Composition containing bioactive peptide and application of composition in treatment of cardiovascular and cerebrovascular diseases

Abstract

The invention provides a composition containing bioactive peptide and application of the composition in treatment of cardiovascular and cerebrovascular diseases, specifically, the specific anticoagulant peptide KN-59 obtained through screening has a strong anticoagulant characteristic, it is identified in experiments that the anticoagulant peptide KN-59 has the effects of prolonging TT and PT in blood, remarkably prolonging APTT and remarkably improving the FIB level in plasma at the same time, and the application of the anticoagulant peptide KN-59 in treatment of cardiovascular and cerebrovascular diseases is achieved. It is shown that the anticoagulant peptide KN-59 can prolong the blood coagulation time and has good antithrombotic and anticoagulant effects.

Description

Technical Field

[0001] The present application relates to the biological field, specifically to the medical field, and more specifically to a composition containing biologically active substances and its application in the treatment of cardiovascular and cerebrovascular diseases. Background Art

[0002] Coagulation is an indispensable physiological function of the human body. Surveys show that in recent years, 1-3 people out of every 1,000 suffer from thrombotic diseases. At present, most clinical anticoagulant and thrombolytic drugs are urokinase, streptokinase, coumarin, heparin, etc., but they are prone to cause side effects such as subcutaneous purpuric hemorrhage. Therefore, the development of anticoagulant, thrombolytic, and antiplatelet aggregation active substances with small side effects has attracted the attention of more and more scholars. In recent years, in addition to extracting anticoagulants from blood, the body fluids of poisons have also become an important source of anticoagulant, thrombolytic, and antiplatelet aggregation active substances: such as hirudin, tick anticoagulant peptides, canine hookworm anticoagulant peptides, snake venom anticoagulant peptides, earthworm fibrinolytic enzymes, and nattokinase. Anticoagulant peptides extracted from natural poisons have high activity, but high toxicity, and scarce resources, which is not conducive to mass production. Therefore, anticoagulant peptides with a wide range of sources have become a hot topic in research.

[0003] The anti-blood coagulation mechanism is that the added substances can prevent prothrombin from forming thrombin, thereby inhibiting the conversion of fibrinogen to fibrin; the fibrinolytic mechanism is to activate plasminogen to generate plasmin by adding plasminogen activators, and plasmin induces fibrin to be converted into fibrin degradation products; the anti-platelet aggregation mechanism is to reduce the platelets attached to the fibrin skeleton by adding anti-platelet aggregation substances.

[0004] Hirudin is the earliest discovered direct thrombin inhibitor. The FDA has approved it for the prevention and treatment of postoperative deep vein thrombosis and heparin-induced thrombocytopenia (HIT) patients who need anticoagulant therapy. Bivalirudin is used for patients with unstable angina undergoing percutaneous transluminal coronary angioplasty (PTICA), and is used in combination with platelet glycoprotein IIb / IIIa inhibitors for patients undergoing percutaneous coronary intervention (PCI) treatment. It can also be used for patients with heparin-induced thrombocytopenia who need anticoagulant therapy. It was approved by the FDA in December 2002. The FDA also approved argatroban in June 2000 and April 2002 for the prevention and treatment of thrombosis in patients with heparin-induced thrombocytopenia, and heparin-induced thrombocytopenia patients treated with PCI, respectively. Ximelagatran is the first oral peptide anticoagulant developed in the past 50 years. Due to its hepatotoxicity, the FDA has not approved it for marketing in the United States. It is only used for short-term administration in Europe. In February 2006, Astrazeneca announced that it would withdraw Ximelagatran (Exanta) from the market. Dabigatran is currently in Phase III clinical trials for anticoagulant therapy after total hip replacement and treatment of venous thromboembolism. New oral anticoagulants have also made significant progress. For example, oral anticoagulants such as dabigatran, apixaban, and rivaroxaban have obvious anticoagulant effects, do not require frequent testing, and have little impact on patients with long-term use. Therefore, the application of these new anticoagulants has gradually replaced traditional anticoagulants such as warfarin and become a more ideal clinical drug. There are also reports that a new thrombolytic peptide GLSHTG (hereinafter referred to as: GLS) has been discovered. It has the functions of clearing human vascular garbage, regenerating vascular structure, and restoring vascular elasticity. It is broad-spectrum, highly efficient, and non-toxic, making it possible to extend the life of human cardiovascular and cerebrovascular systems.

[0005] In addition, the preparation of anticoagulant, thrombolytic, and platelet aggregation-inhibiting peptides at home and abroad has laid the foundation for the prevention and treatment of thrombotic diseases. However, such substances are prone to cause side effects such as subcutaneous bleeding in patients. Therefore, the study of safe bioactive peptides with anticoagulant, thrombolytic, and platelet aggregation-inhibiting functions has become a new research direction. The development of a new anticoagulant peptide for preventing thrombosis has broad research and development prospects. Summary of the invention

[0006] The present invention provides a novel anticoagulant peptide, anticoagulant peptide KN-59, whose amino acid sequence is shown in SEQ ID NO:1.

[0007] Furthermore, the anticoagulant peptide KN-59 provided by the present invention has good anticoagulant properties and safety.

[0008] Specifically, the anticoagulant peptide KN-59 can also be modified or conservatively substituted or homologously substituted while still maintaining the corresponding biological activity.

[0009] Specifically, the polypeptide can form a pharmaceutically acceptable salt with any non-toxic organic or inorganic acid. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and acidic metal salts, such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include monobasic, dibasic and tribasic carboxylic acids. Examples of these acids include, for example, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid and sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Salts of the carboxyl terminal amino acid moiety include non-toxic carboxylates formed with any suitable inorganic or organic base. By way of example, these salts include alkali metal salts, such as sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IIIA, including aluminum; and organic primary, secondary and tertiary amines, such as trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethyleneamine, N,N'-dibenzylethylenediamine, dihydroabietylamine, N-(lower)alkylpiperidines and any other suitable amines.

[0010] The anticoagulant peptide KN-59 of the present invention can be produced by biological fermentation or solid phase synthesis. As known in the field of solid phase peptide synthesis, many amino acids have functions that need to be protected during chain preparation. The use and selection of suitable protecting groups are within the capabilities of those skilled in the art and will depend on the presence of other protected amino acid residues on the amino acid to be protected and the peptide. The selection of such side chain protecting groups is critical because it must be a side chain protecting group that is not removed by cutting during the cutting process of the protecting group of the amino moiety. For example, the suitable side chain protecting group of lysine is benzyloxycarbonyl and substituted benzyloxycarbonyl, and the substituent is selected from halogen (e.g., chlorine, bromine, fluorine) and nitro (e.g., 2-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 3,4-dichlorobenzyloxycarbonyl), tosyl, tert-pentyloxycarbonyl, tert-butyloxycarbonyl and diisopropylmethoxycarbonyl. The alcoholic hydroxyl groups of threonine and serine can be protected by acetyl, benzoyl, tert-butyl, trityl, benzyl, 2,6-dichlorobenzyl or benzyloxycarbonyl. A preferred protecting group is benzyl.

[0011] These groups can be removed by methods known in the art. Typically, the removal of protecting groups is performed after the peptide chain synthesis is complete, but the protecting groups can be removed at any other suitable time.

[0012] Furthermore, the present invention also provides a pharmaceutical composition comprising the bioactive peptide, the anticoagulant peptide KN-59.

[0013] Further, since polypeptides may be denatured by gastric acid when orally administered, the active ingredients of the composition for oral administration should be coated or formulated to prevent degradation in the stomach. In addition, the composition can be administered using a specific device capable of transporting the active ingredient into target cells.

[0014] Composition of the present disclosure can be prepared into the form of pharmaceutical composition for preventing or treating inflammatory diseases, and the pharmaceutical composition also includes suitable carriers, excipients or diluents commonly used to prepare pharmaceutical compositions. Specifically, the pharmaceutical composition can be formulated into oral dosage forms according to conventional methods, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external dosage forms, suppositories or sterile injections. In the present invention, the carrier, excipient and diluent included in the pharmaceutical composition can include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and minerals. These preparations can be prepared by using commonly used diluents or excipients, such as fillers, extenders, adhesives, wetting agents, disintegrants, surfactants, etc. Examples of solid preparations for oral administration include tablets, pills, powders, granules, capsules. Etc. These solid preparations are prepared by mixing extracts or their fractions with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate or talcum can also be used. As liquid preparations for oral administration, for example: suspensions, internal solutions, emulsions, syrups, etc. can be cited. In addition to liquid paraffin or water, liquid preparations can include commonly available simple diluents, various excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration can include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate. The base of the suppository preparation can be witepsol, polyethylene glycol, tween61, cocoa butter, lauryl alcohol butter, glycerin gelatin and the like.

[0015] Further, suitable carriers include, but are not limited to, buffers containing succinate, phosphate, borate, HEPES, citrate, histidine, imidazole, acetate, bicarbonate and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight polypeptides, including but not limited to polypeptides of less than about 10 residues; proteins, including but not limited to serum albumin, gelatin or immunoglobulins; hydrophilic polymers, including but not limited to polyvinylpyrrolidone; amino acids, including but not limited to glycine, glutamine, asparagine, arginine, histidine or histidine derivatives, methionine, glutamic acid or lysine; monosaccharides, disaccharides and other carbohydrates, including but not limited to trehalose, sucrose, glucose, mannose or dextrin; chelating agents, including but not limited to EDTA and disodium ethylenediaminetetraacetate; divalent metal ions, including but not limited to zinc, cobalt or copper ; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counterions, including but not limited to sodium and sodium chloride; fillers, such as microcrystalline cellulose, lactose, corn starch and other starches; binders; sweeteners and other flavoring agents; colorants; and / or nonionic surfactants, including but not limited to Tween™ (including but not limited to Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), Pluronics™ and other pluronic acids, including but not limited to pluronic acid F68 (poloxamer 188) or PEG. Suitable surfactants include, for example, but not limited to, polyethers based on poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO) or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO) or combinations thereof.

[0016] The pharmaceutical composition of the present disclosure can be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to any medical treatment or prevention, and the effective dose level can be determined depending on the following factors, including the severity of the disease, the activity of the drug, the age, weight, health status and gender of the patient, sensitivity to the drug, the administration time of the composition of the present invention, the route of administration and the excretion rate, the duration of treatment, the drug used simultaneously or in combination with the composition of the present invention, and other factors known in the medical field. The pharmaceutical composition of the present disclosure can be administered alone or in combination with other known therapeutic agents for sarcopenia. Taking all of the above factors into account, it is important to administer the composition in a minimum amount that can show the maximum effect without causing side effects.

[0017] Further, the anticoagulant dosage of the anticoagulant peptide KN-59 composition of the present invention is 0.1 mg / kg to 250 mg / kg of patient weight per day, depending on the patient, the severity of the thrombocytosis disorder to be treated and the selected peptide derivative. The appropriate dosage for a particular patient can be easily determined. Preferably, 1-4 doses are given per day, and 5-100 mg of active compound is usually given per dose.

[0018] Anticoagulant therapy is applicable to the treatment and prevention of various thrombotic conditions, particularly coronary and cerebrovascular diseases. Those skilled in the art will readily appreciate the need for anticoagulant therapy. As used herein, the term "patient" refers to a mammal, such as a primate, including humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice.

[0019] Although some peptide derivatives may survive passage through the intestine following oral administration, Applicants prefer non-oral administration, e.g., subcutaneously, intravenously, intramuscularly or intraperitoneally; administration by depot injection; preparation by implant; or application to mucous membranes, e.g., those of the nose, throat and bronchial tubes, e.g., in an aerosol can containing a peptide derivative of the invention in spray or dry powder form.

[0020] For parenteral administration, the compound can be administered as an injectable dose of a solution or suspension of the compound in a physiologically acceptable diluent together with a pharmaceutical carrier, which can be a sterile liquid, such as water and oil, with or without the addition of surfactants and other pharmaceutically acceptable diluents. Acceptable adjuvants. Examples of oils that can be used for these preparations are oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil and mineral oil. Typically, water, saline, aqueous dextrose solutions and related sugar solutions, ethanol and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0021] The compound can be administered in the form of a reservoir injection or implant formulation, which can be formulated in a manner that allows for sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as a reservoir injection or implant. Implants can use inert materials such as biodegradable polymers or synthetic silicones.

[0022] Beneficial Effects

[0023] The present invention provides a composition comprising bioactive peptides and an application thereof in treating cardiovascular and cerebrovascular diseases. Specifically, the specific anticoagulant peptide KN-59 obtained by screening has a strong anticoagulant property. In a mouse experiment, it was identified that the anticoagulant peptide has the ability to prolong TT and PT in the blood, and can also significantly prolong APTT, and can significantly increase the FIB level in plasma, indicating that the anticoagulant peptide KN-59 can prolong the coagulation time of normal mice and has good antithrombotic and anticoagulant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Effects of each group on the coagulation time of the mouse tail tip DETAILED DESCRIPTION

[0025] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0026] Example 1 Design and synthesis of anticoagulant peptides

[0027] A random 12-peptide library XXXXXXXXX was designed, where X was a random composition of Fmoc-protected D-Gly, D-Ala, D-Val, D-Leu, D-Ile, D-Phe, D-Pro, D-Tyr, D-Ser, D-Cys, D-Thr, D-Trp, D-Met, D-Glu, D-Gln, D-Asp, D-Asn, D-Lys, D-Arg, and D-His. Tech Bio was commissioned to perform in situ peptide chip synthesis and in vitro thrombus lysis assay experiments, and 7 peptides with good anticoagulant properties were identified, as shown in Table 1. The peptides were all artificially synthesized and set aside.

[0028] Table 1 Anticoagulant peptides screened

[0029]

[0030]

[0031] Example 27 Further determination of the anticoagulant peptide activity

[0032] Take 300 μL Tyrode-HEPES buffer, perform the base test, and automatically adjust the platelet aggregation instrument transmittance to 0%. Then take 300 μL mouse platelet suspension in a cuvette, add 10 μL of 7 kinds of polypeptide drugs (concentration of 5 mg / mL) screened in Example 1, incubate at 37°C for 10 minutes, and quickly add 10 μL platelet aggregation inducer (ADP) at the end of preheating to test the platelet aggregation rate within 5 minutes. Among them, 0.9% sodium chloride solution is used as a blank control solution, and aspirin of the same concentration is tested as a positive drug. All experiments are tested in parallel in 3 groups. All experiments are completed within 4 hours after the platelets are prepared. The inhibition rate of drug on platelet aggregation is calculated by the following formula: inhibition rate (%) = [(maximum aggregation rate of blank group-maximum aggregation rate of test group) / maximum aggregation rate of blank group] × 100%. SPSS is used for statistical analysis, and the calculation results are expressed as IC50 values. All experiments are repeated 3 times independently, and the results are expressed as the average of the 3 results. The results are shown in Table 2.

[0033] Table 2 Inhibition rate of various anticoagulant peptides on platelet aggregation

[0034] Peptide name Inhibition rate (%) KN-5 40.57±0.17# KN-19 51.74±0.22# KN-46 62.49±0.24# KN-59 93.64±0.48## KN-91 70.51±0.34# KN-151 49.53±0.17# KN-197 66.42±0.23# Positive Control 86.32±0.87## Blank control 1.27±0.03

[0035] As can be seen from Table 2, the anticoagulant peptide KN-59 has the best anticoagulant effect, with a significant difference compared with the blank control (P<0.01), and a similar effect compared with the positive control. This peptide was selected for subsequent experiments.

[0036] Example 3 In vivo anticoagulation test of anticoagulant peptide KN-59

[0037] Kunming mice were randomly divided into 5 groups, half male and half female, with 10 mice in each group, namely normal group, low-, medium- and high-dose groups of anticoagulant peptide KN-59 (1, 5, 10 mg / kg), and positive control group (aspirin 1 mg / kg). The normal group was gavaged with an equal amount of normal saline, and the rest of the feeding conditions were the same for each group. The drugs were administered by gavage continuously for 7 days, once a day.

[0038] One hour after the last intragastric administration, cut off the 1 cm tail tip of the mouse to allow the blood to flow out automatically. After the first drop of blood was wiped dry with filter paper, two drops were dripped on both ends of a clean glass slide. The No. 6 needle used for drying was slowly moved inward along the outer edge of the blood drop until the No. 6 needle could pick up the fibrin filaments. This was calculated as the mouse tail tip coagulation time. The results were as follows: Figure 1 shown.

[0039] from Figure 1It can be seen that compared with the normal saline group (129.1±3.2) s, the low, medium and high dose groups of peptide can significantly prolong the blood coagulation time of the normal mouse tail. The coagulation time of the high dose group reached (198.3±6.4) s, which was also significantly improved compared with the positive control group (145.3±2.8) s.

[0040] One hour after the last oral administration, the mouse eyeballs were removed with ophthalmic forceps and blood was collected, anticoagulated with 3.8% sodium citrate and centrifuged at 1500 r / min for 5 min. The thrombin time TT, PT, fibrinogen (FIB) level, and activated partial prothrombin time (APTT) of the mice were measured according to the instructions of the kit. The results are shown in Table 3.

[0041] Table 3 Effects of peptides on four coagulation indexes in normal mice

[0042] Group TT(s) PT(s) APPT(s) FIB(g / L) Normal group 24.53±0.84 13.51±0.55 28.10±0.84 43.01±2.76 Low dose group 37.37±0.96# 16.13±0.77 36.58±1.14 69.47±3.17## Medium dose group 46.53±1.27## 24.36±1.21# 45.51±1.53# 87.45±4.53## High dose group 53.17±1.54## 33.54±1.45## 54.17±1.87## 98.75±5.37## Positive control group 36.78±1.03# 15.99±0.86 30.84±1.03 67.14±2.17##

[0043] From the results in Table 3, it can be seen that compared with the normal group, the anticoagulant peptide KN-59 can prolong TT and PT, and can also significantly prolong APTT, and can significantly increase the FIB level in plasma, and all of them are dose-related, indicating that the anticoagulant peptide KN-59 can prolong the coagulation time of normal mice and has good antithrombotic and anticoagulant effects. After the detection, there was no significant difference in the organs of mice in each dose group compared with the normal group, indicating that the anticoagulant peptide has good safety.

[0044] Example 4 Safety Experiment of Anticoagulant Peptide KN-59

[0045] Umbilical vein endothelial cells in the logarithmic phase (Shanghai Kanglang Biotechnology Co., Ltd., catalog number: KL-01221) were obtained, digested with trypsin, centrifuged and resuspended, and the cell concentration was adjusted to 1×10 7 L -1, inoculated in 96-well plates, 200 μL of cell suspension was added to each well, the control group was cultured in DMEM medium with a volume fraction of 10% fetal bovine serum, and the experimental groups 1-3 were added with complete medium with final concentrations of 1 mg / mL, 3 mg / mL, and 5 mg / mL of anticoagulant peptide KN-59, respectively, with 5 parallel samples. After conventional culture in the incubator for 72 hours, 20 μL MTT solution was added to each well, and the liquid in the well was discarded after continued culture for 4 hours, 150 μL DMSO was added, and the absorbance (A value) was measured at a wavelength of 592 nm by a microplate reader after mixing, and the relative cell proliferation rate was calculated, and the relative cell proliferation rate (%) = absorbance value of the experimental group / absorbance value of the control group × 100%. The toxicity reaction was graded according to the relative cell proliferation rate: when the relative proliferation rate was ≥100%, it was grade 0; between 80%-99% was grade 1; between 50%-79% was grade 2; between 30%-49% was grade 3; between 0-29% was grade 4. The results showed that when its mass concentration was 1 mg / mL, the cytotoxicity level was grade 0; when its mass concentration was 3 mg / mL, the cytotoxicity level was still grade 0; when its mass concentration was 5 mg / mL, the cytotoxicity level was grade 1. These results showed that the anticoagulant peptide KN-59 had no obvious cytotoxicity and had good safety.

[0046] Based on the above description, it will be appreciated by those skilled in the art that the present invention can be implemented in different specific forms without changing its essential features. Therefore, it should be understood that the above embodiments are not restrictive, but exemplary in all respects. The scope of the present invention is limited by the appended claims rather than by the preceding description thereof, and therefore all changes and modifications falling within the scope and range of the claims, or the equivalents of these scopes and ranges are intended to be covered by the claims.

Claims

1. An anticoagulant peptide, characterized in that The anticoagulant peptide is KN-59, and its amino acid sequence is shown in SEQ ID NO:

4.

2. A pharmaceutical composition for inhibiting thrombosis in cardiovascular and cerebrovascular diseases, characterized in that The active peptide KN-59 has an amino acid sequence as shown in SEQ ID NO:

4.

3. Use of the anticoagulant peptide KN-59 as claimed in claim 1 in the preparation of a medicament for inhibiting thrombosis.

4. The use as claimed in claim 3, which is a drug for preventing cerebral infarction.

5. The use as claimed in claim 3, wherein the medicine further contains a pharmaceutically acceptable carrier.

Images