KR20190130366A - Liposome Comprising Chitosan Coating and Phycocyanin Therein, and Pharmaceutical Composition Comprising the Same for Treatment of Brain Diseases for Nasal Administration - Google Patents

Abstract

The present invention is a pharmaceutical composition for the treatment of brain diseases for nasal administration and to liposomes comprising chitosan coating and phycocyanin supported therein. The chitosan coated on the surface of the liposome of the present invention binds positively with the sialic acid of the nasal mucosa, which is positively charged, and can effectively deliver phycocyanin into the brain through the nasal route.

Description

Liposomes comprising chitosan coating and phycocyanin supported therein, and pharmaceutical compositions for treating nasal diseases comprising the same, and pharmaceutical composition comprising the same for treatment of brain diseases for nasal Administration}

The present invention is a chitosan coating; And phycocyanin supported therein; It relates to a liposome comprising a and a pharmaceutical composition for the treatment of brain diseases for nasal administration comprising the same.

The death rate from cerebrovascular disease (death deaths per 100,000 population) reaches 73.8, the second-largest after cancer, with a high incidence in older people, and a high mortality rate once developed. Cerebrovascular diseases can be broadly classified into two types. first

First, hemorrhagic brain disease can be seen in cerebral hemorrhage and the like, and second, ischemic brain disease caused by occlusion of cerebrovascular vessels. The hemorrhagic brain disease is mainly caused by traffic accidents, etc., ischemic brain disease is a disease that occurs frequently in older people. The ischemic brain disease can be subdivided into thrombosis, embolism, transient ischemic attack, and infarction, and ischemic cerebrovascular disease is mainly due to thrombus and embolism. The blood vessels that supply blood flow to the pathological abnormalities have occurred. In the case of transient cerebral ischemia in the cerebrum, oxygen and glucose supply to brain tissues are blocked, resulting in a decrease in ATP and edema in neurons, resulting in extensive brain damage. Neuronal death occurs after a significant period of time after cerebral ischemia, which is called delayed neuronal death. Delayed neuronal fossa was observed in a transient forebrain ischemic model using Mongolian gerbil. (Kirino T, Sano K. Acta Neuropathol., 62, 201-208, 1984; Kirino T. Brain Res., 239, 57-69, 1982).

There are two major neuronal death mechanisms by cerebral ischemia. First, excess glutamate accumulates outside the cell by cerebral ischemia, and such glutamate flows into the cell, resulting in excessive accumulation of intracellular calcium.

Neuronal cell death (Kang TC et al., J. Neurocytol., 30, 945-955, 2001), which causes neuronal death, and the other is due to the sudden oxygen supply during ischemia-reperfusion. Oxidative neuronal death mechanism caused by damage to DNA and cytoplasm due to the increase (Won MH et al., Brain Res., 836, 70-78, 1999; Sun AY, Chen YM, J. Biomed. Sci., 5 , 401-414, 1998; Flowers F, Zimmerman J J. 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 in cerebral ischemia or to reveal the mechanism of substances. However, it is still effective to suppress neuronal cell death caused by cerebral ischemia.

There is almost no substance.

Clinically available drugs in ischemic brain disease are rare, and the only currently approved drugs are antithrombotic agents (including anticoagulants, antiplatelets, thrombolytics). Antiplatelet or anticoagulants such as ticlopidine, cilostazole and prostacycline often have headaches, palpitations and liver side effects that limit their use. In addition, the FDA-issued tissue plasminogen activator is a substance that induces rapid supply of oxygen and glucose by melting blood clots that induce cerebral ischemia by thrombolytics, and thus does not directly protect nerve cells. Need to use In addition, due to the characteristics of thrombolytics, when used excessively or frequently, the wall of the blood vessel becomes thin and eventually causes hemorrhagic cerebrovascular disease. At this time, necrosis occurs along with free radicals that occur excessively during reperfusion. For this reason, studies are currently being conducted to protect nerve tissue by simultaneously administering an antioxidant when tPA is administered. However, this neuroprotection is refactored

The need arises for a considerable period of time, and there is considerable demand for the development of antioxidant-containing delivery systems to extend neuroprotection.

The blood-brain barrier (BBB) acts as a physical barrier to the passage of substances, which is important as a route of agent administration 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 for brain injury (William, M. P. Pharm. Res. 24, 1733-1744 (2007)). This type of delivery delivers the drug from the nose to the brain through the neuroepithelium, bypasses the blood-brain barrier (BBB), and avoids 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, olfactory neuroepithelial lipophilic drug carriers, and liposomes are typical of lipophilic drug carriers (Illum, L, Eur J. Pharm.

11, 1-18 (2000)). Liposomes have been used to prevent oxidation and degradation of encapsulated drugs because of their lipophilic and mucosa-adhesive properties (Corace, G. et al., J. Liposome. Res. 24, 323-335 (2014)). The drug half-life of liposomes was found to be at least four times higher than nasal drugs administered intra-nasally (Jaafari, M. R. Iran. J. Pharm. Res. 4, 3-11 (2005)). Liposomes can be designed to have specific functions to enhance or release 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 of improving the safety of liposomes, and the incorporation of cholesterol into the phospholipid vesicles makes the lipid bilayer structure harder and more stable (Johnson, S. M. BBA-Biomembranes 307, 27-41 (1973)). The hydroxyl group of cholesterol interacts with the hydrophilic head of phospholipids, and its steroid backbone, which leads to the regulation of strength and the strength of lipid bilayers, interacts with hydrophobic phospholipid chains (Maria-Lucia, B. Drug. Deliv. Transl. Res. 5, 231-242 (2015)). For this reason, cholesterol increases solute retention and serum-induced stability inside liposome transporters. In addition, cholesterol accounts for 20-25% of brain cell membranes. Liposomes containing cholesterol have been reported to 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 route affects the physical stability and biological efficacy of liposomes.

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

Meanwhile, the inventor of the present invention has developed a pharmaceutical composition (KR 10-2017-0158383) for preventing or treating brain diseases, including liposomes carrying phycocyanin or phycoerythrin as an active ingredient, but the influence of phosphatidyl choline in its components. Because of the negative charge on the surface was difficult to administer nasal.

An object of the present invention is to provide a pharmaceutical composition for treating brain diseases, including liposomes and the like so that phycocyanin bypasses the blood-brain barrier and is effectively delivered into the brain through the nasal route.

In order to solve the above problems, the inventor of the present invention completed the present invention by preparing a liposome that can be effectively coated with sialic acid of the nasal mucosa is coated with chitosan on the outside. Therefore, the problem solving means of this invention is as follows.

1. chitosan coating; And phycocyanin supported therein; Liposomes comprising the.

2. A pharmaceutical composition for treating brain disease for nasal administration comprising liposomes of 1.

The chitosan coated on the surface of the liposome of the present invention binds strongly with sialic acid of the nasal mucosa, which is positively charged and negatively charged, thereby effectively delivering phycocyanin into the brain through the nasal route.

1 is a simplified view of the chitosan-coated liposome of the present invention.
Figure 2 shows a TEM image of liposomes coated with uncoated liposomes (A) and 0.1% concentration of chitosan prepared in Examples of the present invention.
Figure 3 shows the size and zeta potential of the uncoated liposomes prepared in the present invention, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes by Z-analyzer. Will be simplified.
Figure 4 shows the cytotoxicity measurement results of the uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes prepared in Examples of the present invention.
Figure 5 shows the results of measuring the antioxidant efficacy of the uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes prepared in Examples of the present invention.
6 is a simplified process of Experimental Example 3 of an embodiment of the present invention.
FIG. 7 is a photograph showing cerebral infarct size when uncoated liposomes prepared in Examples of the present invention, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes. .
FIG. 8 shows the results of confirming the cerebral infarct size of FIG.
FIG. 9 shows behavioral test results when mice were treated with uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes prepared in Examples of the present invention. will be.
10 is an expression of catalase when mice were treated with uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes prepared in Examples of the present invention. The measurement results are shown.
Figure 11 Measurement of the expression level of BDNF when the mice treated with uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes prepared in Examples of the present invention The results are shown.
12 shows three kinds of inflammatory factors when uncoated liposomes prepared in Examples of the present invention, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes to mice. TNF-α, IL-6, IL-1β) expression results are shown.
FIG. 13 shows phosphopacans and neurocanes when treated with uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes prepared in Examples of the present invention. The expression level measurement result of the is shown.

The present invention is a chitosan coating; And phycocyanin supported therein; It provides a liposome comprising a.

The form of liposomes supported with phycocyanin of the present invention and coated with chitosan is shown in FIG. 1. Picocyanine (C-Pc) is supported inside the liposome, the outside is surrounded by chitosan.

In the present invention, the "liposomes" refer to vesicles comprising at least one bilayer separating the inner aqueous phase from the outer aqueous environment.

In the present invention, the "phycocyanin" refers to a peptide compound including "phycocyanobilin" represented by Formula 1 below:

[Formula 1]

The phycocyanin is a blue pigment protein contained in algae, which is characterized by being bound to the protein by a peptide bond without containing a metal.

In the present invention, the liposomes include phospholipids, or phospholipids and sterols.

In the present invention, the phospholipid refers to a lipid including phosphorus. Phospholipids available include phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidic acid. , Preferably phosphatidyl choline.

In the present invention, the sterol is a kind of steroid, and refers to steroid alcohol. Sterols that may be used include cholesterol, coprostanol, cytosterol and ergosterol, preferably cholesterol.

The sterol is included in the liposome in an amount of 0 to 50% (w / w), but preferably in an amount of 10 to 40% (w / w), more preferably 20 to 33% (w / w). Included in content.

The size of the liposome is 80 to 150 nanometers (nanometer), preferably 80 to 130 nanometers, more preferably 90 to 110 nanometers.

In the present invention, the "chitosan" refers to a material obtained by deacetylating chitin. Chitosan allows the pharmaceutical composition of the present invention to bind well with sialic acid of the nasal mucosa and is biodegradable and suitable for use in pharmaceutical compositions. The chitosan may vary in molecular weight, but preferably 5 to 19 kDa. In the case of using low molecular weight chitosan, solubility is increased as compared with the use of high molecular weight chitosan, and the bactericidal effect against pathogenic bacteria is high.

The present invention provides a pharmaceutical composition for treating brain diseases for nasal administration comprising the liposomes.

In the present invention, the brain disease may be at least one selected from the group consisting of stroke, stroke, cerebral hemorrhage, cerebral infarction, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, lethargy and shock brain injury.

“Treatment” in the context of the present invention includes the cure of the disease as well as the partial cure, improvement and alleviation of the condition.

The pharmaceutical composition of the present invention is for nasal administration. Nasal refers to the space in the nasal cavity, which is separated by left and right by the nasal septum, and the pharmaceutical composition of the present invention, which is administered nasal, passes through the nasal mucosa and is delivered into the brain. At this time, the sialic acid group is present in the nasal mucosa has a negative charge, and the chitosan on the surface of the pharmaceutical composition of the present invention has a positive charge, so that the pharmaceutical composition of the present invention can be effectively combined with the nasal mucosa. Accordingly, the pharmaceutical composition of the present invention can be efficiently delivered to the brain.

The pharmaceutical compositions of the present invention may further comprise conventional carriers, excipients and / or diluents suitable for formulation for nasal administration.

The pharmaceutical compositions of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers. The dosage of the pharmaceutical composition of the present invention may vary depending on the body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, and those skilled in the art It may be administered at a dosage that is contemplated.

Example

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example-Preparation of Liposomes Coated with Chitosan of the Present Invention

Phosphatidylcholine and 0.01 mmol of cholesterol were dissolved in 1 ml of chloroform and vacuum-treated for 3 hours to form a film. Thereafter, 20 μg / ml of phycocyanin was added and hydrated through a pipette, followed by freeze-thawing for 10 minutes at room temperature with a deepfreezer. The resultant was passed through a membrane of 100 nm pore size 30 times using an extruder to complete a liposome of a constant size. Separately, the solution was prepared by dissolving it in acetic acid so that the concentration of chitosan was 0.001, 0.01, 0.1 and 0.5%, and then dropping it in a liposome suspension for 1 hour while stirring to obtain four liposomes coated with different concentrations of chitosan. It was. Uncoated liposomes were also prepared for use as controls.

Experimental Example 1. Characterization of the prepared chitosan coated liposomes

The morphology of the prepared chitosan coated liposomes was confirmed by TEM image. TEM images of uncoated liposomes and liposomes coated with 0.1% chitosan are shown as A and B of FIG. 2, respectively. The scale bar in FIG. 2 is 50 nm. As a result of negative staining with uranyl acetate, a white layer was found in B of FIG. 2, indicating that the liposome was coated with positive chitosan.

The size and zeta potential of the uncoated liposomes, 0.001% chitosan coated liposomes, 0.01% chitosan coated liposomes, 0.1% chitosan coated liposomes and 0.5% chitosan coated liposomes were confirmed by Z-analyzer, which is shown in FIG. 3. From the size measurement results, it was confirmed that the liposomes of about 90 nm increased in size due to the coating of chitosan, and from the result of the zeta potential measurement, the charge changed positively as the chitosan concentration was increased.

Experimental Example 2. Confirmation of cytotoxicity and antioxidant efficacy of the prepared chitosan coated liposomes

The cytotoxicity and antioxidant efficacy of the prepared liposomes were confirmed through in vitro experiments. Neurons, astrocytes (astrocytes) were used, and cells were directly cultured from 1 day old rats. Culture conditions were 3 × 10 ⁴ cells / well (24 well plates), 5% CO ₂ incubator and 37 ° C., and the medium was DMEM-High glucose (89%), fetal bovine serum (10%) and penicillin-streptomycin (1%) was used.

4 shows the results of confirming the cytotoxicity according to the concentration of the prepared liposomes. From this it was confirmed that the liposome of the present invention does not have cytotoxicity and is suitable for use as a pharmaceutical composition. 5 shows the results of confirming the antioxidant efficacy according to the concentration of the prepared liposomes. In order to confirm the antioxidant efficacy, the oxidative stress condition was set with hydrogen peroxide, and cell activity was measured under the oxidative stress condition. While the cell activity was decreased by hydrogen peroxide treatment in the control group not treated with liposomes, the cells pre-treated with liposomes were shown to have less damage, and from this, it was confirmed that the liposomes of the present invention had oxidative efficacy.

Experimental Example 3. Confirmation of the brain disease treatment effect of the prepared chitosan coated liposome

The MCAO model was developed to describe the local ischemic condition in stroke and 8-week-old rat reperfusion after thrombolytic dissolution. MCAO model is to block the arterial blood to the brain for 1 hour, then to damage the brain tissue by releasing, 6 hours after surgery was administered the liposome of the present invention. Two days after the liposome treatment, the degree of nerve recovery was confirmed by evaluation of cerebral infarct size through behavioral test (mNSS test) and TTC (triphenyltetraazolium chloride) staining. Raise), inflammatory factors (TNF-α, IL-6, IL-1β) neurotrophic factor (BDNF) and Glia scar (neurocans, phosphacans) by checking the expression level of each neuroprotective effect was confirmed. Evaluation of cerebral infarction size was performed by extracting the brains of mice, sliced at 2 mm intervals, and stained using TTC reagent. This experimental process is simplified and shown in FIG. 6.

The photo of the change in cerebral infarct size according to the experiment is shown in FIG. 7, and the specific infarct volume change is shown in the graph of FIG. 8. From this, it was confirmed that the chitosan coating of the present invention helped to increase the pharmacological effect of liposomes. Behavior test results according to the experiment is shown in FIG.

The result of confirming the expression level of catalase by recovering the brain tissue is shown in FIG. 10, and the result of confirming the expression level of BDNF is shown in FIG. It was confirmed that the chitosan-coated liposomes of the present invention have antioxidative effect and thus have low expression of antioxidant enzymes, and have an effect of restoring damage after cerebral ischemia and reperfusion. Results of confirming the inflammatory factor expression of the brain tissue is shown in Figure 12. In all three types of inflammatory factors, the expression level was reduced compared to the control group, and it was confirmed that the chitosan-coated liposome of the present invention had an anti-inflammatory effect. The result of measuring the expression level of glia scar is shown in FIG. It was confirmed that the value was increased in the liposome coated with chitosan, from which the activation of scar formation factor proceeds smoothly after brain injury, and it can be seen that the therapeutic effect is higher.

Claims (7)

Chitosan coatings; And phycocyanin supported therein; Liposomes comprising the. The liposome of claim 1, wherein the liposome comprises phospholipids or phospholipids and sterols. The method of claim 2, wherein the phospholipid is phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol, and phosphatidic acid. liposomes selected from the group consisting of acid). The liposome according to claim 2, wherein the sterol is at least one selected from the group consisting of cholesterol, coprostanol, cytosterol and ergosterol. The liposome of claim 1, wherein the chitosan has a molecular weight of 5 to 19 kDa. A pharmaceutical composition for treating cerebral disease for nasal administration comprising the liposome of any one of claims 1 to 5. According to claim 6, wherein the brain disease is stroke or stroke, cerebral hemorrhage, cerebral infarction, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury is at least one selected from the group consisting of Pharmaceutical composition for treating brain disease.

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