thereof, an aliphatic hydrocarbon group or an alkyl group, or is substituted or unsubstituted with a substituent in which two or more of the substituents listed above are linked together.

[0017] In many cases, the general substituent is selected from the group consisting of deuterium, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aralkyl, alkoxy, aryloxy, amino, silanyl, alkenyl, cycloalkenyl, heteroalkenyl, alkynyl, aryl, heteroaryl, acyl, carboxylic acid, ether, ester, nitrile, isonitrile, thio, sulfinyl, sulfonyl, and phosphino.

[0018] "Halogen", "halogen atom" in the sense of the present invention are used interchangeably and refer to fluorine, chlorine, bromine or iodine.

[0019] The aliphatic hydrocarbon or alkyl substituted by the above groups is preferably the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, cyclopentyl, n-hexyl, neohexyl, cyclohexyl, n-heptyl, cycloheptyl, n-octyl, cyclooctyl, 2-ethylhexyl, cyclohexenyl, heptenyl, cycloheptenyl, octenyl or cyclooctenyl.

[0020] According to a specific embodiment of the present invention, the DR8 cyclic peptide analog has the following structure:

[0021]

[0022] The present invention connects cysteine ​​through a hexane chain, thereby changing the steric hindrance of the polypeptide, forming a hydrophobic microenvironment around itself as a hydrophobic side chain, promoting the interaction with non-polar free radicals and unsaturated fatty acids, increasing the interaction between the peptide and the lipid target or the membrane lipid bilayer, and facilitating the polypeptide to enter the cell and play a better role. In addition, the present invention introduces a hexane chain between the two cysteines at the 4th and 8th positions to form a ring, which can effectively prevent the recognition of proteases in the body, has higher drug stability, can prolong the half-life in the body, and helps to improve the durability and efficacy of the anti-fibrosis drug prepared.

[0023] The DR8 cyclic peptide analogues screened out through chemical modification in the present invention have better biological activity against renal fibrosis diseases; compared with the polypeptide DR3penA 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.

[0024] The present invention also provides the use of the above DR8 cyclic peptide analogs in preparing medicines for treating fibrotic diseases.

[0025] Preferably, the fibrotic disease includes renal fibrotic disease, pulmonary fibrotic disease, and hepatic fibrotic disease.

[0026] The DR8 cyclic peptide analogues of the present invention have an improving effect on renal fibrosis diseases and can be used to directly or indirectly treat diseases characterized by renal fibrosis. Among them, renal fibrosis diseases include obstructive nephropathy, diabetic nephropathy, nephrosclerosis, nephritis, renal tumors, and renal fibrosis diseases caused by various factors such as bad living habits, adverse drug reactions, and infection by pathogenic microorganisms.

[0027] In addition to the improvement effect on renal fibrosis, the DR8 cyclic peptide analogues of the present invention also have similar improvement effects on pulmonary fibrosis and liver fibrosis.

[0028] The present invention also provides a medicine for treating fibrotic diseases, wherein the medicine has a DR8 cyclic peptide analog as a main component and further comprises a pharmaceutically acceptable carrier or excipient.

[0029] Another object of the present invention is to provide a composition, which contains the above-mentioned DR8 cyclic peptide analogs and also includes a pharmaceutically acceptable carrier or excipient. The carrier can be a carrier that can reduce drug degradation and loss and reduce side effects, such as micelles, microemulsions, gels, etc.; the excipient can be a material added to make the drug into a suitable dosage form, such as a buffer, an excipient for lyophilization, etc. The liquid formula is generally a buffer, an isotonic solution and an aqueous solution. Specifically, a pharmaceutical composition is added with a DR8 cyclic peptide analog as an active ingredient and a pharmaceutically acceptable carrier and / or excipient is added to support the drug composition.

[0030] The drug 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. According to the administration method adopted, the polypeptide analog drug combination of the present invention can be supported in various suitable dosage forms, which contain at least one effective amount of the polypeptide analog of the present invention and at least one pharmaceutically acceptable pharmaceutical carrier.

[0031] Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions that can be used for injection. The pharmaceutical composition containing the polypeptide analogs of the present invention can be prepared into a solution or a lyophilized powder for parenteral administration. Before use, a suitable solvent or other carrier can be added to reconstitute the powder. The liquid formula is generally PBS buffer, physiological saline isotonic solution, and aqueous solution.

[0032] The dosage of the polypeptide analogs 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, and the like.

[0033] The present invention provides a method for preparing the above-mentioned DR8 cyclic peptide analog, comprising the following steps:

[0034] Step 1: using the Fmoc solid phase synthesis method, synthesizing the linear peptide resin corresponding to the DR8 cyclic peptide analog from the carboxyl end to the amino end;

[0035] Step 2: cutting the linear peptide resin synthesized in step 1, removing the full protection of the peptide chain, and collecting the cutting solution containing the DR8 linear analog;

[0036] Step 3: purifying the cleavage solution obtained in step 2 to obtain a DR8 linear analog;

[0037] Step 4: The DR8 linear analogue obtained in step 3 was dissolved in 250 μL 1M Tris-HCl (pH=9.0), and the same molar amount of the thianthrenium salt was dissolved in 750 μL DMSO; mixed, and stirred at room temperature for 1 h to react;

[0038] Step 5: Purify the reaction solution obtained in step 4 to obtain the DR8 cyclic peptide analog.

[0039] Preferably, in step 1, the resin used in the Fmoc solid phase synthesis method is MBHA resin.

[0040] Preferably, in step 2, the polypeptide resin synthesized in step 1 is cut using a cutting solution, wherein the cutting solution consists of TFA:Tis:H2O:βME in a volume ratio of 90:6.5:2.5:1.

[0041] Beneficial effects of the present invention:

[0042] The present invention provides a DR8 cyclic peptide analog, and studies the biological activity of the DR8 cyclic peptide analog, including constructing an in vitro cell model of renal fibrosis and an in vivo animal model to evaluate the in vitro anti-fibrotic activity and inhibitory effect of the DR8 cyclic peptide analog and the in vivo therapeutic effect, and also includes studying the cytotoxicity, serum stability, and pharmacokinetics of the DR8 cyclic peptide analog. The results show that compared with the polypeptide DR3penA, the DR8 cyclic peptide analog obtained by the present invention has better activity indicators and stability in all aspects. This proves that the DR8 cyclic peptide analog of the present invention can be used to prepare a drug for preventing renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings 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 creative work.

[0044] Figure 1 The survival rate of mouse embryonic fibroblasts affected by DR8 cyclic peptide analogs.

[0045] Figure 2 The results of Western blot detection of the effects of DR8 cyclic peptide analogs on the expression of Fibronectin and α-SMA in the mouse embryonic fibroblast model and the corresponding statistical graphs.

[0046] Figure 3 The results of Western blot detection of the effect of cyclic peptide 79 on the expression of Fibronectin and α-SMA in the mouse embryonic fibroblast model and its corresponding statistical graph.

[0047] Figure 4The results of Western blot detection of the effect of cyclopeptide 79 on the expression of Collagen I, MMP2 and Vimentin in the mouse embryonic fibroblast model and its corresponding statistical graph.

[0048] Figure 5 The mRNA expression levels of Fibronectin, CollagenI, α-SMA, TGF-β1, Vimentin and MMP2 in the mouse embryonic fibroblast model detected by qPCR were detected by cyclic peptide 79.

[0049] Figure 6 is the effect of cyclic peptide 79 on the survival rate of human renal tubular epithelial cells.

[0050] Figure 7 The results of Western blot detection of the effect of cyclic peptide 79 on the expression of Fibronectin and α-SMA in the human renal tubular epithelial cell model and its corresponding statistical graph.

[0051] Figure 8 The results of Western blot detection of the effect of cyclic peptide 79 on the expression of Collagen I, MMP2, E-cadherin and Vimentin in the human renal tubular epithelial cell model and its corresponding statistical graph.

[0052] Fig. 9 The effect of cyclic peptide 79 on the mRNA expression levels of Fibronectin, Collagen I, α-SMA, E-cadherin, Vimentin and MMP2 in the human renal tubular epithelial cell model detected by qPCR.

[0053] Fig.10 The results of Western blot detection of the effects of cyclic peptide 79 and polypeptide DR3penA on the expression of Fibronectin and α-SMA in the human renal tubular epithelial cell model and their corresponding statistical graphs.

[0054] Fig.11 The results of Western blot detection of the effects of cyclic peptide 79 and polypeptide DR3penA on the expression of Fibronectin, Collagen I, E-cadherin, Vimentin, MMP2 and α-SMA in mouse kidneys and their corresponding statistical graphs.

[0055] Fig.12 The results of qPCR detection of the effects of cyclic peptide 79 and polypeptide DR3penA on the expression of Fibronectin, CollagenI, TGF-β1, α-SMA, E-cadherin, Vimentin and MMP2 in mouse kidneys.

[0056] Fig.13 The results of Western blot detection of the effects of cyclopeptide 79 and Captopril on the expression of Fibronectin, Collagen I, E-cadherin, MMP2, Vimentin and α-SMA in mouse kidneys and their corresponding statistical graphs.

[0057] Fig.14 The results of qPCR detection of the effects of cyclotide 79 and Captopril on the expression of Fibronectin, Collagen I, TGF-β1, α-SMA, E-cadherin, Vimentin and MMP2 in mouse kidneys.

[0058] Fig.15 These are the results of H&E and immunohistochemical staining of kidney tissue pathological sections of mice in each group 7 days after administration of cyclotide 79.

[0059] Fig.16 These are the in vitro serum stability results of cyclic peptide 79 and polypeptide DR3penA.

[0060] Fig.17 The in vivo half-life results of cyclic peptide 79 and polypeptide DR3penA. DETAILED DESCRIPTION

[0061] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0062] The specific meanings of the abbreviations used in the present invention are as follows:

[0063] dAla (abbreviated a) is D-alanine, Ile (abbreviated I) is isoleucine, Cys (abbreviated C) is cysteine, Pro (abbreviated P) is proline, Gln (abbreviated Q) is glutamine, Arg (abbreviated R) is arginine, Asn (abbreviated N) is asparagine, dAsn (abbreviated n) is D-asparagine, Asp (abbreviated D) is aspartic acid, dAsp (abbreviated d) is D-aspartic acid, His (abbreviated H) is histidine, and α-(4-pentenyl)-Ala (abbreviated X) is α-(4-pentenyl)-alanine.

[0064] Materials and Methods

[0065] 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.

[0066] 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. The data were expressed as mean ± SD, and significant differences were analyzed using one-way ANOVA 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.

[0067] Example 1 Design and Synthesis of Cyclic Peptide

[0068] Step 1: The linear peptide was synthesized by Fmoc solid phase synthesis from the carboxyl end to the amino end. The amino acid sequence is as follows:

[0069] C12-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2.

[0070] The synthesis specifically comprises the following steps:

[0071] (1) Activation of MBHA resin: Weigh the resin, add appropriate amount of DCM and swell on a shaker for 30 min. After draining, add DMF to wash three times, each time for 3 min.

[0072] (2) Indole test: Add ninhydrin: pyridine: phenol = 1:2:1 indole test reagent into a test tube, dip a small amount of resin into it, and boil it in boiling water for 3 minutes. If the indole test result is yellow, it indicates that the resin is normal;

[0073] (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;

[0074] (4) Indole test: Add ninhydrin: pyridine: phenol = 1:2:1 indole test reagent to a test tube, take a small amount of resin into the test tube, and boil it in boiling water for 3 minutes. If the indole test result is blue-purple, it proves that the protecting group has been removed;

[0075] (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 hour, drain the solvent, wash with DMF for 3 minutes, and repeat 3 times;

[0076] (6) Indole test: If the indole test result is yellow, it indicates that the condensation is successful;

[0077] (7) Repeat steps (3), (4), (5), and (6) in the order of the amino acid sequence in the compound (Cys, dAla, Gln, Pro, Cys, Asn, His, dAsp, C12 chain alkyl acid) until all the amino acids in the compound to be synthesized are condensed to obtain a polypeptide resin.

[0078] Step 2: Cleavage of polypeptide chain: After peptide chain synthesis, deprotection was performed, and the resin was washed with DCM for 3 min, and repeated twice; washed with methanol for 3 min; washed with DCM for 3 min; and finally washed with methanol for 3 min, and repeated twice. The resin was dried to powder.

[0079] Prepare a cutting solution of TFA: Tis: H2O: βME = 90: 6.5: 2.5: 1 (V / V / V / V), add it to the resin for cutting for 3 hours, and 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°C refrigerator overnight, and then freeze-dry it to obtain a crude peptide.

[0080] Step 3: Preparation and purification of linear peptides:

[0081] (1) Weigh about 40 mg of crude peptide and dissolve it in deionized water to prepare a peptide solution. After it is completely dissolved, use a 0.45 μm filter to remove insoluble substances in the peptide solution, and add 0.1% TFA to the elution solvent (acetonitrile and deionized water);

[0082] (2) High performance liquid chromatography using a C18 reverse preparative column with 100% acetonitrile flushed until the spectrum is stable, 30% acetonitrile initial concentration for equilibrium, and injection after setting the flow gradient;

[0083] (3) After injection, the absorption peak at 220 nm was detected, the main peak was collected, and finally the collected components were marked, sealed with plastic wrap, pierced with vent holes, frozen in a -80°C refrigerator overnight, and then freeze-dried to obtain a linear polypeptide compound;

[0084] (4) After freeze-drying, a small amount of the compound was dissolved and eluted with 5%-95% acetonitrile / deionized water on a C18 reverse analytical column for 30 min. The purity was statistically analyzed by integrating the peak area of ​​the chromatogram at 220 nm. 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 1068.4831 and the theoretical m / z was 1068.4892, confirming that the synthesized and purified product was the target product.

[0085] Step 4: Preparation and purification of cyclic peptide:

[0086] (1) The linear peptide obtained in step 3 was dissolved in 250 μL 1M Tris-HCl (pH=9.0), and the same molar amount of thianthrenium salt was dissolved in 750 μL DMSO; mixed and stirred at room temperature for 1 h to react;

[0087] Structure of thianthrenium salts.

[0088] (2) The obtained reaction solution was purified by high performance liquid chromatography to obtain a DR8 cyclic peptide analogue having the following structure:

[0089]

[0090] (3) After freeze-drying, a small amount of the compound was dissolved and eluted with 5%-95% acetonitrile / deionized water on a C18 reverse analytical column for 30 min. The purity was statistically calculated by integrating the peak area of ​​the chromatogram at 220 nm. The purity of the compound was >95%, and 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 1150.5771, and the theoretical m / z was 1150.5748, confirming that the synthesized and purified product was the target product.

[0091] Table 1 Structure, theoretical m / z, measured m / z and purity of the cyclic peptide synthesized in Example 1

[0092]

[0093] The compounds in Table 2 were prepared by similar synthetic methods to those in the above examples:

[0094] Table 2 Structure, theoretical m / z, measured m / z and purity of the compounds synthesized with reference to Example 1

[0095]

[0096] Example 2 Evaluation of cytotoxicity of DR8 cyclic peptide analogs on NIH-3T3 cells as test substances

[0097] NIH-3T3 cell line was selected to study and observe the cytotoxicity of cyclic peptide DR8 (labeled as cyclic peptide 79) and the polypeptide analogs shown in Table 2 on NIH-3T3 cells.

[0098] NIH-3T3 cells were inoculated into 96-well plates and cultured with DMEM medium containing 10% FBS + 1% double antibody at 37°C, 5% CO2 for 24 h. 0, 10, 20, 40, 80 and 160 μM of the test substances were added. After incubation for 48 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 an enzyme reader.

[0099] Figure 1The results show the survival rate of NIH-3T3 cells. The results show that in the concentration range of 0-160 μM, after the DR8 cyclic peptide analogs 30, 36, 31, 37, 66 and 67 acted on the cells, the cell survival rate remained basically the same as that of the control group, with no significant difference, indicating that there was no significant toxicity to the cells. In the concentration range of 10-160 μM, after the DR8 cyclic peptide analogs 68, 69, 70, 71 and 83 acted on the cells, the cell survival rate remained significantly lower than that of the control group, indicating that there was greater toxicity to the cells. In the concentration range of 0-40 μM, after the cyclic peptide DR8 acted on the cells, the cell survival rate remained basically the same level, with no significant difference, indicating that the cyclic peptide DR8 had no significant toxicity to the cells at this concentration. At concentrations of 80 and 160 μM, the cyclic peptide DR8 significantly reduced the cell survival rate, indicating that the cyclic peptide DR8 above 80 μM was significantly toxic to the cells.

[0100] Example 3 Detection of the activity of DR8 cyclic peptide analogs on fibrosis of NIH-3T3 cells in vitro

[0101] The experiment used NIH-3T3 cell line to study and observe the effects of cyclic peptide DR8 (labeled as cyclic peptide 79) and the polypeptide analogs shown in Table 2 on the expression of fibronectin and α-smooth muscle actin (α-SMA) in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0102] NIH-3T3 cells were seeded in 6-well plates using DMEM (Gibco) medium containing 10% FBS and 1% double antibody, cultured at 37°C, 5% CO2 for 24 h, replaced with serum-free medium and cultured for 12 h, and then 5 ng / mL TGF-β1 and 10 μM cyclic peptide DR8 or the polypeptide analogs shown in Table 2 were added to the cells for 48 h, and then the total cell protein was extracted. The protein expression levels of Fibronectin and α-SMA were detected by Western blot.

[0103] Control group (labeled as Ctrl): TGF-β1 and DR8 cyclic peptide analogs were not added to the culture medium;

[0104] Induction group (labeled with TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0105] DR8 cyclic peptide analogue group (labeled as cyclic peptide 79 or the names of the polypeptide analogues shown in Table 2): 5 ng / mL TGF-β1 and 10 μM DR8 cyclic peptide analogue (cyclic peptide 79 prepared in Example 1 and the polypeptide analogues shown in Table 2) were added to the culture medium.

[0106] Figure 2After NIH-3T3 cells were treated with TGF-β1 and DR8 cyclic peptide analogs, the protein expression of Fibronectin, α-SMA and GAPDH was detected by Western blot, and the relative expression levels of Fibronectin and α-SMA proteins were analyzed.

[0107] Depend on Figure 2 It can be seen that the cyclic peptide DR8 and the polypeptide analogs shown in Table 2 can significantly inhibit the expression of Fibronectin and α-SMA proteins. Among them, the activity of analog 67 cyclized by hexane chain is better than that of analog 66 and 37 cyclized by butane chain and octane chain. Figure 1 It was found that the activity of analogs 68, 69, 70 and 71 increased significantly with the extension of the fatty acid chain, while the cytotoxicity also increased significantly. The cyclic peptide DR8 synthesized by combining the cyclization strategy with the fatty acid modification strategy has good activity and low cytotoxicity. The anti-fibrosis effect will be further investigated using the cyclic peptide DR8 as an example.

[0108] Example 4 Investigating the activity of DR8 cyclic peptide analogs on fibrosis of NIH-3T3 cells in vitro

[0109] The experiment used NIH-3T3 cell line to study and observe the effect of cyclic peptide DR8 (labeled as cyclic peptide 79) on the expression of Fibronectin, α-SMA, type I collagen (Collagen I), matrix metalloproteinase (MMP2) and vimentin (Vimentin) in NIH-3T3 cells induced by TGF-β1.

[0110] NIH-3T3 cells were seeded in 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% double antibody, cultured at 37°C, 5% CO2 for 24 h, replaced with serum-free medium and cultured for 12 h. After 5 ng / mL TGF-β1 and 1, 2, 5 and 10 μM cyclic peptide DR8 were added to the cells for 48 h, and then the total protein and total RNA of the cells were extracted. Western blot was used to detect the protein expression levels of Fibronectin, α-SMA, Collagen I, MMP2 and Vimentin, and qPCR was used to detect the mRNA expression levels of Fibronectin, Collagen I, α-SMA, TGF-β1, Vimentin and MMP2.

[0111] Control group (labeled as Ctrl): TGF-β1 and cyclic peptide DR8 were not added to the culture medium;

[0112] Induction group (labeled with TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0113] Cyclic peptide DR8 group (labeled as cyclic peptide 79): 5 ng / mL TGF-β1 and 1, 2, 5 and 10 μM cyclic peptide DR8 (cyclic peptide 79 prepared in Example 1) were added to the culture medium.

[0114] Figure 3 After TGF-β1 and cyclic peptide DR8 acted on NIH-3T3 cells, the protein expression results of Fibronectin, α-SMA and GAPDH were detected by Western blot, and the relative expression levels of Fibronectin and α-SMA proteins were analyzed. Figure 3 It can be seen that TGF-β1 stimulates NIH-3T3 cells to express Fibronectin and α-SMA proteins in large quantities, and the cyclic peptide DR8 can inhibit the expression of Fibronectin and α-SMA proteins in a dose-dependent manner, and the effect is strongest at 2μM.

[0115] Figure 4 After TGF-β1 and cyclic peptide DR8 acted on NIH-3T3 cells, the protein expression results of Vimentin, MMP2, Collagen I and GAPDH were detected by Western blot, and the relative expression levels of Vimentin, MMP2 and Collagen I proteins were analyzed. Figure 4 It can be seen that TGF-β1 stimulates NIH-3T3 cells to express Vimentin, MMP2 and Collagen I proteins in large quantities, and cyclic peptide DR8 can effectively inhibit the expression of Vimentin, MMP2 and Collagen I proteins.

[0116] Figure 5 After TGF-β1 and cyclic peptide DR8 acted on NIH-3T3 cells, qPCR was used to detect the mRNA expression levels of Fibronectin, CollagenI, α-SMA, TGF-β1, Vimentin and MMP2. Figure 5 It can be seen that TGF-β1 stimulated a significant increase in the mRNA levels of Fibronectin, Collagen I, α-SMA, TGF-β1, Vimentin and MMP2 in NIH-3T3 cells, and cyclic peptide DR8 could effectively inhibit the increase in the mRNA levels of these genes.

[0117] Depend on Figure 3 , Figure 4 and Figure 5 It can be seen that the cyclic peptide DR8 obtained in Example 1 can inhibit the expression of fibrotic proteins and genes in NIH-3T3 cells induced by TGF-β1 in a dose-dependent manner.

[0118] Example 5 Investigating the cytotoxicity of DR8 cyclic peptide analogs to HK-2 cells

[0119] HK-2 cell line was selected to study and observe the cytotoxicity of cyclic peptide DR8 on HK-2 cells. HK-2 cells were inoculated into 96-well plates and cultured at 37°C, 5% CO2 for 24 hours with DMEM / F12 medium containing 10% FBS + 1% double antibody. 0, 10, 20, 40, 80 and 160 μM cyclic peptide DR8 were added. After incubation for 24 hours, 10 μL CCK-8 solution was added and incubated for 2 hours. The absorbance at a wavelength of 450 nm was detected by an enzyme reader.

[0120] Figure 6 is the survival rate of HK-2 cells. The results show that within the concentration range of 0-160 μM, after the cyclic peptide DR8 acts on the cells, the cell survival rate remains basically at the same level with no significant difference, indicating that the cyclic peptide DR8 has no significant toxicity to the cells.

[0121] Example 6 Investigating the activity of DR8 cyclic peptide analogs on HK-2 cell fibrosis in vitro

[0122] HK-2 cell line was selected to study and observe the effect of cyclic peptide DR8 on the expression of Fibronectin, Collagen I, E-cadherin, MMP2, Vimentin and α-SMA in HK-2 cells induced by TGF-β1. HK-2 cells were inoculated in 6-well plates with DMEM / F12 (Gibco) medium containing 10% FBS and 1% double antibody, cultured at 37°C, 5% CO2 for 24 hours, replaced with serum-free medium for 12 hours, and then 5ng / mL TGF-β1 and 1, 2, 5 and 10μM cyclic peptide DR8 were added to the cells for 24 hours to extract total cell protein and total RNA, Western blot was used to detect the protein expression levels of Fibronectin, α-SMA, Collagen I, MMP2, E-cadherin and Vimentin, and qPCR was used to detect the mRNA expression levels of Fibronectin, Collagen I, α-SMA, E-cadherin, Vimentin and MMP2.

[0123] Control group (labeled as Ctrl): TGF-β1 and cyclic peptide DR8 were not added to the culture medium;

[0124] Induction group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0125] Cyclic peptide DR8 group (labeled as cyclic peptide 79): 5 ng / mL TGF-β1 and 1, 2, 5 and 10 μM cyclic peptide DR8 (cyclic peptide 79) were added to the culture medium.

[0126] Figure 7 After TGF-β1 and cyclic peptide DR8 acted on HK-2 cells, the protein expression results of Fibronectin, α-SMA and GAPDH were detected by Western blot, and the relative expression levels of the proteins were analyzed. Figure 7 It can be seen that TGF-β1 stimulates HK-2 cells to express Fibronectin and α-SMA proteins in large quantities, and the cyclic peptide DR8 can inhibit the expression of Fibronectin and α-SMA proteins in a dose-dependent manner, and the effect is strongest at 5μM.

[0127] Figure 8 After TGF-β1 and cyclic peptide DR8 acted on HK-2 cells, the protein expression results of Collagen I, MMP2, E-cadherin, Vimentin and GAPDH were detected by Western blot, and the relative expression levels of the proteins were analyzed. Figure 8 It can be seen that TGF-β1 stimulates HK-2 cells to express Vimentin, MMP2 and Collagen I proteins in large quantities, and cyclic peptide DR8 can effectively inhibit the expression of Vimentin, MMP2 and Collagen I proteins, and upregulate the expression of E-cadherin.

[0128] Fig. 9 After TGF-β1 and cyclic peptide DR8 acted on HK-2 cells, qPCR was used to detect the mRNA expression levels of Fibronectin, Collagen I, α-SMA, E-cadherin, Vimentin and MMP2. Fig. 9 It can be seen that TGF-β1 stimulated the mRNA levels of Fibronectin, Collagen I, α-SMA, Vimentin and MMP2 in HK-2 cells to increase significantly, and the cyclic peptide DR8 can effectively inhibit the increase in the mRNA levels of these genes and upregulate the mRNA level of E-cadherin.

[0129] Depend on Figure 7 , Figure 8 and Fig. 9 It can be seen that the cyclic peptide DR8 obtained in Example 1 can inhibit the expression of fibrotic proteins and genes in HK-2 cells induced by TGF-β1 in a dose-dependent manner.

[0130] Example 7 Investigating the in vitro inhibitory effects of DR8 cyclic peptide analogs and DR3penA on fibrosis

[0131] HK-2 cell line was selected to study and observe the effects of the test substances DR8 cyclic peptide analogs and DR3penA on the expression levels of fibrosis-related proteins in different cells.

[0132] HK-2 cells were plated in 6-well plates and cultured in DMEM / F12 medium containing 10% FBS + 1% double antibody at 37°C, 5% CO2 for 24 hours, then changed to serum-free medium and cultured for 12 hours. After 5 ng / mL TGF-β1 and 1 or 5 μM test substances were added to HK-2 cells for 24 hours, the total cell protein was extracted, and the protein expression levels of Fibronectin and α-SMA were detected by Western blot.

[0133] Control group (labeled as Ctrl): TGF-β1 and test substances were not added to the culture medium;

[0134] Induction group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the culture medium;

[0135] In the experimental group (labeled with the name of the test substance, DR3penA or cyclic peptide 79), 5 ng / mL TGF-β1 and 1 or 5 μM of the test substance were added to the culture medium.

[0136] Fig.10 The results of protein expression of α-SMA, Fibronectin and GAPDH were detected by Western blot after TGF-β1 and the test substances acted on HK-2 cells, and the relative protein expression levels were analyzed. The results showed that the relative expression levels of the two proteins Fibronectin and α-SMA in HK-2 cells acted on by cyclic peptides DR8 and DR3penA in the experimental group were significantly lower than those in the TGF-β1-induced group, indicating that cyclic peptides DR8 and DR3penA can significantly inhibit the effect of TGF-β1-induced renal fibrosis in HK-2 cells. Compared with DR3penA, the relative protein expression levels of α-SMA and Fibronectin were significantly lower after 1 and 5 μM cyclic peptide DR8 acted on HK-2 cells, indicating that cyclic peptide DR8 has stronger anti-fibrotic activity than DR3penA.

[0137] Example 8 Investigating the therapeutic effects of DR8 cyclic peptide analogs and DR3penA on improving renal fibrosis in mice

[0138] (1) Test substances: cyclic peptides DR8 and DR3penA.

[0139] (2) Thirty male C57BL / 6J mice (6 weeks old, weighing approximately 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 6 groups, with 5 mice in each group.

[0140] Groups: sham operation group (Sham), model group (UUO), cyclopeptide 79 group (0.5 mg / kg), and DR3penA group (0.5 mg / kg).

[0141] (3) Establishment of renal fibrosis model: UUO (unilateral ureteric obstruction, abbreviated as UUO) induced renal fibrosis in mice

[0142] After intraperitoneal injection of 1% sodium pentobarbital solution for anesthesia, the left ureter of the model mice was separated, the ureter near the renal pelvis was ligated with 4-0 suture, and the ureter in the middle of the ligation was cut; the renal fibrosis model induced by UUO is characterized by hydronephrosis, thinning of the renal cortex, dilatation of renal tubules, and infiltration of inflammatory cells. After the UUO model mice were treated with the test substance, their kidneys were pathologically analyzed. In the sham operation group, only the left ureter was separated without ligation.

[0143] (4) Cyclic peptide DR8 group: model mice were intraperitoneally injected with 0.5 mg / kg cyclic peptide DR8 daily;

[0144] DR3penA group: model mice were intraperitoneally injected with 0.5 mg / kg compound DR3penA daily;

[0145] UUO group: mice were model mice; mice in the UUO group and the sham operation group were intraperitoneally injected with the same amount of sterile PBS solution. Samples were collected 7 days after administration.

[0146] Fig.11 The expression of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin, Vimentin and GAPDH proteins in the kidney tissue of each group of mice after 7 days of administration and the statistical diagram of the relative expression obtained by analysis show that in UUO model mice, after treatment with the test substance, the level of fibrosis-related proteins in the kidney was significantly reduced, indicating that the test substance has a good effect in improving renal fibrosis. In addition, the therapeutic effect of cyclic peptide DR8 is significantly better than that of DR3penA.

[0147] Fig.12 The gene expression of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin and Vimentin in the kidney tissue of each group of mice after 7 days of administration shows that in UUO model mice, after treatment with the test substance, the gene level of fibrosis-related proteins in the kidney was significantly reduced, indicating that the test substance has a good effect in improving renal fibrosis. In addition, the therapeutic effect of cyclic peptide DR8 is significantly better than that of DR3penA.

[0148] Example 9 Investigating the therapeutic effects of DR8 cyclic peptide analogs and captopril on improving renal fibrosis in mice

[0149] (1) Test substance: cyclic peptide DR8.

[0150] (2) Thirty-six male C57BL / 6J mice (6 weeks old, weighing approximately 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 6 groups, with 6 mice in each group.

[0151] Grouping: sham operation group (Sham), model group (UUO), captopril positive control group (Captopril, 20 mg / kg), medication groups: cyclic peptide DR8 (0.05 mg / kg), cyclic peptide DR8 (0.25 mg / kg), cyclic peptide DR8 (1.25 mg / kg).

[0152] (3) The renal fibrosis model was established in the same manner as in Example 7.

[0153] (4) In the drug administration group, the model mice were intraperitoneally injected with 0.05, 0.25 or 1.25 mg / kg of cyclic peptide DR8 daily; in the Captopril group, the model mice were intraperitoneally injected with 20 mg / kg of captopril; the UUO group was the model mice, and the UUO group and the sham operation group were intraperitoneally injected with the same amount of sterile PBS solution. The samples were collected 7 days after drug administration.

[0154] Fig.13 The expression of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin, Vimentin and GAPDH proteins in the kidney tissue of each group of mice 7 days after administration and the statistical graph of relative expression obtained by analysis show that in UUO model mice, the levels of fibrosis-related proteins in the kidneys were significantly reduced after treatment with cyclic peptide DR8, indicating that cyclic peptide DR8 has a good effect in improving renal fibrosis.

[0155] Fig.14 The gene expression of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin and Vimentin in the kidney tissue of each group of mice after 7 days of administration shows that in the UUO model mice, after treatment with cyclic peptide DR8, the level of fibrosis-related proteins in the kidney was significantly reduced, indicating that cyclic peptide DR8 has a good effect in improving renal fibrosis. From the above results, it can be seen that cyclic peptide DR8 can inhibit and treat UUO-induced renal fibrosis and renal injury, and the therapeutic effect is significantly better than that of the positive drug captopril. In addition, the dosage of cyclic peptide DR8 (1.25 mg / kg) is lower than that of captopril (20 mg / kg), which has a significant protective effect.

[0156] Fig.15 These are the results of H&E and immunohistochemical staining of kidney tissue pathological sections of each group of mice 7 days after administration in Example 7.

[0157] H&E staining: paraffin sections were baked in a 60°C 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 with warm water twice (2 min each). Staining: hematoxylin drip staining (3 min) → rinse with tap water twice (2 min each) → 0.5% hydrochloric acid alcohol differentiation (2 s) → tap water washing → blueing with dilute ammonia water (5-10 s) → tap water washing → 5% eosin staining (5 s). Dehydration and transparency: 95% ethanol I (15 s) → 95% ethanol II (15 s) → anhydrous ethanol I (15 s) → anhydrous ethanol II (15 s) → air-drying → xylene transparency twice (2 min each) → after air-drying, seal the slide with neutral gum.

[0158] Immunohistochemistry: Paraffin sections were baked in a 60°C 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 with warm water twice (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 color development → hematoxylin counterstaining → tap water washing. Dehydration and transparency: 95% ethanol I (15 s) → 95% ethanol II (15 s) → anhydrous ethanol I (15 s) → anhydrous ethanol II (15 s) → air drying → xylene transparency twice (2 min each) → air drying, neutral gum sealing.

[0159] Fig.15 The results of H&E staining showed that compared with the sham operation group, the kidneys in the UUO group were significantly damaged, with pathological changes such as tubular dilatation, tubular epithelial cell shedding, and inflammatory cell infiltration. After 7 days of treatment with cyclic peptide DR8, tubular dilatation was significantly reduced, and inflammatory cell infiltration was reduced, showing a significant therapeutic and improvement effect. Fig.15 The results of immunohistochemical staining showed that Fibronectin and α-SMA were expressed in large quantities in the kidneys of the UUO group. After 7 days of treatment with cyclic peptide DR8, the expression level decreased significantly, indicating that the renal fibrosis of mice was improved by treatment.

[0160] Example 10 Comparison of serum stability of DR8 cyclic peptide analogs and polypeptide DR3penA

[0161] Blood was collected from C57BL / 6J mice after anesthesia, and the samples were allowed to stand at room temperature for 30 minutes, centrifuged at 5000rpm / min for 30 minutes, and the upper serum was collected and frozen at -80℃. The frozen serum was thawed and incubated at 37℃ for 2 minutes before the experiment. An appropriate amount of the test substance was weighed and prepared into a mother solution with a concentration of 10mM. 855μL of mouse serum and 45μL of 10mM test substance were fully mixed and incubated at 37℃. 70μL of samples were taken at different time points (0h, 3h, 6h, 10h, 24h, 48h, 72h), and 70μL of ice acetonitrile was immediately added. After vortexing, the samples were allowed to stand on ice for 10 minutes. The samples were centrifuged at 13000g for 15 minutes, and the supernatant was collected.

[0162] C18 reverse analytical column was used for gradient elution with 5%-95% acetonitrile / deionized water for 30 min. 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] Fig.16 Figure 2 is the content curve of cyclic peptide DR8 and polypeptide DR3penA in serum. The content of cyclic peptide DR8 in serum is still higher than 50% after 24 hours, and the half-life is 27.03 hours, while the half-life of DR3penA is only 2.91 hours, indicating that the stability of cyclic peptide DR8 is significantly better than that of DR3penA.

[0164] Example 11 Comparison of the pharmacokinetics of DR8 cyclic peptide analogs and DR3penA polypeptide

[0165] C57BL / 6J mice were injected with 2.4mmol / kg cyclic peptide DR8 (2.8mg / kg) and DR3penA (2.5mg / kg) through the tail vein, and blood was collected from the eyeball at different time points (0min, 5min, 10min, 20min, 30min, 45min, 60min, 120min, 240min), mixed with 200μL 2mg / kg heparin sodium, centrifuged at 4℃2000rpm for 10min, 100μL of upper plasma was taken, 300μL of methanol was added and vortexed, centrifuged at 4℃15000rpm for 10min, 300μL of supernatant was concentrated, and the precipitate was resuspended in 100μL 5% acetonitrile solution, centrifuged at 4℃15000rpm for 5min, and the supernatant was aspirated for LC-MS detection.

[0166] Fig.17 Figure 2 is the drug-time curve of cyclic peptide DR8 and DR3penA in vivo. The concentration of cyclic peptide DR8 in vivo is much higher than that of DR3penA, with a half-life of 1.08h, while the half-life of DR3penA is 0.19h, indicating that the action time and concentration of cyclic peptide DR8 in vivo are significantly improved compared with DR3penA.

[0167] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A DR8 cyclic peptide analogue, characterized in that: Its structure is as follows: R1-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2, where R1 is a C1-C30 alkyl group, The 4th amino acid and the 8th amino acid are cyclized via a chain alkyl group.

2. The DR8 cyclic peptide analogue according to claim 1, characterized in that R1 is a C6-C16 alkyl group.

3. The DR8 cyclic peptide analogue according to claim 1, characterized in that The 4th amino acid and the 8th amino acid are cyclized via a substituted or unsubstituted C4-C12 chain alkyl group.

4. A DR8 cyclic peptide analogue, characterized in that: Its structure is as follows:

5. Use of the DR8 cyclic peptide analogue according to any one of claims 1 to 4 in the preparation of a medicament for treating fibrotic diseases.

6. The use according to claim 5, characterized in that The fibrotic diseases include renal fibrotic diseases, pulmonary fibrotic diseases, and hepatic fibrotic diseases.

7. A drug for treating fibrotic diseases, characterized in that: The drug has the DR8 cyclic peptide analogue described in any one of claims 1 to 4 as a main component.

8. The drug for treating fibrotic diseases according to claim 7, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

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