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

Abstract

The invention discloses a pharmaceutical composition containing bioactive peptide and application of the pharmaceutical composition in treatment of cardiovascular and cerebrovascular diseases. The pharmaceutical composition comprises a specifically designed bioactive peptide, and the peptide has multiple functions of resisting inflammation, resisting oxidation, promoting angiogenesis and the like. The composition can accurately inhibit inflammatory response in cardiovascular and cerebrovascular diseases, eliminate free radical damage caused by oxidative stress, and promote repair and regeneration of damaged blood vessels and myocardium. Experimental results show that the pharmaceutical composition significantly improves the treatment efficiency of cardiovascular and cerebrovascular diseases, and has significant anti-inflammatory and antioxidant effects. The bioactive peptide disclosed by the invention has the advantages of high safety, remarkable treatment effect and the like, and is suitable for treating various cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a pharmaceutical composition comprising a bioactive peptide and application thereof in treating cardiovascular and cerebrovascular diseases. Background Art

[0002] Globally, cardiovascular and cerebrovascular diseases have become one of the main causes of death and disability, especially atherosclerosis, coronary heart disease and stroke, which seriously affect people's quality of life. At present, the commonly used treatments in clinical practice mainly include statins, antiplatelet drugs, vasodilators, etc. However, these drugs are often accompanied by many side effects during long-term use, such as muscle damage, liver damage and bleeding risks. In addition, the pathogenesis of cardiovascular and cerebrovascular diseases is complex, involving multiple factors such as inflammatory response, oxidative stress, endothelial damage, thrombosis, etc. The single target action mode of existing drugs is often insufficient in complex pathological environments, making it difficult to fully control the progression of the disease. There is an urgent need to develop new drugs with multi-target effects.

[0003] In recent years, bioactive peptides have gradually become emerging candidate drugs for the treatment of cardiovascular and cerebrovascular diseases due to their unique advantages in regulating cell function, promoting tissue repair, anti-inflammatory and antioxidant effects. Bioactive peptides are small molecule chains composed of amino acids. They have a simple structure, high stability, good targeting and selectivity, and can not only directly act on the cardiovascular system, but also regulate inflammatory responses, angiogenesis and other processes through a variety of signaling pathways. However, traditional bioactive peptide drugs have some problems, such as short half-life in the body, easy enzymatic degradation and low bioavailability, which limit their clinical application.

[0004] In response to these problems, research in recent years has gradually focused on how to improve the stability and efficacy of bioactive peptides. For example, the structure of peptides can be optimized through chemical modification or genetic engineering to enhance their metabolic stability in the body; at the same time, nano drug delivery systems, such as nanoparticles or liposomes, are used to encapsulate bioactive peptides in carriers, which can not only effectively prevent the rapid degradation of peptides in the body, but also achieve targeted release and sustained action of drugs. Nanoparticle drug delivery systems have become an important means to enhance the efficacy of bioactive peptides due to their unique sustained release properties, good biocompatibility and targeting. In addition, nanoparticles can achieve efficient delivery to specific tissues or lesions by adjusting particle size and surface modification, further enhancing the therapeutic effect of drugs.

[0005] Therefore, the development of an efficient bioactive peptide pharmaceutical composition that can regulate cardiovascular and cerebrovascular pathological processes through multiple pathways and multiple targets has important clinical application value and potential. Summary of the invention

[0006] The purpose of the present invention is to provide a pharmaceutical composition containing a bioactive peptide, which has the characteristics of high efficiency, low toxicity, and sustainable release, and can effectively treat cardiovascular and cerebrovascular diseases. The present invention improves the stability and bioavailability of the drug by optimizing the carrier and administration method of the bioactive peptide, and significantly improves the therapeutic effect of cardiovascular and cerebrovascular diseases.

[0007] Therefore, on one hand, the present invention discloses a bioactive peptide, which is a bifunctional bioactive peptide, and its amino acid sequence is CRDKEVGGGFLRKV.

[0008] In another aspect, the present invention also discloses a pharmaceutical composition comprising a bioactive peptide, wherein the pharmaceutical composition comprises a nanoparticle drug comprising the bioactive peptide.

[0009] Preferably, the average particle size of the nanoparticle drug of the present invention is 150±20 nm, and the polymerization index (PDI) is 0.15.

[0010] Preferably, the encapsulation efficiency of the bioactive peptide in the nanoparticle drug of the present invention is 80%, and the drug loading is 12%.

[0011] Preferably, the pharmaceutical composition of the present invention further comprises an anti-VCAM-1 single domain antibody, wherein the amino acid sequence of the anti-VCAM-1 single domain antibody is as shown in SEQ ID NO.1.

[0012] Preferably, the IC50 of the anti-VCAM-1 single domain antibody of the present invention is 8 nM.

[0013] Preferably, the nanoparticle drug and the anti-VCAM-1 single domain antibody in the pharmaceutical composition of the present invention are mixed in equal mass ratios.

[0014] Preferably, the dosage of the pharmaceutical composition of the present invention is 1 mg / kg body weight.

[0015] In another aspect, the present invention also discloses the use of the bioactive peptide in preparing medicines for treating cardiovascular and cerebrovascular diseases.

[0016] In another aspect, the present invention also discloses the use of the anti-VCAM-1 single domain antibody in the preparation of drugs for treating cardiovascular and cerebrovascular diseases.

[0017] The bioactive peptides of the present invention and the pharmaceutical compositions prepared thereon have shown significant therapeutic effects in treating cardiovascular and cerebrovascular diseases, have the characteristics of high efficiency, low toxicity and sustained effects, and have broad clinical application prospects.

[0018] The present invention transforms the bioactive peptides to make them have higher targeting and stability. The present invention adopts nanoparticle carriers and combines sustained-release technology to achieve long-term stable release of bioactive peptides. The pharmaceutical composition of the present invention not only exerts anti-inflammatory, anticoagulant and other effects by directly acting on the cardiovascular and cerebrovascular systems, but also promotes endothelial repair and blood flow regulation, thereby alleviating and treating cardiovascular and cerebrovascular diseases in multiple ways. Among them, the anti-VCAM-1 single-domain antibody disclosed in the present invention is used to inhibit inflammatory reactions and vascular lesions. The single-domain antibody has the ability to specifically bind to VCAM-1, can interfere with vascular inflammatory signal transduction, and thus reduce cardiovascular and cerebrovascular damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 SDS-PAGE detection results of anti-VCAM-1 single domain antibody.

[0020] Figure 2 Western blot detection results of anti-VCAM-1 single domain antibody. DETAILED DESCRIPTION

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0022] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0023] Example 1: Design and synthesis of peptides

[0024] 1. Based on an in-depth analysis of the mechanisms related to cardiovascular and cerebrovascular diseases, a bifunctional bioactive peptide was designed, whose amino acid sequence is CRDKEVGGGFLRKV. The peptide achieves the effect of simultaneously targeting endothelial inflammation and oxidative stress by connecting amino acid fragments with different functions. The experimental results show that the peptide can significantly improve cardiovascular protection in vivo and reduce inflammatory response after myocardial infarction.

[0025] 2. Synthesis and purification of peptides

[0026] (1) Solid-phase peptide synthesis: Using the Fmoc solid-phase synthesis method, amino resin is used as a solid phase carrier, and amino acid monomers are sequentially connected to the resin to synthesize the target peptide. The specific steps are as follows:

[0027] Deprotection: First, the Fmoc protecting group was removed by 20% piperidine solution to expose the amino group.

[0028] Amino acid coupling: Each time, an Fmoc-protected amino acid monomer (such as Fmoc-Gly) is reacted with diisopropylcarbodiimide (DIC) as a coupling reagent and coupled to a solid support. After the coupling is completed, the unreacted reagent is washed with N-methylimidazole (NMI) and the peptide chain is formed through amide bond connection.

[0029] Repeat the cycle: each amino acid is coupled in turn according to the designed sequence until the synthesis of the target peptide is completed.

[0030] Cleavage and deprotection: The peptide was cleaved from the resin using trifluoroacetic acid and the protecting groups of the amino acid side chains were removed to obtain the crude peptide.

[0031] (2) Purification: The crude peptide was purified by high performance liquid chromatography. A reverse phase C18 column was used with water and acetonitrile as the mobile phases, and the peptide was purified by gradient elution at a flow rate of 1.0 mL / min. During the purification process, the peptide peak was monitored by a UV detector at a wavelength of 214 nm, and the target peptide peak was collected.

[0032] Gradient elution conditions: The initial conditions were 95% water and 5% acetonitrile, and the linear gradient was increased to 50% acetonitrile within 30 minutes. The target peptide peak was finally obtained and this fraction was collected.

[0033] (3) Purity identification: Mass spectrometry (MALDI-TOF) and liquid chromatography-mass spectrometry (LC-MS) were used to confirm the molecular weight of the purified bioactive peptides, confirming that they were consistent with expectations, and the purity was greater than 98% as detected by HPLC.

[0034] Example 2: Preparation and testing of nanopharmaceutical compositions containing bioactive peptides

[0035] In order to enhance the stability and targeting of bioactive peptides in vivo, this study used poly(lactic-co-glycolic acid) (PLGA) nanoparticles as drug carriers. PLGA is a polymer material with good biocompatibility and degradability, and the drug release rate can be controlled by adjusting its molecular weight and emulsification conditions.

[0036] The specific preparation steps are as follows:

[0037] 1. Materials and reagents: poly(lactic acid-co-glycolic acid) (PLGA, molar ratio of lactic acid to acetic acid 50:50, molecular weight 15 kDa), bioactive peptide (prepared in Example 1), polyvinyl alcohol (PVA, 88% hydrolyzed), dichloromethane (DCM), deionized water or ultrapure water.

[0038] 2. Nanoparticle preparation method

[0039] (1) Dissolve 50 mg of PLGA in 5 mL of dichloromethane as the organic phase and 2 mg of the bioactive peptide in 1 mL of deionized water as the aqueous phase.

[0040] (2) The aqueous phase was added dropwise into the organic phase, and ultrasonic emulsification was performed for 5 minutes using an ultrasonic emulsifier (power 100 W, frequency 40 kHz) to form colostrum.

[0041] (3) The colostrum was quickly added to 5 mL of an aqueous solution containing 1% PVA, and ultrasonic emulsification was continued for 3 minutes to form a stable oil-in-water (W / O / W) double emulsion system.

[0042] (4) The double emulsion solution was stirred at room temperature for 4 hours to evaporate the dichloromethane to obtain a nanoparticle suspension.

[0043] (5) The nanoparticles were washed three times with deionized water and centrifuged at 10,000 rpm for 15 min each time to remove excess PVA and unencapsulated peptides.

[0044] 3. Drying of nanoparticles: freeze-dry the final nanoparticle suspension to obtain powdered PLGA-bioactive peptide nanoparticles.

[0045] 4. Characterization of Nanoparticles

[0046] (1) Particle size and distribution: Dynamic light scattering (DLS) was used to measure the particle size and distribution of the nanoparticles. The results showed that the average particle size of the nanoparticles was 150±20 nm, the distribution was uniform, and the polymerization index (PDI) was 0.15, indicating that the nanoparticles had good dispersibility.

[0047] (2) Encapsulation efficiency and drug loading: High performance liquid chromatography (HPLC) was used to determine the encapsulation efficiency and drug loading of the nanoparticles. The results showed that the encapsulation efficiency of the bioactive peptide was 80% and the drug loading was 12%. This indicates that the bioactive peptide was successfully encapsulated in the PLGA nanoparticles and had a high drug loading capacity.

[0048] (3) Drug release curve: The nanoparticles were suspended in phosphate buffer (pH 7.4), and the drug release experiment was carried out at 37°C. Samples were taken at regular intervals and the amount of peptide released was detected by HPLC. The results showed that the drug release of the nanoparticles showed a sustained release characteristic, and about 40% of the bioactive peptide was released within 72 hours, showing good sustained release performance.

[0049] 5. In vitro functional testing

[0050] (1) Endothelial cell proliferation experiment: Human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates. After the cells adhered to the wall, a blank control group (no drug added), a bioactive peptide group (10 μg / mL) and a nanoparticle drug group (10 μg / mL of equal peptide) were given respectively. The cell proliferation rate was detected after 48 hours using a CCK-8 kit. The results showed that the cell proliferation rate of the nanoparticle drug group was 180% of that of the control group (P<0.01), which was significantly better than the 150% of the free peptide group, indicating that the nanocarrier significantly improved the biological activity and proliferation-promoting effect of the peptide.

[0051] (2) Cell migration experiment: The effect of nanomedicine on the migration ability of HUVECs was detected by scratch test. After the cells were spread all over the 6-well plate, they were scratched with a sterile pipette tip, and the control group, free peptide group and nanoparticle drug group were added respectively. After 24 hours, the scratch repair was observed under a microscope and the cell migration distance was measured. The results showed that the scratch repair rate of the nanoparticle drug group was 80%, while that of the free peptide group was 60%, and that of the control group was only 35%, indicating that the nanoparticle drug has a stronger ability to promote cell migration and wound healing.

[0052] (3) Anti-inflammatory experiment: ELISA was used to detect the inhibitory effect of peptide drugs on inflammatory factors (such as TNF-α and IL-6) in HUVECs stimulated by LPS. After stimulating the cells with LPS (10 ng / mL) for 6 hours, LPS (10 ng / mL) was added to each experimental group, and the levels of inflammatory factors in the cell supernatant were detected after 24 hours. The results showed that the levels of TNF-α and IL-6 in the nanoparticle drug group decreased by 45% and 40%, respectively, and the free peptide group decreased by 30%, while there was no significant change in the control group, indicating that the anti-inflammatory effect of the nanoparticle drug was significantly enhanced.

[0053] Example 3: Preparation and testing of anti-VCAM-1 single domain antibodies

[0054] 1. Select an adult alpaca (weight 65kg). Use recombinant human VCAM-1 (ab173991) as the antigen and adjust the concentration to 1mg / mL. The first subcutaneous injection is 1mL of vaccine (containing 500μg antigen + complete Freund's adjuvant mixed emulsification). The second and third vaccinations are separated by two weeks, using incomplete Freund's adjuvant, and the amount of antigen remains the same.

[0055] 2. Ten days after the last immunization, collect 100 mL of blood at 4000 rpm for 10 minutes, and obtain serum after separation.

[0056] 3. Screening and cloning of single domain antibodies

[0057] (1) RNA extraction: Add TRIzol reagent to serum, usually 1 ml TRIzol for every 200 μl serum, mix TRIzol and serum thoroughly, gently invert several times to ensure uniformity. Incubate at room temperature for 5 minutes to ensure that cells and proteins are completely lysed and RNA is released. Add 200 μl chloroform to every 1 ml TRIzol, cover the tube tightly, and shake vigorously for 15 seconds. The solution should turn milky white at this time. Let it stand at room temperature for 2-3 minutes. Centrifuge at 12000g for 15 minutes at 4°C. At this time, the solution should be separated into three layers: the upper aqueous phase (containing RNA), the middle layer (protein) and the lower organic phase (DNA and lipids). Carefully transfer the upper colorless aqueous phase to a new RNase-free centrifuge tube. Add an equal volume of isopropanol to the transferred supernatant, gently invert to mix, incubate at room temperature for 10 minutes, and centrifuge at 12000g and 4°C for 10 minutes. RNA will precipitate as a white precipitate. Carefully remove the supernatant, gently wash the pellet with 1 ml of pre-cooled 75% ethanol, centrifuge at 7500g, 4°C for 5 minutes, remove the ethanol, and air dry the pellet. Dissolve the RNA pellet in an appropriate amount of RNase-free water (usually 20-50 μl), incubate for 5 minutes and gently mix to ensure that the RNA is completely dissolved. Use NanoDrop or other spectrophotometer to detect RNA concentration and purity. The ideal A260 / A280 value should be between 1.8-2.0.

[0058] (2) cDNA synthesis:

[0059] Reaction system: contains 10 μL RNA (1000 ng), 5 μL primer (10 μM), 4 μL dNTPs (10 mM), 2 μL reverse transcriptase, 1 μL reverse transcription buffer, total volume 20 μL.

[0060] The PCR amplification procedure is as follows:

[0061] Pre-denaturation: 95°C, 5 minutes

[0062] Denaturation: 95°C, 30 seconds

[0063] Annealing: 55°C, 30 seconds

[0064] Extension: 72°C, 1 min

[0065] Number of cycles: 35 times

[0066] Final extension: 72°C, 10 min

[0067] 4. Antibody expression and purification

[0068] The PCR product was cloned into the pGEM-T easy cloning vector, transformed into Escherichia coli (DH5α), and selected for screening on LB plates containing antibiotics. BL21 (DE3) cells were used, with an induction concentration of 0.5 mM IPTG, a temperature of 37°C, and an induction time of 4 hours. The cells were disrupted by ultrasound and the supernatant was collected. Affinity purification was performed using a Ni-NTA column, with a washing buffer (containing 20 mM imidazole) and an elution buffer (containing 250 mM imidazole). Samples were taken to detect the purity of the antibody, and the electrophoresis conditions were 12% polyacrylamide gel. The results were shown (as shown in Figure 2A). Figure 1 The target band is at 15 kDa and the purity is about 96%.

[0069] 5. Single domain antibody testing

[0070] (1) Western Blot: The experiment was performed using recombinant human VCAM-1 (ab173991). The results showed that ( Figure 2 ), the single domain antibody in this study can specifically bind to the target protein.

[0071] (2) ELISA assay: Antigen was coated in a 96-well plate, and different concentrations of single-domain antibodies were added. After incubation, secondary antibodies were added, and the OD450nm value was measured after the reaction was developed (as shown in Table 1). After calculation, the IC50 of the single-domain antibody was 8nM. However, the IC50 of the commercial monoclonal antibody (Anti-VCAM1 antibody (ab134047)) detected by the same method was 12nM. This shows that the single-domain antibody in this study has better binding activity than the commercial monoclonal antibody.

[0072] Table 1 ELISA test results

[0073]

[0074] (3) Amino acid sequence of the single-domain antibody: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.1 by sequencing the single-domain antibody.

[0075] Example 4: In vivo efficacy experiment of polypeptide composition

[0076] 1. Animal model establishment: SD rats (250-300 g) were randomly divided into three groups, 3 rats in each group, namely control group, polypeptide group 1 (containing the nanoparticle drug of Example 2 + single domain antibody of Example 3) and polypeptide group 2 (containing the nanoparticle drug prepared in Example 2). The acute myocardial infarction model was established by left coronary artery ligation, and cardiac function was evaluated by echocardiography 24 hours after surgery.

[0077] 2. Drug administration: After surgery, equal doses of peptides were injected through the tail vein for 7 consecutive days, once a day, with a dose of 1 mg / kg body weight (wherein the nanoparticle drug in peptide group 1 and the single domain antibody were mixed in equal mass ratios).

[0078] 3. Cardiac function assessment: Echocardiography was used to evaluate the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of rats on the 7th and 14th days after surgery. The results showed that the LVEF of peptide group 1 was significantly improved to 65% (P<0.01), which was significantly higher than 50% of peptide group 2, while the control group was only 30%. The results of LVFS were consistent with the above results.

[0079] The above results show that the polypeptide drug composition prepared in this study has better therapeutic effect than a single polypeptide.

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A bioactive peptide, characterized in that: The bioactive peptide is a bifunctional bioactive peptide, and its amino acid sequence is CRDKEVGGGFLRKV.

2. A pharmaceutical composition comprising a bioactive peptide, characterized in that: The pharmaceutical composition comprises a nanoparticle drug comprising the bioactive peptide according to claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that The average particle size of the nanoparticle drug is 150±20 nm, and the polymerization index is 0.

15.

4. The pharmaceutical composition according to claim 2, characterized in that The encapsulation rate of the bioactive peptide in the nanoparticle medicine is 80%, and the drug loading is 12%.

5. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition further comprises an anti-VCAM-1 single domain antibody, wherein the amino acid sequence of the anti-VCAM-1 single domain antibody is shown in SEQ ID NO.

1.

6. The pharmaceutical composition according to claim 5, characterized in that The IC50 of the anti-VCAM-1 single domain antibody is 8 nM.

7. The pharmaceutical composition according to claim 5, characterized in that The nanoparticle drug and the anti-VCAM-1 single domain antibody in the pharmaceutical composition are mixed in equal mass ratios.

8. The pharmaceutical composition according to any one of claims 2 or 5, characterized in that: The dosage of the pharmaceutical composition is 1 mg / kg body weight.

9. Use of the bioactive peptide according to claim 1 in the preparation of drugs for treating cardiovascular and cerebrovascular diseases.

10. Use of the anti-VCAM-1 single domain antibody according to claim 5 in the preparation of a drug for treating cardiovascular and cerebrovascular diseases.

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