T8 synthetic product and application thereof

Abstract

The invention relates to application of a T8 synthetic product and application of the T8 synthetic product, namely (Thr-Lys-Pro-Arg-AAn) 8-(Lys-AAn) 4-(Lys-AAn) 2-Lys-AAn, wherein AA is a natural amino acid peptide chain, and N is a natural number. The branched polypeptide synthetic product has the effect of comprehensively improving the immunocompetence of an organism, and can be prepared into a clinical anti-infection and anti-tumor treatment medicine.

Description

Technical Field

[0001] The present invention relates to the use of an artificially synthesized functional 8-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. Technical Background

[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 has the ability to 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 1 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 can be prepared by artificial peptide synthesis using a short peptide of N-threonine-lysine-proline-arginine-C (N-Thr-Lys-Pro-Arg-C). Since the functional peptide is small in molecule, it is easily degraded after entering the body as a drug and it is difficult to achieve the expected efficacy. In order to retain the efficacy of the functional peptide, improve the efficacy, and increase its metabolic half-life in the body, the structural design of the functional peptide is improved. Using the technology of solid phase peptide synthesis, lysine can be connected to the solid phase resin, and lysine can be further connected on this basis. Since lysine contains two amino groups, amino acids can be condensed and connected on the two amino groups of a lysine to form a branched polypeptide. Connecting 2 rounds of lysine can form a 4-branch skeleton, and connecting 3 rounds of lysine can form an 8-branch skeleton. Finally, the synthesized branched polypeptide is broken off from the resin. Thr-Lys-Pro-Arg

[0004]

[0005] The synthesized 8-branched polypeptide can have the following structural forms:

[0006] 1. Connect the N-threonine-lysine-proline-arginine-C (N-Thr-Lys-Pro-Arg-C) functional peptide to the branch end of the 8-branched polypeptide skeleton (Multiple Antigen Peptide, MAP8). The 8-branched polypeptide skeleton (MAP8) can be synthesized independently or purchased commercially. The functional peptide is usually synthesized at the branch end sequence of the polypeptide skeleton using the Fmoc or Boc synthesis method to produce a functional 8-branched polypeptide synthetic product (Thr-Lys-Pro-Arg-)8-(Lys)4-(Lys)2-Lys containing the N-Thr-Lys-Pro-Arg-C peptide.

[0007] 2. The N-Thr-Lys-Pro-Arg-C functional peptide segment and the branch end of the polypeptide backbone can be connected by a peptide chain formed by N natural amino acids to produce (Thr-Lys-Pro-Arg-AAN)8-(Lys)4-(Lys)2-Lys, where N is a natural number. The purpose of the functional 8-branched polypeptide synthesis product produced in this way is to reduce the steric hindrance effect that may be generated between the functional peptide segment and the backbone.

[0008] 3. Synthesize multiple functional peptides linearly at the branch ends of the polypeptide backbone, such as (Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg-)8-(Lys)4-(Lys)2-Lys to produce a functional 8-branched polypeptide synthesis product. This method may improve the function of the polypeptide product.

[0009] 4. The 8-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, and the functional 8-branched polypeptide synthesis product (Thr-Lys-Pro-Arg)8-(Lys)4-(Lys)2-Lys-AAN is generated after cleavage from the resin, where N is a natural number. This synthesis method is to reduce the steric hindrance effect that may occur between the backbone molecule and the solid phase resin.

[0010] 5. The functional peptide and the 8-branched polypeptide backbone form (Thr-Lys-Pro-Arg-AAN)8-(Lys-AAN)4-(Lys--AAN)2-Lys-AAN, AA is a peptide chain formed by connecting N natural amino acids, and N is a natural number. Adding N natural amino acid peptide chains for connection is to reduce the steric hindrance effect between the functional peptides.

[0011] The functional 8-branched polypeptide product (Thr-Lys-Pro-Arg-AAN)8-(Lys-AAN)4-(Lys-AAN)2-Lys-AAN prepared by polypeptide 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 and as an immunopotentiator for treating infectious diseases and improving resistance to diseases.

[0012] The finished product can also be developed into clinical medicine as an immunopotentiator to improve the patient's immune resistance to disease, and to be used for anti-infection, anti-virus and tumor prevention and treatment.

[0013] The purity of the synthetic product after purification can reach more than 98%, which meets the purity quality requirements of the State Food and Drug Administration for drug registration. The synthetic product is dissolved in water for injection as the main drug raw material, and is filled into sterile, endotoxin-free, and pyrogen-free medicinal ampoules or vials 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 volume of the synthetic product can be determined according to the treatment needs, and the filling specifications can be between 100ug-5mg / ml / bottle. The route of administration can be intramuscular injection, the injection method can be 1 time / day to 1 time / week, the clinical dosage can be 250ug / time or 1mg / time, 12 weeks as a course of treatment, or the dosage and treatment plan can be adjusted according to the condition. 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, and a small dosage and high efficacy. It can be used as an anti-infection and anti-tumor therapeutic drug in clinical practice. DETAILED DESCRIPTION

[0014] 1. [Dosage form] Lyophilized powder for injection.

[0015]

Specification

[0016] Prescription composition:

[0017]

[0018] Preparation process:

[0019] Solution preparation: Operate in a class 100 sterile room.

[0020] Preparation of phosphate buffer:

[0021] ①0.2mol / L NaH2PO4: Accurately weigh 17.8g of sodium dihydrogen phosphate (NaH2PO4·2H2O), dissolve it in water for injection and make up to 500ml, shake well and set aside;

[0022] ②0.2mol / L Na2HPO4: Accurately weigh 35.82g of disodium hydrogen phosphate (Na2HPO4·12H2O), dissolve it in water for injection and make up to 500ml, shake well and set aside;

[0023] ③10mmol / L phosphate buffer (pH6.8): Take 51ml 0.2mol / L NaH2PO4 solution and 49ml 0.2mol / L Na2HPO4 solution, mix them evenly, and dissolve them to 2000ml. Adjust the pH to 6.8 with phosphoric acid and set aside.

[0024] Preparation of liquid medicine:

[0025] Accurately weigh the prescribed amount of mannitol and the synthetic product, dissolve them in 10 mmol / L phosphate buffer (pH 6.8) and make them into 1000 ml. Accurately weigh 0.2% (W / V) of the prepared liquid volume of activated carbon, add it to the above solution, and stir for 15 minutes; take the above solution and filter it twice to remove carbon, then filter it under positive pressure with a 0.2 μm microporous filter membrane, and store it at 4°C for later use.

[0026] Packaging and Freezing:

[0027] Operate in a sterile room; take the above-mentioned fine filtrate to determine the content, adjust the filling volume, and take 1m l Packed in 3ml medicinal ampoules; the product is put into a freeze dryer at 20℃, pre-frozen for 3 hours to make the product temperature reach -40℃ (the plate temperature is pre-cooled to -20℃), kept warm for 1 hour, and the condenser temperature is refrigerated to -55℃; turn on the vacuum pump to make the vacuum degree in the drying oven reach about 8.0×102mBar, heat the plate layer and increase the temperature by 5℃ per hour; after 6 hours, the plate temperature is constant at -10℃, the product temperature increases by about 3℃ per hour with the plate temperature, and the vacuum degree is maintained at about 6.0×10-2mBar. Dry for about 8 hours, the product temperature reaches the eutectic point of about -15℃, and the water layer of the product disappears; then continue to heat the plate layer at a rate of 5℃ per hour, after 7 hours, the plate temperature reaches 25℃, the product temperature reaches 25℃ after about 14 hours, and is kept warm for 3 hours. The ultimate vacuum degree of the drying oven is about 6.0×10-2mBar; stop the machine, seal the ampoule and obtain.

[0028] Process Validation:

[0029] 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.

[0030] 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.

[0031] Sources and quality specifications of raw materials and auxiliary materials:

[0032] Raw materials, quality specifications, sources, uses Synthetic products Mannitol Sodium dihydrogen phosphate Disodium hydrogen phosphate Water for injection Activated carbon for injection Conforms to the provisional standard CP.200089 CP.20001058

[0033] Page GB1263-86 CP.2000428 Page CP.2000465 Page Homemade Beijing Pharmaceutical Factory Suzhou Sinochem Pharmaceutical Co., Ltd. Suzhou Sinochem Pharmaceutical Co., Ltd. Beijing Pharmaceutical Factory Beijing Dali Activated Carbon Factory Main drug Proppant Buffer salt Buffer salt Solvent Adsorbent Packaging specifications: The finished product is white freeze-dried powder injection, 1mg / ampoule, 10 ampoules / box Storage: Store at 2-8℃ away from light, valid for 3 years.

[0034] 2. [Dosage form] Water for injection.

[0035]

Specification

[0036] Prescription composition:

[0037] The amount of raw materials and excipients used in the synthesis product is 1g / 1000ml of water for injection, and a total of 1000 bottles are prepared.

[0038] Preparation process:

[0039] Solution preparation: Operate in a class 100 sterile room.

[0040] 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 with a 0.2 μm microporous filter membrane, take 1 ml and dispense it into a 3 ml medicinal ampoule and seal it.

[0041] 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.

[0042] Packaging specifications: 1mg / ml / ampoule, 10 ampoules / box.

[0043] Storage: Store at 2-8℃ away from light. Valid for 1 year.

[0044] The preparation method can be appropriately modified without being restricted to this.

[0045] Example 1

[0046] Hepatitis B patients are given subcutaneous injection of the synthetic drug freeze-dried powder (1 mg / ampoule). The treatment plan is 1 mg / time / week, and 12 weeks is a course of treatment. The drug in the ampoule can be dissolved with 1 ml of injection water, and the solution can be drawn out of the ampoule with a syringe before subcutaneous injection. Closely observe the patient's reaction and condition. If the patient is found to have symptoms such as palpitations, sweating, fatigue, tremors, etc., the injection should be stopped immediately.

[0047] Example 2

[0048] Patients after tumor resection should be given subcutaneous injection of this drug, 1 mg / time / week, for a course of 12 weeks. If the patient has symptoms such as palpitations, sweating, fatigue, tremor, etc., the injection should be stopped immediately.

Claims

1. A functional peptide segment consisting of N-threonine-lysine-proline-arginine-C (N-Thr-Lys-Pro-Arg-C), which is connected to an 8-branched polypeptide backbone (Lys)4-(Lys)2-Lys- or a purchased commercial 8-branched backbone (MultipalAntigen peptide, MAP8) to produce a functional 8-branched polypeptide synthetic product (Thr-Lys-Pro-Arg)g-(Lys)4-(Lys)2-Lys.

2. The functional peptide segment and the branch ends of the 8-branched polypeptide backbone can be connected through a peptide chain formed by N natural amino acids (AA) to produce a functional 8-branched polypeptide synthetic product (Thr-Lys-Pro-Arg-AAn)8-(Lys)4-(Lys)2-Lys, where N is a natural number.

3. Multiple functional peptide segments can be linearly connected and then connected to the branch ends of the 8-branched polypeptide backbone to produce a functional 8-branched polypeptide synthetic product.

4. The 8-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). The functional 8-branched polypeptide synthesis product (Thr-Lys-Pro-Arg)8-(Lys)4-(Lys)2-Lys-AAn produced by cleavage with the resin, where N is a natural number.

5. The functional peptide segment and the 8-branched skeleton form (Thr-Lys-Pro-Arg-AAn)8-(Lys-AAn)4-(Lys-AAN)2-Lys-AAN, AA is a peptide chain connected by N natural amino acids, and N is a natural number.

6. The prepared functional 8-branched polypeptide synthesis product has the effects of resistance to degradation and improving the body's immune ability, and can be prepared into oral or injectable medicines to improve the body's immune ability.

7. It can be prepared into veterinary medicine and used as an immunopotentiator to treat infectious diseases and improve resistance.

8. It can be prepared into clinical medicine as an immunopotentiator for anti-infection, anti-virus and prevention and treatment of tumors.

Images