T4 synthetic product and application thereof

Abstract

The invention discloses a 4-branched peptide, which belongs to the technical field of medicines, and the structural formula of the 4-branched peptide is (Thr-Lys-Pro-Arg) 4-(Lys) 2-Lys, the medicine is a polypeptide medicine, and a degradation product is free amino acid which can be directly absorbed and utilized by an organism, so that the medicine is small in toxic and side effects, good in safety, small in use dosage and high in curative effect, and can be clinically used as an anti-infection and anti-tumor treatment medicine.

Description

Technical Field

[0001] The present invention relates to the use of an artificially synthesized functional four-branched polypeptide product, which has the effect of improving the body's immune ability and has the potential to be developed into an anti-infection and anti-tumor clinical drug. The present invention belongs to the field of medicine. Background Art

[0002] The spleen is an important organ of the body, which is responsible for the physiological functions of hematopoiesis, blood storage, endocrine and immunity. The spleen produces a short peptide with a structure of N-threonine-lysine-proline-arginine-C, which has strong anti-infection and anti-tumor effects. The short peptide can quickly and specifically bind to macrophages and monocytes, increase their activity and contact with T lymphocytes, thereby improving the killing effect of macrophages on foreign invaders or self-mutated cells, increasing the activity of natural killer cells (NK cells), promoting the cytotoxic effect of T lymphocytes, and enhancing the ability of the body's T lymphocytes and other immune cells to recognize and kill foreign invading pathogens or self-mutated cells (such as tumor cells). Therefore, the short peptide has important clinical application value in anti-infection and anti-tumor. Summary of the invention

[0003] The functional peptide segment can be prepared by artificial peptide synthesis using a short peptide of N-Thr-Lys-Pro-Arg-C. Since the functional peptide segment is small in size, it is easily degraded after entering the body as a drug and is difficult to achieve the expected efficacy. In order to retain the efficacy of the functional peptide segment, improve the efficacy, and increase its metabolic half-life in the body, the structural design of the functional peptide segment is improved.

[0004] Using the technology of solid phase peptide synthesis, lysine can be connected to the solid phase resin, and then lysine can be connected on this basis. Since lysine contains two amino groups, amino acids can be condensed and connected on the two amino groups of one lysine to form a branched polypeptide. Connecting 2 rounds of lysine can form a 4-branched skeleton, and connecting 3 rounds of lysine can form an 8-branched skeleton. Finally, the synthesized branched polypeptide is cleaved from the resin.

[0005] Thr-Lys-Pro-Arg

[0006] >Lys cleavage

[0007] Thr-Lys-Pro-Arg

[0008] >Lys-resin

[0009] Thr-Lys-Pro-Arg

[0010] >Lys

[0011] Thr-Lys-Pro-Arg

[0012] The synthetic branched peptides can have the following structural forms:

[0013] 1. Connect the N-Threonine-Lysine-Proline-Arginine-C (N-Thr-Lys-Pro-Arg-C) functional peptide to the branch ends on the 4-branched polypeptide backbone (Multipal Antigen peptide, MAP4). You can synthesize the 4-branched polypeptide backbone (MAP4) yourself or purchase a commercial product. Usually, the functional peptide is synthesized at the branch end sequence of the polypeptide backbone using the Fmoc or Boc synthesis method to produce a functional 4-branched polypeptide synthesis product (Thr-Lys-Pro-Arg) containing the N-Thr-Lys-Pro-Arg-C peptide. 4 -(Lys)z-Lys.

[0014] 2. The N-Thr-Lys-Pro-Arg-C functional peptide segment can be connected to the branch end of the polypeptide backbone through a peptide chain formed by N natural amino acids (AA) (Thr-Lys-Pro-Arg-AAw) 4 -(Lys)z-Lys, N is a natural number. The purpose of the functional four-branched peptide synthesis product produced in this way is to reduce the steric hindrance effect that may occur between the functional peptide segment and the skeleton.

[0015] 3. Synthesize multiple functional peptides linearly at the branch ends of the polypeptide backbone, such as (Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg-) 4 -(Lys)z-Lys to produce a functional 4-branched peptide synthesis product. This method may improve the function of the peptide product.

[0016] 4. The four-branched polypeptide backbone connected to the functional peptide segment can be connected to the solid phase resin through a peptide chain formed by N natural amino acids (AA), and the functional four-branched polypeptide synthesis product (Thr-Lys-Pro-Arg) is generated by breaking with the resin. 4 -(Lys)z-Lys-AAn, N is a natural number. This synthesis method is to reduce the steric hindrance effect that may occur between the skeleton molecule and the solid phase resin.

[0017] 5. Functional peptide and 4-branched skeleton (Thr-Lys-Pro-Arg-AAv) 4 -(Lys-AAw)z-Lys-AAv, AA is a peptide chain formed by connecting N natural amino acids, and N is a natural number. The purpose of adding N natural amino acid peptide chains for connection is to reduce the steric hindrance effect between functional peptide segments.

[0018] The functional 4-branched polypeptide product prepared by peptide synthesis according to the above design has the effect of improving the body's immune ability. The synthetic product can be used as a veterinary drug to improve the immune ability of livestock, as an immunopotentiator to treat infectious diseases and improve resistance to diseases. The synthetic product can also be developed into a clinical drug to improve the patient's immune resistance to disease, and as an immunopotentiator can be used for anti-infection, anti-virus and tumor prevention and treatment. The purity of the purified synthetic product can exceed 98%, which meets the national purity quality requirements for drug registration. The synthetic product is dissolved in water for injection as the main drug raw material, and is filled into a sterile, endotoxin-free, and pyrogen-free medicinal ampoule or vial through aseptic filtration. After filling, it can be directly sealed into a water injection dosage form or sealed into a freeze-dried powder injection dosage form after freeze drying. The filling amount of the synthetic product can be determined according to the treatment needs, and the filling specifications can be between 50ug-2mg / ml / bottle. The administration route can be oral or intramuscular injection, the injection method can be 1 time / day to 1 time / week, the dosage can be 250ug / time or 2mg / time, 12 weeks is a course of treatment, or the dosage and treatment plan can be adjusted according to the condition.

[0019] Since this drug is a polypeptide drug, the degradation product is free amino acids that can be directly absorbed and utilized by the body, so it has small toxic side effects, good safety, small dosage and high efficacy. It can be used as an anti-infection and anti-tumor therapeutic drug in clinical practice. DETAILED DESCRIPTION

[0020]

Dosage form

[0021]

Specification

[0022] Prescription composition:

[0023]

[0024] Preparation process:

[0025] Solution preparation: Operate in a Class 100 sterile room. Preparation of phosphate buffer:

[0026] ①0.2mol / LNaH 2 PO;: Accurately weigh sodium dihydrogen phosphate (NaH 2 PO 4 ·2H 2 O) 17.8g, add water for injection and dissolve to 500ml, shake well and set aside;

[0027] ②0.2mol / LNa 2 HPO;: Accurately weigh disodium hydrogen phosphate (Na 2 HPO4 12H 2 0) 35.82g, dissolve in water for injection and make up to 500ml, shake well and set aside;

[0028] ③10mmol / L phosphate buffer (pH6.8): take 51ml0.2mol / LNaH 2 PO 4 Solution, 49m10.2mol / LNaHPO 2 Mix the solution and dilute to 2000 ml, adjust the pH to 6.8 with phosphoric acid and set aside.

[0029] Preparation of liquid medicine:

[0030] According to the formula, accurately weigh the prescribed amount of mannitol and synthetic product, dissolve them in 10mmol / L phosphate buffer (pH6.8) and make them into 1000ml. Accurately weigh 0.2% (W / V) of the prepared liquid volume of activated carbon and add it to the above solution, stir for 15 minutes; take the above solution and filter it twice to remove carbon, then filter it under positive pressure with a 0.2μm microporous filter membrane, and store it at 4℃ for later use.

[0031] Packaging and Freezing:

[0032] Operate in a sterile room; take the above-mentioned fine filtrate to determine the content, adjust the filling volume, take 1ml and divide it into 3ml medicinal ampoules; put the product into a freeze dryer at 20℃, pre-freeze for 3 hours to make the product temperature reach -40℃ (the plate temperature is pre-cooled to -20℃), keep warm for 1 hour, and refrigerate the condenser temperature to -55℃; turn on the vacuum pump to make the vacuum degree in the drying box reach about 8.0×10°mBar, heat the plate layer, and increase the temperature by 5℃ per hour; after 6 hours, the plate temperature reaches The temperature of the product rises by about 3°C ​​per hour along with the plate temperature, and the vacuum is maintained at about 6.0×10°mBar. After drying for about 8 hours, the product temperature reaches the eutectic point of about -15°C, and the water layer of the product disappears. Then the plate layer is heated up at a rate of 5°C per hour. After 7 hours, the plate temperature reaches 25°C, and the product temperature reaches 25°C after about 14 hours. After keeping warm for 3 hours, the ultimate vacuum of the drying oven is about 6.0×10°mBar. Stop the machine and seal the ampoule.

[0033] Process Validation:

[0034] According to the prescription preparation process, three batches of freeze-dried powder for injection were prepared, and the qualified rate of the finished product was greater than 90%. Random sampling was conducted to test the physical and chemical properties, related substances, content, heat source, and bacterial content of the finished product.

[0035] It is required that the content of preparations in different batches is basically consistent with the labeled amount, that there is no obvious change in the relevant substances before and after preparation, and that the sterility, pyrogen-free, vascular irritation, hemolysis and allergy tests are all negative.

[0036] Sources and quality specifications of raw materials and auxiliary materials:

[0037]

[0038]

[0039] Packaging specifications: The finished product is white freeze-dried powder injection, 1 mg / ampoule, 10 ampoules / box.

[0040] Storage: Store at 2-8℃ away from light. Valid for 3 years.

[0041]

Dosage form

[0042]

Specification

[0043] Prescription composition:

[0044]

[0045] Preparation process:

[0046] Solution preparation: Operate in a class 100 sterile room.

[0047] Accurately weigh the synthetic product, dissolve it in sterile water for injection and make sure it is dissolved in 1000 ml. After positive pressure sterile filtration using a 0.2 μm microporous filter membrane, take 1 ml and dispense it into a 3 ml medicinal ampoule and seal it.

[0048] When packaging the finished product, visually inspect whether the seal is neat and smooth, and check the appearance, color, and clarity of the contents. Randomly check the finished product to see if the filling volume, content, related substances, bacteria, and pyrogens must meet the quality inspection standards, and the vascular irritation, hemolysis, and allergy tests must all be negative.

[0049] Packaging specifications: 1mg / ml / ampoule, 10 ampoules / box.

[0050] Storage: Store at 2-8℃ away from light. Valid for 1 year.

[0051] The preparation method can be appropriately modified without being restricted to this.

[0052] Example 1

[0053] Animal pharmacodynamic model test setup:

[0054] Six-week-old C57 mice were taken, regardless of gender, with a body weight of 18 grams. A control group, a lead group, and a remodeling group were set up, with 10 mice in each group. 1x10° mouse melanocytoma (B16 tumor strain) was implanted subcutaneously in the shoulder blade of the mouse forelimb. After 7 days, soybean-sized tumors were observed and drug administration began. The drug was subcutaneously injected into the mice once a week according to lug / g body weight. The control group was saline. The lead group was the N-Thr-Lys-Pro-Arg-C polypeptide product, and the remodeling group was the 4-branched polypeptide synthesis product of N-Thr-Lys-Pro-Arg-C. The experiment was terminated after 4 weeks of administration. The tumor weight of each group of animals was measured.

[0055] Tumor inhibition test results:

[0056] Group Dosage Tumor weight at the end of the experiment (g Control group (normal saline) 200ul / pc 5.34+1.838 Lead Group lug / g body weight 3.90+2.614 Restructuring Group 1ug / g body weight 0.664+0.337

[0057] At the end of the experiment, no mice in each group died and no weight loss occurred. No tumor tissue liquefaction or abnormality was found in the autopsy. The formula for calculating the tumor inhibition rate is: (tumor weight of the control group - tumor weight of the reconstructed group) / tumor weight of the control group = 87.56%

[0058] P<0.001

[0059] Result evaluation: The modified product significantly improved the tumor inhibition effect compared with the lead product.

[0060] Example 2

[0061] Patients who have undergone tumor resection are given subcutaneous injection of the synthetic drug lyophilized powder (1 mg / ampule). The dosage is 1 mg / time / week, and a course of treatment is 12 weeks. The drug in the ampoule can be dissolved with 1 ml of injection water and then

[0062] After the syringe draws the solution out of the ampoule, inject it subcutaneously into the patient. Observe the patient's reaction closely. If the patient has symptoms such as palpitations, sweating, fatigue, tremors, etc., stop the injection immediately.

Claims

1. A 4-branched peptide, characterized in that The structural formula of the 4-branch peptide is: (Thr-Lys-Pro-Arg)4-(Lys)2-Lys.

2. Use of the four-branched peptide according to claim 1 in the preparation of drugs for improving immunity.

3. Use of the four-branched peptide according to claim 1 in the preparation of anti-tumor drugs.

Images