KR20170027684A - Combination composition comprising choline alfoscerate, and memantine - Google Patents

Abstract

The present invention relates to a composition for treating or ameliorating cognitive impairments, which is useful for treating cerebropathia and contains choline alfoscerate and memantine as active ingredients. The combination of two types of anti-dementia drugs including choline alfoscerate and memantine shows high therapeutic effects on dementia, and drug compliance increases by reducing the number or an amount of doses.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition comprising choline alfoscerate and memantine,

The present invention relates to a composite composition containing choline alfoscerate and memantine, which is useful for the treatment of brain diseases.

Recently, dementia such as senile dementia and Alzheimer's disease has become a serious social problem. Geriatric dementia is also commonly used to refer to Alzheimer's disease. Alzheimer's disease is likely to affect older people (20% of all people over 80 years of age suffering from Alzheimer's disease).

Alzheimer's disease (AD) is an irreversible progressive disorder in which brain cells (neurons) are destroyed and cause loss of cognitive function, mainly memory, judgment and reasoning, control of motor function and pattern recognition. At the advanced stages of the disease, all memory and mental functions can be lost. People with Alzheimer's disease have problems with memory, judgment, and thinking, and they do not allow people to work or do everyday activities. Neuronal death occurs gradually over a period of years. This is associated with senile dementia, which is a psychological devastation associated with old age (a loss of intellectual ability). Two major types of senile dementia have been identified by systemic dystrophy (type of Alzheimer's) and vascular problems (mainly stroke).

Currently, there is no cure for Alzheimer's disease, but there are drugs that offer the benefit of symptoms.

Among them, L-α-glycerylphosphorylcholine (GPC), which is known as choline alfoscerate, has a structure represented by the following general formula (1), and a semisynthetic derivative of lecithin As a precursor of cholinergic system, it is effective for improvement of perception ability and improvement and treatment of brain function. In particular, choline alfoscerate is a drug used in the treatment of secondary symptoms and degenerative or degenerative brain syndromes due to cerebrovascular damage, including senile cognitive impairment such as memory loss, confusion, disorientation, and impaired concentration; Feelings and behavioral changes such as emotional anxiety, irritability, and irritability; It is known to be useful for senile depression.

Choline alfoscerate acts directly on damaged brain cells through 45% of the BBB (Blood Brain Barrier) according to high bioavailability and separates into choline and glycerophosphate in the body, which not only improves neurotransmission but also helps repair damaged cell membrane structure . In addition, it is a precursor of biomaterials and 85% is excreted in carbon dioxide.

Although choline alfoscerate is an important compound for the production of new acetylcholine in the brain, it is naturally present in all cells of the body, but decreases as the aging progresses, resulting in decreased perception, vascular dementia, neurodegeneration in Alzheimer's disease And the like.

Also, memantine has been a very long-approved drug. Initially (in 1978), memantine went on the market in Germany for the treatment of Parkinson's disease, stiffness and other neurological disorders. Memantine then blocks N-methyl-D-aspartate (NMDA) receptors in a non-competitive manner (Bormann, Eur. J. Pharmacol. 166: 591-592 1989) Have been shown to be effective in preventing cognitive and histological damage in preclinical models of Alzheimer's disease (Parsons et al. Neuropharmacology 38: 735-767 1999); Rammes et al. Curr. Neuropharmacol. 6: 55-78 2008). Its efficacy has also been demonstrated in clinical trials of vascular dementia and Alzheimer's disease (Raina et al. Ann. Intern. Med. 148: 379-397 2008). Currently, memantine is an approved and widely used drug for the treatment of Alzheimer's disease. Its usual therapeutic dose is 20 mg / day in clinical practice and this should only reach a gradual dose-elevation. Its therapeutic window is narrow and typical side effects for NMDA antagonists, such as anxiety, chaos, or more severe illness may occur in the case of too fast a dose-up or higher dose of administration. Nonetheless, side effects of memantine are rare when administered according to the recommended dosing regimen: anxiety (1.3%), nausea (0.9%), dizziness (0.8%), fatigue (0.4% Drugs of Today 40: 685-695 2004). In animal studies it is difficult to determine which dose corresponds to the human therapeutic dose. However, the range of effective anti-Alzheimer's doses of memantine in mice can be estimated to be 5 to 30 mg / kg / day by oral administration based on plasma concentration data or cognitive and neurological effects in animal models of Alzheimer's disease (Dong et al. Neuropsychopharmacology 33: 3226-3236 2008); Minkeviciene et al. J Pharmacol. Exp. Ther. 31: 677-682 2004); Rammes et al. Curr. Neuropharmacol. 6: 55-78 2008).

In recent years, it has been attempted to use two or more agents together for the treatment of dementia of a better severity. When memantine was given to moderate to severe AD patients receiving donepezil, the AD symptoms were unexpectedly more relaxed compared to AD patients who received placebo. That is, patients with moderate to severe Alzheimer type dementia who had been administered hydrochloric acid donepezil were further administered with methanthine hydrochloride or placebo by the blind test method, followed by administration of donepezil hydrochloride and mennitine hydrochloride It was reported that the cognitive ability and the daily life action were improved in the administration group as compared with the placebo administration group (Non-Patent Document 1: Non-Patent Document 1 is hereby incorporated by reference in its entirety). In addition, the concept of a formulation containing an acetylcholinesterase inhibitor and an NMDA receptor antagonist has been disclosed (Patent Documents 1 and 2; Patent Documents 1 and 2 thus disclose that, as a background art of this specification, do).

However, when a single commercially available product is used in combination with two or more medicines, problems such as adherence to medicines and the burden of caregivers arise. In other words, most of the Alzheimer type dementia patients suffer not only the loss of cognitive ability but also the difficulty of swallowing, which has a great influence on the patient's own compliance with the medication, and the care burden of the caregiver. For example, one dose of donepezil hydrochloride should be administered once a day, and then mennitine hydrochloride should be administered twice a day, once per tablet. Therefore, the dosage adherence .

Accordingly, it is thought that it is effective to prepare a combined preparation for compliance with medicines and to reduce the nursing burden. However, in the case of a combination preparation, since each of the main components has different solubilities or pKa, It may be very difficult to control the release of the drug simultaneously. In particular, it is well known in the art that the mechanism of formation of the polymer matrix depends on a number of process parameters that directly affect drug release characteristics, since choline alfoscerate and memantine are water soluble and permeable , There is a very great concern that such an arbitrary deformation in matrix formation will almost lead to deformation of both released drug and absorbed drug.

WO 03/101457 A1 US Publication 2004/0087658 A

 Pierre N. Tariot et al., &Quot; Memantine Treatment in Patients with Moderate to Severe Alzheimer Disease Already Receiving Donepezil a Randomized Controlled Trial ", JAMA, Vol. 291, No.3, p. 317-324

It is an object of the present invention to provide a composition having high therapeutic effect on dementia by using choline alfoscerate and memantine in combination with two anti-dementia drugs.

It is another object of the present invention to provide a composition and a preparation method thereof which can improve the dosage adherence by reducing the number of administrations and dosages.

It is still another object of the present invention to provide a method of solving problems such as precipitation and the like caused when mixing solid mannitol with choline alfoscerate in liquid form and solving the problems of uniformity of contents and difficulties in the manufacturing process of choline alfoscerate and memantine And to provide a method for producing two kinds of composite composition.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art,

Choline alfoscerate, and memantine as an active ingredient.

The present invention also provides a composition for treating or improving a cognitive dysfunction, which further comprises a plasticizer.

In the present invention, the plasticizer may be at least one selected from the group consisting of acetyl tributyl citrate, acetyl triethyl citrate, benzyl benzoate, chlorobutanol, dibutyl phthalate, dibutyl sebacate, non-ethyl phthalate, dimethyl phthalate, glycerin, mannitol, Characterized in that it is at least one member selected from the group consisting of an oil, a lanolin alcohol, a proprololum lanolin alcohol, a polypropylene glycol, propylene glycol, polyvinylpyrrolidone, sorbitol, triacetin, tributyl citrate, A composition for treating or improving cognitive dysfunction.

Further, in the present invention, the plasticizer is concentrated glycerin, and provides a composition for treating or improving a cognitive dysfunction.

In the present invention, the plasticizer is contained in an amount of 1 to 20% by weight based on the total weight of the composition.

Further, in the present invention, there is provided a composition for treating or improving cognitive dysfunction, wherein the formulation is a capsule.

Further, the present invention provides a method for preparing a composition for treating or improving cognitive dysfunction, which comprises dissolving choline alfoscerate and memantine in one or more plasticizers selected from propylene glycol and glycerin.

The present invention has an effect of improving not only the therapeutic effect on dementia but also the administration frequency and dose of the dementia drug of choline alfoselate and memantine to improve the adherence of medication.

Fig. 1 is a photograph of a water maze in which a cue used in a probe test is installed.
FIG. 2 is a diagram for setting each range for inputting the area values of Arena, Quadrants, and Platform in the Ethovision XT program to record the moving path and distance of the laboratory animal in the probe test.
3 is a graph showing statistical results of the moving speed of an experimental animal measured during a probe test.
FIG. 4 is a graph showing statistical results of an experimental animal crossing (platform crossing) measured in a probe test.
FIG. 5 is a graph showing the results of time in target quadrand of experimental animals measured during probe test.
6 is a graph showing the path of the experimental animals moved in the water tank for 60 seconds during the probe test.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for treating or improving cognitive dysfunction, which comprises choline alfoscerate and memantine as an active ingredient.

Choline alfoscerate, and memantine as an active ingredient is a novel combination that is not conventionally known, as the inventors have learned. That is, the inventors of the present invention have found that when choline alfoscerate and memantine, which are known as anti-dementia drugs, are administered separately, when administered in combination, synergy effect And the present invention has been accomplished. In addition, choline alfoscerate is a precursor of biomaterial and has an advantage free from side effects.

Further, the composition of the present invention is characterized by further comprising a plasticizer for the pharmaceutical aspect and dissolution characteristics.

Choline alfoscerate as an active ingredient is water-soluble but has a very high viscosity, so that it is difficult to mix the composition and it is very difficult to ensure homogeneity. In addition, even if the dispersion is formed well at an early stage, it tends to precipitate with a specific gravity over time, and this tendency is a factor that hinders elution characteristics and storage stability at a later time. Therefore, in order to prevent such tendency in advance not only from the pharmaceutical point of view but also from the viewpoint of the effect to prevent the homogeneity of the content and the reduction of the therapeutic effect by mixing and completely dissolving the two components which are not dispersed, .

The plasticizer is preferably contained within a range of 1 to 20% by weight based on the total weight of the composition. If it is less than 1% by weight, it may be difficult to achieve the intended purpose. If it is more than 20% by weight, not only an undesirable influence on the elution characteristics, but also a large amount of the active ingredient, There is a possibility that instability of the preparation itself may be caused.

The plasticizer that can be used in the present invention is not particularly limited, and any of the pharmaceutical plasticizers known to those skilled in the art can be used. Non-limiting examples thereof include acetyltributyl citrate, acetyltriethyl citrate, benzyl benzoate, chlorobutanol, dibutyl phthalate, dibutyl sebacate, nonethyl phthalate, dimethyl phthalate, glycerin, mannitol, mineral oil lanolin alcohol At least one selected from the group consisting of propylene glycol, propanol, lanolin alcohol, propolol lanolin alcohol, polypropylene glycol, propylene glycol, polyvinylpyrrolidone, sorbitol, triacetin, tributyl citrate, and triethyl citrate, , And / or propylene glycol is preferable from the viewpoint of pharmaceutical properties or dissolution characteristics. When these plasticizers are used, the main component completely dissolves and shows a clear liquid phase.

Hereinafter, the present invention will be described in more detail by way of examples. It is to be understood that the following embodiments are for the purpose of illustration only and are not intended to limit the scope of the present invention.

Example

After mixing 400 g of choline alfoscerate and 25 g of purified water, the mixture is heated with warm water. Approximately 50 g of a plasticizer was added thereto and stirred. Then, 10 g of memantine hydrochloride was added to prepare a composition in which the active ingredient was dissolved. As shown in Table 1 below, the plasticizer was varied.

Plasticizer Dissolution Flowability Appearance Example 1 Concentrated glycerin O Ha Transparent state Example 2 PG O Ha Transparent state Example 3 PEG400 X - Memantine hydrochloride does not dissolve,
Oil remains floating on the surface Example 4 Sorbitol O Ha It does not melt quickly in early days, Example 5 Concentrated glycerin (45 g)
+
Water (remaining amount) O Prize Transparently soluble in light yellow Example 6 Concentrated glycerin (45 g)
+
Aqueous sodium docusate solution (balance) O medium Dissolve in dark yellow Example 7 PG (45 g)
+
Water (remaining amount) O Prize Almost colorless and transparent Example 8 PG (45 g)
+
Aqueous sodium docusate solution (balance) O medium Little color but slightly opaque Example 9 PEG400 (45 g)
+
Water (remaining amount) O medium Dissolve in dark yellow Example 10 PEG400 (45 g)
+
Aqueous sodium docusate solution (balance) O Ha The most opaque dissolution Example 11 Sorbitol (45 g)
+
Water (remaining amount) O Prize Most transparent and most flowable Example 12 Sorbitol (45 g)
+
Aqueous sodium docusate solution (balance) O medium Slightly opaque compared to adding water * PG: Propylene glycol
* PEG400: PEG-400
* Tokseite sodium aqueous solution: Dissolve 4.8 g of docusate sodium in 50 g of water.

The solubility of choline in memantine hydrochloride was the most favorable, and the sorbentol> glycerin> PG in order to compare the flow rate and dissolution rate of plasticizer and water. In addition, when water was added, the dissolution rate of memantine hydrochloride was fast and the flowability was improved, so that the leakage and defective rate of capsules could be lowered when the soft capsule was filled.

For statistical observation, three of the best prescriptions among the prescriptions of Table 1 were separately prepared and observed for 4 days in sunlight to confirm stability. In the experimental method, the content of petridish was contained and visual properties of browning degree were visually observed.

Prescription Initial state After 1 day Two days later Final Characteristics Main + sorbitol Transparent contents + ++ Light brown Week + glycerin Transparent contents + + Transparent contents Main + propylene glycol Transparent contents + + Transparent contents Note: Choline alfoscerate + memantine + water
+: Good
++: light brown
+++: dark brown

As shown in Table 2, there was a change in properties during exposure to sunlight, and it was found that propylene glycol was used as a plasticizer in stabilizing the formulation.

Example  13

Soft capsules containing choline alfoscerate and memantine hydrochloride were prepared as described in Table 3 below.

Example  14

A soft capsule was prepared in the same manner as in Example 13, except that sorbitol was used in addition to concentrated glycerin as a plasticizer (excipient).

Example  15

Soft capsules were prepared in the same manner as in Example 13, except that PG (propylene glycol) was used in addition to concentrated glycerin as a plasticizer (excipient).

Comparative Example

Comparative Example  One

After mixing 400 g of choline alfoscerate and 25 g of purified water, the mixture is heated with warm water. 50 g of concentrated glycerin as a plasticizer was added and stirred to prepare a composition.

Comparative Example  2

25 g of purified water with hot water and 50 g of concentrated glycerin were added and stirred and mixed with 10 g of memantine hydrochloride, followed by stirring to prepare a composition.

Comparative Example  3

25 g of purified water and 50 g of concentrated glycerin were added and stirred to prepare a composition.

Experimental Example

Cytotoxicity experiment - MTT  analysis

Experimental group: Each of Example 5 and Comparative Examples 1 to 3 was dissolved in PBS buffer (pH 7.4) so as to have a concentration of 10 mM

* Amyloid beta protein: The amyloid beta protein to be processed on the outside of the cell is purchased from U.S. Peptide Inc., Aβ1-42 protein.

Cell culture: PC12 cells derived from rat pheochromocytoma were selected for the neuronal cell model. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotic. NGF (nerve growth factor; Sigma) of 50 ng / ) Medium to differentiate into neurons by replacing the medium.

For the MTT assay, it was added to 30μM of Aβ _{1 -42} in the cultured cells and cultured in a 5% CO _2, conditions of 37 ℃. After 24 hours, a solution of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) was added to each well to a final concentration of 0.5 mg / Lt; / RTI > The formazan precipitate formed by the reduction of MTT was dissolved in dimethyl sulfoxide (DMSO), and the absorbance at 570 nm was measured using an ELISA instrument. The cytotoxicity of each sample was calculated as a relative value with the absorbance measured by the solvent alone as 100% and the absorbance measured with the 0.9% Triton X-100 in the completely destroyed cell as 0%. In this case, the lower the degree of MTT reduction, the lower the cell activity. The control group was prepared by adding 30 [mu] M of A [beta] _{i- 42} to the solvent.

Cell viability of each group was obtained by measuring the degree of MTT reduction as described above, and the results are shown in Table 4.

cell viability (%) Control group 82 Example 5 94 Comparative Example 1 85 Comparative Example 2 84 Comparative Example 3 82

Compared with the control group, the cell activity recovery effect of Example 5 and Comparative Examples 1 and 2 was confirmed, and the recovery effect of Example 5 was the most remarkable.

Elution characteristics evaluation

In order to evaluate the dissolution characteristics, Examples 13 to 15 were used as a control. As a control group of glycyrrhizin soft capsules (TM) (400 mg) (manufactured by Daewoong Pharmaceutical Co., Ltd.) and as a control group of memantine hydrochloride (Manufactured by Lundbeck, Korea) was used.

* Elution experiment condition

- Test apparatus: second method, paddle method

- Eluent: Water (purified water)

- Leaching temperature: 37 ℃

- Rotation speed of paddle: 50rpm

- Detector: Exothermic absorptiometer (measuring wavelength: 270 nm)

- Amount of Test Solution: 500 ml

The results were as shown in Table 5 (choline alfoselate elution) and Table 6 (memantine hydrochloride elution).

5 minutes 10 minutes 15 minutes Control group 3.5 ± 0.6 39.8 ± 11.8 96.2 ± 5.5 Example 13 0 ± 0 39.7 ± 8 101.2 ± 8 Example 14 0 ± 0 37.8 ± 0 101.2 ± 5.9 Example 15 0.5 ± 1 45.9 ± 11.6 100.9 ± 0.5

5 minutes 10 minutes 15 minutes 30 minutes Control group 16.9 ± 29.2 54.4 ± 4.2 58.4 ± 2.8 61.4 ± 1.1 Example 13 0 ± 0 42.5 ± 10.8 67.3 ± 4.2 81.9 ± 4.1 Example 14 0 ± 0 50.1 ± 6.3 66.7 ± 8.9 83.7 ± 7.2 Example 15 0 ± 0 43.2 ± 9.7 71.5 ± 6.1 91.1 ± 1.3

As shown in Tables 5 and 6, it was confirmed that the respective components were smoothly eluted well.

Evaluation of simple memory ability by measuring passive avoidance performance

- Preparation of experimental animals: ICR male mice weighing 18-20 g were used. In the breeding room, 24 ± 1 ° C ventilation was activated and the intensity of light was adjusted by 12 hours (6:00 - 18:00). During the breeding, water and feed were freely consumed. The above-prepared Example 5 and Comparative Examples 1 to 3 were used as a test solution, and physiological saline was used as a reference solution. The test solution was administered to the experimental group and the control solution was orally administered once a day for 1 week.

Through passive avoidance response measurements, mice with or without administration of the composition of the present invention were studied and their simple memory ability after dementia induction was evaluated.

① Experimental apparatus: Measured using manual avoidance experiment. The device is divided into two compartments, with a partition with a guillotine door in the middle. One of the two compartments is illuminated by a light of intensity 10 and the other is shaded to darken. The bottom consists of a grid that can give an electric shock.

② Study: After 30 minutes of the last drug administration, ethanol is administered. After 1 hour of ethanol administration, the mice are placed on the illuminated side and the guillotine door is opened to enter the dark compartment after 30 seconds of searching time. Rats prefer dark places, so they immediately go dark. Exclude rats that do not enter the darkness within 120 seconds of opening the guillotine door. Measure the time until the guillotine door opens and the mouse enters the dark side. Once the rat has entered the dark side, the gyrotinous door closes and a current of 0.6 mA flows through the grid floor for 5 seconds (learning trial).

③ Memory test: After 24 hours, the test is performed in the same way as the study. In this case, a latency of 300 seconds after the 30-second seek time and a dark time is measured, which indicates that the longer the learning and memory of the passive avoidance is, the testing trial.

Table 7 shows the results of the memory-enhancing effect test.

Reach time (s) Control group 162 Example 5 37 Comparative Example 1 100 Comparative Example 2 95 Comparative Example 3 152

As shown in Table 7, the effect of Example 5 on the short-term memory improvement was higher than that of the control or Comparative Example 3, and particularly, it was superior to Comparative Examples 1 and 2, The synergistic effect of choline alfoscerate and memantine was confirmed.

Water maze  Assessment of cognitive impairment animal model memory ability

After the drug was administered as shown in Table 8 below, animal model memory ability test evaluation was carried out.

1) Preparation stage

<Establishment of water tanks for environment improvement of water tank maze>

 We set up a water tank environment to be used for probe test.

· The height of the water tank was such that the height of the water level in the water tank was such that the leg of the animal could not reach the bottom of the water tank, Using paint, the paint was released so that the bottom of the water tank could not be seen.

As shown in FIG. 1, a marker in the shape of a figure is installed on the four sides (East, West, North, South) of the water tank and the experimental animal memorizes the marker installed in the water tank, Always installed in the same location.

· The indoor lighting was shielded to prevent water from reflecting on the water surface in the water tank.

· The noise around the water tank was removed.

<Water adaptation training>

Prior to the probe test, water adaptation training of the experimental animals was carried out as follows.

· Two times a day, water adaptation training was conducted after 6 hours of water adaptation training in the morning.

· Being careful not to let the nose of the test animal enter the water first, the leg was put in the state of looking at the wall side of the tank, and water adaptation training was performed for 60 seconds.

After 60 seconds, the animals were taken out of the water tank, the water was wiped off with a dry towel, and the training was completed by placing in a cage.

&Lt; Training of location information of escape pods, learning, and adaptation training for escape pods &

Prior to the probe test, training on adaptive training was performed on the location information of the esophagus of the experimental animals, learning, and the escape zone as described below.

· A 15 cm diameter cylinder was used as an escape pod, and the height of the water surface was set 1 cm lower than the height of the escape pod so that the experimental animals could see the esophagus sufficiently during swimming.

• The location of the escape pods is determined by the location of the south-west (SW), north-west (NW), north-east (NE), center (C), south- Respectively.

• The release position of the experimental animal is changed to the south (S), north (N), south (S), east (E), west Respectively.

· Three hours and 30 minutes before the training for the location information, learning and escape of the escape area, 50 μl of saline was injected intraperitoneally in the control group and 50 μl (1 mg / kg) scopolamine hydrobromide (Sigma Aldrich) was intraperitoneally injected. After 30 minutes, 100 μl of saline was orally administered to the control and experimental group 1, and the experimental groups 2 to 4 were orally administered with choline alfoscerate or memantine, a dementia treatment drug, alone or in combination.

· When the animal was released into the water, the esophagus was turned back and put into the water tank while looking at the wall.

· An experimental animal climbed up to the esophagus within 60 seconds and stayed on for more than 5 seconds, and the experiment was finished and it was trained to learn and memorize the surrounding markers and environment for 30 seconds.

· If the animal can not find the escape pod within 60 seconds, it can be traced to the esophagus by using the hand, and then it is trained to learn and memorize the surrounding markers and environment for 30 seconds on the esophagus.

After the training was completed, the animals were taken out of the water tank, the water was wiped off with a dry towel, and the training was completed by placing it in a cage.

<Training to find refugees by markers>

The esophagus was placed below the southwest (SW) surface, and the animals were not trained to visit the esophagus, but were trained to visit the esophagus for 5 days.

During the 5 - day training, the position of the escape pod is always the same in the SW position.

In the 5-day training, 50 μl of saline was intraperitoneally injected into the control group 3 hours and 30 minutes before each test, and 50 μl (1 mg / kg) of scopolamine hydrobromide (Sigma Aldrich) Intraperitoneal injection. After 30 minutes, 100 μl of saline was orally administered to the control and experimental group 1, and the experimental groups 2 to 4 were orally administered with choline alfoscerate or memantine, a dementia treatment drug, alone or in combination.

· When the animal was released into the water, the esophagus was turned back and put into the water tank while looking at the wall.

· An experimental animal climbed up to the esophagus within 60 seconds and stayed on for more than 5 seconds, and the experiment was finished and it was trained to learn and memorize the surrounding markers and environment for 30 seconds.

· If the animal can not find the escape pod within 60 seconds, it is guided to the esophagus using the hand, and then it is trained to learn and memorize the surrounding markers and environment for 30 seconds on the esophagus.

2) Probe test

Then, using the Ethovision XT (Noldus, Netherlands) with the escape rod removed in the water tank, the movement route and distance of the animal were recorded, and the movement speed, the platform crossing and the time in target quadrant The results were evaluated to evaluate the learning ability and spatial memory ability of experimental animals.

As shown in Fig. 2, the arena range in the water tank maze is the total area of the water surface, and the range of the quadrants is a total of four zones in which the arenas are divided into fan-shaped areas. The area of each area, the range of the platform, and the measured area values of these were entered into the Ethovision XT (Noldus, Netherlands) program.

· Three hours and 30 minutes before the probe test, 50 μl of saline was injected intraperitoneally in the control group and 50 μl (1 mg / kg) of scopolamine was injected intraperitoneally in the experimental groups 1 to 4. After 30 minutes, 100 μl of saline was orally administered to the control and experimental group 1, and the experimental groups 2 to 4 were orally administered with choline alfoscerate or memantine, a dementia treatment drug, alone or in combination.

The release positions of the experimental animals were changed in the order of North (N), East (E), South-east (SE), and North-west (NW). When the animal was released into the water, the position where the escape pod was located was turned back and the wall inside the tank was looked at.

After 60 seconds and the experiment was finished, the animals were taken out of the water tank, the water was wiped off with a dry towel, and the experiment was completed in a cage.

① Movement speed measurement result

- Control: 18.39 +/- 2.56 cm / s

- Experimental group 1: 16.80 + - 2.61 cm / s

- Experimental group 2: 20.04 + - 2.80 cm / s

- Experimental group 3: 19.99 + - 2.62 cm / s

- Experimental group 4: 20.23 + - 2.43 cm / s

FIG. 3 is a graph showing the results of measuring the velocity (cm / s) of the control group and the experimental group measured by the probe test.

As a result of measuring the speed of the control group and the experimental group, there was no significant difference (p <0.05) between the speeds of the groups, and no difference in mobility was observed between the control group and each test group.

② Result of platform crossing measurement

- Control group: 5.2 ± 1.4 times

- Experimental group 1: 1.2 ± 1.09 times

- Experimental group 2: 4.8 ± 1.92 times

- Experimental group 3: 3.75 ± 1.89 times

- Experimental group 4: 5 ± 1.58 times

 FIG. 4 is a graph showing the results of measuring the number of times that an experimental animal passed through a place where the esophagus was installed for 60 seconds during the probe test.

The control group passed a total of 5.2 ± 1.4 times for 60 seconds during the probe test and passed the most place where the esophagus was installed compared to the other test groups.

Experimental group 1 had the lowest of 1.2 ± 1.09 times among all experimental groups for 60 seconds and showed a significant difference (p <0.01) from the control group.

There were statistically significant differences (p <0.05) in the number of platform crossings between experimental group 1 and experimental group 2 ~ 4. Further, when the choline alfoscerate and memantine are co-administered, the measurement result closest to the control group can be confirmed.

③ Time in target quadrant measurement result

- Control group: 38.61 + - 22.08%

- Experimental group 1: 18.22 + - 11.05%

- Experimental group 2: 25.37 ± 7.27%

- Experimental group 3: 29.96 ± 15.04%

- Experimental group 4: 34.90 + - 5.42%

FIG. 5 shows the result of measuring the time spent in the SW quadrant in which the experimental animals were installed for 60 seconds during the probe test.

The control group remained at 38.61 ± 22.08% in the south-west (SW) quadrant among the four quadrants and remained in the SW zone for the longest period of time compared to the other quadrants.

Experimental group 1 remained in the SW zone for only 18.22 ± 11.05% of the 60 seconds of the probe test, and remained in the SW zone for the shortest time compared to the other quadrants.

The time spent in the SW quadrant of the experimental group 2 was not significantly different from that of the experimental group 1 (p> 0.05).

There was no significant difference (p> 0.05) between group 1 and group 1 in SW quadrant.

Experimental group 4 remained in the SW zone for 34.90 ± 5.42% of the 60 seconds of the probe test, and there was a significant difference (p <0.05) from the experimental group 1.

In addition, the time in target quadrant of the experimental group 4 was significantly different (p <0.05) compared to the experimental group 2.

FIG. 6 shows the results of the probe test in which the experimental animals in each experimental group showed the movement path in the water bath for 60 seconds.

The control group moved in a straight direction toward the escape platform immediately after entering the water tank, and it was found that there was the most movement in the SW quadrant zone where the escape platform was located.

Experimental group 1 showed no swinging behavior along the wall of the tank without showing any particular directionality.

In experiment group 2 ~ 4, it was found that most of the movement routes appeared around the SW quadrant where the escape zone was located, unlike experiment group 1, and it was confirmed that the escape zone was moved repeatedly to the place where the escape zone was located and the central part of the tank.

As noted above, the ability of cognitive impairment-induced animal models of cognitive impairment induced by cognitive impairment and the effects of coincidental administration of choline alfoscerate, memantine, or choline alfoscerate and memantine, Through the probe test using a water maze measuring ability, the platform crossing measurement results showed the results similar to those of the control group when concurrent administration of choline alfoscerate or memantine was individually administered I could confirm.

In addition, in the time in target quadrant measurement, significant improvement was observed in the spatial memory ability and the learning ability only in the group administered with choline alfoscerate and memantine (experimental group 4). Particularly, statistically significant differences were observed between the choline alfoscerate-only group and the choline alfoscerate and memantine-co-administered groups, so that choline alfoscerate or memantine alone And memantine were shown to have the most significant effect of improving spatial perception and learning ability without any adverse effects (the phenomenon of abnormal behavior, insomnia, diarrhea, dietary disturbance, etc. was observed throughout the entire experiment) ).

Claims (7)

Choline alfoscerate, and memantine as an active ingredient. The composition for treating or improving cognitive dysfunction according to claim 1, further comprising a plasticizer. The method of claim 2, wherein the plasticizer is selected from the group consisting of acetyl tributyl citrate, acetyl triethyl citrate, benzyl benzoate, chlorobutanol, dibutyl phthalate, dibutyl sebacate, non-ethyl phthalate, dimethyl phthalate, glycerin, mannitol, Characterized in that it is at least one selected from the group consisting of lanolin alcohol, proprolatum lanolin alcohol, polypropylene glycol, propylene glycol, polyvinylpyrrolidone, sorbitol, triacetin, tributyl citrate, and triethyl citrate A composition for treating or ameliorating dysfunction. The composition for treating or improving cognitive dysfunction according to claim 2, wherein the plasticizer is propylene glycol and / or glycerin. [Claim 2] The composition for treating or improving cognitive dysfunction according to claim 2, wherein the plasticizer is contained within a range of 1 to 20% by weight based on the total weight of the composition. The composition according to claim 1, wherein the formulation is a capsule. Choline alfoscerate, and memantine in one or more plasticizers selected from propylene glycol and glycerin. &Lt; Desc / Clms Page number 20 &gt;

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