KR101898528B1 - A pharmaceutical composition comprising liposome containing phycocyanin or phycoerythrin as an active ingredient for brain disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating brain diseases, which comprises a liposome carrying phycocyanin or phycoerythrin as an active ingredient. The pharmaceutical compositions for the prevention or treatment of brain diseases according to the present invention are excellent in thermochemical stability, size mono-dispersity, stability and efficiency of phycocyanin encapsulation, excellent in cell absorption, biocompatibility and antioxidant effect The neuroprotective time window of picocyanin, which is an active ingredient, can be further increased than that of conventional drugs, thereby protecting brain nerve cells from ischemia-reperfusion injury of the brain. Accordingly, it is useful for the prevention or treatment of brain diseases associated with the brain nerve cells.

Description

A pharmaceutical composition comprising liposome containing phycocyanin or phycoerythrin as an active ingredient for brain disease}

The present invention relates to a pharmaceutical composition for preventing or treating brain diseases, a composition for drug delivery, and a food composition, including liposomes carrying phycocyanin or phycoerythrin as an active ingredient.

The mortality rate from cerebrovascular disease (the number of deaths per 100,000 population) reaches 73.8, which is second only to cancer, and the incidence rate is particularly high in the elderly, and once it occurs, the mortality rate is very high. Cerebrovascular diseases can be broadly classified into two types. First, it is a hemorrhagic brain disease that can be seen in cerebral hemorrhage, and the second is an ischemic brain disease that is caused by obstruction of cerebrovascular vessels. The hemorrhagic brain disease is mainly manifested by traffic accidents, and the ischemic brain disease is a disease that frequently appears in elderly people. The ischemic brain disease can be subdivided into thrombosis, em-bolism, transient ischemic attack, and lacune. A pathological abnormality has occurred in the blood vessels that supply blood to the body. When transient cerebral ischemia is induced in the cerebrum, the supply of oxygen and glucose to the brain tissue is blocked, resulting in a decrease in ATP and edema in nerve cells, resulting in extensive damage to the brain. Neuronal death occurs after a significant amount of time after cerebral ischemia, which is called delayed neuronal death. Delayed neuronal cell death was observed in an experiment using a transient forebrain ischemic model using a Mongolian gerbil. After 4 days of induction of cerebral ischemia for 5 minutes, neuronal cell death was observed in the CA1 region of the hippcampus. (Kirino T, Sano K. Acta Neuropathol., 62, 201-208, 1984; Kirino T. Brain Res., 239, 57-69, 1982).

There are two major mechanisms of neuronal death due to cerebral ischemia. First, excessive glutamate is accumulated outside the cells due to cerebral ischemia, and this glutamate is introduced into the cells, eventually causing neuronal death due to excessive intracellular calcium accumulation (Kang TC et al., J. Neurocytol., 30, 945-955, 2001), and the other is an oxidative neuronal cell death mechanism caused by damage to DNA and cytoplasm due to an increase in radicals in vivo due to sudden oxygen supply during ischemia-reperfusion ( Won MH et al., Brain Res., 836, 70-78, 1999; Sun AY, Chen YM, J. Biomed. Sci., 5, 401-414, 1998; Flowers F, Zimmerman JJ. New Horiz., 6 , 169-180, 1998). Based on the above mechanism studies, many studies have been conducted to search for substances that effectively inhibit neuronal cell death during cerebral ischemia, or to reveal the mechanism of substances. However, there are few substances that effectively inhibit neuronal death due to cerebral ischemia.

Clinically available drugs for ischemic brain disease are scarce, and the only drugs currently licensed are antithrombotic drugs (including anticoagulants, antiplatelet drugs, and thrombolytic drugs). Antiplatelet drugs such as ticlopidine, cilostazole, and prostacycline, or anticoagulants, often have headaches, palpitations, and liver-borne side effects, so their use is limited. In addition, the FDA-approved commercially available tissue plasminogen activator is a thrombolytic agent that dissolves blood clots that cause cerebral ischemia and induces rapid supply of oxygen and glucose. Need to use In addition, due to the characteristic of a thrombolytic agent, when used excessively or frequently, the walls of blood vessels become thin, resulting in hemorrhagic cerebrovascular disease, so caution is required in use. At this time, nerve necrosis occurs along with free radicals that occur excessively during reperfusion. For this reason, studies are being conducted to protect the nervous tissue by administering an antioxidant at the same time when tPA is administered. However, there is a need for such neuroprotection to proceed for a considerable time during reperfusion, and thus there is a significant demand for the development of a delivery system including antioxidants for prolonging neuroprotection.

The blood-brain barrier (BBB) acts as a physical barrier to the passage of substances, and is important as a route of administration of agents for the treatment of central nervous system disorders (Xie, Y. et al., J. Control Release 105 , 106-119 (2005)). Nasal administration is known to be effective against brain injury (William, M. P. Pharm. Res. 24, 1733-1744 (2007)). This type of delivery is the delivery of drugs from the nose to the brain through the olfactory neuroepithelium (neuroepithelium), bypassing the blood-brain barrier (BBB), and avoiding first-pass metabolism of the liver (Candace, LG et al., J. Pharm. Sci. 94, 1187-1195 (2005)). Drug delivery through the nasal route includes routes through the nasal mucosa, mucosal epithelium, and olfactory neuroepithelium, lipophilic drug carriers are suitable candidates for nasal administration, and liposomes are typical of lipophilic drug carriers (Illum, L, Eur. J. Pharm. Sci. 11, 1-18 (2000)). Liposomes have been used to prevent oxidation and degradation of encapsulated drugs because of their lipophilic and mucosal-adhesion properties (Corace, G. et al., J. Liposome. Res. 24, 323-335 (2014)). It was confirmed that the drug half-life of liposomes was at least four times higher than that of non-drugs administered intranasally (Jaafari, M. R. Iran. J. Pharm. Res. 4, 3-11 (2005)). Liposomes can be designed to have specific functions of enhancing or releasing target-delivery by synthesizing target-specific lipids, using adjuvants, and coating appropriate antibodies and polymers (Gregoriadis, G. Trends Biotechnol). 13, 527-537 (1995)). Cholesterol supplementation is a conventional method to improve the safety of liposomes, because the incorporation of cholesterol into the phospholipid endoplasmic reticulum makes the lipid bilayer structure more rigid and stable (Johnson, S. M. BBA-Biomembranes 307, 27-41 (1973)). The hydroxyl group of cholesterol interacts with the hydrophilic head of the phospholipid, and its steroid backbone, which results in the regulation of the strength and the strength of the lipid bilayer, interacts with the hydrophobic phospholipid chain (Maria-Lucia, B. Drug. Deliv. Transl. Res. 5, 231-242 (2015)). For these reasons, cholesterol increases the retention of solutes entrapped inside liposome vesicles and serum-induced stability 17. In addition, cholesterol accounts for 20-25% of the brain cell membrane. It has been reported that liposomes containing cholesterol enhance internalization by SH-SY5Y (human neuroblastoma cell line) and Schwann cells (Lee, S. Y. et al., Plos One 8 (2013)). However, the physical and biological environment associated with the nasal delivery pathway affects the physical stability and biological efficacy of liposomes.

C-Phycocyanin (C-Pc) is a protein obtained from blue-green algae such as spirulina, which is known to have antioxidant and ROS scavenger, anti-inflammatory and antibacterial effects (Romay, C. Et al., Curr. Protein Pept. Sci. 4, 207-216 (2003)).

Accordingly, the present inventors have studied methods for effectively protecting brain nerve cells and preventing or treating brain diseases related thereto. As a result, when phycocyanin, an antioxidant, is supported on the liposome, and the liposome is composed of a certain amount of cholesterol , When ischemic stroke reperfusion, the present invention was completed by confirming that the liposome of the present invention is effective in prolonging the brain tissue protection time.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating brain diseases comprising liposomes carrying phycocyanin or phycoerythrin as an active ingredient.

In order to achieve the above object, the present invention is a pharmaceutical composition for preventing or treating brain diseases comprising a liposome carrying phycocyanin or phycoerythrin as an active ingredient, wherein the liposome is a phospholipid Or a sterol, wherein the sterol is contained in an amount of 20 to 33% (w/w) of the liposome, and the liposome is for administration 2 to 10 hours after brain injury. Provide the appropriate composition.

The pharmaceutical composition for preventing or treating brain diseases according to the present invention has high thermochemical stability, size mono-dispersity, phycocyanin encapsulation stability and efficiency, and excellent cell absorption, biocompatibility and antioxidant effects. Thus, the neuroprotective time window of phycocyanin, which is an active ingredient, can be increased more than that of conventional drugs, so that the effect of protecting nerve cells in the brain due to ischemia-reperfusion injury in the brain is excellent. Accordingly, it is useful for the prevention or treatment of brain diseases related to the neurons of the brain.

1 is 0% (w/w) cholesterol-liposome-0 mg/ml C-phycocyanin (C-Pc) (a), 20% (w/w) cholesterol-liposome-0 mg/ml Figure confirming the TEM image of C-phycocyanin (C-Pc) (b), 20% (w/w) of cholesterol-liposome -1mg/ml of C-phycocyanin (C-Pc) (c) to be.
FIG. 2 shows 0% (w/w) cholesterol (a), 20% (w/w) cholesterol (b), and 50% (w/w) cholesterol without C-phycocyanin (C-Pc). (c) shows the size distribution of the liposomes containing. In addition, C-phycocyanin (C-Pc) is supported, and 0% (w/w) cholesterol (d), 20% (w/w) cholesterol (e), 50% (w/w) cholesterol (f ) Is a diagram showing the size distribution of liposomes containing.
Figure 3 is a diagram showing the results measured using differential scanning calorimetry (DSC) of liposomes containing 0%, 11.1%, 20% and 33.3% (w/w) cholesterol, respectively.
Figure 4 shows the degree of ejection of C-phycocyanin (C-Pc) from liposomes containing 0%, 11.1%, 20%, 33.3% and 42.8% (w/w), cholesterol, respectively, time (0, 1, 3, 5, 24, 48 and 72 hours).
5 shows 0% (w/w) cholesterol liposomes and C-phycocyanin supported by PBS, free C-phycocyanin (C-Pc), and C-phycocyanin (C-Pc) in Neuro2a cells. (C-Pc) A diagram showing the result of confirming the absorption level of each Neuro2a cell after 1 hour (a) or 10 hours (b) by treating the supported 20% (w/w) cholesterol liposome through fluorescence intensity.
6 is a 0% (w/w) cholesterol liposome and C-phycocyanin (C-) supported by PBS, free C-phycocyanin (C-Pc), and C-phycocyanin (C-Pc). Pc) confirmed the survival rate of the cells treated with the supported 20% (w/w) cholesterol liposome (a), and confirmed the survival rate of the cells treated with each of the above conditions under the conditions treated with _{hydrogen peroxide (H 2} O _{2 ).} It is a diagram showing the result (b).
Figure 7 is PBS, free (free) C-phycocyanin (C-Pc), 0% cholesterol liposome, C-phycocyanin (C-Pc) supported 0% (w / w) cholesterol liposome and C-pico A diagram showing the results of confirming the mRNA expression level of MnSOD (a) or catalase (b) in cells treated with 20% (w/w) cholesterol liposomes carrying cyanine (C-Pc).
8 is a schematic experimental schematic diagram of processing the liposome of the present invention 2 hours after the induction time point of the middle cerebral artery occlusion (MCAO) animal model of the present invention (a), the infarct volume of the animal model according to each condition (infarct volumes) is a view showing the result (b) and the result (c) showing the average of the infarct volumes of the animal model according to each condition.
9 is a schematic experimental schematic diagram of processing the liposome of the present invention 6 hours after the induction time point of the middle cerebral artery occlusion (MCAO) animal model of the present invention (a), the infarct volume of the animal model according to each condition (infarct volumes), (b), the average of the infarct volumes of the animal model according to each condition (c), and the results of analyzing the neurological deficits of mice in each treatment group (mNSS Value) is a diagram showing (d).
Figure 10 is a schematic diagram schematically showing the liposome and administration method of the present invention (a), is a diagram showing the characteristic (b) according to the cholesterol content.

Hereinafter, the present invention will be described in detail.

Terms that are not otherwise defined in the present specification have the meanings commonly used in the technical field to which the present invention belongs.

The present inventors believe that the pharmaceutical composition for preventing or treating brain diseases comprising liposomes carrying Phycocyanin or phycoerythrin of the present invention as an active ingredient is a neuroprotective time window As a result, it was found for the first time that even if administered after a certain period of time after the onset of ischemic stroke, the ejection amount of phycocyanin or phycoerythrin, a neuroprotective substance, was maintained continuously and the infarct volume was significantly reduced.

As an aspect, as a pharmaceutical composition for preventing or treating brain diseases comprising a liposome carrying phycocyanin or phycoerythrin as an active ingredient, the liposome comprises a phospholipid or a sterol, and the sterol Is contained in an amount of 20 to 33% (w/w) of the liposome, and the liposome provides a pharmaceutical composition for preventing or treating brain diseases, characterized in that for administration 2 to 10 hours after brain injury.

In the present invention, "liposome" refers to a vesicle comprising at least one bilayer separating the inner aqueous phase from the outer aqueous environment.

In the present invention, the "phycocyanin" is represented by the structure of the following formula (1), and is a substance contained in algae such as red algae, blue algae, and cryptozoic acid together with phycoerythrin, and is covalently bonded to phycocyanobilin (straight chain The tetrapyrrole derivative of the phase) refers to a blue pigment protein called chromogen. “Picoerythrin” is called a protein that is borrowed together with phycocyanin, and photosynthesis proceeds by transferring light energy to chlorophyll a.

[Formula 1]

The liposome contains a phospholipid or a sterol, and the phospholipid is phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol, and phosphatidyl inositol. It is one type selected from the group consisting of phosphatidic acid, preferably phosphatidyl choline, but is not limited thereto.

In the present invention, the "phosphatidyl choline" is the most representative phospholipid, which means that choline phosphate is bound to diglyceride. It is widely distributed in the flora and fauna, and accounts for about 1/2 of lipids in animal tissues. It is a major constituent of biomembrane lipids, is important as an efficient source of choline, and is required for the activity of various enzymes.

The sterol is at least one selected from the group consisting of cholesterol, coprostanol, sitosterol, and ergosterol, preferably cholesterol, but is not limited thereto.

In the present invention, the "cholesterol" is a representative sterol found in vertebrates. In the case of humans, it is present in a free form or an ester form with fatty acids as a constituent of almost all cells, especially in the brain, spinal cord, and nervous tissue. On the other hand, most of other steroids such as sex hormones, adrenal corticosteroids, bile acids, and vitamin D are biosynthesized from cholesterol.

The sterol is included in an amount of 0 to 50% (w/w) of the liposome, but is preferably in an amount of 10 to 40% (w/w), more preferably of 20 to 33% (w/w). Content, but is not limited thereto.

The size of the liposome is 80 to 150 nanometers (nanometer), preferably 80 to 130 nanometers, more preferably 90 to 110 nanometers, but is not limited thereto.

The liposomes are intranasal administration, oral administration, intracutaneous injection, subcutaneous injection, intramuscular injection, intravenous injection, and intraarterial injection. (intraarterial injection), rectal application (rectal administration), application to oral mucous membrane (application to oral mucous membrane) and external application administration to perform one or more administrations selected from the group consisting of administration, preferably nasal Administration, but is not limited thereto.

In the present invention, the "nasal administration" is a formulation intended to be administered directly to the nostril, and there is a risk of causing seizures or loss of consciousness in the case of conventional intramuscular injection, whereas nasal administration is much safer than the method and There is an advantage of being easy to apply to patients who do not have.

The brain disease is at least one selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury, preferably stroke, and more preferably Is ischemic stroke, but is not limited thereto.

The liposome is one or more types of damage selected from the group consisting of ischemia-reperfusion injury, ischemic cell injury, and oxidative stress, preferably brain damaged due to ischemia-reperfusion injury To protect the nerve cells of, but is not limited thereto.

The liposome is for administration 2 to 10 hours after brain injury, preferably 2 to 6 hours, but is not limited thereto. When ischemic stroke occurs and reperfusion occurs 4 hours after (6 hours after ischemic stroke onset), when administered intranasally, it has the effect of prolonging the neuroprotective action time of phycocyanin carried in liposomes, which is excellent for the protection of nerve cells in the brain. Shows the effect.

The liposome may contain a liposome alone, or may further contain a substance that is effective in protecting nerve cells of the brain and thus brain disease as an active ingredient, and in addition to the active ingredient, additional ingredients depending on the formulation, method of use, and purpose of use. That is, it may further include a pharmaceutically acceptable or nutritionally acceptable carrier, excipient, diluent or subcomponent.

In more detail, the pharmaceutical composition for preventing or treating brain diseases, including liposomes carrying phycocyanin or phycoerythrin as an active ingredient, in addition to the active ingredient, additional nutrients, vitamins, electrolytes, Flavors, colorants, heavy thickeners, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. can do.

As the carrier, excipient, and diluent, all conventional ones can be used, for example lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, calcium carbonate, dextrin, propylene glycol, liquid paraffin And one or more selected from the group consisting of physiological saline, but is not limited thereto. The components can be added independently or in combination to the liposome.

The liposome according to the present invention may be used alone as a pharmaceutical composition for preventing or treating brain diseases, or may be used in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The liposome according to the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may be the weight, age, sex, and health of the patient. The range varies depending on the condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dose of the liposome of the present invention is 0.01 to 10000 mg/kg, preferably 1 to 20 mg/kg, and is preferably administered in divided doses from 1 to 3 times a day.

Hereinafter, the examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, those of ordinary skill in the art to which the present invention pertains. It will be self-evident to you.

Experimental Example 1. Liposome Preparation

1-1. Preparation of liposomes carrying C-Phycocyanin (C-Pc)

Each liposome was prepared according to the cholesterol content in the liposome and whether C-phycocyanin (C-Pc) was supported.

Specifically, liposomes were prepared using a film hydration method (Romay, C. et al., Inflamm. Res. 47, 36-41 (1998)). L-α-phosphatidylcholine and cholesterol (Chol; Sigma Aldrich, US) obtained from egg yolk (PC, Sigma Aldrich, US) in different contents (0, 11.1, 20, 33.3, 42.8 and 50% (w/w) )). The lipid solution was dried in a vacuum chamber for 3 hours to form a lipid film. To prepare a 10 mM liposome solution, the film was hydrated with 1 ml of PBS with or without 20 μg/ml of C-phycocyanine (C-Pc), and then peeled off from the bottom of the vial. It was vortexed. Liposomes for in vivo experiments were prepared to contain 1 mg/ml of C-phycocyanin (C-Pc). In order to destroy the multi-layered liposome, freezing/thawing was performed 10 times using a -80 degree freezer and a 20 degree water bath. The liposome solution was extruded through a 100 nm pore polycarbonate membrane using an extruder (Avanti Polar Lipids, U.S.). Before the cells were treated with liposomes, liposomes passed through a 0.22 μm filter were sterilized and used.

1-2. Differential scanning calorimetry (DSC)

After adding cholesterol, the change in transition temperature of liposomes carrying C-phycocyanin (C-Pc) was measured using a differential scanning calorimeter (calorimeter (Netzsch, Germany)). Liposome samples (2 mg) of each condition were placed in an aluminum pan and sealed with an aluminum lid. The differential scanning calorific value was measured over a range of 20 to 50 degrees at a heating rate of 5 degrees/minute using an empty pan as a reference.

1-3. Liposome size and zeta potential

In order to measure the size and zeta potential of liposomes, a liposome solution was prepared by diluting 5 times with distilled water. Particle size, polydispersity indices, and zeta potential were analyzed through dynamic light scattering using a laser diffraction size analyzer (Mastersizer 2000, Malvern, UK). For each standard liposome sample, the above procedure was performed 3 times.

1-4. Transmission electron microscopy (TEM)

The liposome morphology was measured using TEM (JEM 2100F, JEOL, Japan). The liposome samples of each condition were diluted 10 times, dropped on a TEM grid, stained with 1% uranyl acetate for 10 minutes, and washed 3 times with distilled water. The grid was placed in an artificial chamber overnight, and liposomes were observed using TEM.

1-5. C-phycocyanine (C- PC ) Liposomal Encapsulation efficiency and in vitro C-phycocyanin (C- PC ) Liposomal Check the emission profile

C-phycocyanin (C-Pc) was used as a reference, and the amount of C-phycocyanin (C-Pc) encapsulated in a liposome was measured. In order to separate the liposomes from the solution, the liposome vesicles were obtained by centrifugation at 20,000 g for 30 minutes. The supernatant containing free C-phycocyanin (C-Pc) and unstable liposomes was collected, and BCA protein analysis was performed using a BCA assay kit (Thermo Scientific, US). The supernatant of liposomes without C-phycocyanin (C-Pc) was used as a control. The ratio of encapsulation efficiency (EE%) was calculated using Equation 1 below, and samples under each condition were measured three times. Samples of each of the above conditions were prepared under dark conditions.

Profile release of C-phycocyanin (C-Pc) in vitro is 0, 6, 12, 24, 36, 48, 60, respectively, in 9 ml of PBS (pH 7.4) and 1 ml of liposome solution at 37 degrees. Alternatively, it was treated under conditions of 72 hours. Each time, 1 ml of a solution was taken and centrifuged under the conditions of 20,000 g and 30 minutes. BCA protein analysis was performed on the supernatant containing the ejected C-phycocyanin (C-Pc).

1-6. Cell culture

Neuro2A cells (mouse neuroblastoma cell line) were obtained from ATCC (American Type Culture Collection) (USA), and DMEM containing 10% fetal bovine serum (FBS, Gibco, UK) and 1% penicillin-streptomycin (Gibco, UK) (Dulbecco's modified Eagle's medium) (Gibco, UK) was cultured _{in a 37°C, 5% CO 2 incubator.} For the differentiation of cells, 2% FBS and 20 μM retinoic acid were treated for 4-5 days. Liposomes were sterilized by filtering with a 0.22 μm filter before being treated with cells.

1-7. Uptake level and cytotoxicity of C-phycocyanin (C-Pc)-carrying liposomes

Specifically, uptake of C-phycocyanin (C-Pc)-carrying liposomes to Neuro2a cells was observed using a confocal microscope (LSM 510 META, Carl Zeiss, and Germany). Neuro2a cells were treated with C-phycocyanin (C-Pc)-carrying liposomes containing 5 μg/ml of C-phycocyanin (C-Pc) for 3 hours. In addition, as a positive control or a negative control, free C-phycocyanin (C-Pc) or PBS was treated on Neuro2a cells. After culturing the cells with or without C-phycocyanin (C-Pc), the medium was removed, the cells were removed with PBS, and then fixed with cold 4% paraformaldehyde for 15 minutes, and then Hoechst 33342 dye (Thermo Fisher, MA USA). The nuclei were imaged using a 405 nm laser and a BP 420-480 nm emission filter, and an LP 615 nm emission filter was used for C-phycocyanin (C-Pc) detection.

The cytotoxic effect of liposomes on Neuro2a cells was analyzed using MTT assay. In Neuro2a cells cultured by the cell culture method of Experimental Example 1-6, 500 μM liposomes were cultured for 1, 3, or 5 days under conditions of 37 degrees. MTT analysis was performed for 1 hour at 37 degrees using 100 μl MTT solution. The MTT solution was removed, and formazan crystals were dissolved with 200 μl of dimethyl sulfoxidase. Optical density (OD) was analyzed at 540 nm using an ELISA reader (Tecan, Austria).

1-8. How to check the antioxidant effect of C-phycocyanin (C-Pc)-carrying liposomes

In order to evaluate the antioxidant effect of C-phycocyanin (C-Pc)-carrying liposomes, Neuro2a cell viability was performed under oxidation conditions induced with _{hydrogen peroxide (H 2} O _{2 ).} Neuro2a cells were cultured by the cell culture method of Experimental Example 1-6, and liposomes supported or non-supported with C-phycocyanin (C-Pc) were pretreated for 1 day, and 200 μM H ₂ O ₂ was used for 24 hours. Processed. Cell viability was measured using the MTT assay of Experimental Examples 1-7. qRT-PCR was used to measure the mRNA level of antioxidant enzymes in Neuro2a cells treated with C-phycocyanin (C-Pc)-carrying liposomes. Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN, Germany). cDNA was synthesized using RNA QuantiFast Reverse Transcription Kit (Qiagen, Germany), and qRT-PCR was used as QuantiFast SYBR Green PCR Kit (Qiagen, Germany) and Rotor-Gene Q PCR unit (Qiagen, Germany). PCR conditions were performed in 40 cycles, 5 minutes at 95 degrees, 10 seconds at 95 degrees, and 30 minutes at 60 degrees. Beta-actin was used as a housekeeping gene. The sequence of the primers is as follows: beta-actin is 5'-GTACTCTGTGTGGATCGGTGG-3' (SEQ ID NO: 1, forward) and 5'-AACGCAGCTCAGTAACAGTCC-3' (SEQ ID NO: 2, reverse), MnSOD is 5'-ACCGAGGAGAAGTACCACGA-3 '(SEQ ID NO: 3, forward) and 5'-TGGGTTCTCCACCACCCTTA-3' (SEQ ID NO: 4, reverse), Catalase is 5'-GCGGATTCCTGAGAGAGTGG-3' (SEQ ID NO: 5, forward) and 5'-TGTGGAGAATCGAACGGCAA-3' (sequence Using number 6, reverse).

1-9. Animal model and nasal administration

Middle cerebral artery occlusion (MCAO) was performed according to national guidelines for the use of laboratory animals. Male Sprague dawley (SD) rats (DBL, Korea) were placed under a condition of 22 ± 2 degrees under a 12 hour light-dark cycle, and fed and drinking water were provided. It was used as a protocol provided by INHA-IACUC (Institutional Animal Care and Use Committee of Inha University (Republic of Korea)), and all animal procedures were blinded.

The MCAO rat model was prepared by the method described in Johnson, S. M, et al. (BBA-Biomembranes 307, 27-41 (1973)). 8-week-old SD rats were anesthetized using 3% isoflurane (BKpharm, South Korea), and 1.5% isoflurane was maintained using a face mask. During the operation, the rectal temperature was maintained at 37 degrees using a heat pad. After the animal is positioned in the supine position on the thermal pad, a small midline neck incision is made, while maintaining the vagal nerve, a common carotid artery (CCA). ), internal carotid artery (ICA) and external carotid artery (ECA) were exposed. The ECA was tied with a silk suture and the MCA was blocked by inserting a silicone-coated 4-0 nylon filament (AILEE, South Korea) into the right ICA. The inserted filament was fixed at the corresponding position using a silk suture, and the CCA was connected to a clamp (JEUNG DO BIO, South Korea). The skin of the neck was closed and sutured for 1 hour, and then the inserted filament was pulled and blood was reperfused through the MCA. An 8.34 μm sample containing liposomes of 0% or 20% cholesterol, liposomes of 0% or 20% cholesterol carrying C-phycocyanin (C-Pc); And free (free) C-phycocyanin (C-Pc) was intranasally administered twice to each nostril at a total dose of 33.36 μl for 6 hours after reperfusion. After 2 days, the brain of MCAO rats was dissected and the coronal plane was separated using a rat brain slicer (Zivic, USA) to provide a 2 mm thick section. The sections were stained with TTC (2, 3, 5-triphenyltetrazolium chloride) to measure infarct volumes.

Example 1.Cholesterol content and C-phycocyanin (C- PC ) Depending on whether it is loaded or not Liposomal Determine size, zeta potential and polydispersity indices

The size, zeta potential, and polydispersity indices of liposomes according to cholesterol content and C-phycocyanin (C-Pc) support were confirmed.

Specifically, in order to confirm the shape and size of liposomes under each condition, as in Experimental Example 1-1, L-α-phosphatidyl choline was included, and C-phycocyanin (C-Pc) was 0 or 1 mg/ml The TEM method shown in Experimental Example 1-4 is supported by and shows the shape and size of each liposome consisting of different cholesterol content (%) (0, 11.1, 20, 33.3, 42.8 and 50% (w/w)). It was observed using. Of the results, 0% (w/w) cholesterol-liposome-0 mg/ml C-phycocyanin (C-Pc) (a), 20% (w/w) cholesterol-liposome-0 mg/ml C-phycocyanin (C-Pc) (b), 20% (w/w) cholesterol-liposome-1 mg/ml C-phycocyanin (C-Pc) (c) is shown in FIG. 1. .

In addition, in order to confirm the size, polydispersity index, and zeta potential of liposomes under each condition, as in Experimental Example 1-1, L-α-phosphatidyl choline was included, and C-phycocyanine (C-Pc) was 0 Alternatively, it is supported at 1 mg/ml, and the size, polydispersity index, and zeta potential of each liposome composed of different cholesterol content (%) (w/w) (0, 11.1, 20, 33.3, 42.8 and 50) were tested in Experimental Example 1- The measurement using the method of 3 and the results are shown in Table 1 below, and the results of confirming the size distribution of some of the liposomes in each condition are shown in FIG. 2.

[Table 1]

In addition, as in Experimental Example 1-1, including L-α-phosphatidyl choline, and different cholesterol content (%) (w / w) (0, 11.1, 20 and 33.3) of each liposome consisting of a transition temperature change experiment It was confirmed by the differential scanning calorimetry (DSC) of Example 1-2. The results are shown in FIG. 3.

As shown in Figure 1, 0% (w/w) cholesterol-liposome-0 mg/ml C-phycocyanin (C-Pc) (a), 20% (w/w) cholesterol-liposome- TEM of 0 mg/ml C-phycocyanine (C-Pc) (b), 20% (w/w) cholesterol-liposome-1 mg/ml C-phycocyanine (C-Pc) (c) As a result of checking the image, it was confirmed that the size of the liposome was not affected by C-phycocyanin (C-Pc), and the size increased according to the content of cholesterol.

In addition, as shown in Table 1 and Figure 2, it was confirmed that the liposome under all conditions is smaller than the size of 126nm. It was confirmed that liposomes containing 50% (w/w) cholesterol increased about 24.97% in size compared to liposomes not containing cholesterol. As the cholesterol content increased, the liposome size also increased, and the size was about 90 to 130 nm. It was confirmed that the size range was formed. In addition, it was confirmed that the loading of C-phycocyanin (C-Pc) did not affect the size of the liposome. In addition, it was confirmed that the polydispersity index (PIs) of the liposome was 0.2 or less in the liposome under the cholesterol condition of 42.8% content or less, and was uniformly distributed. In addition, it was confirmed that the zeta potential was 50% (w/w) cholesterol liposomes -17 mV, and 0% (w/w) cholesterol liposomes 7.97 mV, and the zeta potential decreased as the cholesterol content increased. . On the other hand, it was confirmed that when C-phycocyanin (C-Pc) was supported, the zeta potential of liposomes increased.

In addition, as shown in Figure 3, in the liposome containing 0% (w / w) and 11.1% (w / w) cholesterol, the transition temperature was measured at 7.1 degrees and -6.9 degrees, respectively, transition peaks (transition peaks) It was confirmed that it appeared. On the other hand, it was confirmed that no transition peak appeared in liposomes containing 20% (w/w) and 33.3% (w/w) cholesterol.

Example 2. Cholesterol content and C-phycocyanin (C- PC ) Depending on whether it is loaded or not Liposomal Encapsulation efficiency and release efficiency check

The encapsulation efficiency and release efficiency of liposomes according to cholesterol content and C-phycocyanin (C-Pc) support were confirmed.

Specifically, in order to confirm the encapsulation efficiency of liposomes under each condition, C-phycocyanine (C-Pc) was supported at 0 or 1 mg/ml by the method of Experimental Example 1-5, and different cholesterol contents (%) The encapsulation efficiency (EE (%)) and release efficiency of each liposome consisting of (w/w) (0, 11.1, 20, 33.3, 42.8 and 50) were confirmed. The results are shown in Table 2 and FIG. 4.

[Table 2]

As shown in Table 2, the encapsulation efficiency of liposomes carrying C-phycocyanin (C-Pc) and having cholesterol contents of 0, 11.1, 20, 33.3 and 42.8% (w/w), respectively, was 63.95±0.53, respectively, and It was confirmed that they were 73.14±1.60, 75.47±0.81, 72.28±0.35, and 65.72±2.04, and it was confirmed that the higher the cholesterol content, the lower the encapsulation efficiency gradually. Therefore, it was confirmed that the encapsulation efficiency is most efficient when the cholesterol content is 20% (w/w).

In addition, as shown in Figure 4, as time passes, the ejection of C-phycocyanin (C-Pc) per hour from liposomes containing 20% (w/w) cholesterol contains different amounts of cholesterol. It was confirmed that it lasts until the latest than the liposome.

Example 3. Confirmation of liposome absorption level, cytotoxicity and antioxidant effect according to cholesterol content

The absorption level of liposomes, cytotoxicity, and antioxidant effects were confirmed according to the cholesterol content.

3-1. Confirmation of liposome absorption level according to cholesterol content

Specifically, in order to confirm the liposome absorption level of each condition for cells, as a positive control or negative control, free C-phycocyanin (C-Pc) or PBS was treated on Neuro2a cells. In addition, after treatment with liposomes containing 0% (w/w) or 20% (w/w) cholesterol loaded with C-phycocyanin (C-Pc) on Neuro2a cells, after each 1 hour or 10 hours The liposome absorption level of each cell was observed, and the absorption rate over time was compared after 1 hour or 10 hours. The results are shown in FIG. 5.

As shown in FIG. 5A, 1 hour after cell treatment, C-phycocyanin (C-Pc) was supported rather than 0% (w/w) cholesterol liposome supported by C-phycocyanin (C-Pc). By confirming that the fluorescence intensity was high in 20% (w/w) cholesterol liposome, it was confirmed that the absorption level for Neuro2a cells was high.

In addition, as shown in b of Figure 5, after 10 hours of cell treatment, it was confirmed that the fluorescence intensity was still high in 20% (w/w) cholesterol liposomes carrying C-phycocyanin (C-Pc), The other control group confirmed that the fluorescence intensity was slightly higher than after 1 hour of the cell treatment.

Therefore, the absorption rate over time of 1 hour or 10 hours is different from that of liposomes in 20% (w/w) cholesterol liposomes carrying C-phycocyanin (C-Pc), even after 1 hour of cell treatment. It was confirmed that the fluorescence intensity was higher than that of the group, and the absorption rate was confirmed to be faster than that of the other groups. In addition, 10 hours after cell treatment showed the same fluorescence intensity as after 1 hour, it was confirmed that absorption persistence was also superior to other groups.

3-2. Confirmation of cytotoxicity of liposomes according to cholesterol content

Specifically, in order to determine whether the liposome cytotoxicity of each condition for cells, as a positive control or negative control, free C-phycocyanin (C-) using the MTT assay method of Experimental Example 1-7. Pc) or PBS was treated on Neuro2a cells. In addition, liposomes containing 0% (w/w) or 20% (w/w) cholesterol loaded with C-phycocyanin (C-Pc) were treated with Neuro2a cells for 1, 3, or 5 days. Cell viability was confirmed by culturing, and further, hydrogen peroxide (H ₂ O ₂ ) was treated for 24 hours to confirm the cell viability of each of the above conditions under the induced oxidation conditions. The results are shown in FIG. 6.

As shown in FIG. 6, when liposomes containing 20% (w/w) cholesterol carrying C-phycocyanin (C-Pc) were treated with Neuro2a cells, the cell viability was 0 as the time passed. It was confirmed that it was higher than the group treated with liposomes containing% (w/w) cholesterol (a). In addition, liposomes without phycocyanin _{were found to have significantly lower cell viability under oxidizing conditions treated with hydrogen peroxide (H 2} O ₂ ), whereas phycocyanin was supported and 0% (w/w) and In cells treated with liposomes containing 20% (w/w) cholesterol, it was confirmed that the cell viability was higher than that of the non-phycocyanin group (b).

3-3. Checking the antioxidant effect of liposomes according to cholesterol content

Specifically, in order to confirm the antioxidant effect of liposomes under each condition on cells, free C-phycocyanin (C-Pc) was used as a positive control or negative control using the method of Experimental Example 1-8. ) Or PBS was treated on Neuro2a cells. In addition, liposomes containing 0% (w/w) or 20% (w/w) cholesterol loaded with C-phycocyanin (C-Pc) were treated with Neuro2a cells and then _{converted into hydrogen peroxide (H 2} O ₂ ). In the induced oxidation conditions, the mRNA expression level of MnSOD or catalase, an antioxidant enzyme in cells according to each condition, was measured. The results are shown in FIG. 7.

As shown in FIG. 7, the mRNA expression level of MnSOD or catalase in the group treated with liposomes containing 20% (w/w) cholesterol supported by C-phycocyanin (C-Pc) was 1.7379 and catalase. It was confirmed that represents an expression level of 1.6485. In addition, it was confirmed that the mRNA expression level of MnSOD or catalase was significantly expressed as in the free C-phycocyanin (C-Pc) treatment group, which is a positive control.

Example 4. Middle cerebral artery occlusion, MCAO ) For animal models Liposomal Neuroprotective effect check

In an animal model of middle cerebral artery occlusion (MCAO) that causes ischemic stroke, the neuroprotective effect according to cholesterol content and C-phycocyanin (C-Pc) loading was confirmed.

Specifically, the method of Experimental Examples 1-9 was carried out, and as shown in FIG. 8A or 9B, blood was reperfused after 1 hour from the time point in which the middle cerebral artery occlusion (MCAO) was induced in the animal model. , 2 hours or 6 hours after the MCAO induction time point, PBS was used as a control, and 20% (w/w) cholesterol liposomes, C-phycocyanin (C-Pc) supported 0% (w/w) or 20% (w/w) cholesterol liposomes; And free (free) C-phycocyanin (C-Pc), respectively, was administered to the mice intranasally, and then infarct volumes were measured 48 hours after MCAO induction. The results are shown in FIGS. 8 and 9.

As shown in Figure 8, 2 hours after MCAO induction, in mice treated with 20% (w/w) cholesterol liposomes carrying C-phycocyanin (C-Pc) of the present invention, a positive control, free ) It was confirmed that the infarct volumes were significantly reduced as in the group treated with C-phycocyanin (C-Pc).

In addition, as shown in Figure 9, 6 hours after MCAO induction, mice treated with 20% (w/w) cholesterol liposomes carrying C-phycocyanin (C-Pc) of the present invention were free as a positive control. (free) C-phycocyanin (C-Pc) at a level of one-third compared to mice, compared with 0% (w/w) cholesterol liposome mice carrying C-phycocyanin (C-Pc) As a result, it was confirmed that the infarct volumes were significantly reduced to a level of 1/2.

Through the above results, as shown in FIG. 10A, C-phycocyanin (C-Pc) is supported 4 hours after the occurrence of ischemic-reperfusion injury or 6 hours after the occurrence of middle cerebral artery occlusion (cause of ischemic stroke). When liposomes containing one cholesterol and within 100 nm in size are administered nasally, the neuroprotective time window of C-phycocyanin (C-Pc), a neuroprotective substance, can be increased to protect brain nerve cells. Confirmed. In particular, as summarized in b of FIG. 10, in the cholesterol content of 0%, 11.1%, 20%, 33.3%, 42.8% and 50% (w/w), 20%, 33.3% and 42.8% (w/w ) It was confirmed that the content of cholesterol conditions was more stable in thermochemical stability and size mono-dispersity. Among them, it was confirmed that the cholesterol condition of 20% (w/w) content had high drug encapsulation stability and efficiency. As such, it was confirmed that the cholesterol condition of the 20% (w/w) content was superior to other contents in cell absorption, biocompatibility, and antioxidant effects.

In particular, when ischemic stroke occurs and reperfusion occurs 4 hours after the onset of ischemic stroke (6 hours after the onset of ischemic stroke), when the liposome in the cholesterol condition of the 20% (w/w) content is nasal administration, the phycocyanin supported on the liposome is As it has the effect of increasing the neuroprotective time window, it has been confirmed that the effect of protecting the nerve cells of the brain due to ischemia-reperfusion injury of the brain is excellent.

<110> Inha University Research and Business Foundation <120> A pharmaceutical composition comprising liposome containing protein from marine microorganism as an active ingredient for brain disease <130> 1-191 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for beta-actin <400> 1 gtactctgtg tggatcggtg g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for beta-actin <400> 2 aacgcagctc agtaacagtc c 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MnSOD <400> 3 accgaggaga agtaccacga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MnSOD <400> 4 tgggttctcc accaccctta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Catalase <400> 5 gcggattcct gagagagtgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Catalase <400> 6 tgtggagaat cgaacggcaa 20

Claims (7)

A pharmaceutical composition for the prevention or treatment of brain diseases, which comprises a liposome carrying phycocyanin or phycoerythrin as an active ingredient,
Wherein the liposome comprises phospholipids and cholesterol, wherein the cholesterol is contained in an amount of 20 to 33% (w / w) of the liposome,
Wherein the liposome is administered to the nasal cavity 2 to 7 hours after brain injury. The method according to claim 1,
The phospholipid is selected from the group consisting of phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidic acid. Or a pharmaceutically acceptable salt thereof. delete The method according to claim 1,
Wherein the liposome has a size of 80 to 150 nanometers. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; delete The method according to claim 1,
Wherein the brain disease is at least one selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain damage. A pharmaceutical composition. The method according to claim 1,
The liposome is characterized in that it protects brain nerve cells damaged by one or more kinds of damage selected from the group consisting of ischemia / reperfusion injury, ischemic cell injury and oxidative stress Or a pharmaceutically acceptable salt thereof.

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