Application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs

Abstract

This invention relates to the field of pharmaceutical technology, providing the application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs. This invention applies short peptide derivatives and their self-assembled materials to the preparation of anti-inflammatory drugs, effectively inhibiting inflammation-related cytokines with good safety and broad application prospects. The results of the examples show that the short peptide derivatives of this invention can effectively inhibit the release of pro-inflammatory cytokines, especially Nap-F, which exhibits the best anti-inflammatory effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation, as a natural protective response of organisms to infection, injury, and other harmful stimuli, is a complex and crucial physiological mechanism. This process involves alterations in vascular permeability, recruitment of immune cells, and the release of inflammatory mediators, all aimed at eliminating irritants and promoting the repair of damaged tissues. Although inflammation may be beneficial to the body in the short term, its long-term chronicity is closely associated with a variety of serious diseases, including cardiovascular disease, cancer, autoimmune diseases, and neurodegenerative diseases.

[0003] Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and other immunosuppressants are commonly used medications for treating inflammation. However, these drugs are often accompanied by numerous adverse reactions and potential drug resistance issues. Therefore, the search for safer and more effective anti-inflammatory drugs has become an important direction in medical research. Summary of the Invention

[0004] In view of this, the present invention provides the application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs. The present invention has found that short peptide derivatives with structures shown in Formulas I to III can effectively inhibit inflammation-related cytokines and have good safety profiles, thus showing broad application prospects in the preparation of anti-inflammatory drugs.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs, wherein the short peptide derivatives are one or more compounds with structures shown in Formulas I to III: Formula I; Formula II; Formula III.

[0006] Preferably, the self-assembly material of the short peptide derivative is a short peptide derivative hydrogel.

[0007] Preferably, the preparation method of the short peptide derivative hydrogel includes the following steps: The short peptide derivative and buffer solution are mixed, and the pH of the resulting mixture is adjusted to 6-8. The mixture is then heated to dissolve the short peptide derivative, and after cooling, the short peptide derivative hydrogel is obtained.

[0008] Preferably, the buffer solution is PBS buffer.

[0009] Preferably, the reagent used to adjust the pH value of the mixture is a sodium carbonate solution.

[0010] Preferably, the anti-inflammatory drug comprises an active ingredient and pharmaceutically acceptable excipients; the active ingredient is a short peptide derivative and / or its self-assembled material.

[0011] Preferably, the dosage form of the anti-inflammatory drug includes injection, powder, granules, capsules, or tablets.

[0012] Preferably, the anti-inflammatory drug is administered by injection or oral administration.

[0013] This invention provides the application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs. The short peptide derivatives are one or more compounds with structures shown in Formulas I to III. This invention has found that the short peptide derivatives with structures shown in Formulas I to III can effectively inhibit inflammation-related cytokines and have good safety profiles, showing broad application prospects in the preparation of anti-inflammatory drugs. The results of the examples show that the short peptide derivatives of this invention can effectively inhibit the release of pro-inflammatory cytokines such as IL-β1, TNF-α, IL-6, IL-18, IL-23, and IL-8. In particular, the short peptide derivative with the structure shown in Formula II (Nap-F) exhibits the best anti-inflammatory effect. Attached Figure Description

[0014] Figure 1 The results show the secretion levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-18, IL-23, IL-8) in LPS-stimulated THP-1 cells after treatment with short peptide derivative hydrogels. Detailed Implementation

[0015] This invention provides the application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs, wherein the short peptide derivatives are one or more compounds with structures shown in Formulas I to III: Formula I; Formula II; Formula III.

[0016] In this invention, the short peptide derivative with the structure shown in Formula I is Nap-G. D F, denoted as Nap-GF; the short peptide derivative of the structure shown in Formula II is Nap- D F, denoted as Nap-F; the short peptide derivative of the structure shown in Formula III is Ac- D F D F is denoted as Ac-FF.

[0017] The present invention does not have any special requirements for the preparation method of the short peptide derivatives with the structures shown in Formulas I to III. The synthesis can be carried out by methods well known to those skilled in the art. In the specific embodiments of the present invention, the FMOC-solid phase synthesis method is used.

[0018] In this invention, the self-assembly material of the short peptide derivative is a short peptide derivative hydrogel.

[0019] In this invention, the preparation method of the short peptide derivative hydrogel preferably includes the following steps: The short peptide derivative and buffer solution are mixed, and the pH of the resulting mixture is adjusted to 6-8. The mixture is then heated to dissolve the short peptide derivative, and after cooling, the short peptide derivative hydrogel is obtained.

[0020] In this invention, the buffer solution is preferably PBS buffer, and the pH value of the PBS buffer is preferably 6-8, specifically 7. The volume ratio of the short peptide derivative to the buffer solution is preferably 1 mg: 300-500 μL. In a specific embodiment of this invention, it is preferable to first add the short peptide derivative to 400 μL of PBS buffer, adjust the pH value to the target value, and then use PBS buffer to bring the volume to 500 μL. The reagent used to adjust the pH value of the mixture is preferably sodium carbonate solution. In this invention, sodium carbonate solution is preferably used to adjust the pH value of the mixture to 7. In this invention, the heating is defined as heating to boiling. This invention does not have special requirements for the heating time, as long as the short peptide derivative is completely dissolved. The cooling is specifically cooling to room temperature. During the heating process, the short peptide derivative undergoes self-assembly, and a hydrogel is formed upon cooling.

[0021] In this invention, the anti-inflammatory drug preferably includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient is the short peptide derivative and / or its self-assembled material described in the above scheme; this invention does not have special requirements for the pharmaceutically acceptable excipients, and any excipients well known to those skilled in the art can be used, specifically pharmaceutically acceptable carriers, excipients, etc.

[0022] In this invention, the dosage form of the anti-inflammatory drug includes injection, powder, granules, capsules or tablets; the administration method of the anti-inflammatory drug is injection or oral administration.

[0023] In this invention, the anti-inflammatory drug may specifically be an anti-arthritis drug, an anti-enteritis drug, or an anti-hepatitis drug.

[0024] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] The sources of the formulations involved in the following examples are as follows: Culture medium, RMPI 1640, purchased from Thermo Fisher Scientific, sterile; Fetal bovine serum, purchased from Thermo Fisher Scientific, was sterile; 2-cl-Trt resin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an activity of 1.2 mmol / mL; N,N-Diisopropylethylamine (hereinafter referred to as DIEPA), purchased from Sigma-Aldrich, 99% purity; Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (hereinafter referred to as HBTU), purchased from Jier Biochemical (Shanghai) Co., Ltd., with a purity of 98%; Trifluoroacetic acid (hereinafter referred to as TFA), purchased from Sigma-Aldrich, with a purity of 99%; Triisopropylsilane (hereinafter referred to as TIS) was purchased from Sigma-Aldrich and has a purity of 99%. Naphthaleneacetic acid (hereinafter referred to as Nap) and amino acids were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., with a purity of 98%. LEGENDplex™ Multifactor Flow Cytometry Kit was purchased from BioLegend. LPS (TLR4 activator) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Acetic acid (hereinafter referred to as Ac) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. All other reagents were commercially available analytical grade reagents.

[0026] Comparative Example 1 The short peptide derivative Nap- was prepared using the FMOC-solid phase synthesis method. D F D F (structure as follows, denoted as Nap-FF) The specific steps are as follows: 1) Weigh 0.5 mmol of 2-cl-Trt resin into a solid-phase synthesizer, add 10 mL of anhydrous dichloromethane (hereinafter referred to as DCM), place it on a shaker and shake for 5 min to allow the 2-cl-Trt resin to fully swell; 2) Use a syringe bulb to remove the DCM completely from the solid-phase synthesizer containing 2-cl-Trt resin; 3) Dissolve 0.75 mmol of Fmoc-protected amino acid (phenylalanine) in 10 mL of anhydrous DCM, add 0.75 mmol of DIEPA, then transfer to the solid-phase synthesizer mentioned above, add another 0.75 mmol of DIEPA, and react at room temperature for 1 h. 4) Sealing: Remove the reaction solution in the solid-phase synthesizer with a rubber bulb, then wash with 10 mL of anhydrous DCM for 1 min each time, for a total of 5 washes. Add 20 mL of the prepared anhydrous DCM-DIEPA-methanol mixed solution (the volume ratio of anhydrous DCM, DIEPA and methanol is 17:1:2) and react at room temperature for 10 min. 5) Remove the reaction solution from the solid-phase synthesizer using a rubber bulb. First, wash with anhydrous DCM, using 10 mL of DCM each time for 1 min, for a total of 5 washes. Then wash with N,N-dimethylformamide (hereinafter referred to as DMF), using 10 mL of DMF each time for 1 min, for a total of 5 washes. Add 10 mL of DMF containing 20% ​​(volume percentage) piperidine and react for 25 min. Then react with 10 mL of DMF containing 20% ​​(volume percentage) piperidine for 5 min. Then wash with DMF, using 10 mL of DMF each time for 1 min, for a total of 5 washes. Proceed to the next step of the reaction. 6) Add 1 mmol of the second Fmoc-protected amino acid (phenylalanine), 1.5 mmol of HBTU, 2 mmol of DIEPA and 10 mL of LDMF, and add the prepared solution to the solid-phase synthesizer mentioned above. React for 2 hours. 7) Repeat steps 5) and 6) to add naphthaleneacetic acid for end capping; then wash 5 times with DMF and 5 times with dichloromethane before proceeding to the next reaction. 8) Add 10 mL of the TFA-TIS-H2O mixed solution (where the volume fraction of TFA is 95%, the volume fraction of TIS is 2.5%, and the volume fraction of H2O is 2.5%) to the solid-phase synthesizer mentioned above, react for half an hour, cut the product off the 2-cl-Trt resin, concentrate under vacuum, remove the solvent, and obtain the crude product, which is then separated and purified by HPLC.

[0027] The structural characterization data are as follows: 1H NMR (400 MHz, DMSO) δ 8.28 (dd, J = 24.1, 8.2 Hz, 2H), 7.88 – 7.71(m, 3H), 7.59 (s, 1H), 7.51 – 7.41 (m, 2H), 7.28 – 7.10 (m, 11H), 4.59 (td, J = 9.7, 4.1 Hz, 1H), 4.46 (td, J = 8.2, 5.4 Hz, 1H), 3.53 (dd, J = 34.4, 14.1Hz, 2H), 3.00 (dtd, J = 22.6, 13.9, 7.0 Hz, 3H), 2.74 (dd, J = 13.7, (10.1 Hz, 1H). [M+1] = 481.2 Take 1mg Nap- D F D F was placed in a glass bottle, 400 μL of PBS solution (pH=7.0) was added, the pH was adjusted to 7.0 with sodium carbonate solution, and the volume was brought up to 500 μL with PBS solution. The mixture was heated to boiling to completely dissolve the compound, and then cooled to room temperature to obtain the short peptide derivative hydrogel.

[0028] Example 1 The short peptide derivative Nap-G was prepared using the FMOC-solid phase synthesis method. D F (Formula I, Nap-GF) is prepared using the same method as in Example 1, except that the types of amino acids used are changed, and it is prepared according to the structure shown in Formula I.

[0029] Formula I.

[0030] The structural characterization data are as follows: 1 H NMR (400 MHz, DMSO) δ 8.26 (t, J = 5.6 Hz, 1H), 8.15 (d, J = 8.0Hz, 1H), 7.84 (ddd, J = 31.5, 17.8, 15.0 Hz, 4H), 7.54 – 7.38 (m, 3H), [M+1] = 391.2. Take 1 mg Nap-GD F was placed in a 1.5 mL glass bottle, 400 μL of PBS solution (pH=7.0) was added, the pH was adjusted to 7.0 with sodium carbonate solution, and the volume was brought up to 500 μL with PBS solution. The mixture was heated to boiling to completely dissolve the compound, and then cooled to room temperature to obtain the short peptide derivative hydrogel.

[0031] Example 2 The short peptide derivative Nap- was prepared using the FMOC-solid phase synthesis method. D F (Formula II, Nap-F) is prepared using the same method as in Example 1, except that the types of amino acids used are changed, and it is prepared according to the structure shown in Formula II.

[0032] Formula II.

[0033] The structural characterization data are as follows: 1 H NMR (400 MHz, DMSO) δ 8.43 (d, J = 8.1 Hz, 1H), 7.89 – 7.74 (m,3H), 7.66 (s, 1H), 7.52 – 7.42 (m, 2H), 7.31 – 7.12 (m, 6H), 4.46 (td, J [M + 1] = 334.1. Take 1mg Nap- D F was placed in a 1.5 mL glass bottle, 400 μL of PBS solution (pH=7.0) was added, the pH was adjusted to 7.0 with sodium carbonate solution, and the volume was brought up to 500 μL with PBS solution. The mixture was heated to boiling to completely dissolve the compound, and then cooled to room temperature to obtain the short peptide derivative hydrogel.

[0034] Example 3 Short peptide derivatives Ac- were prepared using the FMOC-solid phase synthesis method. D F D F(Ac-FF) was prepared using the same method as in Example 1, except that the types of amino acids used were changed and acetic acid was used as the end-capping group. It was prepared according to the structure shown in Formula III.

[0035] Formula III.

[0036] The structural characterization data are as follows: 1 H NMR (400 MHz, DMSO) δ 8.21 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 8.6Hz, 1H), 7.39 – 7.07 (m, 11H), 4.56 – 4.40 (m, 2H), 3.32 (s, 1H), 3.12 – 2.86(m, 3H), 2.67 (dd, J = 13.8, 10.2 Hz, 1H). [M+1] =355.2. Take 1mg Ac- D F D F was placed in a 1.5 mL glass bottle, 400 μL of PBS solution (pH=7.0) was added, the pH was adjusted to 7.0 with sodium carbonate solution, and the volume was brought up to 500 μL with PBS solution. The mixture was heated to boiling to completely dissolve the compound, and then cooled to room temperature to obtain the self-assembled material.

[0037] Test Example 1 THP-1 cell culture: (1) THP-1 cells were quickly removed from the cryogenic storage and immediately placed in a preheated water bath to thaw; transferred to a cell handling table and prepared RPMI 1640 medium containing 10% FBS and 1% penicillin antibiotics; centrifuged to remove the cryopreservation solution, resuspended the cells in fresh medium, and then seeded into pre-labeled culture containers.

[0038] (2) After 48 hours, change the medium, centrifuge to collect cells, resuspend them and transfer them to a new container, and replenish the culture medium.

[0039] THP-1 cell stimulation: After passaged THP-1 cells twice, the cells were collected by centrifugation, resuspended in fresh culture medium, and seeded into 24-well plates at 5 × 10⁶ cells per well. 5 Cells were cultured in a cell culture incubator for 24 hours. Then, 10 ng LPS was added to each well for 24 hours of stimulation. Next, 50 μg of short peptide derivative hydrogel was added to the culture medium. After incubation for 24 hours, the supernatant was collected by centrifugation for cytokine detection. The LPS-treated group without the short peptide derivative hydrogel served as the positive control, while the group without both the short peptide derivative hydrogel and LPS served as the negative control.

[0040] Cytokine detection: (1) Preparation of cytokine standards 1) Transfer a bottle of lyophilized cytokine standard beads to a 15 mL conical centrifuge tube and label it as the highest concentration standard (5000 pg / mL). 2) Dilute the standard with 2 ml of Assay Diluent and allow to equilibrate at room temperature for at least 15 minutes; 3) Gently mix the standard sample with the nozzle; do not vortex or violently shake it. 4) Take 9 flow cytometer tubes of 12×75mm and label them with serial dilution ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 respectively; 5) Add 300 μL of Assay Diluent to each tube; 6) Starting with the highest concentration standard, add 300 μL of serial dilution solution to each tube to serially dilute the standard (take 300 μL of solution from the highest concentration standard tube to the 1:2 tube, mix by pipetting, then take 300 μL of solution from the 1:2 tube to the 1:4 tube, mix by pipetting, and so on, up to the 1:256 tube).

[0041] (2) Hybrid microspheres 1) Determine the number of experimental samples (including all standards, negative controls and test samples).

[0042] 2) Each type of capture microsphere needs to be vortexed for 5-15 seconds before mixing.

[0043] 3) Take an appropriate amount of capture microspheres at a rate of 10 μL / sample, mix all microspheres in a single flow cytometer tube, and label it "mixed microspheres".

[0044] 4) Mix thoroughly by vortexing.

[0045] (3) Suspended microspheres 1) Centrifuge the mixed microspheres at room temperature and 200 g for 5 minutes; 2) Carefully aspirate the supernatant and add the same volume of serum enhancement solution as the "mixed microspheres" in step 1), then vortex to mix. 3) Incubate at room temperature in the dark for 30 minutes.

[0046] (4) Sample incubation 1) Take the fully vortexed and resuspended "mixed microspheres" and add 50 μL to each experimental tube; 2) Add 50 μL of PE-labeled cytokine detection antibody to all experimental tubes; 3) Incubate at room temperature in the dark for 3 hours.

[0047] (5) On-machine testing 1) Add 1 mL of washing buffer to each tube to wash the sample, and centrifuge at 200 g for 5 minutes; 2) Carefully aspirate or gently discard the supernatant, and add 300 μL of washing buffer to each tube to resuspend the cells.

[0048] (6) Data Analysis The THP-1 cytokine secretion data were calculated based on the standard curve using the LEGENDplex™ analysis software.

[0049] Figure 1 The results show the secretion levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-18, IL-23, IL-8) in LPS-stimulated THP-1 cells after treatment with short peptide derivative hydrogels. Figure 1 The results showed that Ac-FF, Nap-F, and Nap-GF short peptide derivative hydrogels inhibited LPS-induced pro-inflammatory cytokine secretion to varying degrees, with Nap-F showing the best anti-inflammatory effect by significantly inhibiting the secretion of each pro-inflammatory factor. In contrast, NaP-FF in Comparative Example 1 had virtually no inhibitory effect on pro-inflammatory cytokine secretion, demonstrating that differences in the amino acid composition, terminal modifications, and sequence length of short peptides significantly affect their interaction with cells. This invention, by controlling the structure of short peptides, can specifically regulate the anti-inflammatory activity of short peptide derivatives, enabling them to effectively intervene in LPS-induced inflammatory response pathways and achieve better anti-inflammatory effects.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of short peptide derivatives and their self-assembled materials in the preparation of anti-inflammatory drugs, characterized in that, The short peptide derivative is one or more of the compounds with structures shown in Formulas I to III: Equation I; Formula II; Formula III.

2. The application according to claim 1, characterized in that, The self-assembly material of the short peptide derivative is a short peptide derivative hydrogel.

3. The application according to claim 2, characterized in that, The preparation method of the short peptide derivative hydrogel includes the following steps: The short peptide derivative and buffer solution are mixed, and the pH of the resulting mixture is adjusted to 6-8. The mixture is then heated to dissolve the short peptide derivative, and after cooling, the short peptide derivative hydrogel is obtained.

4. The application according to claim 3, characterized in that, The buffer solution is PBS buffer.

5. The application according to claim 3, characterized in that, The reagent used to adjust the pH value of the mixture is sodium carbonate solution.

6. The application according to claim 1, characterized in that, The anti-inflammatory drug comprises an active ingredient and pharmaceutically acceptable excipients; the active ingredient is a short peptide derivative and / or its self-assembled material.

7. The application according to claim 1, characterized in that, The dosage forms of the anti-inflammatory drugs include injections, powders, granules, capsules, or tablets.

8. The application according to claim 1, characterized in that, The anti-inflammatory drug is administered by injection or orally.

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