WO2024096424A1 - Pharmaceutical composition for preventing or treating ischemic diseases, containing liposome having qk peptide loaded on surface thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating ischemic diseases, the pharmaceutical composition containing liposome nanoparticles in which an angiogenic QK peptide is loaded on the surface of a liposome and a drug having ROS regulatory ability is loaded inside the liposome. According to the present invention, the QK peptide is loaded on the surface of the liposome and thus can be more accurately delivered to a desired ischemic site, and the QK peptide targets a VEGF receptor in the ischemic site and thus exhibits excellent targeting. In addition, liposomes labeled with isotopes or fluorescence can be traced and quantitatively analyzed, thus enabling appropriate treatment through monitoring.

Description

Pharmaceutical composition for the prevention or treatment of ischemic disease comprising a liposome with QK peptide loaded on the surface of the liposome

The present invention relates to a pharmaceutical composition for the prevention or treatment of ischemic disease comprising liposome nanoparticles in which QK peptide with angiogenic ability is loaded on the surface of a liposome and a drug with ROS control ability is loaded inside the liposome.

Ischemic disease includes cardiovascular diseases caused by obstruction of blood flow, and includes ischemic myocardial infarction and ischemic peripheral vascular disease. Among these ischemic diseases, cardiovascular disease is competing for the leading cause of death in Korea, and in addition, cerebrovascular disease and lower extremity ischemic disease are among the three major ischemic diseases. In Korea, which has entered an era of westernization of lifestyle and aging, the incidence of such ischemic diseases is increasing. Among peripheral vascular diseases, peripheral artery disease that occurs in the arteries at the extremities of the extremities is steadily increasing. The incidence of coronary artery disease in patients with peripheral artery disease is as high as 30-50%, and it is the main cause of death in patients with diabetes. Drugs and surgery are used to treat peripheral artery disease. Stents are available for large blood vessels, but their use in small peripheral blood vessels is limited. In addition, although thrombolytics or anticoagulants are prescribed, there is no cure for peripheral arterial occlusive diseases such as severe limb ischemia, and if treatment is not possible, amputation is performed. Despite these various treatments, treatment results are poor. In ischemic diseases, cell death is the biggest cause of death and sequelae. Research on this is actively conducted in regenerative medicine, protein, and gene therapy, but most of them fail at the clinical trial stage. These biopharmaceuticals circulate throughout the body, causing unwanted effects. There is a risk of side effects such as cancer metastasis, worsening of diabetic retinopathy, and rupture of arteriosclerotic atherosclerosis by causing angiogenesis in other parts of the body that are not present.

Recently, as the mechanism of angiogenesis has been discovered, attempts have been made to treat ischemic diseases by increasing collateral blood flow by inducing angiogenesis by administering genes or proteins of factors related to angiogenesis in ischemic areas. . Among them, the most important factor is VEGF, and numerous efforts are being made to deliver it, but the method of administering angiogenic recombinant proteins such as VEGF is 1) angiogenic because the injected angiogenic protein moves to other tissues or loses its activity. In order to achieve the effect, a large amount of highly purified protein must be used, which incurs enormous costs. 2) It is desirable to continuously administer the protein in small doses to form collateral blood vessels, but there is a problem that frequent injections are required. In order to solve the above problems, angiogenic proteins can be accurately delivered only to the ischemic area to exert local effects, while the biodistribution of the injected material can be accurately monitored and the angiogenic protein can be accurately delivered only to the ischemic area. It is necessary to prepare it in a dosage form.

The present inventors have completed a liposome that is accurately delivered to the desired ischemic area and has the dual effect of inducing new blood vessel formation and removing ROS from the ischemic area.

The technical problem to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating ischemic disease, which includes liposomes loaded with angiogenic peptides on the surface and loaded with a reactive oxygen species (ROS) scavenger inside. will be.

In order to achieve the above technical problem, an embodiment of the present invention is a method for preventing or treating ischemic disease, which includes a liposome loaded with a angiogenic peptide on the surface and loaded with a reactive oxygen species (ROS) scavenger inside. Provides a pharmaceutical composition for use.

In the present invention, the angiogenic peptide may be a QK peptide represented by SEQ ID NO: 1, and the QK peptide may target the VEGF receptor. SEQ ID NO: 1 is indicated as KLTWQELYQLKYKGI. Additionally, the QK peptide may be bound to the liposome surface through a linker.

The reactive oxygen species scavenger is a statin-based drug such as Simvastatin, Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, and Pravastatin. and Rosuvastatin.

In the present invention, the reactive oxygen species scavenger may be Simvastatin or ibuprofen, an anti-inflammatory drug (NSAID).

In the present invention, the composition may be labeled with a fluorescence or isotope to track movement in vivo during treatment. In the present invention, technesium is used as an isotope, but it is not limited thereto.

In the present invention, the composition may be characterized as inducing blood vessel formation in the ischemic area and removing reactive oxygen species. According to the present invention, both effects of inducing blood vessel formation and removing active oxygen can be obtained simultaneously, and furthermore, the effect of enabling tracking and quantification of drugs through fluorescence or isotope labeling can also be obtained.

In the present invention, the liposome contains lipid and cholesterol, and the lipid may be one selected from the group consisting of DPPC, DSPC, and HSPC, and the size of the liposome may be 100 to 500 nm.

In the present invention, the ischemic disease may be characterized as being selected from the group consisting of myocardial infarction, middle cerebral artery stenosis disease, lower extremity ischemia, and cerebral infarction.

In another embodiment, the present invention includes the steps of a) preparing a liposome composed of lipid and cholesterol and carrying a ROS scavenger; and b) binding a angiogenic peptide to the surface of the liposome. More specifically, a) preparing a liposome composed of DPPC, DSPE-PEG-2000-NH ₂ and cholesterol and carrying simvastatin; and b) binding the QK peptide represented by SEQ ID NO: 1 to the surface of the liposome, wherein the molar ratio of DPPC, DSPE-PEG-2000-NH ₂ , cholesterol, and simvastatin is 16.5 to 17.5: 0.5 to 1.5: 1. : Can be 0.7 to 1.7, preferably 17:1.01:1:1.209.

In the present invention, step b) connects the SPDP linker to the surface of the liposome to generate liposome-SPDP, connects the GKRKC linker to the QK peptide to generate QK-GKRKC, and then reacts the liposome-SPDP with QK-GKRKC to create liposome-SPDP. It may be characterized by binding the QK peptide to.

According to the present invention, the QK peptide is mounted on the surface of liposomes and can be delivered to the desired ischemic area more accurately, and the QK peptide targets the VEGF receptor in the ischemic area, thereby providing excellent targeting. In addition, liposomes labeled with isotopes or fluorescence can be tracked and quantitatively analyzed, enabling appropriate treatment through monitoring.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 shows the results of confirming the drug peak after injection of the prepared simvastatin liposome.

Figure 2 shows the results of measuring the size of liposome nanoparticles using a particle size analyzer.

Figure 3 shows the results of treating HUVEC cells with NBD-QK-LP and observing intracellular uptake using a fluorescence microscope.

Figure 4 shows the results of microscopic observation of blood vessel formation after sample treatment in HUVEC cells.

Figure 5 shows the results of confirming whether ROS is generated by treating HUVEC cells with H ₂ O ₂ and samples and treating them with DCFDA 2 hours later.

Figure 6 is a gamma camera image after intravenous injection of Tc-99m DTPA-QK-LP and Tc-99m DTPA-LP into normal mice.

Figure 7 shows gamma camera imaging results obtained up to 60 minutes after intravenous injection of Tc-99m simvastatin loaded QK-LP into a mouse lower extremity ischemia model.

Figure 8 shows the results of intramuscularly injecting simvastatin loaded QK-LP and QK-LP into an ischemic disease model and checking whether blood flow increased 6 hours later using a gamma camera.

The terms used in this specification are merely used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In the specification of the present invention, 'including' a certain element means that other elements may be further included, rather than excluding other elements, unless specifically stated to the contrary.

In the present invention, liposomes loaded with angiogenic QK peptide and loaded with a drug capable of controlling ROS were completed, and it was confirmed that a composition containing the same exhibits an angiogenic effect in an ischemic area and a ROS removal effect. This feature is because the present invention uses liposome nanoparticles that are not toxic to the human body, conjugates QK peptides with an angiogenic effect to the surface of the nanoparticles, and carries a drug capable of removing ROS inside the nanoparticles. Furthermore, tracking and quantitative analysis of nanoparticles is possible by labeling the nanoparticles with isotopes or fluorescence, and the QK peptide conjugated to the surface of the liposome targets the VEGF receptor, so it has excellent targetability and can be monitored.

According to the present invention, in ischemic areas, VEGF receptor is expressed in large quantities to create new blood vessels. By attaching the QK peptide targeting this to the surface of the liposome, it is better delivered to the ischemic area. In addition, the form in which the drug is carried inside liposomes has a problem in that it is difficult to control drug release in the body. For example, there are cases where the drug is released before it reaches the desired area, or the drug is released before it is used, making it difficult to use it as a drug. In the present invention, the QK peptide is mounted on the surface of the liposome rather than inside the liposome, so there is no problem in controlling drug release, and the drug can be accurately delivered to the area desired for treatment by targeting the VEGF receptor in the ischemic area.

The QK peptide used in the present invention is a VEGF mimetic pentadecapeptide designed by adjusting the sequence of the VEGF165 protein. It has a helix structure even in water and binds to the VEGF receptor to induce proliferation of endothelial cells, activate VEGF-dependent cell signaling, and form capillaries. and has properties that promote organization (Proc Natl Acad Sci USA 2005, D'Andrea LD et al.). The original sequences of positions 14 to 28 of VEGF165 and QK are as follows:

In the present invention, Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate, Pierce) was added to the prepared liposome, reacted at room temperature, separated with PD-10, and SH-functional Peptide ( CKRKG-QK: CKRKGKLTWQELYQLKYKGI, Peptron, Daejeon) was reacted and bound to the end of SPDP. CKRKG is an abbreviation for Cystein-lysine-arginine-lysine-glycine and was used as an intermediate linker to bind QK peptide to the liposome surface. It acts to bind the thiol of Cysteine to the disulfide of SPDP on the liposome surface. I do it. At this time, CKRKG can be modified into a form that is soluble in water and can be easily removed from the cell, and in addition to CKRKG, any hydrophilic peptide linker that ends in cysteine can be used without limitation. In addition, as a linker for connecting a peptide and a liposome, any dual reactive linker, in addition to SPDP, where one side can bind to the amine terminal and the other side can react with thiol, can be used without limitation. For example, SMCC (succinimidyl trans 4-(maleimidylmethyl)cyclohexane-1-carboxylate) can be used as a linker in which one side reacts with amines and the other side reacts with thiols.

Ischemic tissue is a state in which the oxygen concentration is lower than normal (hypoxia). Due to vascular constriction, reactive oxygen species (ROS) such as hydrogen peroxide are excessively generated and oxidative stress is increased, making the disease worse. Additionally, excessively produced ROS gradually breaks down vascular cells, causing blood vessels to lose elasticity, increase the risk of blood clots, and greatly increase the risk of diseases related to blood vessels, such as arteriosclerosis and angina pectoris. When reperfusion of ischemic tissue occurs, the cells do not recover but rather deteriorate. This is because the sudden supply of oxygen generates a large amount of ROS, resulting in tissue damage (destruction of protein, lipid, and DNA, which are major components within cells) or inflammation. I do it. In particular, H ₂ O ₂ , the most abundant form of ROS generated during I/R (ischemia/reperfusion) injury, induces the release of proinflammatory cytokines and apoptosis, and excessive tissue damage leads to death.

In the present invention, the reactive oxygen species scavenger is a drug capable of removing reactive oxygen species, and Simvastatin was used in the examples of the present invention, but is not limited thereto. Statin drugs, such as Simvastatin, are the most widely used drugs for cardiovascular diseases and have the effect of lowering cholesterol. Research is being conducted on their therapeutic effects by applying them to various diseases in numerous fields. Among them, ROS control Research on the effects is also continuously being conducted.

Simvastatin, which has ROS control ability, used in the present invention is a drug with very strong hydrophobic properties and was encapsulated in the liposome membrane. Even drugs other than Simvastatin used in the present invention may be similar types of statin drugs. Hydrophobic statin drugs are encapsulated in the liposome membrane by the same mechanism as Simvastatin, and other hydrophilic statin drugs are encapsulated inside the liposome. You can do it. For example, hydrophobic statin drugs include Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, and Mevastatin, and hydrophilic statin drugs include Pravastatin and Rosuvastatin.

Additionally, when manufacturing liposomes according to the present invention, ibuprofen as an anti-inflammatory drug (NSAIDs) can be encapsulated. Ibuprofen is another drug that can be used to control ROS and can be manufactured in the following manner, and similar NSAIDs other than ibuprofen can also be encapsulated in the same manner. The preparation of ibuprofen-encapsulated liposomes is as follows: Prepare lipids in the following mol% ratio (HSPC or DPPC, DSPE-PEG-2000, cholesterol 17:1.01:1), and dissolve 1-2 mg of ibuprofen in methanol. and mixed with lipids (lipids and reagents purchased from Sigma and Avanti). Lipids are dissolved using chloroform and methanol in a 9:1 ratio, evaporated at 45°C for 2 hours to remove the organic solvent, and then vacuum dried for 2 hours. Add 1 mL of PBS to the thin film, stir at 60°C for 1 hour to hydrate it, briefly apply ultrasound, and then use an extruder to make the film uniform in size. The extruder temperature is above 60℃ and filters are used in the order of 400nm-200nm-100nm. Ibuprofen that did not enter the liposome particles is separated with PD-10 (cytiva), and the remaining drug is removed by centrifugation using an ultracentrifugal filter tube (Merck) with a Mw of 10,000 (6000 rpm, 40 min). Encapsulation was confirmed by RP-HPLC (Reverse phase C18 column, acetonitrile:water, 8:2 ratio, 0.8 mL/min, 220 nm), and ibuprofen encapsulation efficiency was determined by preparing standards for each concentration of ibuprofen and measuring ROI on each HPLC. Check and create a standard curve. At this time, ibuprofen was contained at 76 μg/mL.

In the present invention, the lipid used to manufacture liposomes was DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), which is a 16:0 phosphochlorine, but in addition to this, DSPC (1,2-distearoyl-sn-glycero- 3-phosphocholine), HSPC (L-α-phosphatidylcholine, hydrogenated (Soy)), etc. can be used, and are not limited to these and can be used as long as they can be easily selected by a person skilled in the art to which the present invention pertains.

In the present invention, the composition may be a pharmaceutical composition. For administration, the composition of the present invention may contain pharmaceutically acceptable salts, carriers, excipients, diluents, solubilizers, etc. The pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., hydrochloric acid, There are salts of inorganic acids such as phosphoric acid and sulfuric acid, and salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotate acid, and acetylsalicylic acid. There are amino acid salts such as lysine, arginine, and guanidine. There are also salts of organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyltrimethyl ammonium, and benzethonium that can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts meant in the present invention are not limited to these salts listed. The carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Examples include cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and solubilizers include poloxamer and labrasol. Not limited.

The pharmaceutical composition of the present invention can be prepared into a pharmaceutical formulation using methods well known in the art. In preparing a dosage form, the active ingredient may be mixed or diluted with a carrier or encapsulated in a carrier in the form of a container. When the pharmaceutical composition of the present invention is prepared in a dosage form for oral administration, it can be formulated into, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. . The pharmaceutical composition may be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, intrathecally or subcutaneously. Dosage forms for oral administration may be tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions, but are not limited thereto. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but are not limited thereto. The pharmaceutical composition may, if necessary, contain additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavors or sweeteners.

Hereinafter, the present invention will be described in detail through examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

Liposome preparation

Lipids were prepared at the following mol% ratio (DPPC, DSPE-PEG-2000-NH ₂ , Cholesterol, Simvastatin 17:1.01:1:1.209). Lipids and reagents were purchased from Sigma and Avanti. Chloroform and methanol were dissolved in a 1:1 ratio and evaporated at 45°C for 2 hours to remove the organic solvent, followed by vacuum drying for 2 hours. 1 mL of PBS was added to the thin film and stirred at 55°C for 1 hour to hydrate it, followed by ultrasonic waves (1 min) and then uniformly sized using an extruder. The extruder temperature was above 55°C, and filters were used in the order of 400nm-200nm-100nm. Simvastatin that did not enter the liposome particles was separated using PD-10 (cytiva) and concentrated again by centrifugation using an ultrafiltration tube (molecular weight 10000, 6000 rpm, 40 min). Encapsulation was confirmed by RP-HPLC (Reverse phase C18 column, acetonitrile:ammonium acetate, pH 4.6, 20 mM, 4:6 ratio, 240 nm), and encapsulation efficiency was determined by preparing standards for each concentration of Simvastatin and using HPLC for each. Check the ROI on the image and create a standard curve. The NH ₂ functional group outside the liposome was confirmed by ninhydrine assay.

Preparation of liposomes loaded with QK peptide (QK-LP)

Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate, Pierce) was added to the prepared liposome, reacted at room temperature, separated with PD-10, and then SH-functional Peptide (CKRKG-QK: CKRKG KLTWQELYQLKYKGI, Peptron, Daejeon) was reacted and bound to the end of SPDP. Peptide binding was confirmed by RP-HPLC at 343 nm for pyridine 2-thione released during reaction (Reverse phase C18 column, 0.1% TFA in ACN/0.1% TFA in water, gradient 97:3 to 30:70). The remaining peptide was confirmed at 220 nm and dually confirmed at 343 nm.

In vitro HUVEC cell binding test of QK-LP

When preparing liposomes, a small amount of NBD-DSPE was added to prepare them in a form with a fluorescent dye attached, and their binding to endothelial cells was confirmed depending on the presence of peptide QK in the cells. Simvastatin was not encapsulated during the preparation of this liposome. HUVEC cells, which are human vascular endothelial cells, were seeded at 1*105 cells in a 24 well plate, treated with bare liposome (LP), QK-LP, etc., and uptake was confirmed using a fluorescence microscope.

In vitro HUVEC cell angiogenesis assay

HUVEC cells, which are human vascular endothelial cells, were distributed in a 10cm dish (Lonza, EBM-2 buffer) and cultured at 37°C in a CO2 incubator (Thermo Forma, USA) while maintaining a 5% CO2 concentration. On the day of the experiment, matrigel was placed in a 96 well plate and hardened, and then 1*104 cells were seeded. Then, VEGF protein (Lonza), QK-LP, LP, etc. were treated at different concentrations, and the degree of blood vessel formation was confirmed under a microscope for up to 6 hours.

Confirmation of in vitro ROS removal ability

HUVEC cells were seeded at 1*105 cells in a 24 well plate, and the cells were treated with H ₂ O ₂ (100 mM) at various concentrations and then treated with liposomes loaded with Simvastatin (2 hours) to determine whether they had the ability to control ROS. . The degree of ROS generation was confirmed using DCFDA (Dichlorofluorescin Diacetate) reagent.

Technesium labeling of liposomes

Tertiary distilled water was purged with nitrogen for about 40 minutes to lower the oxygen concentration before use. After dissolving SnCl2 (1 mg) in 1N HCl (100 μL), 900 μL of nitrogen-purged water was added, and after sufficiently dissolving, free Tc-99m was added to the liposome sample contained in PBS, and then 5 μL of tin solution was added to label it. . After 10 minutes of reaction at room temperature, the pH was checked and the labeling efficiency was confirmed using ITLC-sg (Bioscan, TLC scanner). On silica gel, liposomes stayed at Rf = 0, and unlabeled free Tc-99m showed Rf = 1. The isotope-labeled reaction was separated again with PD-10.

Ischemic disease modeling and drug administration

Ischemia is induced by tying and resewing the femoral artery of the hind limb of a 9-10 week old male C57BL6 (OrientBio) mouse. The success of ischemia induction is determined by intravenous injection of free Tc-99m and the degree of blood flow through images on a gamma camera. I confirmed it by looking at it. Simvastatin loaded QK-LP drug was injected into three areas centered on the ischemic area, and blood flow was reevaluated 6 hours later to evaluate the degree of recovery in the ischemic area.

Example 1. Preparation and confirmation of effect of liposome loaded with Simvastatin (Simva-LP) and liposome loaded with QK peptide (Simva-QK-LP)

First, as a result of checking the liposome particles prepared by the above method, the Simvastatin encapsulation rate in the liposome was 76%. After final separation of the free drug, HPLC was performed under the above conditions, and the encapsulation rate was calculated based on the peak seen at 16.8 minutes and calculated in proportion to the initially administered drug amount (Figure 1). As a result of measuring the size of liposome nanoparticles using a particle size analyzer, the liposome loaded with Simvastatin (Simva-LP) showed an average of 148 nm, and the liposome finally conjugated to QK (Simva-QK-LP) showed an average of 168 nm. This was confirmed (Figure 2).

Afterwards, HUVEC cells were treated with NBD-QK-LP, and cellular uptake was observed using a fluorescence microscope. As a result, it was confirmed that QK-conjugated liposomes showed significantly different uptake compared to liposomes without QK when treated in HUVEC cells. Fluorescence was clearly maintained even after 24 and 48 hours after ingestion, and remained in the cytoplasm even after 72 hours. Fluorescence was maintained and overall binding to endothelial cells was confirmed (Figure 3).

In addition, in order to confirm the pro-angiogenic effect, blood vessel formation was confirmed using matrigel, and it was confirmed that tubes were clearly formed in the case with QK peptide compared to the case without (Figure 4). In Figure 4, it can be seen that when HUVEC cells were treated with LP-SPDP-QK, blood vessels were formed more densely compared to when LP-SPDP was treated without QK peptide.

Example 2. Confirmation of ROS removal effect

When QK-LP loaded with Simvastatin was treated with HUVEC cells, it was performed under the assumption that the ROS effect could be shown as a receptor target of QK peptide. As a result, compared to liposomes loaded with only Simvastatin without QK peptide (LP-SPDP-SIM), QK It was confirmed that the peptide-loaded liposome (LP-SPDP-SIM-QK) showed an excellent effect in H ₂ O ₂ removal (Figure 5). Through this, it can be seen that the QK peptide loaded on the surface of the liposome targets the VEGF receptor and is well guided to the ischemic area and has an excellent effect in removing ROS from the ischemic area.

Example 3. Confirmation of the effect of QK-LP injection containing Simvastatin in lower extremity ischemia model mice

DTPA-QK-LP and DTPA-LP were injected into normal mice, and the whole body distribution was photographed 1 hour later (acquisition time 5 min, 14.8 MBq). In the image confirming the whole body distribution, it was confirmed that the liposome loaded with QK peptide stayed in the whole body for a longer period of time compared to the liposome without QK peptide (Figure 6). In Figure 6, the left side shows DTPA-QK-LP, and the right side shows DTPA-LP. At this time, Simvastatin was not encapsulated, and a small amount of DSPE-PEG-DTPA was used during liposome preparation for technesium labeling.

Meanwhile, ischemia was induced by tying off the femoral artery of the hind limb of the mouse, and QK-LP loaded with technesium-labeled Simvastatin was injected intravenously (acquisition time 3 min, 14.8 MBq). As a result, systemic distribution was confirmed, showing ischemia. It was confirmed that there was an accumulation pattern in the area over time (Figure 7). The arrow in Figure 7 indicates the ischemia induction area. At this time, DSPE-PEG-DTPA was not used in the liposome because DSPE-PEG-DTPA partially interferes with the encapsulation of Simvastatin, and due to the characteristics of Tc-99m, phosphate groups, etc. can be sufficiently labeled, so Tc-99m radiolabeled Simvastatin loaded QK-LP was used. As shown in Figure 7, it was confirmed that the labeled QK-LP was targeted and delivered well to the ischemia induction site as time passed after injection.

In addition, after tying the aorta and creating ischemia, the drug was injected around the ischemia (at 3 locations) and checked for up to 6 hours. As shown in Figure 8, the QK-LP treated group and Simvastatin-loaded group were compared to the untreated group. In the QK-LP treatment group, blood flow toward the legs and toes below the ischemic region was shown to be more increased than in the control group (acquisition time 3 min, 16.2 MBq).

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as single may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

The present invention relates to a pharmaceutical composition for the prevention or treatment of ischemic disease comprising liposome nanoparticles in which QK peptide with angiogenic ability is loaded on the surface of a liposome and a drug with ROS control ability is loaded inside the liposome. According to the present invention, the QK peptide is mounted on the surface of liposomes and can be delivered to the desired ischemic area more accurately, and the QK peptide targets the VEGF receptor in the ischemic area, thereby providing excellent targeting. In addition, isotope- or fluorescently-labeled liposomes are useful because they allow for tracking and quantitative analysis, allowing for appropriate treatment through monitoring.

Claims (13)

1. A pharmaceutical composition for preventing or treating ischemic disease, comprising liposomes loaded with angiogenic peptides on the surface and loaded with a reactive oxygen species (ROS) scavenger inside.
2. According to clause 1,

   A pharmaceutical composition for preventing or treating ischemic disease, wherein the angiogenic peptide is QK peptide represented by SEQ ID NO: 1.
3. According to clause 2,

   The QK peptide is a pharmaceutical composition for preventing or treating ischemic disease, characterized in that it targets the VEGF receptor.
4. According to clause 2,

   A pharmaceutical composition for preventing or treating ischemic disease, wherein the QK peptide is bound to the surface of a liposome through a linker.
5. According to clause 1,

   The reactive oxygen species scavengers include Simvastatin, Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pravastatin, and Rosuvastatin. A pharmaceutical composition for preventing or treating ischemic disease, characterized in that it is selected from the group consisting of (Rosuvastatin).
6. According to clause 1,

   A pharmaceutical composition for preventing or treating ischemic disease, wherein the reactive oxygen species scavenger is simvastatin or ibuprofen.
7. According to clause 1,

   A pharmaceutical composition for the prevention or treatment of ischemic disease, characterized in that the composition is labeled with a fluorescence or isotope so that movement in the body can be tracked during treatment.
8. According to clause 1,

   The composition is a pharmaceutical composition for preventing or treating ischemic disease, characterized in that it induces blood vessel formation in the ischemic area and removes reactive oxygen species.
9. According to clause 1,

   The liposome contains lipid and cholesterol, and the lipid is selected from the group consisting of DPPC, DSPC, and HSPC. A pharmaceutical composition for preventing or treating ischemic disease.
10. According to clause 1,

    A pharmaceutical composition for preventing or treating ischemic disease, wherein the liposome has a size of 100 to 500 nm.
11. According to clause 1,

    A pharmaceutical composition for preventing or treating ischemic diseases, wherein the ischemic disease is selected from the group consisting of myocardial infarction, middle cerebral artery stenosis, lower extremity ischemia, and cerebral infarction.
12. a) preparing a liposome composed of DPPC, DSPE-PEG-2000-NH ₂ and cholesterol and carrying simvastatin; and

    b) binding the QK peptide represented by SEQ ID NO: 1 to the surface of the liposome;

    A method for producing a pharmaceutical composition for preventing or treating ischemic disease, characterized in that the molar ratio of DPPC, DSPE-PEG-2000-NH ₂ , cholesterol, and simvastatin is 16.5 to 17.5: 0.5 to 1.5: 1: 0.7 to 1.7.
13. According to clause 12,

    In step b), liposome-SPDP is created by linking the SPDP linker to the surface of the liposome, QK-GKRKC is created by linking the GKRKC linker to the QK peptide, and then the liposome-SPDP and QK-GKRKC are reacted to form the QK peptide on the liposome surface. A method for producing a pharmaceutical composition for preventing or treating ischemic disease, characterized in that it combines.

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