N-terminal 1-position phosphorylation modified derivative of NPFF as well as preparation method and application of N-terminal 1-position phosphorylation modified derivative

Abstract

The invention discloses an N-terminal 1-position phosphorylation modified derivative of NPFF as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The derivative is Phe at the amino terminal of the NPFF subjected to phosphorylation modification, and the amino acid sequence of the Phe at the amino terminal of the NPFF subjected to phosphorylation modification is PEG (Polyethylene Glycol)-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Ph-NH2. The N terminal of neuropeptide FF (NPFF) is modified by adopting NPFF (N terminal 1 position) phosphorylation, and the NPFF-PEG derivative is generated. The derivative shows remarkable anti-inflammatory activity in a mouse foot swelling inflammation model and a survival curve model under the stimulation of lipopolysaccharide (LPS).

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an N-terminal 1-phosphorylated derivative of NPFF, and a preparation method and application thereof. Background Art

[0002] Neuropeptide FF (NPFF), as a neuromodulator inherent in mammals, is composed of eight amino acid residues (sequence FLFQPQRFamide). It belongs to the RFamide peptide family. The notable feature of this family is that the C-terminus of its molecular chain contains RFamide structure. The physiological effects of NPFF are mediated by its endogenous receptor NPFFR2 (NPFF receptor 2), which belongs to the large protein family of G protein-coupled receptors (GPCR). NPFF exhibits diversified physiological functions in the body. It is not only deeply involved in the transmission of pain signals, but also has an important impact on key physiological activities such as blood pressure regulation, feeding behavior, endocrine system balance and body temperature regulation. In view of the wide distribution of NPFF in the body and its core role in multiple biological processes, in-depth research on NPFF and its receptors is of great significance for revealing the mysteries of life activities and developing new drugs to treat pain, cardiovascular diseases, metabolic disorders, etc., so research work in this field has attracted much attention.

[0003] Protein drugs, represented by peptides, have shown broad application prospects in the medical field due to their high biological activity and specificity. However, the challenges faced by such drugs, such as poor water solubility, poor stability and short plasma half-life, have significantly restricted the breadth and depth of their clinical applications. In order to overcome these limitations, scientists have explored a variety of strategies, among which the N-terminal 1 position of NPFF, referred to as NPFF (N-terminal 1 position), NPFF (N-terminal 1 position) phosphorylation and covalent coupling with drugs can affect biological activity and bring possible therapeutic applications, thereby improving the therapeutic effect. As a cutting-edge strategy for the improvement of peptide drugs, NPFF (N-terminal 1 position) phosphorylation has shown great potential. Although NPFF itself has significant anti-inflammatory properties, its inherent easy degradation and short half-life have become the main bottlenecks hindering its widespread development and application as a clinical drug. It is worth noting that to date, there are few studies or reports on direct N-terminal phosphorylation modification of NPFF to enhance its stability and efficacy. Therefore, this field has become a new direction worth exploring in the innovative research and development of peptide drugs. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned background technology, the present invention provides a derivative of NPFF phosphorylated at position 1 of the N-terminus and a preparation method and application thereof. The method uses NPFF (position 1 of the N-terminus) to modify the N-terminus of neuropeptide FF (NPFF) by phosphorylation to generate NPFF-PEG derivatives. The derivatives exhibited significant anti-inflammatory activity in a mouse foot swelling inflammation model and a survival curve model under lipopolysaccharide (LPS) stimulation.

[0005] The first object of the present invention is to provide a derivative of NPFF with phosphorylation modification at position 1 of the N-terminus, wherein the derivative is Phe at the amino terminus of NPFF with phosphorylation modification, and the amino acid sequence of Phe at the amino terminus of NPFF with phosphorylation modification is: PEG-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2.

[0006] Preferably, the derivative has the following structural formula: .

[0007] The second object of the present invention is to provide a method for preparing a derivative of NPFF with phosphorylation modification at position 1 of the N-terminus, comprising the following steps: S1. Place the wang resin in N,N-dimethylamide to swell; S2, the swollen resin, Fmoc-protected Phe, dicycloethylcarbodiimide and 4-dimethylaminopyridine are uniformly dissolved in DMF for reaction, and then end-capped with acetic anhydride to obtain an end-capped compound; S3, adding the capped compound to a piperidine DMF solution with a volume fraction of 18%, then draining, and adding a piperidine DMF solution with a volume fraction of 18% again to obtain a protected amino acid through reaction; then dissolving the protected amino acid and o-benzotriazole-tetramethyluronium hexafluorophosphate in DMF, and adding 4-methylmorpholine to carry out a condensation reaction; Repeat S2 to S3, and condense the amino acids of the following sequence in order from the C-terminal to the N-terminal residue sequence: Phosphate-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 to obtain a condensate; The condensate, NH2-(CH2CH2O)8-CH2CH2COOH and HBTU were dissolved in DMF, and then NMM was added to react to remove the Fmoc protecting group, and then cleaved to obtain a cleavage solution containing the peptide; The lysate was post-processed to obtain the N-terminal 1-phosphorylated derivative of NPFF.

[0008] Preferably, the lysis process comprises: Prepare lysis solution: add ultrapure water, triisopropylsilane and 1,2-ethanedithiol to trifluoroacetic acid and mix well; wherein the volume ratio of trifluoroacetic acid, ultrapure water, triisopropylsilane and 1,2-ethanedithiol is 94:2:1:3; The product with the Fmoc protecting group removed was immersed in the lysis buffer for 100 to 200 minutes.

[0009] Preferably, after the lysis, the method further comprises: drying the lysate in a chlorine environment; washing the dried sample with ether for multiple times, and volatilizing the dried sample at room temperature until it is completely dry; and then purifying the dried sample with high performance liquid chromatography; During purification, the mobile phase was a mixture of 0.15% trifluoroacetic acid and acetonitrile in a volume ratio of 50:50.

[0010] Preferably, the obtained end-capping compound, the product after condensation reaction and the product after removal of Fmoc protecting group are all washed in DMF and methanol for multiple cycles to remove impurities.

[0011] The third object of the present invention is to provide an N-terminal 1-phosphorylated derivative of NPFF for use in the preparation of analgesics or anti-inflammatory drugs, characterized in that the derivative is prepared into tablets, capsules or injections according to conventional pharmacy.

[0012] The fourth object of the present invention is to provide an anti-inflammatory tablet comprising the following components in parts by weight: 40.0 parts of NPFF-PEG, 175.0 parts of a filler, 10.0 parts of a disintegrant, 5.0 parts of a binder, and 3.0 parts of a lubricant; Among them, NPFF-PEG is a derivative of the NPFF provided above that is phosphorylated at position 1 of the N-terminus.

[0013] The fifth object of the present invention is to provide an anti-inflammatory soft capsule, comprising the following components by weight: 85.0 parts of NPFF-PEG, 85.0 parts of filler, 5.0 parts of binder, and 5.0 parts of lubricant; Among them, NPFF-PEG is a derivative of the NPFF provided above that is phosphorylated at position 1 of the N-terminus.

[0014] The sixth object of the present invention is to provide an anti-inflammatory injection, comprising the following components by weight: 120.0 parts of NPFF-PEG, 1.0 parts of citric acid, 1.0 parts of sodium citrate, 16.0 parts of sodium chloride, and 2000 parts of water for injection; Among them, NPFF-PEG is a derivative of the NPFF provided above that is phosphorylated at position 1 of the N-terminus.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a derivative of NPFF phosphorylated at position 1 of the N-terminus and a preparation method thereof, and further explores its potential application in anti-inflammatory treatment. The present invention uses NPFF (position 1 of the N-terminus) phosphorylation to modify the N-terminus of neuropeptide FF (NPFF) to generate an NPFF-PEG complex.

[0016] The present invention uses a carefully designed experimental scheme, a mouse foot swelling inflammation model and an LPS (lipopolysaccharide) stimulation survival curve model to comprehensively evaluate the anti-inflammatory activity of NPFF (N-terminal position 1) phosphorylation. The experimental results show that NPFF (N-terminal position 1) phosphorylation exhibits excellent anti-inflammatory properties. In a mouse foot swelling model simulating an inflammatory environment, 14 mg / kg and 28 mg / kg doses of NPFF (N-terminal position 1) phosphorylation effectively inhibited 41.25% and 50.74% of the inflammatory response, respectively (after 12 hours of observation). This significant effect demonstrates the powerful ability of NPFF (N-terminal position 1) phosphorylation in controlling local inflammation. Furthermore, in the more severe LPS stimulation survival curve model, NPFF (N-terminal position 1) phosphorylation also exhibited its protective effect. When given at a dose of 1 mg / kg to mice in the LPS stimulation group (LPS dose of 25 mg / kg), NPFF (N-terminal position 1) phosphorylation significantly prolonged the survival time of the test animals, especially on the 14th day of the experiment, the survival rate was increased by 75% compared with the untreated group. This finding highlights the potential therapeutic value of NPFF (N-terminal position 1) phosphorylation in responding to systemic inflammatory challenges. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The mouse foot swelling inflammation model was used to evaluate the anti-inflammatory activity of NPFF (position 1 at the N-terminus). VS Control, * P<0.05, **** P<0.0001. NPFF (position 1 at the N-terminus) phosphorylation has significant anti-inflammatory activity.

[0018] Figure 2 The LPS-stimulated survival curve experiment was used to evaluate the anti-inflammatory activity of NPFF (position 1 at the N-terminus). NPFF (position 1 at the N-terminus) phosphorylation has significant anti-inflammatory activity. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0020] In a first aspect, the present invention provides a derivative of NPFF that is phosphorylated at position 1 of the N-terminus, wherein the derivative is Phe at the amino terminus of NPFF that is phosphorylated, and the amino acid sequence of Phe at the amino terminus of NPFF that is phosphorylated is: PEG-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2.

[0021] The structural formula of the derivative is as follows: .

[0022] The second aspect of the present invention provides a method for preparing a derivative of NPFF with phosphorylation modification at position 1 of the N-terminus, comprising the following steps: S1. Place the wang resin in N,N-dimethylamide to swell; S2, the swollen resin, Fmoc-protected Phe, dicycloethylcarbodiimide and 4-dimethylaminopyridine are uniformly dissolved in DMF for reaction, and then end-capped with acetic anhydride to obtain an end-capped compound; S3, adding the capped compound to a piperidine DMF solution with a volume fraction of 18%, then draining, and adding a piperidine DMF solution with a volume fraction of 18% again to obtain a protected amino acid through reaction; then dissolving the protected amino acid and o-benzotriazole-tetramethyluronium hexafluorophosphate in DMF, and adding 4-methylmorpholine to carry out a condensation reaction; Repeat S2 to S3, and condense the amino acids of the following sequence in order from the C-terminal to the N-terminal residue sequence: Phosphate-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 to obtain a condensate; The condensate, NH2-(CH2CH2O)8-CH2CH2COOH and HBTU were dissolved in DMF, and then NMM was added to react to remove the Fmoc protecting group, and then cleaved to obtain a cleavage solution containing the peptide; The lysate was post-processed to obtain the N-terminal 1-phosphorylated derivative of NPFF.

[0023] The cracking process includes: Prepare lysis solution: add ultrapure water, triisopropylsilane and 1,2-ethanedithiol to trifluoroacetic acid and mix well; wherein the volume ratio of trifluoroacetic acid, ultrapure water, triisopropylsilane and 1,2-ethanedithiol is 94:2:1:3; The product with the Fmoc protecting group removed was immersed in the lysis buffer for 100 to 200 minutes.

[0024] After the lysis, the method further includes: drying the lysis solution in a chlorine environment; washing the dried sample with ether for multiple times, and volatilizing the dried sample at room temperature until it is completely dry; and then purifying the dried sample with high performance liquid chromatography; During purification, the mobile phase was a mixture of 0.15% trifluoroacetic acid and acetonitrile in a volume ratio of 50:50.

[0025] The obtained end-capping compound, the product after the condensation reaction and the product after the Fmoc protecting group is removed are all washed in DMF and methanol for multiple cycles to remove impurities.

[0026] The third aspect of the present invention provides an N-terminal 1-phosphorylated derivative of NPFF for use in the preparation of analgesics or anti-inflammatory drugs. The derivative is prepared into tablets, capsules or injections according to conventional pharmacy.

[0027] The fourth aspect of the present invention provides an anti-inflammatory tablet comprising the following components in parts by weight: 40.0 parts of NPFF-PEG, 175.0 parts of a filler, 10.0 parts of a disintegrant, 5.0 parts of a binder, and 3.0 parts of a lubricant.

[0028] The fifth aspect of the present invention provides an anti-inflammatory soft capsule, comprising the following components in parts by weight: 85.0 parts of NPFF-PEG, 85.0 parts of a filler, 5.0 parts of a binder, and 5.0 parts of a lubricant.

[0029] The sixth aspect of the present invention provides an anti-inflammatory injection, comprising the following components in parts by weight: 120.0 parts of NPFF-PEG, 1.0 parts of citric acid, 1.0 parts of sodium citrate, 16.0 parts of sodium chloride, and 2000 parts of water for injection.

[0030] Among them, NPFF-PEG is a derivative of the NPFF provided above that is phosphorylated at position 1 of the N-terminus.

[0031] The present invention uses NPFF (N-terminal 1 position) phosphorylation to modify the N-terminus of neuropeptide FF (NPFF) to generate NPFF-PEG derivatives. The derivatives exhibit significant anti-inflammatory activity in a mouse foot swelling inflammation model and a survival curve model under lipopolysaccharide (LPS) stimulation. The molecular formula of the compound is C 54 H 77 N 14 O 14 P, molecular weight 1226.30.

[0032] The mouse foot swelling inflammation model is used as a biological platform to evaluate anti-inflammatory activity. The basic principle is that carrageenan is used as a standard inflammatory substance. By injecting it subcutaneously in the plantar of mice, an inflammatory response can be induced, resulting in an increase in exudate at the site of inflammation, which in turn increases the weight of the injected foot. By accurately weighing and comparing the weight difference between the inflamed and control sides of the foot, the degree of inflammation and the actual efficacy of the anti-inflammatory drug to be tested can be quantitatively evaluated. This method provides a reliable and sensitive experimental means for evaluating the in vivo activity of anti-inflammatory drugs.

[0033] Inhibition rate = (foot swelling value of the drug group - foot swelling value of the blank group) / (foot swelling value of the blank group) × 100% The LPS-stimulated survival curve model is used to evaluate the anti-inflammatory activity of animals. Its principle is based on LPS (a large molecular endotoxin covalently bound by lipids and polysaccharides) as an inflammatory factor that can trigger a strong immune response. LPS (24 mg / kg) was injected intraperitoneally into mice, followed by different drugs, and the changes in the survival time of the mice were observed. Drugs that prolong the survival time of mice are considered to have anti-inflammatory activity, thereby realizing the detection of the anti-inflammatory effect of drugs.

[0034] The present invention verifies the anti-inflammatory effect through phosphorylation of NPFF (position 1 of the N-terminus) and through the mouse foot swelling inflammation and LPS survival curve model. The experimental results show that NPFF (position 1 of the N-terminus) phosphorylation exhibits significant anti-inflammatory activity in both models: in the foot swelling model, the inflammation was reduced by 23.19% and 34.41% (12 hours) at doses of 14 mg / kg and 28 mg / kg, respectively; in the LPS model, the 1 mg / kg dose significantly increased the survival rate of LPS-stimulated mice to 68.75% (14 days). This discovery not only expands the application prospects of NPFF (position 1 of the N-terminus) phosphorylation as an anti-inflammatory drug, but also provides a new potential drug option for the treatment of inflammation, which has important medical value.

[0035] Among them, the NPFF (N-terminal 1 position) phosphorylated compound refers to the derivative modified by phosphorylation at the N-terminal 1 position of NPFF.

[0036] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0037] Example 1 The preparation of phosphorylated NPFF (position 1 at the N-terminus), specifically a method for preparing a derivative of NPFF phosphorylated at position 1 at the N-terminus, comprises the following steps: Synthesis method: TBTU+DIEA+protected amino acid, all triple feed, modification on the amino group of Phe at the N-terminus.

[0038] The specific embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. For method steps in the embodiments where specific experimental conditions are not explicitly mentioned, the standard operating procedures recommended by the manufacturer or conventional conditions generally recognized in the industry will be followed for execution.

[0039] The solid phase peptide synthesis method was used to prepare the phosphorylated derivative of NPFF (N-terminal position 1), the steps are as follows: Step 1: Resin swelling. Add N,N-dimethylformamide (DMF) to the reaction vessel, then add Wang resin and shake for 23 minutes to allow the resin to swell fully.

[0040] Step 2: Grafting amino acids. After removing the solvent by filtration, add Fmoc-protected Phe (3.5 times the molar amount required for synthesizing peptides) to the reaction system, then add dicycloethylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) equivalent to 11 times the molar amount of synthesized peptides, then dissolve in DMF and shake for 32 minutes to promote the reaction. Subsequently, acetic anhydride is used for end-capping.

[0041] Step 3: Deprotection: After removing DMF, add 18% piperidine DMF solution by volume, drain for 8 minutes, add 18% piperidine DMF solution by volume again, and react for 18 minutes.

[0042] Step 4: Detection. After removing the piperidine DMF solution, carefully pick up a small amount of resin particles with a sterile pipette and place it in a blank test tube, then add one drop each of ninhydrin, potassium cyanide and phenol solution. Heat the test tube at 104-108°C for 5 minutes. If the solution turns dark blue, it indicates a positive result.

[0043] Step 5: Washing: Wash three times with DMF, methanol, and DMF in sequence to ensure that impurities are removed.

[0044] Step 6: Condensation reaction. The protected amino acid (3 times the molar amount of the synthetic peptide) and o-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) were dissolved in DMF and added to the reaction vessel. Subsequently, 4-methylmorpholine (NMM) (12 times the molar amount of the peptide) was quickly added to carry out the condensation reaction for 32 minutes.

[0045] Step 7: Washing: Wash twice with DMF (9 ml / g), then wash four times with methanol (9 ml / g), and finally wash twice with DMF (9 ml / g) to ensure that impurities are fully removed.

[0046] Step 8: Cyclic operation. Repeat the process from step 2 to step 7, and condense the following sequence of amino acids in the order from the C-terminal to the N-terminal residue: Phosphate-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 Step 9: After confirming that NPFF is the main peak by mass spectrometry, add NH2-(CH2CH2O)8-CH2CH2COOH (3.5 times the molar amount of NPFF) and HBTU (5.5 times the molar amount of the peptide), both dissolved in DMF, and then add NMM (18 times the molar amount of NPFF) to react for 32 minutes. The detection method in step 4 was used, and the result showed colorless, indicating that the Fmoc protecting group was successfully removed.

[0047] Step 10: Perform multiple solvent washing and draining. First, wash three times with DMF (9 ml / g), then wash three times with methanol (9 ml / g), wash three times with DMF (9 ml / g), and finally wash three times with DMF (9 ml / g). After all washings are completed, drain for 28 minutes.

[0048] Step 11: Cut the peptide on the resin. Specifically, mix 94% trifluoroacetic acid (V / V) with 8 ml per gram of resin as the cutting solution base, and then add 2% ultrapure water (V / V), 1% triisopropylsilane (V / V) and 3% 1,2-ethanedithiol (V / V) to it. Then, soak the resin with the cutting solution for 145 minutes.

[0049] Step 12: Blow dry and wash. Blow dry the lysate in a chlorine environment. Then, wash the dried sample 8 times with ether and evaporate it naturally at room temperature (24°C) until it is completely dry.

[0050] Step 13: Preparation of dry powdered peptide crude product. The washed sample was purified by high performance liquid chromatography, and the mobile phase was a mixture of 0.15% trifluoroacetic acid and acetonitrile in a volume ratio of 50:50. Through this step, the pure phosphorylated derivative of NPFF was successfully prepared, and its structural formula is as follows:

[0051] From the above structural formula, it can be seen that the mass spectrometry molecular weight of NPFF (position 1 at the N-terminus) phosphorylation is 1177.27, which is consistent with the theoretically calculated molecular weight, indicating that the phosphorylation of NPFF (position 1 at the N-terminus) was correctly identified.

[0052] Example 2. Mouse foot swelling inflammation model was used to evaluate the anti-inflammatory activity of NPFF (position 1 at the N-terminus) phosphorylation.

[0053] 1) Experimental animals: The experimental mice were raised in a clean environment, with the ambient temperature maintained between 22-25°C, the humidity controlled between 48% and 52%, and sufficient light from 8:00 to 20:00 every day. There were 6 mice in each cage, and they were free to eat and drink.

[0054] 2) Experimental groups: The experiment was divided into four groups: a blank control group (Vehicle), a positive control group (DEX, dose of 5 mg / kg), and two different doses of NPFF (N-terminal 1 position) phosphorylation groups (14 mg / kg and 28 mg / kg, respectively). Each group contained 11 mice to ensure the consistency of experimental conditions.

[0055] 3) Mouse foot swelling inflammation model: Healthy mice aged 6-8 weeks were selected and randomly divided into several groups, each containing 11 mice. Each group of mice received different types of intraperitoneal injections of drugs. 28 minutes later, 23 μl of 1% (W / V) carrageenan was injected into the subcutaneous tissue of the right toe of the mouse as an inflammatory factor, while the same amount of 0.9% saline was injected subcutaneously into the left toe as a control. At 1, 2, 3, 4, 5, 6, and 12 hours after the injection of carrageenan, the thickness of the left and right feet of the mouse was measured and recorded using a vernier caliper. By calculating the difference in thickness between the right and left feet, the swelling thickness (unit: mm) was obtained to evaluate the inhibitory effect of the drug on the inflammatory response.

[0056] exist Figure 1 In the mouse foot swelling inflammation model shown in the figure, the NPFF (N-terminal 1 position) phosphorylation drug showed an inflammation inhibition effect of 23.19% at a dose of 14 mg / kg; when the dose was increased to 28 mg / kg, the inflammation inhibition rate increased to 34.41% (12 hours).

[0057] Example 3: The anti-inflammatory activity of NPFF-PEG was evaluated using a survival curve experiment induced by LPS (lipopolysaccharide) stimulation.

[0058] 1) Experimental animals: The experimental mice were placed in a clean-grade breeding environment, with the ambient temperature strictly controlled at 22℃±2℃ and the humidity maintained at 50%~55%. The light cycle was set from 08:00 to 20:00 every day to ensure that the mice had a regular circadian rhythm. The number of mice per cage was controlled at 6, and sufficient free food and water were provided.

[0059] 2) Experimental grouping: The experiment was divided into two groups, including a positive control group (treated with LPS 24 mg / kg only) and a drug group (treated with LPS 24 mg / kg and NPFF (N-terminal 1 position) phosphorylated 1 mg / kg at the same time). Each group contained an equal number of 11 mice to ensure the reliability and comparability of the experimental results.

[0060] 3) LPS stimulation survival curve experiment: Healthy mice aged 6-8 weeks were selected and randomly assigned to different experimental groups, with 11 mice in each group. Each group received corresponding drug treatment according to the experimental design. In the following 0 to 14 days, key physiological indicators such as the activity, behavior patterns and weight changes of the mice were closely monitored. At the end of the experiment, the mice were humanely treated and the survival curve was analyzed to evaluate the effects of different treatments on the survival of the mice.

[0061] See also Figure 2 As shown, in the LPS-induced survival challenge model, the mouse group treated with 1 mg / kg phosphorylated NPFF (position 1 at the N-terminus) significantly prolonged its survival time. Compared with the LPS stimulation group (LPS 24 mg / kg) that did not receive phosphorylated NPFF (position 1 at the N-terminus), its 14-day survival rate increased to 68.75%, while the survival rate of the control group was only 36.36%, indicating that NPFF-PEG has a significant protective effect on LPS-induced survival stress.

[0062] The following specific examples will further illustrate the potential applications and advantages of phosphorylated NPFF (N-terminal position 1) as an active ingredient in the development of anti-inflammatory and analgesic drugs.

[0063] Example 4. Preparation of tablets Formula: NPFF-PEG (purity greater than 95%) 40.0g, filler 175.0g, disintegrant 10.0g, binder 5.0g, lubricant 3.0g, totaling 233.0g.

[0064] Process: 1000 tablets were prepared according to the conventional process for preparing tablets, each tablet containing 50 mg of NPFF-PEG.

[0065] Dosage and Administration: Once a day, 3 tablets each time.

[0066] Example 5. Preparation of soft capsules Formula: NPFF-PEG (purity greater than 94%) 85.0g, filler 85.0g, adhesive 5.0g, lubricant 5.0g, totaling 180.0g.

[0067] Process: 1000 capsules are prepared according to the conventional process for preparing capsules, each capsule containing 50 mg of NPFF-PEG.

[0068] Dosage and administration: Once a day, 3 capsules each time.

[0069] Example 6. Preparation of injection Formula: NPFF-PEG (purity greater than 94%) 120.0g, citric acid 1.0g, sodium citrate 1.0g, sodium chloride 16.0g, water for injection 2000ml.

[0070] Process: According to the conventional process for preparing injections, a total of 1000 injections of 2 ml were prepared, each containing 120 mg of NPFF-PEG.

[0071] Dosage and administration: Once a day, 2 sticks each time.

[0072] The fillers, disintegrants, binders, lubricants and other excipients in the above embodiments are all the most common excipients in pharmacy. NPFF-PEG is a derivative of the NPFF provided above with phosphorylation modification at position 1 of the N-terminus.

[0073] In summary, the present invention not only successfully prepared NPFF (N-terminal position 1) phosphorylated as a new anti-inflammatory drug candidate, but also verified its significant anti-inflammatory effect through rigorous experiments. The application of NPFF (N-terminal position 1) phosphorylated as an anti-inflammatory drug provides a new strategy and idea for the treatment of inflammatory diseases, indicating that it has broad development prospects and potential market value in the field of medicine, especially in the research and development of anti-inflammatory drugs.

[0074] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0075] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A derivative of NPFF with phosphorylation modification at position 1 of the N-terminus, characterized in that: The derivative is Phe at the amino terminus of phosphorylated NPFF, and the amino acid sequence of Phe at the amino terminus of phosphorylated NPFF is: PEG-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2.

2. The N-terminal 1-phosphorylated derivative of NPFF according to claim 1, characterized in that: The structural formula of the derivative is as follows: 。 3. A method for preparing a derivative of NPFF with phosphorylation modification at position 1 of the N-terminus according to claim 1 or 2, characterized in that: The following steps are involved: S1. Place the wang resin in N,N-dimethylamide to swell; S2, the swollen resin, Fmoc-protected Phe, dicycloethylcarbodiimide and 4-dimethylaminopyridine are uniformly dissolved in DMF for reaction, and then end-capped with acetic anhydride to obtain an end-capped compound; S3, adding the capped compound to a piperidine DMF solution with a volume fraction of 18%, then draining, and adding a piperidine DMF solution with a volume fraction of 18% again to obtain a protected amino acid through reaction; then dissolving the protected amino acid and o-benzotriazole-tetramethyluronium hexafluorophosphate in DMF, and adding 4-methylmorpholine to carry out a condensation reaction; Repeat S2 to S3, and condense the amino acids of the following sequence in order from the C-terminal to the N-terminal residue sequence: Phosphate-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 to obtain a condensate; The condensate, NH2-(CH2CH2O)8-CH2CH2COOH and HBTU were dissolved in DMF, and then NMM was added to react to remove the Fmoc protecting group, and then the peptide-containing cleavage solution was obtained by cleavage; The lysate was post-processed to obtain the N-terminal 1-phosphorylated derivative of NPFF.

4. The method for preparing the derivative of NPFF with phosphorylation modification at position 1 of the N-terminus according to claim 3, characterized in that: The lysis process includes: Prepare lysis solution: add ultrapure water, triisopropylsilane and 1,2-ethanedithiol to trifluoroacetic acid and mix well; wherein the volume ratio of trifluoroacetic acid, ultrapure water, triisopropylsilane and 1,2-ethanedithiol is 94:2:1:3; The product with the Fmoc protecting group removed was immersed in the lysis buffer for 100 to 200 minutes.

5. The method for preparing the derivative of NPFF with phosphorylation modification at position 1 of the N-terminus according to claim 3, characterized in that: After the lysis, the method further includes: drying the lysis solution in a chlorine environment; washing the dried sample with ether for multiple times, and volatilizing the dried sample at room temperature until it is completely dry; and then purifying the dried sample with high performance liquid chromatography; During purification, the mobile phase was a mixture of 0.15% trifluoroacetic acid and acetonitrile in a volume ratio of 50:

50.

6. The method for preparing the derivative of NPFF with phosphorylation modification at position 1 of the N-terminus according to claim 3, characterized in that: The obtained end-capping compound, the product after the condensation reaction and the product after the Fmoc protecting group is removed are all washed in DMF and methanol for multiple cycles to remove impurities.

7. Use of a derivative of NPFF modified by phosphorylation at position 1 of the N-terminus according to claim 1 or 2 in the preparation of analgesic drugs or anti-inflammatory drugs, characterized in that: The derivative is prepared into tablets, capsules or injections according to conventional pharmacy.

8. An anti-inflammatory tablet, characterized in that: The composition comprises the following components by weight: 40.0 parts of NPFF-PEG, 175.0 parts of filler, 10.0 parts of disintegrant, 5.0 parts of binder and 3.0 parts of lubricant; Wherein, NPFF-PEG is a derivative of NPFF provided in claim 1 or 2 that is phosphorylated at position 1 of the N-terminus.

9. An anti-inflammatory soft capsule, characterized in that: The composition comprises the following components by weight: 85.0 parts of NPFF-PEG, 85.0 parts of filler, 5.0 parts of adhesive, and 5.0 parts of lubricant; Wherein, NPFF-PEG is a derivative of NPFF provided in claim 1 or 2 that is phosphorylated at position 1 of the N-terminus.

10. An anti-inflammatory injection, characterized in that: The invention comprises the following components in parts by weight: 120.0 parts of NPFF-PEG, 1.0 parts of citric acid, 1.0 parts of sodium citrate, 16.0 parts of sodium chloride, and 2000 parts of water for injection; Wherein, NPFF-PEG is a derivative of NPFF provided in claim 1 or 2 that is phosphorylated at position 1 of the N-terminus.

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