Application of PMRFamide short peptide in anti-inflammatory

Abstract

This invention belongs to the field of biomedical technology and relates to the application of PMRFamide short peptide in anti-inflammation. The amino acid sequence of PMRFamide is Pro-Met-Arg-Phe. This invention provides a method for preparing this peptide, including sequentially linking amino acids using a solid-phase synthesis method, obtaining a crude peptide through resin cleavage and precipitation, followed by purification by reversed-phase high-performance liquid chromatography and lyophilization to finally obtain a high-purity PMRFamide peptide product. This method is mature, has a high yield, and is easy to scale up for production. This invention reveals the significant effect of PMRFamide short peptide in inhibiting the release of key inflammatory mediators, exhibiting highly efficient and sustained potential anti-inflammatory activity. This tetrapeptide has a simple structure and good biocompatibility, providing a foundation for the development of novel and safe peptide anti-inflammatory drugs and showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of PMRFamide short peptide in anti-inflammatory effects. Background Technology

[0002] As a classic inflammatory mediator, nitrogen (NO) has received widespread attention. Under inflammatory stimulation, activated macrophages activate the transcription of inducible nitric oxide synthase, producing large amounts of NO to mediate the inflammatory response. NO's role in the inflammatory response is like a double-edged sword: under normal physiological conditions, NO exerts an anti-inflammatory effect; however, in chronic inflammation, macrophages are continuously activated, producing large amounts of NO that exert physiological toxicity, further promoting the occurrence and development of inflammation, ultimately leading to damage to tissues and organs. It is closely related to the onset and progression of chronic inflammatory diseases such as arthritis, cardiovascular disease, and diabetes. However, the main target of commonly used anti-inflammatory nonsteroidal anti-inflammatory drugs (NSAIDs) in clinical practice is cyclooxygenase, but these drugs have many side effects and are often accompanied by off-target effects. Currently, there are no ideal anti-inflammatory interventions to alleviate excessive NO production. Therefore, discovering substances that can moderately inhibit excessive NO production during macrophage inflammation and reduce the inflammatory response may provide a breakthrough for the intervention of related chronic inflammatory diseases, and is of great significance for restoring health and improving prognosis.

[0003] RFamide neuropeptides are a family of neuropeptides with a C-terminal phenylalanine amidation structure, widely distributed in invertebrates and vertebrates, and involved in regulating various physiological processes such as pain, stress, feeding, immunity, and inflammatory responses. Among them, the mammalian neuropeptide FF (NPFF) and its analogues, as well as various RFamide peptides from invertebrates such as insects, have been shown to influence the activation of immune cells and the release of inflammatory mediators through G protein-coupled receptor-mediated signaling pathways, exhibiting potential anti-inflammatory activity. However, a series of challenges remain in the process of translating them into clinical applications. (1) Poor stability and short half-life make it easily degraded by proteases in vivo, resulting in low bioavailability and limiting its clinical application scenarios.

[0004] (2) The synthesis is difficult and costly, the solid-phase synthesis efficiency is low, the protection group strategy is complex, the steps are cumbersome and the production cost is high when the production scale is large.

[0005] (3) Insufficient specificity and selectivity: Some anti-inflammatory peptides lack cell or receptor specificity in inhibiting inflammatory signaling pathways, which may affect normal immune function or trigger non-target effects.

[0006] (4) Some exogenous peptides may induce immune responses, and long-term use may pose potential safety risks.

[0007] (5) Some peptide drugs often require complex formulation processes (such as liposomes and nanoparticle encapsulation) to improve stability and targeting, which further increases the research and development and production costs. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides the application of PMRFamide short peptide in anti-inflammatory applications. The amino acid sequence of PMRFamide in this invention is proline (Pro)-methionine (Met)-arginine (Arg)-phenylalanine (Phe), and its preparation method is as follows: Step 1: Mix 2-chlorotriphenylmethyl chloride resin (2-Cl-Trt-Cl) with dichloromethane (DCM) at a loading of 0.4-0.6 mmol / g, swell at 23-27℃ for 50-70 min, filter to remove the filtrate, and obtain swollen 2-Cl-Trt-Cl resin.

[0009] Fmoc-Phe-OH was mixed with DCM and then mixed with the swollen 2-Cl-Trt-Cl resin. N,N-diisopropylethylamine (DIPEA) was added dropwise at a rate of 2-3% total volume / min under nitrogen atmosphere at 23-27℃. After the addition was complete, the mixture was stirred at 100-200 rpm for 4-6 h. The filtrate was removed by filtration. The product was washed alternately with DCM and methanol, and then unreacted sites were blocked with blocking solution. After 25-35 min, the mixture was filtered, and the precipitate was the resin loaded with Fmoc-Phe-.

[0010] Preferably, the ratio of 2-Cl-Trt-Cl, Fmoc-Phe-OH, DIPEA, resin-swellable DCM, amino acid-dissolved DCM, DCM washing, methanol washing, and sealing liquid is (4-6) kg:(4-5) kg:(2-3) L:(150-250) L:(40-60) L:(200-250) L:(150-200) L:(80-120) L.

[0011] Most preferably, the sealing liquid is a methanol-DCM solution with a methanol to DCM volume ratio of 1:(4-5).

[0012] Step 2: Repeat the following steps to sequentially connect arginine, methionine, and proline to obtain resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe: a) Mix the resin obtained in the previous step with piperidine-DMF solution, stir at 24-26℃ and 100-150 rpm for 15-25 min, filter to remove the filtrate, and wash the product with DMF for later use. b) Mix the next Fmoc-protected amino acid to be linked, 1-hydroxy-7-azabenzotriazole (HOAt), and DMF, then add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), and stir at 60-90 rpm for 30-60 s to obtain a pre-activated solution; c) Mix the pre-activated solution with the product from a), adjust the pH to 8-9 with DIPEA, stir at 120-150 rpm for 50-70 min, filter, remove the filtrate, and wash the precipitate with DMF.

[0013] Preferably, the ratio of the resin, piperidine-DMF solution, pre-activated solution and DMF used during washing is 1 kg: (8-12) L: (1-2) L: (10-20) L.

[0014] Preferably, the ratio of Fmoc-protected amino acids, HOAt, HATU and DMF in the pre-activated solution is (0.8-1.2) mol:(0.8-1.2) mol:(0.8-1.2) mol:(0.2-0.4) L.

[0015] Preferably, the volume ratio of piperidine to DMF in the piperidine-DMF solution is (2-3):(7-8).

[0016] Step 3: Wash the resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe sequentially with DCM and methanol, dry at 20-25℃ and ≤50 Pa for 8-12 h, then mix with the cutting solution, stir at 23-27℃ and 60-80 rpm for 2.5-3.5 h, filter, wash the product with TFA, and combine all the filtrates and washing solutions to obtain the peptide dispersion.

[0017] Preferably, the ratio of resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe, cutting fluid, DCM washing amount, methanol washing amount and TFA washing amount is (4-6) kg:(45-55) L:(200-250) L:(150-170) L:(1-3) L.

[0018] Preferably, the cutting fluid is TFA, deionized water and triisopropylsilane (TIS) in a volume ratio of (90-100):(2-3):(2-3).

[0019] Step 4: Under stirring at 300-400 rpm, add the peptide dispersion dropwise to methyl tert-butyl ether (MTBE) at a rate of 1-2% of total volume / min. Let it stand at 0-4℃ for 1.5-2.5 h, remove the supernatant, centrifuge at 10000-20000 rpm for 10-20 min, wash the precipitate with MTBE, and dry it at 20-35℃ for 12-24 h to obtain PMRFamide peptide.

[0020] Preferably, the volume ratio of the peptide dispersion, the amount of MTBE mixture, and the amount of MTBE washing is 1:(8-10):(2-4).

[0021] Step 5: Mix PMRFamide peptide with TFA aqueous solution and purify using preparative reversed-phase high-performance liquid chromatography. Chromatographic conditions: C18 bonded silica gel as packing material, mobile phase A as TFA aqueous solution, mobile phase B as TFA-acetonitrile solution, gradient elution, the proportion of mobile phase B linearly increases from 10% to 50% within 30 min, detection wavelength is 220 nm, and the main peak fraction is collected.

[0022] The collected main peak fractions were combined and concentrated to 10-20% of the original volume at ≤35℃ and (-0.095)-(-0.085)MPa. Then, a freeze-drying protectant was added, stirred and dissolved, frozen at (-50)-(-45)℃ for 20-24 h, and dried at 20-25℃ for 7-9 h to obtain high-purity PMRFamide peptide.

[0023] Preferably, the ratio of PMRFamide peptide, TFA aqueous solution, and lyophilization protectant is 1 kg:(8-12) L:(40-60) mg. Most preferably, the lyophilization protectant is mannitol.

[0024] Most preferably, the volume concentration of TFA in the TFA aqueous solution is 0.1-0.2%, and the volume concentration of TFA in the TFA-acetonitrile solution is 0.1-0.2%.

[0025] The present invention has the following advantages: (1) PMRFamide exhibits significant NO inhibition at extremely low concentrations (0.001-1 µM), indicating its potential for potent anti-inflammatory effects. Compared to many anti-inflammatory peptides that require higher concentrations to be effective, the high efficacy of PMRFamide means lower dosage, less risk of side effects, and more economical medication costs.

[0026] (2) PMRFamide not only has a rapid onset of action, but also maintains its anti-inflammatory effect for a relatively long period of time. This characteristic is superior to many existing peptide anti-inflammatory agents with short half-lives that require frequent administration, which is beneficial for designing long-acting treatment regimens.

[0027] (3) PMRFamide, as a short peptide containing only four amino acids, has a simple solid-phase synthesis route with mild reaction conditions, high yield, and easy scale-up. The optimized washing, cutting, precipitation, and purification process in this invention can stably obtain high-purity products, overcome the problems of numerous side reactions and difficult purification in the synthesis of long peptides, and significantly reduce production costs and process complexity.

[0028] (4) The PMRFamide sequence is derived from the natural peptide structural framework. Its amino acid composition consists of common components in the body and does not contain non-natural or modified residues. It is expected to have low immunogenicity and good biosafety, making it suitable for further development into an anti-inflammatory therapeutic agent. Attached Figure Description

[0029] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 The anti-inflammatory properties of PMRFamide at different concentrations.

[0031] Figure 2 The anti-inflammatory capacity of PMRFamide at different times. Detailed Implementation

[0032] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1 Raw materials: Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Pro-OH, 2-Cl-Trt-Cl with a loading of 0.5 mmol / g, DCM, DMF, HATU, DIPEA, methanol, HOAt, piperidine-DMF solution (volume ratio 2:8), TFA, TIS, water, MTBE, and anhydrous ethanol.

[0034] The blocking solution consists of methanol and DCM solution in a volume ratio of 1:4.

[0035] Cutting fluid: The volume ratio of TFA, deionized water and TIS is 95:2.5:2.5.

[0036] The volume concentration of TFA in the TFA aqueous solution is 0.15%.

[0037] The volume concentration of TFA in the TFA-acetonitrile solution is 0.15%.

[0038] Preparation method: Step 1: The ratio of 2-Cl-Trt-Cl, Fmoc-Phe-OH, DIPEA, resin swelling DCM, amino acid dissolving DCM, DCM washing, methanol washing, and sealing solution is 5kg:4.5kg:2.5L:200L:50L:225L:160L:100L.

[0039] Mix 2-Cl-Trt-Cl with DCM, swell at 25°C for 60 min, filter to remove the filtrate, and obtain swollen 2-Cl-Trt-Cl resin; Fmoc-Phe-OH was mixed with DCM and then mixed with swollen 2-Cl-Trt-Cl resin. DIPEA was added dropwise at a rate of 2.5% total volume / min under nitrogen atmosphere at 25°C. After the addition was complete, the mixture was stirred at 150 rpm for 5 h. The filtrate was removed by filtration. The product was washed alternately with DCM and methanol, and then unreacted sites were blocked with blocking solution. After 30 min, the mixture was filtered, and the precipitate was the resin loaded with Fmoc-Phe-.

[0040] Step 2: The ratio of resin, piperidine-DMF solution, pre-activation solution, and DMF used during washing is 1 kg: 10 L: 1.5 L: 15 L. The ratio of Fmoc-protected amino acids, HOAt, HATU, and DMF in the pre-activation solution is 1 mol: 1 mol: 1 mol: 0.3 L.

[0041] Repeat the following steps to sequentially connect arginine, methionine, and proline to obtain resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe: a) Mix the resin obtained in the previous step with piperidine-DMF solution, stir at 25°C and 125 rpm for 20 min, filter to remove the filtrate, and wash the product with DMF for later use. b) Mix the next Fmoc-protected amino acid to be linked, HOAt and DMF, then add HATU, stir at 75 rpm for 45 s to obtain a pre-activated solution; c) Mix the pre-activated solution with the product from a), adjust the pH to 8-9 with DIPEA, stir at 135 rpm for 660 min, filter, remove the filtrate, and wash the precipitate with DMF.

[0042] Step 3: The ratio of resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe, cutting fluid, DCM washing volume, methanol washing volume, and TFA washing volume is 5kg:50L:225L:160L:2L.

[0043] Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe was washed sequentially with DCM and methanol, dried at 23°C and 42 Pa for 10 h, then mixed with the cutting fluid, stirred at 25°C and 70 rpm for 3 h, filtered, and the product was washed with TFA. All filtrates and washings were combined to obtain a peptide dispersion.

[0044] Step 4: The volume ratio of peptide dispersion, MTBE mixture, and MTBE washing agent is 1:9:3.

[0045] Under stirring at 350 rpm, the peptide dispersion was added dropwise to MTBE at a rate of 1.5% total volume / min. After standing at 2℃ for 2 h, the supernatant was removed, and the mixture was centrifuged at 15000 rpm for 15 min. The precipitate was washed with MTBE and dried at 27℃ for 28 h to obtain PMRFamide peptide.

[0046] Step 5: The ratio of PMRFamide peptide, TFA aqueous solution and mannitol is 1kg:10L:50mg.

[0047] PMRFamide peptide was mixed with TFA aqueous solution and purified by preparative reversed-phase high-performance liquid chromatography. Chromatographic conditions: C18 bonded silica gel as packing material, TFA aqueous solution as mobile phase A, TFA-acetonitrile solution as mobile phase B, gradient elution, the proportion of mobile phase B linearly increased from 10% to 50% within 30 min, detection wavelength was 220 nm, and the main peak fraction was collected.

[0048] The collected main peak fractions were combined and concentrated to 15% of the original volume at 26℃ and -0.09 MPa. Then, a freeze-drying protectant was added, stirred to dissolve, and frozen at -49℃ for 22 h. After drying at 21℃ for 8 h, high-purity PMRFamide peptides were obtained.

[0049] Experimental Example 1 Experimental procedure: Log-phase mouse monocytes (RAW264.7) were resuspended in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 cells / mL. Add 180 µL of the above cell suspension to each well of a 96-well plate and incubate overnight at 37°C in a 5% CO2 incubator.

[0050] The PMRFamide prepared in Example 1 was dissolved in sterile water to prepare concentration gradients of 0.001 µM, 0.01 µM, 0.1 µM, and 1 µM, respectively. The cell culture supernatant was discarded, and 180 µL of fresh culture medium was added to each well. 20 µL of the above-mentioned peptide solutions of different concentrations were added for pretreatment for 15 min. 20 µL of LPS solution (final concentration 1 µg / mL) was added to each well, and the cells were cultured for another 24 h.

[0051] Cell supernatant was collected, and 100 µL of supernatant was mixed with an equal volume of Griess reagent (1% sulfonamide + 0.1% NEDD + 2.5% H3PO4) in a 96-well plate. The mixture was incubated at room temperature in the dark for 10 min. The absorbance was measured at 540 nm using a microplate reader. The NO concentration was calculated based on the NaNO2 standard curve. The results are shown in the figure. Figure 1 The blank group served as a blank control.

[0052] Experimental Example 2 Experimental Procedure: Similar to Example 1, RAW264.7 cells were seeded in 96-well plates and cultured overnight. 0.01 µM PMRFamide solution was added to each well for pretreatment for 15 min. After adding 20 µL LPS (final concentration 1 µg / mL), the supernatant was collected at 12 h, 24 h, and 48 h. Similar to Example 1, the NO concentration in the supernatant at each time point was detected using the Griess reagent method. The results are shown below. Figure 2 .

[0053] LPS group: LPS only; blank group: no LPS, no medication.

[0054] Depend on Figure 1-2 It can be seen that PMRFamide can significantly inhibit LPS-induced NO production at 0.001 µM, and its inhibitory effect can be maintained for at least 48 hours at the extreme concentration of 0.01 µM, indicating that the peptide has potent and sustained anti-inflammatory activity.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of PMRFamide short peptide in anti-inflammatory effects, characterized in that, The amino acid sequence of the PMRFamide short peptide is Pro-Met-Arg-Phe, and the preparation method of the PMRFamide short peptide is as follows: Step 1: Fmoc-Phe-OH is mixed with DCM and then mixed with swollen 2-Cl-Trt-Cl resin. DIPEA is added dropwise and stirred for 4-6 h. The filtrate is removed by filtration. The product is washed alternately with DCM and methanol and then a blocking solution is added. After filtration, the precipitate is the resin loaded with Fmoc-Phe-. Step 2: Arginine, methionine and proline are sequentially linked to the resin loaded with Fmoc-Phe- to obtain resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe. Step 3: Wash the resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe sequentially with DCM and methanol, dry, mix and stir with the cutting fluid, filter, wash the product with TFA, and combine all the filtrates and washing liquids to obtain a peptide dispersion. The ratio of the resin-loaded Fmoc-Pro-Met(Trt)-Arg(Pbf)-Phe, cutting fluid, DCM washing amount, methanol washing amount and TFA washing amount is (4-6) kg:(45-55) L:(200-250) L:(150-170) L:(1-3) L; Step 4: Add the peptide dispersion dropwise to MTBE, let stand at 0-4℃, remove the supernatant, centrifuge to collect the precipitate and wash with MTBE, dry to obtain PMRFamide peptide; The volume ratio of the peptide dispersion, the amount of MTBE mixture, and the amount of MTBE used for washing is 1:(8-10):(2-4).

2. The application of the PMRFamide short peptide according to claim 1 in anti-inflammatory effects, characterized in that, The process also includes a purification step: PMRFamide peptide is mixed with TFA aqueous solution, purified by preparative reversed-phase high-performance liquid chromatography, the main peak fraction is collected and combined, then concentrated to 10-20% of the original volume, then a freeze-drying protectant is added, stirred, and dried to obtain high-purity PMRFamide peptide. The ratio of PMRFamide peptide, TFA aqueous solution and lyophilization protectant is 1 kg: (8-12) L: (40-60) mg.

3. The application of the PMRFamide short peptide according to claim 2 in anti-inflammatory effects, characterized in that, The freeze-drying protectant is mannitol.

4. The application of the PMRFamide short peptide according to claim 2 in anti-inflammatory effects, characterized in that, The volume concentration of TFA in the TFA aqueous solution is 0.1-0.2%.

5. The application of the PMRFamide short peptide according to claim 1 in anti-inflammatory effects, characterized in that, The method for preparing the swollen 2-Cl-Trt-Cl resin in step one is as follows: 2-Cl-Trt-Cl is mixed with DCM to swell, and the filtrate is removed by filtration to obtain the swollen 2-Cl-Trt-Cl resin. The ratio of 2-Cl-Trt-Cl, Fmoc-Phe-OH, DIPEA, resin swelling DCM, amino acid dissolving DCM, DCM washing, methanol washing, and sealing liquid is (4-6) kg:(4-5) kg:(2-3) L:(150-250) L:(40-60) L:(200-250) L:(150-200) L:(80-120) L.

6. The application of the PMRFamide short peptide according to claim 1 in anti-inflammatory effects, characterized in that, The sealing solution mentioned in step one is a methanol-DCM solution, with a methanol to DCM volume ratio of 1:(4-5).

7. The application of the PMRFamide short peptide according to claim 1 in anti-inflammatory effects, characterized in that, In step two, repeat the following steps sequentially to connect arginine, methionine, and proline: a) Mix the resin obtained in the previous step with piperidine-DMF solution, stir, filter to remove the filtrate, and wash the product with DMF for later use; b) Mix the next Fmoc-protected amino acid to be ligated, HOAt, and DMF, then add HATU and stir to obtain a pre-activated solution; c) Mix the pre-activated solution with the product from a), adjust the pH to 8-9 with DIPEA, stir, filter, remove the filtrate, and wash the precipitate with DMF; The ratio of the resin, piperidine-DMF solution, pre-activation solution and DMF used during washing is 1 kg: (8-12) L: (1-2) L: (10-20) L; The ratio of Fmoc-protected amino acids, HOAt, HATU and DMF in the pre-activated solution is (0.8-1.2) mol:(0.8-1.2) mol:(0.8-1.2) mol:(0.2-0.4) L.

8. The application of the PMRFamide short peptide according to claim 7 in anti-inflammatory effects, characterized in that, The volume ratio of piperidine to DMF in the piperidine-DMF solution is (2-3):(7-8).

9. The application of the PMRFamide short peptide according to claim 1 in anti-inflammatory effects, characterized in that, The cutting fluid mentioned in step three is TFA, deionized water and TIS, with a volume ratio of (90-100):(2-3):(2-3).

10. The application of the PMRFamide short peptide according to claim 1 in anti-inflammatory effects, characterized in that, In step four, the peptide dispersion is added dropwise to MTBE at a rate of 1-2% of total volume / min.

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