Compositions comprising bioactive peptides and use in the treatment of cardiovascular and cerebrovascular diseases

Abstract

This invention provides a composition containing bioactive peptides and its application in the treatment of cardiovascular and cerebrovascular diseases. Specifically, the screened specific anticoagulant peptide KN-59 has strong anticoagulant properties. Experiments have shown that it can prolong TT and PT in the blood, and can also significantly prolong APTT, while significantly increasing the FIB level in plasma. This indicates that the anticoagulant peptide KN-59 can prolong clotting time and has good antithrombotic and anticoagulant effects.

Description

Technical Field

[0001] This application relates to the biological field, specifically the pharmaceutical field, and more specifically to compositions containing bioactive substances and their use in the treatment of cardiovascular and cerebrovascular diseases. Background Technology

[0002] Blood clotting is an indispensable physiological function of the human body. Surveys show that in recent years, 1-3 out of every 1000 people suffer from thrombotic diseases. Currently, clinical anticoagulant and thrombolytic drugs are mostly urokinase, streptokinase, coumarin, and heparin, but they are prone to causing side effects such as subcutaneous purpuric hemorrhage. Therefore, the development of anticoagulant, thrombolytic, and antiplatelet aggregation active substances with fewer side effects has attracted increasing attention from researchers. In recent years, in addition to extracting anticoagulant substances from blood, the bodily fluids of toxic substances have also become important sources of anticoagulant, thrombolytic, and antiplatelet aggregation active substances: such as hirudin, tick anticoagulant peptides, hookworm anticoagulant peptides, snake venom anticoagulant peptides, earthworm fibrinolytic enzymes, and nattokinase. Anticoagulant peptides extracted from natural toxins have high activity but also high toxicity, and resources are scarce, hindering large-scale production. Therefore, research on anticoagulant peptides with widely available sources has become a research hotspot.

[0003] The anti-blood coagulation mechanism is that the added substance can prevent prothrombin from forming thrombin, thereby inhibiting the conversion of fibrinogen to fibrin; the fibrinolysis mechanism is that the added plasminogen activator activates plasminogen to generate plasmin, and plasmin induces fibrin to be converted into fibrin degradation products; the anti-platelet aggregation mechanism is that the added anti-platelet aggregation substance reduces the number of platelets attached to the fibrin backbone.

[0004] Hirudin was the first discovered direct thrombin inhibitor, and the FDA has approved it for the prevention and treatment of postoperative deep vein thrombosis (DVT) and heparin-induced thrombocytopenia requiring anticoagulation therapy in patients with heparin-induced thrombocytopenia (HIT). Bivalirudin is used for patients with unstable angina undergoing percutaneous transluminal coronary angioplasty (PTICA), in combination with platelet glycoprotein IIb / IIIa inhibitors for patients undergoing percutaneous coronary intervention (PCI), and also for patients with heparin-induced thrombocytopenia requiring anticoagulation 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 type II thrombocytopenia and for patients with heparin-induced thrombocytopenia undergoing PCI, respectively. Cimetazidine was the first orally administered peptide anticoagulant developed in nearly 50 years. Due to its hepatotoxicity, it was not approved for marketing in the United States by the FDA and was only used for short-term administration in Europe. In February 2006, AstraZeneca announced the withdrawal of Cimetazidine (Exanta) from the market. Dabigatran is currently in Phase III clinical trials for anticoagulation therapy after total hip replacement surgery and for the treatment of venous thromboembolism. Significant progress has also been made in the development of novel oral anticoagulants. For example, oral anticoagulants such as dabigatran, apixaban, and rivaroxaban have significant anticoagulant effects, do not require frequent monitoring, and have minimal long-term impact on patients. Therefore, these new anticoagulants are gradually replacing traditional anticoagulants such as warfarin, becoming a more ideal clinical application. A novel thrombolytic peptide, GLSHTG (hereinafter referred to as GLS), has also been reported to have been discovered. It has the ability to clear vascular waste, regenerate vascular structure, and restore vascular elasticity. It is broad-spectrum, highly effective, and non-toxic, potentially extending the lifespan of the cardiovascular system.

[0005] Furthermore, the preparation of anticoagulant, thrombolytic, and platelet aggregation-inhibiting peptides both domestically and internationally has laid the foundation for the prevention and treatment of thrombotic diseases. However, these substances are prone to causing side effects such as subcutaneous bleeding in patients. Therefore, researching safe bioactive peptides with anticoagulant, thrombolytic, and platelet aggregation-inhibiting functions has become a new research direction. Developing a novel anticoagulant peptide for thrombosis prevention has broad research and development prospects. Summary of the Invention

[0006] This invention provides a novel anticoagulant peptide, KN-59, whose amino acid sequence is shown in SEQ ID NO:4.

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

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

[0009] Specifically, polypeptides can form pharmaceutically acceptable salts 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, as well as acidic metal salts such as sodium orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include monocarboxylic, dicarboxylic, and tricarboxylic 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 carboxylic acid salts formed with any suitable inorganic or organic base. For example, these salts include alkali metal salts such as sodium and potassium; alkaline earth metals such as calcium and magnesium; Group IIIA light metals including aluminum; and organic primary, secondary, and tertiary amines, such as trialkylamines including triethylamine, procaine, dibenzylamine, 1-ethyleneamine, N,N'-dibenzylethylenediamine, dihydrorosinylamine, N-(lower)alkylpiperidine, and any other suitable amine.

[0010] The anticoagulant peptide KN-59 of the present invention can be produced by bio-fermentation or by solid-phase synthesis. In the field of solid-phase peptide synthesis, many amino acids are known to have functions requiring protection 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 amino acid to be protected and the presence of other protected amino acid residues on the peptide. The selection of such side-chain protecting groups is critical because they must be side-chain protecting groups that are not cleaved during the cleavage of the protecting group of the -amino moiety. For example, suitable side-chain protecting groups for lysine are benzyloxycarbonyl and substituted benzyloxycarbonyl, wherein the substituent is selected from halogens (e.g., chlorine, bromine, fluorine) and nitro groups (e.g., 2-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 3,4-dichlorobenzyloxycarbonyl), toluenesulfonyl, tert-pentyloxycarbonyl, tert-butyloxycarbonyl, and diisopropylmethoxycarbonyl. The hydroxyl groups of threonine and serine can be protected by acetyl, benzoyl, tert-butyl, triphenylmethyl, benzyl, 2,6-dichlorobenzyl, or benzyloxycarbonyl. The preferred protecting group is benzyl.

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

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

[0013] Furthermore, since peptides may be denatured by gastric acid upon oral administration, the active ingredient in compositions intended for oral administration should be coated or formulated to prevent degradation in the stomach. Additionally, specific devices capable of delivering the active ingredient to target cells can be used to administer the composition.

[0014] The compositions disclosed herein can be formulated into pharmaceutical compositions for the prevention or treatment of inflammatory diseases, said pharmaceutical compositions further comprising suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. Specifically, the pharmaceutical compositions can be formulated according to conventional methods into oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and external dosage forms, suppositories, or sterile injections. In this invention, the carriers, excipients, and diluents included in the pharmaceutical compositions may include lactose, dextran, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and minerals. These formulations can be prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Examples of solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms are prepared by mixing an extract or fraction thereof 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 talc may be used. Examples of liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to liquid paraffin or water, liquid dosage forms may include commonly available simple diluents and various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. Dosage forms for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate. The base of suppository formulations can be witepsol, polyethylene glycol, Tween 61, cocoa butter, lauryl butter, glycerin gelatin, etc.

[0015] Furthermore, 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 peptides, including but not limited to peptides with fewer 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; and 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 flavorings; 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 (poloxam 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 compositions disclosed herein can be administered in pharmaceutically effective amounts. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio suitable for any medical treatment or prevention, and the effective dose level may be determined depending on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health status and sex, sensitivity to the drug, the timing of administration of the composition of the present invention, route of administration and excretion rate, duration of treatment, drugs used concurrently or in combination with the composition of the present invention, and other factors known in the medical field. The pharmaceutical compositions disclosed herein can be administered alone or in combination with other known therapeutic agents for sarcopenia. Taking all the foregoing factors into account, it is important to administer the composition in the minimum amount that can exhibit maximum effect without causing side effects.

[0017] Furthermore, the anticoagulant dose of the KN-59 anticoagulant peptide composition of the present invention is from 0.1 mg / kg to 250 mg / kg of patient body weight per day, depending on the patient, the severity of the thrombocytosis condition to be treated, and the peptide derivative selected. The appropriate dose for a particular patient can be readily determined. Preferably, 1-4 doses are administered daily, typically 5-100 mg of the active compound per dose.

[0018] Anticoagulation therapy is suitable for the treatment and prevention of various thrombotic conditions, particularly coronary and cerebrovascular diseases. Those skilled in the art will readily recognize situations requiring anticoagulation therapy. As used herein, the term "patient" refers to mammals, such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice.

[0019] Although some peptide derivatives may survive through the intestine after oral administration, the applicant prefers non-oral administration, such as subcutaneous, intravenous, intramuscular, or intraperitoneal administration; reservoir injection; preparation by implantation; or application to mucous membranes, such as the mucous membranes of the nose, pharynx, and bronchi, for example, in an aerosol can containing the peptide derivatives of the invention in spray or dry powder form.

[0020] For parenteral administration, the compound may be administered as a solution or suspension in a physiologically acceptable diluent in an injectable dose, co-administered with a drug carrier, which may be a sterile liquid, such as water and oil, with or without the addition of surfactants and other pharmaceutically acceptable diluents. Acceptable adjuvants are also acceptable. Examples of oils that can be used in these formulations are petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, and mineral oil. Generally, water, saline, aqueous solutions of dextran 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, 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. The implant can use inert materials, such as biodegradable polymers or synthetic siloxanes.

[0022] Beneficial effects

[0023] This invention provides a composition containing bioactive peptides and its application in the treatment of cardiovascular and cerebrovascular diseases. Specifically, the screened specific anticoagulant peptide KN-59 has strong anticoagulant properties. In mouse experiments, it was identified that it can prolong the TT and PT in the blood, and can also significantly prolong the APTT. At the same time, it can significantly increase the FIB level in the plasma, indicating that the anticoagulant peptide KN-59 can prolong the clotting time of normal mice and has good antithrombotic and anticoagulant effects. Attached Figure Description

[0024] Figure 1 Figure 1 shows the effect of each group on the tail tip clotting time of mice. Detailed Implementation

[0025] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. Unless otherwise specified, the methods, equipment, and materials in the following embodiments are all conventional methods, equipment, and materials in the art and are commercially available.

[0026] Example 1: Design and Synthesis of Anticoagulant Peptides

[0027] A randomized 12-peptide library XXXXXXXXX was designed, where X is a randomized 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. In-situ peptide chip synthesis and in vitro thrombolysis assays were commissioned to Tektronix for initial screening. Seven peptides with good anticoagulant properties were identified, as shown in Table 1. All peptides were then artificially synthesized for future use.

[0028] Table 1. Anticoagulant peptides screened initially

[0029]

[0030]

[0031] Example 27: Further Determination of the Activity of Anticoagulant Peptides

[0032] 300 μL of Tyrode-HEPES buffer was used for substrate testing, and the transmittance of the platelet aggregator was automatically adjusted to 0%. Then, 300 μL of mouse platelet suspension was placed in a cuvette, and 10 μL of a solution of the seven peptide drugs screened in Example 1 (concentration 5 mg / mL) was added. The mixture was incubated at 37°C for 10 min. At the end of the pre-warming, 10 μL of platelet aggregation inducer (ADP) was quickly added, and the platelet aggregation rate was tested within 5 min. 0.9% sodium chloride solution was used as a blank control, and aspirin of the same concentration was used as a positive control. All experiments were performed in triplicate. All experiments were completed within 4 h after platelet preparation. The inhibition rate of the drugs on platelet aggregation was calculated using the following formula: Inhibition rate (%) = [(Maximum aggregation rate of blank group - Maximum aggregation rate of test group) / Maximum aggregation rate of blank group] × 100%. Statistical analysis was performed using SPSS, and the results were expressed as IC50 values. All experiments were performed independently in triplicate, and the results are expressed as the average of the three results. The results are shown in Table 2.

[0033] Table 2. Results of the inhibition rate of various anticoagulant peptides on platelet aggregation.

[0034] polypeptide 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 shown in Table 2, the anticoagulant peptide KN-59 exhibited the best anticoagulant effect, with a highly significant difference compared to the blank control (P<0.01), and its effect was similar to that of 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. The groups were: normal group, low, medium and high dose groups of anticoagulant peptide KN-59 (1, 5 and 10 mg / kg), and positive control group (aspirin 1 mg / kg). The normal group was given an equal volume of physiological saline by gavage. All other feeding conditions were the same for all groups. The mice were given the drug by gavage for 7 consecutive days, once a day.

[0038] One hour after the last gavage, the tip of the mouse's tail (1 cm) was cut off to allow blood to flow out automatically. The first drop of blood was wiped dry with filter paper, and two drops were placed on either end of a clean glass slide. A dry No. 6 needle was then used to slowly pick at the blood droplets from the outer edge inwards until fibrin threads could be picked up. This time was recorded as the tail tip clotting time. The results were as follows: Figure 1 As 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 polypeptide can significantly prolong the coagulation time of blood in the tail of normal mice. The coagulation time of the high dose group reached (198.3±6.4)s, which is also significantly higher than that of the positive control group (145.3±2.8)s.

[0040] One hour after the last gavage, the mice were enucleated with ophthalmic forceps to collect blood. The blood was anticoagulated with 3.8% sodium citrate and centrifuged at 1500 r / min for 5 min. The thrombin time (TT), PT, fibrinogen (FIB) levels, and activated partial thromboplastin time (APTT) were measured according to the kit instructions. The results are shown in Table 3.

[0041] Table 3. Effects of peptides on four coagulation parameters 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] As shown in Table 3, compared with the normal group, anticoagulant peptide KN-59 prolonged TT and PT, and also significantly prolonged APTT, while significantly increasing plasma FIB levels, all in a dose-dependent manner. This indicates that anticoagulant peptide KN-59 can prolong the clotting time of normal mice and has good antithrombotic and anticoagulant effects. Furthermore, after testing, there were no significant differences in organ function between the different dosage groups and the normal group, indicating that the anticoagulant peptide has good safety.

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

[0045] Log-phase umbilical vein endothelial cells (Shanghai Kanglang Biotechnology Co., Ltd., catalog number: KL-01221) were obtained, digested with trypsin, centrifuged, and resuspended to adjust the cell concentration to 1×10⁻⁶. 7 L -1Cells were seeded in 96-well plates, with 200 μL of cell suspension added to each well. The control group was cultured in DMEM medium containing 10% fetal bovine serum (FBS). Experimental groups 1-3 were cultured in complete medium containing the anticoagulant peptide KN-59 at final concentrations of 1 mg / mL, 3 mg / mL, and 5 mg / mL, respectively, with five replicates. After 72 h of routine incubation, 20 μL of LMT solution was added to each well, and the cells were cultured for another 4 h. The liquid in the wells was discarded, and 150 μL of LDMSO was added. After mixing, the absorbance (A value) was measured at 592 nm using a microplate reader. The relative cell proliferation rate was calculated as: Relative cell proliferation rate (%) = (Experimental group absorbance / Control group absorbance) × 100%. The cytotoxicity level was determined based on the relative cell proliferation rate: Grade 0 for a relative proliferation rate ≥100%; Grade 1 for 80%-99%; Grade 2 for 50%-79%; Grade 3 for 30%-49%; and Grade 4 for 0-29%. The results showed that at a concentration of 1 mg / mL, the cytotoxicity level was Grade 0; at a concentration of 3 mg / mL, the cytotoxicity level remained Grade 0; and at a concentration of 5 mg / mL, the cytotoxicity level was Grade 1. These results indicate that the anticoagulant peptide KN-59 has no significant cytotoxicity and exhibits good safety.

[0046] Based on the foregoing description, those skilled in the art will understand that the present invention can be implemented in different specific forms without altering its essential characteristics. Therefore, it should be understood that the above embodiments are not restrictive, but rather exemplary in all respects. The scope of the invention is defined by the appended claims rather than by the foregoing description, and therefore all variations and modifications falling within the scope and equivalents of the claims 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 thrombus formation in cardiovascular and cerebrovascular diseases, characterized by The active ingredient is the anticoagulant peptide KN-59, whose amino acid sequence is shown in SEQ ID NO:

4.

3. The use of the anticoagulant peptide KN-59 as described in claim 1 in the preparation of a drug for inhibiting thrombus formation.

4. The application as described in claim 3, wherein it is used to prepare a drug for preventing cerebral infarction.

5. The application as described in claim 3, wherein the drug further comprises a pharmaceutically acceptable carrier.

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