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

Pharmaceutical composition for preventing or treating ischemic diseases, containing liposome having qk peptide loaded on surface thereof Download PDF

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WO2024096424A1
WO2024096424A1 PCT/KR2023/016556 KR2023016556W WO2024096424A1 WO 2024096424 A1 WO2024096424 A1 WO 2024096424A1 KR 2023016556 W KR2023016556 W KR 2023016556W WO 2024096424 A1 WO2024096424 A1 WO 2024096424A1
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liposome
pharmaceutical composition
preventing
peptide
clause
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PCT/KR2023/016556
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French (fr)
Korean (ko)
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정환정
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전북대학교산학협력단
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/192Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365Lactones
    • A61K31/366Lactones having six-membered rings, e.g. delta-lactones
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • treatment results are poor.
  • 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.
  • 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.
  • 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.
  • ROS reactive oxygen species
  • 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.
  • ROS reactive oxygen species
  • 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.
  • 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.
  • the reactive oxygen species scavenger may be Simvastatin or ibuprofen, an anti-inflammatory drug (NSAID).
  • NSAID anti-inflammatory drug
  • the composition may be labeled with a fluorescence or isotope to track movement in vivo during treatment.
  • technesium is used as an isotope, but it is not limited thereto.
  • 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.
  • the liposome contains lipid and cholesterol
  • the lipid may be one selected from the group consisting of DPPC, DSPC, and HSPC
  • the size of the liposome may be 100 to 500 nm.
  • 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.
  • 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.
  • 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.
  • 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.
  • liposomes labeled with isotopes or fluorescence can be tracked and quantitatively analyzed, enabling appropriate treatment through monitoring.
  • 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.
  • 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.
  • 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.
  • VEGF receptor in ischemic areas, is expressed in large quantities to create new blood vessels.
  • QK peptide targeting this to the surface of the liposome, it is better delivered to the ischemic area.
  • 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.
  • 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:
  • Sulfo-LC-SPDP sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate, Pierce
  • SH-functional Peptide CKRKG-QK: CKRKGKLTWQELYQLKYKGI, Peptron, Daejeon
  • 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.
  • 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.
  • 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.
  • SMCC succinimidyl trans 4-(maleimidylmethyl)cyclohexane-1-carboxylate
  • 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.
  • ROS reactive oxygen species
  • the reactive oxygen species scavenger is a drug capable of removing reactive oxygen species
  • 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.
  • 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.
  • hydrophobic statin drugs include Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, and Mevastatin
  • hydrophilic statin drugs include Pravastatin and Rosuvastatin.
  • 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°C 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).
  • 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 ⁇ g/mL.
  • 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- ⁇ -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.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • HSPC L- ⁇ -phosphatidylcholine, hydrogenated (Soy)
  • the composition may be a pharmaceutical composition.
  • 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,
  • inorganic acids such as phosphoric acid and sulfuric acid
  • 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.
  • amino acid salts such as lysine, arginine, and guanidine.
  • 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.
  • 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.
  • lactose dextrose
  • sucrose 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
  • 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.
  • the active ingredient may be mixed or diluted with a carrier or encapsulated in a carrier in the form of a container.
  • the pharmaceutical composition of the present invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • matrigel was placed in a 96 well plate and hardened, and then 1*104 cells were seeded.
  • VEGF protein Longza
  • QK-LP 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.
  • 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.
  • 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.
  • the Simvastatin encapsulation rate in the liposome was 76%.
  • 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).
  • the liposome loaded with Simvastatin showed an average of 148 nm
  • the liposome finally conjugated to QK showed an average of 168 nm. This was confirmed ( Figure 2).
  • 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).
  • the drug was injected around the ischemia (at 3 locations) and checked for up to 6 hours.
  • the QK-LP treated group and Simvastatin-loaded group were compared to the untreated 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 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.
  • 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.
  • isotope- or fluorescently-labeled liposomes are useful because they allow for tracking and quantitative analysis, allowing for appropriate treatment through monitoring.

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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

QK 펩타이드가 리포좀 표면에 탑재된 리포좀을 포함하는 허혈성 질환의 예방 또는 치료용 약학조성물Pharmaceutical composition for the prevention or treatment of ischemic disease comprising a liposome with QK peptide loaded on the surface of the liposome
본 발명은 신생혈관형성능을 가지는 QK 펩타이드가 리포좀 표면에 탑재되고 ROS 제어능이 있는 약물이 리포좀 내부에 담지된 리포좀 나노입자를 포함하는 허혈성 질환의 예방 또는 치료용 약학조성물에 관한 것이다.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.
허혈성 질환(ischemic disease)은 혈류의 흐름이 막혀 일어나는 심혈관 질환을 포함하는 것으로 허혈성 심근경색(myocardial ischemia) 및 허혈성 말초혈관 질환(peripheral vascular disease)이 이에 해당한다. 이러한 허혈성 질환 중 심혈관질환은 우리나라에서 사망의 수위를 다투고 있으며, 이외에도 뇌혈관질환 및 하지허혈질환이 3대 허혈질환에 속한다. 생활습관의 서구화와 고령화시대로 진입한 우리나라는 이러한 허혈질환 발생률이 높아지고 있다. 말초혈관질환 중 사지 말단 동맥에 발생하는 말초동맥질환이 꾸준히 증가추세이며 말초동맥질환 환자에서 관상동맥질환의 발생율은 30-50%로 높고 당뇨병 환자의 주된 사망원인이 된다. 말초동맥질환의 치료는 약물, 수술 등이 이용되는데 혈관크기가 큰 경우 스텐트가 가능하지만 미세 말초혈관에 사용하기에는 제한적이다. 또한, 혈전용해제나 항혈전응고제 처방을 하지만 중증하지허혈 같은 말초동맥폐색질환은 치료제가 없으며 치료불가능한 경우 절단을 시행하게 된다. 이러한 여러 치료법에도 불구하고 치료 성적은 저조한 실정이다. 허혈성 질환에서는 세포 사멸이 사망과 후유장애의 가장 큰 원인으로, 이에 대한 연구가 재생의학, 단백질, 유전자 치료 등 활발히 이루어지고 있으나 대부분 임상 시험 단계에서 실패하고 있으며 이런 바이오 의약품은 전신적인 순환을 하여 원하지 않는 다른 부위에 혈관신생을 일으켜 암의 전이나, 당뇨병성 망막증의 악화, 동맥경화 죽상종의 파열과 같은 부작용 발생의 위험이 있다.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.
최근에는 혈관신생의 기전이 밝혀짐에 따라 허혈 부위에 혈관신생에 관련한 인자(factor)의 유전자 또는 단백질을 투여하여 혈관생성을 유도함으로써 측부혈류를 증가시켜 허혈성 질환을 치료하고자 하는 시도들이 이루어지고 있다. 그 중 가장 중요한 인자는 VEGF로 이를 전달하기 위한 수많은 노력이 이루어지고 있으나 VEGF와 같은 혈관신생 재조합 단백질을 투여하는 방법은 1) 주사한 혈관신생 단백질은 다른 조직으로 이동하거나 활성을 잃어버리기 때문에 혈관신생의 효과를 보기 위해 고순도의 단백을 다량 사용하여야 하므로 막대한 비용이 들고, 2) 측부혈관의 형성을 위하여 단백질을 적은 용량으로 지속적으로 투여하는 것이 바람직하지만, 자주 주사하여야 하는 문제점이 있다. 위와 같은 문제점을 해결하기 위하여, 신생혈관형성 단백질이 허혈 부위에만 정확하게 전달되어 국소적으로 효과가 발현될 수 있으면서 주입된 물질의 생체 내 분포를 정확하게 모니터링 할 수 있고, 인체의 면역반응을 유발하지 않는 제형으로 제조되는 것이 필요하다.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.
본 발명자들은 원하는 허혈 부위에 정확하게 전달되어 신생혈관 형성을 유도하고 허혈 부위의 ROS를 제거하는 두 가지 효과를 가지는 리포좀을 완성하게 되었다.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.
본 발명이 이루고자 하는 기술적 과제는 표면에 신생혈관형성 펩타이드가 탑재되고, 내부에 활성 산소종(reactive oxygen species: ROS) 소거제가 담지된 리포좀을 포함하는 허혈성 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다. 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.
상기 기술적 과제를 달성하기 위하여, 본 발명의 실시예는 표면에 신생혈관형성 펩타이드가 탑재되고, 내부에 활성 산소종(reactive oxygen specie s: ROS) 소거제가 담지된 리포좀을 포함하는 허혈성 질환 예방 또는 치료용 약학적 조성물을 제공한다. 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.
본 발명에 있어서, 상기 신생혈관형성 펩타이드는 서열번호 1로 표시되는 QK 펩타이드인 것을 특징으로 할 수 있고, 상기 QK 펩타이드는 VEGF 수용체를 표적하는 것을 특징으로 할 수 있다. 서열번호 1은 KLTWQELYQLKYKGI로 표시된다. 또한, 상기 QK 펩타이드는 링커를 통해 리포좀 표면에 결합되는 것을 특징으로 할 수 있다. 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.
상기 활성 산소종 소거제는 스타틴 계열 약물로 심바스타틴(Simvastatin), 아토르바스타틴(Atorvastatin), 세리바스타틴(Cerivastatin), 플루바스타틴(Fluvastatin), 로바스타틴(Lovastatin), 메바스타틴(Mevastatin), 프라바스타틴(Pravastatin) 및 로수바스타틴(Rosuvastatin)로 이루어진 군에서 선택되는 것을 특징으로 할 수 있다. The reactive oxygen species scavenger is a statin-based drug such as Simvastatin, Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, and Pravastatin. and Rosuvastatin.
본 발명에 있어서, 상기 활성 산소종 소거제는 심바스타틴(Simvastatin)일 수 있고, 또는 항염증 약물(NSAIDs)인 이부프로펜(ibuprofen)인 것을 특징으로 할 수 있다. In the present invention, the reactive oxygen species scavenger may be Simvastatin or ibuprofen, an anti-inflammatory drug (NSAID).
본 발명에 있어서, 상기 조성물은 형광 또는 동위원소로 표지되어 치료 중 생체 내 이동을 추적할 수 있는 것을 특징으로 할 수 있다. 본 발명에서는 동위원소로 테크네슘을 사용하였으나 이에 제한되지 않는다.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.
본 발명에 있어서, 상기 조성물은 허혈 부위에 혈관 형성을 유도하고 활성 산소종을 제거하는 것을 특징으로 할 수 있다. 본 발명에 따르면 혈관 형성 유도 및 활성 산소 제거 두 가지 효과를 동시에 얻을 수 있고, 나아가 형광 또는 동위원소 표지를 통해 약물의 추적 및 정량이 가능한 효과도 함께 얻을 수 있다. 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.
본 발명에서, 상기 리포좀은 지질 및 콜레스테롤을 포함하며 상기 지질은 DPPC, DSPC 및 HSPC로 이루어진 군에서 선택되는 하나일 수 있고, 상기 리포좀의 크기는 100 내지 500nm인 것을 특징으로 할 수 있다.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.
본 발명에 있어서, 상기 허혈성 질환은 심근경색, 중뇌동맥협착 질환, 하지허혈 및 뇌경색으로 이루어진 군에서 선택되는 하나인 것을 특징으로 할 수 있다. 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.
본 발명은 또 다른 실시예에서, a) 지질 및 콜레스테롤로 구성되고 ROS 소거제를 담지한 리포좀을 제조하는 단계; 및 b) 상기 리포좀 표면에 신생혈관형성 펩타이드를 결합시키는 단계를 포함하는 허혈성 질환 예방 또는 치료용 약학적 조성물을 제조하는 방법을 제공한다. 보다 구체적으로 a) DPPC, DSPE-PEG-2000-NH2 및 콜레스테롤로 구성되고 심바스타틴을 담지한 리포좀을 제조하는 단계; 및 b)상기 리포좀 표면에 서열번호 1로 표시되는 QK 펩타이드를 결합시키는 단계를 포함하고, 상기 DPPC, DSPE-PEG-2000-NH2, 콜레스테롤 및 심바스타틴의 몰비는 16.5 내지 17.5: 0.5 내지 1.5: 1: 0.7 내지 1.7 일 수 있고, 바람직하게는 17:1.01:1:1.209 이다.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.
본 발명에서 상기 b) 단계는 리포좀 표면에 SPDP 링커를 연결하여 리포좀-SPDP를 생성하고 QK 펩타이드에 GKRKC 링커를 연결하여 QK-GKRKC를 생성한 다음, 리포좀-SPDP과 QK-GKRKC를 반응시켜 리포좀 표면에 QK 펩타이드를 결합시키는 것을 특징으로 할 수 있다.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.
본 발명에 따르면 QK 펩타이드는 리포좀 표면에 탑재되어 원하는 허혈 부위로 보다 정확하게 전달 가능하고, QK 펩타이드는 허혈부위의 VEGF receptor에 타겟함으로써 표적성이 우수한 효과가 있다. 또한, 동위원소 또는 형광을 표지한 리포좀은 추적, 정량 분석이 가능하여 모니터링을 통한 적절한 치료를 할 수 있다.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.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.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.
도 1은 제조된 simvastatin 리포좀을 주입후에 약물 피크를 확인한 결과이다. Figure 1 shows the results of confirming the drug peak after injection of the prepared simvastatin liposome.
도 2는 입도분석기를 이용하여 리포좀 나노입자의 크기를 측정한 결과이다. Figure 2 shows the results of measuring the size of liposome nanoparticles using a particle size analyzer.
도 3은 HUVEC 세포에 NBD-QK-LP를 처리하고 세포내 섭취를 형광현미경으로 관찰한 결과이다. Figure 3 shows the results of treating HUVEC cells with NBD-QK-LP and observing intracellular uptake using a fluorescence microscope.
도 4는 HUVEC세포에 샘플 처리후 혈관 형성을 현미경으로 관찰한 결과이다. Figure 4 shows the results of microscopic observation of blood vessel formation after sample treatment in HUVEC cells.
도 5는 HUVEC세포에 H2O2와 샘플을 처리하고 2시간 후 DCFDA를 처리하여 ROS 생성여부를 확인한 결과이다. 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.
도 6은 Tc-99m DTPA-QK-LP, Tc-99m DTPA-LP를 정상마우스 에 정맥주사 후의 감마카메라 영상이다. Figure 6 is a gamma camera image after intravenous injection of Tc-99m DTPA-QK-LP and Tc-99m DTPA-LP into normal mice.
도 7은 Tc-99m simvastatin loaded QK-LP를 마우스 하지허혈 모델에 정맥주 사 후 60분까지 얻은 감마카메라 영상결과이다. 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.
도 8은 simvastatin loaded QK-LP, QK-LP을 허혈질환 모델에 근육 내 주사하 고 6시간 경과 후에 혈류 증가 여부를 감마카메라로 확인한 결과이다. 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.
본 명세서에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 발명의 명세서에서 어떤 구성요소를 '포함' 한다는 것은 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있다는 것을 의미한다. 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.
본 발명에서는 신생혈관형성 QK 펩타이드가 탑재되고 ROS 제어능이 있는 약물이 담지된 리포좀을 완성하고, 이를 포함하는 조성물이 허혈 부위의 혈관 생성 효과, ROS 제거 효과를 나타내는 것을 확인하였다. 이러한 특징은 본 발명이 인체 독성 없는 리포좀 나노입자 이용하여 신생혈관형성 효과를 갖는 QK 펩타이드를 나노입자 표면에 컨쥬게이트하고, ROS를 제거할 수 있는 약물을 나노입자 내부에 담지한 특징을 갖기 때문이며, 나아가, 나노입자를 동위원소 또는 형광을 표지하여 나노입자의 추적 및 정량 분석이 가능하고 리포좀 표면에 컨쥬게이트된 QK 펩타이드가 VEGF receptor를 표적하므로 표적성이 우수하고 모니터링을 할 수 있기 때문이다. 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.
본 발명에 따르면, 허혈 부위는 신생혈관을 만들어 내기 위해 VEGF receptor가 많이 발현되게 되는데 이것을 표적하는 QK 펩타이드를 리포좀의 표면에 붙임으로써 허혈 부위로 더욱 잘 전달되도록 한다. 또한, 약물이 리포좀 내부에 담지된 형태는 체내에서 약물 방출을 제어하는 데 어려움이 있는 문제점이 있다. 예를 들어, 약물이 원하는 부위까지 가기 전에 먼저 방출되거나, 약물을 사용하기도 전에 약물이 방출되어 약물로서 활용 자체가 어려운 경우도 발생한다. 본 발명은 QK 펩타이드가 리포좀의 내부에 담지된 것이 아니라 리포좀 표면에 탑재된 것으로 약물 방출 제어의 문제점이 없고, 허혈 부위의 VEGF receptor를 표적하여 치료를 원하는 부위로 정확하게 약물이 전달될 수 있다.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.
본 발명에서 사용된 QK 펩타이드는 VEGF165 단백질의 서열을 조정하여 설계된 VEGF mimetic pentadecapeptide로, 물에서도 헬릭스 구조를 띄고 VEGF receptor에 결합하여 내피세포의 증식을 유도하고 VEGF에 의존하는 세포 신호를 활성화하며 모세관 형성 및 조직화를 촉진하는 특성을 갖는다(Proc Natl Acad Sci USA 2005, D'Andrea LD et al.). VEGF165의 14번에서 28번의 원래 서열과 QK의 서열은 아래와 같다: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:
Figure PCTKR2023016556-appb-img-000001
Figure PCTKR2023016556-appb-img-000001
본 발명에서는 제조된 리포좀에 Sulfo-LC-SPDP(sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate, Pierce)를 넣어 실온에서 반응시키고 PD-10으로 분리한 다음, SH-functional Peptide(CKRKG-QK: CKRKGKLTWQELYQLKYKGI, Peptron, Daejeon)를 반응시켜서 SPDP 말단에 결합시켰다. CKRKG는 Cystein-lysine-arginine-lysine-glycine의 약자로 QK 펩타이드를 리포좀 표면에 결합시키기 위한 중간 링커로 사용되었고, Cystein의 티올을 리포좀 표면의 SPDP의 이황화물(disulfide)에 작용하여 결합시키는 역할을 하게 된다. 이 때, CKRKG는 물에 녹는 형태, 세포 내에서 쉽게 탈락될 수 있는 형태로의 변형이 가능하고, CKRKG 이외에도 말단이 cystein으로 끝나는 hydrophilic한 펩타이드 링커라면 제한 없이 사용될 수 있다. 또한, 펩타이드와 리포좀을 연결하기 위한 링커로 SPDP 이외에도 한쪽은 아민 말단에 결합 가능하고 다른 한쪽은 티올에 반응이 가능한 dual reactive 링커라면 제한 없이 사용될 수 있다. 예를 들어, SMCC(succinimidyl trans 4-(maleimidylmethyl)cyclohexane-1-carboxylate)은 한쪽은 아민과 반응하고 다른 한 족은 티올과 반응하는 링커로 사용 가능하다.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.
허혈 조직은 산소농도가 정상적 상태보다 낮은 상태로(hypoxia) 혈관 수축에 의하여 과산화수소와 같은 활성 산소종(reactive oxygen species:ROS)이 과다 발생되고 산화 스트레스를 증가시켜 병을 더 악화시키게 된다. 또한, 과다 생성된 ROS가 혈관세포를 서서히 허물어뜨려 혈관의 탄력을 잃게 하며, 혈전을 잘 생기게 하고, 동맥경화,협심증 등 혈관과 관련된 질병 위험을 크게 증가시킨다. 허혈 조직의 재관류 시 세포가 회복되는 것이 아니고 오히려 악화되는데 이는 산소 공급이 갑자기 이뤄지면서 많은 양의 ROS를 생성하게 되고 결과적으로 조직을 손상시키거나(세포내 주요성분인 단백질 지질 DNA 파괴) 염증을 유발하게 된다. 특히, I/R(허혈/재관류) 손상 동안 생성되는 ROS의 가장 풍부한 형태인 H2O2는 염증유발 사이토카인의 방출과 세포자살을 유도하여 과도한 조직손상은 사망으로 이어지게 된다.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.
본 발명에서, 활성 산소종 소거제는 활성 산소 제거능이 있는 약물로 본 발명의 실시예에서는 Simvastatin이 이용되었으나 이에 제한되지 않는다. Simvastatin 같은 스타틴(statin) 계열 약물은 심혈관 질환에 가장 광범위하게 사용되는 약물로 콜레스테롤을 낮추는 효과가 있고, 수많은 분야에서 다양한 질병에적용하여 치료효과에 대한 연구가 이루어지고 있으며, 그 중 ROS를 제어하는 효과에 대한 연구도 지속적으로 이루어지고 있다. 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.
본 발명에서 사용된 ROS 제어능이 있는 Simvastatin은 매우 강한 hydrophobic 성질을 갖는 약물로 리포좀 막에 봉입되었다. 본 발명에서 사용된 Simvastatin 이외의 약물이라도 비슷한 계열의 스타틴 약물이 사용될 수 있고, hydrophobic한 스타틴 계열 약물은 Simvastatin과 동일한 기전으로 리포좀의 막에 봉입되고, 그 외 hydrophilic한 스타틴 계열 약물은 리포좀 내부에 봉입시킬 수 있다. 예를 들어, hydrophobic한 스타틴 계열 약물로는 Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin 등이 있고, hydrophilic한 스타틴 계열 약물 로는 Pravastatin, Rosuvastatin 등이 있다. 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.
또한, 본 발명에 따른 리포좀을 제조 시 anti-inflammatory drug (NSAIDs)으로 이부프로펜(ibuprofen)을 봉입시킬 수 있다. ibuprofen은 ROS 제어에 사용될 수 있는 또 다른 약물로 다음과 같은 방법으로 제조될 수 있으며, ibuprofen 이외의 비슷한 계열의 NSAIDs 도 동일한 방법으로 봉입시킬 수 있다. ibuprofen이 봉입된 리포좀의 제조는 다음과 같다: 지질을 다음과 같은 mol% 비율로 준비 (HSPC or DPPC, DSPE-PEG-2000, cholesterol 17:1.01:1)하고, ibuprofen은 1~2 mg을 메탄올에 녹여 지질에 같이 혼합한다(지질과 시약은 Sigma와 Avanti에서 구매). 지질은 클로로포름과 메탄올을 9:1 비율로 사용하여 녹이고 evaporation을 45℃에서 2시간 동안 시행하여 유기용매를 제거하고, 이후 진공건조를 2시간 동안 시행한다. 박막에 PBS 1 mL을 넣고 60℃에서 1시간 동안 교반하여 수화를 시키고 초음파를 짧게 시행하고 이어 압출기를 이용하여 크기를 균일하게 제조한다. 압출기의 온도는 60℃ 이상에서 진행하고 필터는 400nm-200nm-100nm 순서대로 사용한다. 리포좀 입자에 들어가지 않은 ibuprofen은 PD-10 (cytiva)으로 분리 후 Mw 10,000의 ultracentrifugal filter tube(Merck)를 이용하여 잔여의 약물을 원심분리로 제거하고 농축한다(6000 rpm, 40 min). 봉입여부는 RP-HPLC로 확인하고(Reverse phase C18 column, acetonitrile:water, 8:2 비율, 0.8 mL/min, 220 nm), ibuprofen 봉입효율은 ibuprofen을 농도별로 standard를 제조하여 각각의 HPLC상의 ROI를 확인하여 standard curve를 만들어 확인한다. 이 때, ibuprofen은 76 μg/mL로 봉입되었다.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.
본 발명에서 리포좀 제조에 사용된 lipid는 DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine)로 16:0의 phosphochloline을 사용하였으나, 이 외에도 DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), HSPC(L-α-phosphatidylcholine, hydrogenated (Soy)) 등이 사용될 수 있으며 본 발명이 속하는 기술분야의 당업자가 용이하게 선택할 수 있는 것이면 이에 국한되지 않고 사용 될 수 있다.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.
본 발명에 있어서, 상기 조성물은 약학 조성물인 것일 수 있다. 본 발명의 조성물은 투여를 위하여, 약학적으로 허용가능한 염, 담체, 부형제, 희석제, 가용화제 등을 포함할 수 있다. 상기 약학적으로 허용가능한 염은 제약업계에서 통상적으로 사용되는 염을 의미하며, 예를 들어 나트륨, 칼륨, 칼슘, 마그네슘, 리튬, 구리, 망간, 아연, 철 등을 비롯한 무기이온의 염과 염산, 인산, 황산과 같은 무기산의 염이 있으며, 그 외에 아스코르브산, 시트르산, 타르타르산, 락트산, 말레산, 말론산, 푸마르산, 글리콜산, 숙신산, 프로피온산, 아세트산, 오로테이트산, 아세틸살리실산과 같은 유기산의 염 등과 라이신, 아르기닌, 구아니딘 등의 아미노산 염이 있다. 또한 약학적인 반응, 정제 및 분리과정에서 사용될 수 있는 테트라메틸 암모늄, 테트라에틸 암모늄, 테트라프로필 암모늄, 테트라부틸 암모늄, 벤질 트리메틸 암모늄, 벤제토늄 등의 유기이온의 염이 있다. 다만, 열거된 이들 염에 의해 본 발명에서 의미하는 염의 종류가 한정되는 것은 아니다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있고, 가용화제로는 폴록사머 및 라브라솔 등을 들 수 있으나, 이에 제한되지 않는다. 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.
본 발명의 약학 조성물은 당업계에 잘 알려진 방법을 사용하여 약학적 제형으로 제조될 수 있다. 제형의 제조에 있어서, 활성 성분을 담체와 함께 혼합 또는 희석하거나, 용기 형태의 담체 내에 봉입시킬 수 있다. 본 발명의 약학 조성물을 경구 투여용 제형으로 제조할 경우, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제로 제제화할수 있다. 상기 약학 조성물은 경구, 직장, 경피, 정맥내, 근육 내, 복강 내, 골수 내, 경막 내 또는 피하로 투여될 수 있다. 경구 투여를 위한 제형은 정제, 환제, 연질 또는 경질 캅셀제, 과립제, 산제, 액제 또는 유탁제일 수 있 으나, 이에 한정되는 것은 아니다. 비경구 투여를 위한 제형은 주사제, 점적제, 로션, 연고, 겔, 크림, 현탁제, 유제, 좌제, 패취 또는 분무제일 수 있으나, 이에 한정되는 것은 아니다. 상기 약학 조성물은 필요에 따라 희석제, 부형제, 활택제, 결합제, 붕해제, 완충제, 분산제, 계면 활성제, 착색제, 향료 또는 감미제 등의 첨가제를 포함할 수 있다.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.
이하, 본 발명의 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail through examples.
단, 하기 실시예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 하기 실시예에 의해 한정되지 아니한다.However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.
리포좀 제조Liposome preparation
지질을 다음과 같은 mol% 비율로 준비하였다(DPPC, DSPE-PEG-2000-NH2, Cholesterol, Simvastatin 17:1.01:1:1.209). 지질과 시약은 sigma와 avanti에서 구매하였다. 클로로포름과 메탄올을 1:1 비율로 사용하여 녹이고 evaporation을 45℃에서 2시간 동안 시행하여 유기용매를 제거하고, 이후 진공건조를 2시간 동안 시행하였다. 박막에 PBS 1 mL을 넣고 55℃에서 1시간 동안 교반하여 수화시키고 초음파를 시행하고(1 min) 이어 압출기를 이용하여 크기를 균일하게 제조하였다. 압출기 온도는 55℃ 이상에서 진행하고 필터는 400nm-200nm-100nm 순서대로 사용하였다. 리포좀 입자에 들어가지 않은 Simvastatin은 PD-10(cytiva)으로 분리하고 재차 ultrafilteration tube (분자량 10000, 6000 rpm, 40 min) 를 이용하여 원심분리로 분리 농축을 하였다. 봉입여부는 RP-HPLC로 확인하고 (Reverse phase C18 column, acetonitrile:ammonium acetate, pH4.6, 20 mM, 4:6 비율, 240 nm), 봉입효율은 Simvastatin을 농도별로 standard를 제조하여 각각의 HPLC상의 ROI를 확인하여 standard curve를 만들어서 확인한다. 리포좀 바깥쪽 NH2 작용기는 ninhydrine assay로 확인하였다. 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.
QK 펩타이드가 탑재된 리포좀의 제조(QK-LP)Preparation of liposomes loaded with QK peptide (QK-LP)
제조된 리포좀에 Sulfo-LC-SPDP(sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate, Pierce)를 넣어 실온에서 반응시키고 PD-10으로 분리한 다음 SH-functional Peptide(CKRKG-QK: CKRKG KLTWQELYQLKYKGI, Peptron, Daejeon)를 반응시켜서 SPDP 말단에 결합시켰다. 펩타이드 결합여부는 반응시 떨어져 나오는 pyridine 2-thione을 RP-HPLC 343 nm에서 확인 하였다(Reverse phase C18 column, 0.1% TFA in ACN/0.1% TFA in water, gradient 97:3에서 30:70). 잔여 펩타이드는 220 nm로 확인하면서 343 nm 를 같이 dual로 확인하였다. 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.
QK-LP의 in vitro HUVEC 세포 결합 테스트In vitro HUVEC cell binding test of QK-LP
리포좀 제조시 NBD-DSPE를 소량 추가하여 형광염료가 달린 형태로 제조하여 세포에서 펩타이드 QK 존재 여부에 따라 내피세포에 결합하는지 확인하였다. 이 리포좀 제조시에는 Simvastatin을 봉입시키지 않았다. 인간 혈관내피세포인 HUVEC 세포를 24 well plate 에 1*105 세포를 seeding 하고 bare liposome(LP), QK-LP 등을 처리하여 uptake여부를 형광현미경을 이용하여 확인하였다. 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 의 혈관 형성 어세이In vitro HUVEC cell angiogenesis assay
인간 혈관 내피세포인 HUVEC 세포를 10cm dish에 풀어(Lonza, EBM-2 buffer) 5% CO2 농도를 유지하며 CO2 인큐베이터(Thermo Forma, USA)에서 37 ℃ 조건으로 배양하였다. 실험 당일 matrigel를 96 well plate에 넣어 굳힌 뒤 1*104 세포를 seeding 하고, 이어 VEGF protein(Lonza), QK-LP, LP 등을 농도별로 처리하고 6시간까지 혈관 형성 정도를 현미경으로 확인하였다.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.
in vitro ROS 제거능 확인Confirmation of in vitro ROS removal ability
HUVEC 세포를 24 well plate 에 1*105 세포를 seeding 하고 세포에 H2O2 (100 mM)를 여러 농도로 처리하고 이어 Simvastatin이 담지된 리포좀을 처리하여 (2시간) ROS 제어 능력이 있는지 확인하였다. ROS 생성정도는 DCFDA(Dichlorofluorescin Diacetate) 시약을 이용하여 확인하였다.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.
리포좀에 테크네슘 표지Technesium labeling of liposomes
3차 증류수를 40분 정도 질소로 PURGE를 시행하여 산소농도를 낮춘 뒤 사용하였다. SnCl2(1 mg)를 1N HCl(100 μL)에 녹인 뒤 질소퍼지한 물을 900 μL를 더하고, 충분히 녹인 뒤 PBS에 담겨진 리포좀 샘플에 free Tc-99m을 넣고 이어서 tin solution을 5 μL추가하여 표지하였다. 실온에서 10분 반응 뒤 pH를 확인하고 ITLC-sg를 이용하여 표지효율을 확인하였다(Bioscan, TLC scanner). silica gel 상에서 리포좀은 Rf=0에 머무르고 표지되지 않은 free Tc-99m은 Rf=1을 나타내었다. 동위원소 표지 반응물을 재차 PD-10으로 분리하였다.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.
허혈질환 모델링 및 약물 투여Ischemic disease modeling and drug administration
9-10주의 male C57BL6(OrientBio) 마우스의 뒷다리의 대퇴부 동맥을 묶은 후 재봉합하여 허혈을 유도하고, 허혈유도의 성공여부는 free Tc-99m을 정맥 내 주사하여 감마카메라상의 이미지를 통해 혈류의 순환 정도를 보고 확인하였다. 허혈부위를 중심으로 3군데로 Simvastatin loaded QK-LP 약물을 주입하고 6시간 경과 후에 혈류를 재평가하여 허혈부위 복구정도를 평가하였다.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.
실시예 1. Simvastatin 담지된 리포좀(Simva-LP), QK 펩타이드가 탑재된 리포좀(Simva-QK-LP)의 제조 및 효과 확인Example 1. Preparation and confirmation of effect of liposome loaded with Simvastatin (Simva-LP) and liposome loaded with QK peptide (Simva-QK-LP)
먼저 상기 방법에 의해 제조된 리포좀 입자를 확인한 결과, 리포좀 내 Simvastatin 봉입율은 76%를 보였다. free의 약물을 최종분리한 다음, 상기 조건으로 HPLC 시행한 후 16.8분에서 보여지는 피크를 토대로 봉입율을 계산하였으며 초기 투여한 약물 양에 비례하여 계산하였다(도 1). 입도분석기를 이용하여 리포좀 나노입자의 크기를 측정한 결과, Simvastatin이 담지된 리포좀(Simva-LP)은 평균 148 nm를 보였고 QK까지 최종 컨쥬게이션된 리포좀(Simva-QK-LP)은 평균 168 nm인 것을 확인하였다(도 2). 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).
이후, HUVEC 세포에 NBD-QK-LP를 처리하고 세포 내 섭취를 형광현미경으로 관찰하였다. 그 결과, QK가 결합된 리포좀의 경우 HUVEC 세포에 처리시 QK 없는 리포좀과는 확연히 다른 섭취를 보임을 확인하였고, 섭취 후 24 시간, 48 시간 경과 후에도 형광이 또렷이 유지되고 이후 72 시간 이후까지도 세포질 안에서 형광이 유지되고 내피세포에 전체적으로 결합되는 양상을 확인할 수 있었다(도 3). 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).
또한, Pro-angiogenic 효과를 확인하기 위하여 혈관 형성을 matrigel를 이용하여 확인한 결과, QK 펩타이드가 있는 경우가 없는 경우에 비하여 선명하게 tube를 형성하는 것을 확인하였다(도 4). 도 4에서 LP-SPDP-QK를 HUVEC 세포에 처리한 경우에 QK 펩타이드가 없는 LP-SPDP 처리한 경우와 비교하여, 보다 조밀하게 혈관이 형성된 것을 확인할 수 있다. 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.
실시예 2. ROS 제거 효과 확인Example 2. Confirmation of ROS removal effect
Simvastatin이 담지된 QK-LP를 HUVEC 세포에 처리시 QK 펩타이드의 리셉터 표적으로 ROS 효과를 보일 수 있다는 가정 하에 시행한 결과, QK 펩타이드 없이 Simvastatin만 담지된 리포좀(LP-SPDP-SIM)과 비교하여 QK 펩타이드가 탑재된 리포좀(LP-SPDP-SIM-QK)이 H2O2 제거에 뛰어난 효과를 나타내는 것을 확인하였다(도 5). 이를 통해, 리포좀 표면에 탑재된 QK 펩타이드가 VEGF receptor를 표적하여 허혈부위로 잘 유도되고 허혈부위의 ROS 제거에 뛰어난 효과를 나타낼 수 있다는 것 을 알 수 있다.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.
실시예 3. 하지허혈 모델 마우스에 Simvastatin이 담지된 QK-LP 주입 효과 확인Example 3. Confirmation of the effect of QK-LP injection containing Simvastatin in lower extremity ischemia model mice
정상 마우스에 DTPA-QK-LP와 DTPA-LP를 주사하고 1 시간 경과 후 전신분포를 촬영하였다(acquisition time 5 min, 14.8 MBq). 전신분포를 확인한 영상으로 QK 펩타이드가 탑재된 리포좀은 QK 펩타이드가 없는 리포좀에 비하여 전신에 좀 더 오랫동안 머무르는 영상을 보이는 것을 확인하였다(도 6). 도 6에서 왼쪽은 DTPA-QK-LP를 주사한 것이고, 오른쪽은 DTPA-LP를 주사한 것이다. 이 때, Simvastatin은 봉입되지 않은 상태이고 테크네슘 표지를 위하여 소량의 DSPE-PEG-DTPA를 리포좀 제조시 같이 사용하였다.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.
한편, 마우스의 뒷다리의 대퇴부 동맥을 묶어 허혈을 유도한 다음, 테크네슘을 표지한 Simvastatin이 담지된 QK-LP를 정맥에 주사한 뒤(acquisition time 3 min, 14.8 MBq) 전신분포를 확인한 결과, 허혈 부위에 시간에 따라 집적되는 양상이 나타나는 것을 확인하였다(도 7). 도 7의 화살표가 허혈유도 부위를 나타낸다. 이 때, 리포좀은 DSPE-PEG-DTPA가 Simvastatin의 봉입을 일부 방해하여 DSPE-PEG-DTPA를 사용하지 않았고, Tc-99m의 특성상 phosphate group 등에 충분히 표지가 가능하여 Tc-99m radiolabeled Simvastatin loaded QK-LP를 사용하였다. 도 7에 나타난 바와 같이, 주사 후 시간이 경과함에 따라 허혈 유도 부위로 타겟되어 표지된 QK-LP가 잘 전달되는 것을 확인하였다. 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.
또한, 대동맥을 묶고 허혈을 만든 후에 허혈 주위로 약물을 주입하고 (3군데) 6시간 까지 확인한 결과, 도 8에 나타난 바와 같이 약물을 처리하지 않은 군에 비하여 QK-LP 처리군과 Simvastatin이 담지된 QK-LP처리군에서 허헐부위 아래 다리와 발가락쪽으로 혈류가 대조군보다 좀 더 증가한 영상을 보였다(acquisition time 3 min, 16.2 MBq).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).
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.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.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.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.
본 발명은 신생혈관형성능을 가지는 QK 펩타이드가 리포좀 표면에 탑재되고 ROS 제어능이 있는 약물이 리포좀 내부에 담지된 리포좀 나노입자를 포함하는 허혈성 질환의 예방 또는 치료용 약학조성물에 관한 것이다. 본 발명에 따르면 QK 펩타이드는 리포좀 표면에 탑재되어 원하는 허혈 부위로 보다 정확하게 전달 가능하고, QK 펩타이드는 허혈부위의 VEGF receptor에 타겟함으로써 표적성이 우수한 효과가 있다. 또한, 동위원소 또는 형광을 표지한 리포좀은 추적, 정량 분석이 가능하여 모니터링을 통한 적절한 치료를 할 수 있어 유용하다. 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. 표면에 신생혈관형성 펩타이드가 탑재되고, 내부에 활성 산소종(reactive oxygen species: ROS) 소거제가 담지된 리포좀을 포함하는 허혈성 질환 예방 또 는 치료용 약학적 조성물. 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. 제 1항에 있어서, According to clause 1,
    상기 신생혈관형성 펩타이드는 서열번호 1로 표시되는 QK 펩타이드인 것을 특징으로 하는 허혈 질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating ischemic disease, wherein the angiogenic peptide is QK peptide represented by SEQ ID NO: 1.
  3. 제 2항에 있어서, According to clause 2,
    상기 QK 펩타이드는 VEGF 수용체를 표적하는 것을 특징으로 하는 허혈성 질 환 예방 또는 치료용 약학적 조성물. The QK peptide is a pharmaceutical composition for preventing or treating ischemic disease, characterized in that it targets the VEGF receptor.
  4. 제 2항에 있어서, According to clause 2,
    상기 QK 펩타이드는 링커를 통해 리포좀 표면에 결합되는 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물. 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. 제 1항에 있어서, According to clause 1,
    상기 활성 산소종 소거제는 심바스타틴(Simvastatin), 아토르바스타틴(Atorvastatin), 세리바스타틴(Cerivastatin), 플루바스타틴(Fluvastatin), 로바스타틴(Lovastatin), 메바스타틴(Mevastatin), 프라바스타틴(Pravastatin) 및 로수바스타틴(Rosuvastatin)로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물. 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. 제 1항에 있어서, According to clause 1,
    상기 활성 산소종 소거제는 심바스타틴(Simvastatin) 또는 이부프로펜(ibuprofen)인 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for preventing or treating ischemic disease, wherein the reactive oxygen species scavenger is simvastatin or ibuprofen.
  7. 제 1항에 있어서, According to clause 1,
    상기 조성물은 형광 또는 동위원소로 표지되어 치료 중 생체 내 이동을 추적할 수 있는 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물. 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. 제 1항에 있어서, According to clause 1,
    상기 조성물은 허혈 부위에 혈관 형성을 유도하고 활성 산소종을 제거하는 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물. 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. 제 1항에 있어서, According to clause 1,
    상기 리포좀은 지질 및 콜레스테롤을 포함하며 상기 지질은 DPPC, DSPC 및 HSPC로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물.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. 제 1항에 있어서, According to clause 1,
    상기 리포좀의 크기는 100 내지 500nm 인 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating ischemic disease, wherein the liposome has a size of 100 to 500 nm.
  11. 제 1항에 있어서,According to clause 1,
    상기 허혈성 질환은 심근경색, 중뇌동맥협착 질환, 하지허혈 및 뇌경색으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 허혈성 질환 예방 또는 치료용 약학적 조성물. 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) DPPC, DSPE-PEG-2000-NH2 및 콜레스테롤로 구성되고 심바스타틴을 담지한리포좀을 제조하는 단계; 및a) preparing a liposome composed of DPPC, DSPE-PEG-2000-NH 2 and cholesterol and carrying simvastatin; and
    b) 상기 리포좀 표면에 서열번호 1로 표시되는 QK 펩타이드를 결합시키는 단계;를 포함하고, b) binding the QK peptide represented by SEQ ID NO: 1 to the surface of the liposome;
    상기 DPPC, DSPE-PEG-2000-NH2, 콜레스테롤 및 심바스타틴의 몰비는 16.5 내지 17.5: 0.5 내지 1.5: 1: 0.7 내지 1.7 인 것을 특징으로 하는 허혈성 질환 예방또는 치료용 약학적 조성물의 제조방법.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. 제 12항에 있어서,According to clause 12,
    상기 b) 단계는 리포좀 표면에 SPDP 링커를 연결하여 리포좀-SPDP를 생성하고 QK 펩타이드에 GKRKC 링커를 연결하여 QK-GKRKC를 생성한 다음, 리포좀-SPDP과QK-GKRKC를 반응시켜 리포좀 표면에 QK 펩타이드를 결합시키는 것을 특징으로 하는, 허혈성 질환 예방 또는 치료용 약학적 조성물의 제조방법.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.
PCT/KR2023/016556 2022-11-04 2023-10-24 Pharmaceutical composition for preventing or treating ischemic diseases, containing liposome having qk peptide loaded on surface thereof WO2024096424A1 (en)

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US20130101628A1 (en) * 2011-04-29 2013-04-25 Northwestern University Novel vegf mimetic peptide-based scaffolds for therapeutic angiogenesis and methods for their use
KR20160141068A (en) * 2015-05-27 2016-12-08 전북대학교산학협력단 Compositions for Prevention or Treatment of Ischemic Disease Comprising Liposome Encapsulated Vascular Endothelial Growth Factor Peptide
US20200262903A1 (en) * 2019-02-15 2020-08-20 Henry J. Smith Treatment of age-related macular degeneration

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US20130101628A1 (en) * 2011-04-29 2013-04-25 Northwestern University Novel vegf mimetic peptide-based scaffolds for therapeutic angiogenesis and methods for their use
KR20160141068A (en) * 2015-05-27 2016-12-08 전북대학교산학협력단 Compositions for Prevention or Treatment of Ischemic Disease Comprising Liposome Encapsulated Vascular Endothelial Growth Factor Peptide
US20200262903A1 (en) * 2019-02-15 2020-08-20 Henry J. Smith Treatment of age-related macular degeneration

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