WO2022010317A1 - Method for preparing donepezil-containing sustained-release plga microspheres - Google Patents

Abstract

The present invention relates to a method for preparing donepezil-containing sustained-release PLGA microspheres and, more specifically, to a method for preparing donepezil-containing sustained-release PLGA microspheres comprising the steps of: i) preparing microsphere droplets by injecting a dispersed phase comprising donepezil or a pharmaceutically acceptable salt thereof, poly(lactic-co-glycolic acid) (PLGA), and an organic solvent into a microfluidic chip; and ii) dispersing the microsphere droplets into a water phase of 15-20℃ comprising a surfactant. The method for producing donepezil-containing sustained-release PLGA microspheres of the present invention can produce sustained-release microspheres having high production reproducibility while containing a high content of donepezil, and thus can be used for production of safe and stable therapeutic agents and the like.

Description

Method for producing sustained-release PLGA microspheres containing donepezil

The present invention relates to a method for producing donepezil-containing sustained-release PLGA microspheres, and specifically, to a method for producing donepezil-containing sustained-release PLGA microspheres, i) donepezil or a pharmaceutically acceptable salt thereof, PLGA (Poly( lactic-co-glycolic acid) and an organic solvent to prepare microfluidic droplets by injecting the dispersed phase into the microfluidic chip; It relates to a method for producing donepezil-containing sustained-release PLGA microspheres comprising the steps of:

Donepezil (donepezil) is a drug used for the treatment of Alzheimer's-type dementia, which has an acetylcholine esterase inhibitory effect. After weekly use and clinical response evaluation, the dosage is increased to 10 mg and administered once a day. The drugs currently in circulation are oral tablet formulations containing donepezil hydrochloride and an orally disintegrating film formulation. Problems with digestive disorders such as diarrhea and nausea are emerging.

Accordingly, a sustained-release injection of donepezil using a biodegradable polymer has been developed to improve patient compliance.

Representative biodegradable polymers used in the development of sustained-release microspheres include poly(lactic-co-glycolic acid, PLGA) and polylactic acid (PLA). PLGA and PLA are Most of them in the living body are decomposed into water and carbon dioxide, which are harmless to the human body by hydrolysis, but have different structures and thus have significantly different properties. It is a homopolymer with long bonds, and poly lactide-co-glycolide is a copolymer in which lactide and glycolide units are cross-linked. The physical properties of PLGA are that of lactide and glycolide. Depends on the composition ratio As the lactide ratio increases, crystallinity increases, strength increases, and the in vivo half-life becomes longer. , each has a different effect on the content and properties of microspheres.

According to previously reported research results, PLGA, as a representative biodegradable polymer used for the development of donepezil sustained-release microspheres, has the advantage of shorter in vivo retention time compared to PLA. It has been known to be unsuitable because it can reduce

As an example related to this, in Biomaterials, 28 (2007) 1882 to 1888, microspheres containing donepezil were prepared by solvent evaporation using PLGA, but the content of the prepared microspheres was 13.2 because of the high solubility of donepezil in aqueous phase. The content was as low as ±2.1%. In order for the microspheres containing the low content of donepezil to be pharmaceutically effective, the administered dose of the microspheres must be excessively increased, which cannot be regarded as an excellent pharmaceutical formulation (Korean Patent Publication No. 19-0064526).

As another example, when PLGA microspheres containing donepezil are prepared by a microfluidic method, the drug and PLGA weight ratio must be limited to 1:9 in the dispersed phase for preparing the microspheres to allow sustained release of the drug without initial rapid drug release. In this case, there is a problem that the dosage of microspheres to show the actual therapeutic effect is very large because the content of the drug is 10% or less (2019 AAPS M0930-02-13).

Accordingly, in the case of microspheres containing donepezil, it is very important to control the content of donepezil inside the microspheres in consideration of an appropriate release rate and dosage, and to simultaneously release sustained release while containing donepezil in a high content. It can be seen that it is a technical task in the art to prepare the PLGA microspheres shown. Accordingly, as a method of increasing the content of donepezil contained in microspheres, a method of reducing the solubility of donepezil in aqueous phase was studied.

As one of the methods of reducing the solubility of donepezil in the aqueous phase, there is a method of adjusting the pH of the external aqueous phase. However, PLGA used in the preparation of microspheres forms a polymer matrix inside the microspheres and acts as a carrier to encapsulate the drug in the microspheres. Since it cannot serve as a carrier, a method of adjusting the pH of the aqueous phase is not suitable as a method for preparing microspheres containing a high content of donepezil (J Control Release. 2007 Oct 8;122(3):338-44) .

Another method of reducing the solubility of donepezil in the aqueous phase is to control the surfactant concentration in the aqueous phase. For example, in Open Pharmaceutical Science Journal, 2016, 3, 182-195, it was confirmed that the concentration of surfactant in the aqueous phase is a factor affecting the drug encapsulation rate and drug release rate of microspheres. The surfactant helps to form stable droplets by adsorbing to the interface between the aqueous phase and the oil phase. At this time, if the concentration of the surfactant is increased, it is possible to form small stable droplets, but by increasing the solubility of the drug in the aqueous phase, the drug is distributed from the inside of the microspheres to the outside, which may lead to a decrease in the drug content. On the other hand, when the concentration of the surfactant is decreased, colloidal stability is lowered and unstable droplets are generated, which may cause aggregation due to adhesion between the droplets. Therefore, a method of reducing the solubility of donepezil by controlling the concentration of the surfactant in the aqueous phase for the purpose of increasing the content of donepezil is not preferable because the range of controlling the concentration of the surfactant is extremely limited.

Another method of reducing the solubility of donepezil in the aqueous phase is to control the volume of the aqueous phase. For example, according to Arabian Journal of Chemistry (2012) 5, 103-108, the content and encapsulation rate of a drug may be adjusted according to a change in the volume of the aqueous phase. When the volume of the aqueous phase decreases during the preparation of the microspheres, collisions between the droplets increase, causing agglomeration or expansion between the droplets, thereby causing aggregation between the droplets. On the other hand, the method of increasing the volume of the aqueous phase can reduce the occurrence of agglomeration by reducing the collision between the droplets that occur during stirring, but it is a fairly counterproductive method in consideration of the size and economy of the reactor in the scale-up step. For the above reasons, a method of controlling the volume of the aqueous phase as a method of reducing the solubility of donepezil in the aqueous phase is not preferable.

Accordingly, there is a need for the development of sustained-release microspheres containing a high content of donepezil using a method other than a method of controlling the pH of the aqueous phase, adjusting the surfactant concentration of the aqueous phase, or controlling the volume of the aqueous phase.

On the other hand, known methods for preparing the microspheres include a solvent evaporation method, a solvent extraction method, a spray drying method, a membrane emulsification method, and a microfluidic method. method), but the previously reported sustained-release microspheres containing donepezil were mainly prepared using a solvent evaporation method or a membrane emulsification method (Korean Patent Application Laid-Open No. 2017-0179678, Korean Patent Publication No. 2019-0064526).

The solvent evaporation method controls the rotation speed per minute of a homogenizer to form droplets through mixing of the aqueous phase and the dispersion (oil phase). The droplets generated in this process are inevitably accompanied by aggregation, expansion, and splitting phenomena due to collisions caused by high shear stress. Reproducibility may be low. In addition, the membrane emulsification method may cause problems in production, such as a polymer forming a film on the surface of the membrane while the dispersed phase passes through the micropores of the membrane, thereby reducing the production yield.

Under this background, the present inventors have developed a method for reducing the solubility of donepezil in aqueous phase by preparing donepezil-containing PLGA microspheres in a microfluidic method using a low-temperature aqueous phase, and thus prepared donepezil-containing PLGA. The present invention was completed by confirming that microspheres contain donepezil in a high content and have excellent sustained-release effect and manufacturing reproducibility.

The present inventors developed a method for producing sustained-release PLGA microspheres containing donepezil that can be used for safe and stable production of therapeutic agents, etc. completed.

One object of the present invention is to provide a method for producing sustained-release PLGA microspheres containing donepezil, comprising: i) donepezil or a pharmaceutically acceptable salt thereof, poly (lactic-co-glycolic acid) (PLGA) and an organic solvent. A sustained-release PLGA microsphere containing donepezil comprising the steps of injecting a dispersed phase into a microfluidic chip to prepare microsphere droplets, and ii) dispersing the microsphere droplets in an aqueous phase of 15 to 25° C. containing a surfactant. To provide a manufacturing method of

The method for producing sustained-release PLGA microspheres containing donepezil of the present invention can produce sustained-release microspheres with high production reproducibility while containing a high content of donepezil, and thus can be used for the manufacture of safe and stable therapeutic agents.

1 is a graph showing the cumulative dissolution rate of donepezil-containing sustained-release PLGA microspheres according to the injected donepezil content.

2 is a view taken with an optical microscope of donepezil-containing sustained-release PLGA microspheres according to the temperature of the aqueous phase.

3 is a graph showing the encapsulation rate of donepezil-containing sustained-release PLGA microspheres according to temperature.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

One aspect of the present invention for achieving the above object is a method for producing sustained-release PLGA microspheres containing donepezil, i) donepezil or a pharmaceutically acceptable salt thereof, PLGA (Poly (lactic-co-glycolic acid)) and injecting a dispersed phase containing an organic solvent into a microfluidic chip to prepare microsphere droplets; Provided is a method for preparing pegil-containing sustained-release PLGA microspheres.

As used herein, the term “donepezil” refers to a compound having the structure of Formula 1 below. Donepezil of the present invention may be used as a therapeutic drug for Alzheimer's disease, but is not limited thereto.

In the present invention, it was attempted to prepare PLGA microspheres specialized for donepezil. Specifically, it is characterized in that PLGA microspheres containing donepezil, in particular, high content of donepezil, while showing sustained-release dissolution.

The term "pharmaceutically acceptable salt" of the present invention refers to a salt in a form that can be used pharmaceutically among salts, which are substances in which a cation and anion are bonded by electrostatic attraction, and is usually combined with a metal salt or an organic base. salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkandiones Non-toxic organic acids such as ates, aromatic acids, aliphatic and aromatic sulfonic acids and sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates Phosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malo Nate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate , hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The term "PLGA" of the present invention is polylactide-co-glycolic acid (Poly (lactic-co-glycolic acid)), the monomers of the lactide unit (Lactic unit) and the glycolide unit (Glycolic unit) are cross-bonded. The PLGA may be a block copolymer or a random copolymer. The composition ratio of the lactide unit and the glycolide unit according to the PLGA of the present invention is 50:50 to 95: It may be 5 days, and specifically, it may be 75:25 to 85:15 when the targeted drug release period is delayed from 1 week to 1 month.

As used herein, the term "organic solvent" is a liquid organic material with good volatility and low miscibility with water that can dissolve donepezil and PLGA, and may be chloroform, ethyl ether, dichloromethane, or a mixture thereof. , may be specifically dichloromethane, but is not limited thereto. The dichloromethane is an organic solvent having a boiling point of 39.6 ° C. as a volatile material, and the solubility in water according to temperature is less than 25 ° C. When the temperature is lower, the solubility of dichloromethane in water increases, and when the temperature is 25 ° C. or more, the temperature is It has a solubility graph in the form of a quadratic function in which the solubility of dichloromethane in water increases as it increases (IUPAC-NIST Solubility Database).

As used herein, the term “dispersed phase” refers to a composition for constituting an internal water phase in the case of water in oil microspheres, and an oil in the case of oil in water microspheres. phase), in the case of water in oil in water microspheres, as a composition for constituting an internal primary water in oil emulsion or primary emulsion, the external phase of the composition for preparing microspheres It refers to a mixture in which the drug and the polymer are dissolved or dispersed in the inner phase except for the (outer phase). The dispersed phase of the present invention may include donepezil or a pharmaceutically acceptable salt thereof, PLGA, and an organic solvent.

In the present invention, the weight ratio of donepezil or a pharmaceutically acceptable salt thereof and PLGA included in the dispersed phase may be 15:85 to 40:60. Although not specifically limited thereto, based on the sum of the weight of donepezil or a pharmaceutically acceptable salt thereof and PLGA, donepezil or a pharmaceutically acceptable salt thereof may be 15% or more, and more specifically, 15 to 40%, 20 to 40%, or 24 to 36%. In the present invention, as described above, while containing donepezil in a high content of 15% or more, the production reproducibility of PLGA microspheres is high, and there is a technical significance in that sustained release can be achieved.

On the other hand, when the ratio of donepezil included in the dispersed phase is very low, it is difficult to commercialize the drug because a single dose of the drug required for long-term drug release is increased. On the other hand, when the ratio of donepezil included in the dispersed phase is excessive, the amount of drug exposed to the surface of the microspheres increases and the initial dissolution of the drug rapidly increases, so that a sufficient sustained-release effect is not obtained.

As used herein, the term “microfluidic chip” refers to a reactor having a micro-channel having a size of a micrometer region, and means a reactor capable of continuously introducing an immiscible continuous phase and a dispersed phase by a pump. If the microfluid can be produced through the input of the continuous phase and the dispersed phase, it corresponds to the microfluidic chip of the present invention regardless of the shape of the reactor. In the method for producing donepezil-containing sustained-release PLGA microspheres of the present invention, in step i), the dispersed phase may be injected into the microfluidic chip simultaneously with the continuous phase through separate passages.

In the present invention, the "continuous phase" refers to a fluid including a surfactant, which is injected into the microfluidic chip for the production of microsphere droplets. After the dispersed phase and the continuous phase are injected into the microfluidic chip in step i) of the present invention, microsphere droplets can be prepared from the contact point between the dispersed and continuous phases inside the microfluidic chip.

As used herein, the term “droplet” refers to droplets of a dispersed phase dispersed in a colloidal form in an aqueous phase. As used herein, the terms “droplet” and “microsphere droplet” have the same meaning and may be used interchangeably. The microsphere droplets of the present invention are intermediate substances obtained in the preparation step of donepezil-containing sustained-release PLGA microspheres. The microsphere droplets of the present invention are prepared by injecting a dispersed phase containing donepezil or a pharmaceutically acceptable salt thereof, PLGA, and an organic solvent into a microfluidic chip, and the prepared microsphere droplets are dispersed in an aqueous phase containing a surfactant to be done Pegil-containing sustained-release PLGA microspheres can be prepared.

As used herein, the term "surfactant" refers to a substance that lowers the interfacial tension between the dispersed phase and the external aqueous phase, and serves to prevent merging and aggregation through collisions between the generated droplets. For example, gelatin, sodium oleate, sodium stearate, sodium lauryl sulfate, anionic surfactants such as sodium dodecyl sulfate (SDS), polyoxyethylene sorbitan fatty esters such as Tween 80, Tween 60 (Polysorbate) , may be a nonionic surfactant such as polyoxyethylene castor oil derivative, polyvinyl alcohol (PVA, polyvinyl alcohol), carboxymethyl cellulose, lecithin, hyaluronic acid, or a mixture thereof, specifically polyvinyl alcohol.

The method for producing donepezil-containing sustained-release PLGA microspheres of the present invention includes dispersing the microsphere droplets obtained in step i) of the present invention in a surfactant-containing aqueous phase at 15 to 25°C.

In order to increase the content of donepezil contained in microspheres by reducing the solubility of donepezil in the aqueous phase, a method of reducing the temperature of the aqueous phase was used in the present invention. Although solubility varies depending on the type of solvent and solute, in most cases, the solubility of a solid solute increases as the temperature of the solvent increases. Therefore, if the temperature of the aqueous phase is decreased during the production of donepezil-containing sustained-release PLGA microspheres, the solubility of donepezil in the aqueous phase decreases, thereby increasing the content of the microspheres.

Specifically, when the temperature of the aqueous phase is less than 15 ° C, dichloromethane (organic solvent) contained in the microsphere droplets diffuses into the aqueous phase and the droplets harden quickly, but the dichloromethane dissolved in the aqueous phase is evaporated and removed, so less than 15 ° C. Since the temperature of is too low, agglomeration may occur due to coalescence between the exposed droplets in the aqueous phase. On the other hand, when the temperature of the aqueous phase exceeds 25° C., as the temperature increases, the dichloromethane contained in the microsphere droplets diffuses into the aqueous phase and the droplets harden rapidly, but the solubility of donepezil contained in the droplets also increases at the same time, The final obtained microsphere content is reduced.

In a specific embodiment of the present invention, the morphological characteristics of the microspheres according to the temperature change of the aqueous phase containing the surfactant were analyzed. As a result, in the case of Comparative Example 3 in which the microsphere droplets were dispersed in the aqueous phase at 10° C., it was confirmed that aggregation between the droplets occurred ( FIG. 2 ), and the temperature of the aqueous phase in step ii) of the present invention was It was confirmed that 15 to 25° C., which can reduce the solubility of the vagina and can stably harden the droplets by diffusing the dichloromethane contained in the droplets into the external aqueous phase, is suitable.

In general, drug release of PLGA microspheres has a tri-phasic profile. First, in the burst phase (or initial phase), the drug that is not encapsulated in the microsphere or the drug on the surface of the microsphere is rapidly diffused and released, and in the second lag phase, the polymer chain is decomposed into oligomers, This is the stage in which channels begin to form, and the drug release is significantly delayed. The third stage, degradation phase (also called erosion phase, or continuous phase, depending on the name), is the phase where the polymer is decomposed and the water channel formed inside is reduced. It refers to the stage in which the drug is released through

Drug release in the burst phase, in which the drug on the surface of microspheres is rapidly diffused, follows Fick's Low and First order kinetics. Drugs located on the surface of microspheres can be easily released from microspheres according to drug solubility without resistance of the polymer matrix.

In the case of the lag phase, the drug release is significantly delayed because the drug on the surface of the microspheres has already diffused and dissipated due to the early drug release in the burst phase, and the internal water channel, which is a pathway for the drug release inside the microspheres, is not sufficiently created.

The drug release in the degradation phase has a property that the diffusion rate is significantly slower than in the burst phase because the diffusion of the drug is affected by the decomposition of the polymer matrix, and thus it is closer to the zero-order kinetic.

The duration of the lag phase is affected by 1) drug content, 2) microsphere internal porosity generated during the manufacturing process, and 3) polymer properties. The higher the drug content inside the microspheres, the shorter the period of the lag phase is because the difference in osmotic pressure inside and outside the microspheres widens and the formation of internal water channels is promoted. The channel formation is accelerated and the lag phase period is shortened. In addition, when the end chain of the polymer is a carboxyl group or when the molecular weight of the polymer is small, the lag phase period is reduced because water is more easily introduced into the microspheres.

For example, in the case of PLGA microspheres containing donepezil, when the drug content was 10% or more, the initial dissolution appeared very quickly with no lag phase observed, and the drug dissolution was terminated before the polymer started to decompose. (asian journal of pharmaceutical sciences 10 (2015) 405-414). For the above reasons, polylactic acid, which has a higher hydrophobicity than PLGA, has been mainly used in the case of having a drug content of 10% or more in order to secure a therapeutically effective drug amount and to expect sufficient sustained-release dissolution during the development of donepezil microspheres.

As used herein, the term "sustained release" means that the drug is slowly released over a long period of time in the body by controlling the release mechanism. Specifically, the sustained release of the present invention is not controlled only by simple diffusion (Fick's law) according to a general concentration gradient, but the release mechanism of the drug by the simple diffusion and the dissolution control effect (Higuchi model) by the polymer matrix is merged with each other It may appear in the form When the drug is released only through simple diffusion, the drug release rate per unit time is expressed as a log value, and the value decreases linearly as time elapses. According to the basic principle of drug release of the sustained-release formulation, the sustained-release pharmaceutical formulation in the form of polymer-based microspheres of the present invention has a relatively low initial burst release rate (%), and after drug release by initial simple diffusion , even if the drug release rate per unit time in the lag phase and the polymer degradation phase is expressed as a log value, the value does not decrease linearly.

In a specific embodiment of the present invention, Examples 1 and 2, both of which are donepezil-containing PLGA microspheres prepared by the method of the present invention, contain a high content of donepezil, but unlike the conventional PLGA microspheres, It was confirmed that the drug could be released in a sufficiently sustained release until the time of polymer degradation, and it was confirmed that the drug had a dissolution pattern corresponding to the sustained release (Table 3).

As used herein, the term “microsphere” refers to spherical particles having a diameter of 1 mm or less.

In the present invention, the method for producing the donepezil-containing sustained-release PLGA microspheres may be characterized in that the content or encapsulation rate of donepezil contained in the donepezil-containing sustained-release PLGA microspheres is uniform. As for the drug of the present invention, the dissolution rate differs significantly depending on the content of the drug of the present invention, the content inside the microspheres is very important, and the uniformity or reproducibility of the production is an important requirement. The rate was adjusted to be uniform.

Specifically, the encapsulation rate of donepezil may have an error rate of 5% or less compared to the average value, but is not limited thereto. The uniformity of the donepezil content or encapsulation rate means that the production reproducibility between production batches is high.

In a specific embodiment of the present invention, as a result of comparing the case where the amount of the injected donepezil was 65 mg and the case of 110 mg, it was confirmed that the dissolution rate and the dissolution pattern greatly changed according to the amount of the injected donepezil (Fig. 1) ) and sustained release, it can be seen that the uniformity of the donepezil content according to the method of manufacturing the microspheres must be guaranteed.

The "encapsulation rate" of the present invention means the percentage of the value obtained by dividing the content of the drug contained in the microspheres by the target content of the drug, that is, the content of the drug contained in the initial dispersion phase.

In a specific embodiment of the present invention, the donepezil content and encapsulation rate of the donepezil-containing sustained-release PLGA microspheres prepared using the microfluidic method and the solvent evaporation method were measured, and the error rate compared to the average value of the encapsulation rate according to the manufacturing method was calculated. did As a result, compared to Comparative Examples 6 to 8 prepared using the solvent evaporation method, Examples 6 to 8 prepared using the microfluidic method had a significantly lower error rate compared to the average value of the encapsulation rate (Table 6) ), it was confirmed that the manufacturing method using the microfluidic method has high reproducibility between batches.

The method for producing donepezil-containing sustained-release PLGA microspheres of the present invention may further include the step of iii) removing the organic solvent from the dispersed microsphere droplets.

The method for removing the organic solvent of the dispersed microsphere droplets in the present invention is a method of removing the organic solvent by stirring by increasing the aqueous phase temperature under reduced pressure, and removing the organic solvent by stirring by increasing the aqueous phase temperature in a controlled vacuum condition It may be selected from a method of removing the organic solvent by stirring by increasing the temperature of the aqueous phase while introducing nitrogen gas into the aqueous phase, or a method of removing the organic solvent by simply stirring by increasing the aqueous phase temperature.

In the present invention, the removal of the organic solvent in step iii) may be due to an increase in the temperature of the aqueous phase, but is not particularly limited as long as it is a temperature range capable of effectively removing the organic solvent. Specifically, the temperature of the prepared donepezil-containing sustained-release PLGA microspheres may be increased to 30 to 45° C., which is below the glass transition temperature and close to the boiling point of the organic solvent.

Step iii) of the present invention may further include replacing the aqueous phase.

The method for producing donepezil-containing sustained-release PLGA microspheres of the present invention may further include, after the organic solvent removal step of step iii), iv) curing the microsphere droplets from which the organic solvent has been removed. The curing of step iv) may be performed at a temperature of 15 to 25° C., but is not limited thereto.

The method for producing the donepezil-containing sustained-release PLGA microspheres of the present invention may further include: iv) curing the microsphere droplets, and v) washing, filtering, and drying the cured microspheres.

In the present invention, the filtration of step v) may be using a membrane filter, but is not limited thereto.

In the present invention, the drying in step v) may be at least one selected from vacuum drying, natural drying, freeze drying, heat drying or blow drying, and specifically, may be freeze drying, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Comparative Example 1. Preparation of PLGA microspheres containing donepezil in which the weight ratio of donepezil and PLGA contained in the dispersed phase was 24.5:75.5 (solvent evaporation method)

A dispersed phase containing donepezil was prepared by dissolving 200 mg of the biodegradable polymer PLGA (DLG85-7A, manufactured by Merck) and 65 mg of donepezil base (manufactured by Neuland Laboratories) in 2 mL of dichloromethane solvent. While injecting the prepared dispersed phase into a 1% polyvinyl alcohol aqueous solution, a homogenizer (Silverson, L5M-A) was used to stir (1,500 rpm) to form microspheres in the form of droplets. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

Comparative Example 2. Preparation of PLGA microspheres containing donepezil in which the weight ratio of donepezil and PLGA contained in the dispersed phase was 35.5:64.5 (solvent evaporation method)

A dispersed phase containing donepezil was prepared by dissolving 200 mg of the biodegradable polymer PLGA and 110 mg of donepezil base in 2 mL of dichloromethane solvent. While injecting the prepared dispersed phase into 1% polyvinyl alcohol aqueous solution, it was stirred (1,500 rpm) using a homogenizer to form microspheres in the form of droplets. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

Example 1. Preparation of donepezil-containing PLGA microspheres in which the weight ratio of donepezil and PLGA contained in the dispersed phase is 24.5:75.5 (microfluidic method)

After dissolving 200 mg of biodegradable polymer PLGA and 65 mg of donepezil base in 2 mL of dichloromethane solvent to prepare a dispersed phase containing donepezil, it was applied to a microfluidic chip (Dolomite, 3D focusing hydrophilic chip) at a flow rate of 0.01 mL/min. injected. At this time, a 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and it was injected simultaneously with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 20 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

Example 2. Preparation of PLGA microspheres containing donepezil in which the weight ratio of donepezil and PLGA contained in the dispersed phase was 35.5:64.5 (microfluidic method)

200 mg of biodegradable polymer PLGA and 110 mg of donepezil base were dissolved in 2 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, a 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and it was injected simultaneously with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 20 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

Comparative Example 3. Preparation of donepezil-containing PLGA microspheres with a temperature of 10° C. of an aqueous phase containing a surfactant (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, a 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and it was simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 10 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution.

As a result of observing the obtained microsphere droplets with an optical microscope, it was confirmed that aggregation between the droplets occurred, as shown in FIG. 2 .

Example 3. Preparation of donepezil-containing PLGA microspheres with a temperature of 15° C. of an aqueous phase containing surfactant (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, the aqueous phase (continuous phase) used 1% polyvinyl alcohol aqueous solution, and was simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 15 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

The results of observing the obtained microsphere droplets with an optical microscope are shown in FIG. 2 .

Example 4. Preparation of donepezil-containing PLGA microspheres with a temperature of 20° C. of an aqueous phase containing a surfactant (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, a 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and it was injected simultaneously with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 20 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

The results of observing the obtained microsphere droplets with an optical microscope are shown in FIG. 2 .

Example 5. Preparation of donepezil-containing PLGA microspheres with a temperature of 25° C. of an aqueous phase containing a surfactant (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, the aqueous phase (continuous phase) used 1% polyvinyl alcohol aqueous solution, and was injected simultaneously with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 25 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

The results of observing the obtained microsphere droplets with an optical microscope are shown in FIG. 2 .

Comparative Example 4. Preparation of PLGA microspheres containing donepezil with a temperature of 30° C. of an aqueous phase containing surfactant (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, the aqueous phase (continuous phase) used 1% polyvinyl alcohol aqueous solution, and was simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 30 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

The results of observing the obtained microsphere droplets with an optical microscope are shown in FIG. 2 .

Comparative Example 5. Preparation of PLGA microspheres containing donepezil with a temperature of 35° C. of an aqueous phase containing surfactant (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, the aqueous phase (continuous phase) used 1% polyvinyl alcohol aqueous solution, and was injected simultaneously with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at 35 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36°C for 2 hours and solidified at a temperature of 20°C for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

The results of observing the obtained microsphere droplets with an optical microscope are shown in FIG. 2 .

Examples 6 to 8. Preparation of PLGA microspheres containing donepezil for verification of content reproducibility between batches (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane solvent to prepare a donepezil-containing dispersed phase, and then injected into the microfluidic chip at a flow rate of 0.01 mL/min. At this time, the aqueous phase (continuous phase) used 1% polyvinyl alcohol aqueous solution, and was simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, and the microfluidic droplets formed inside the microfluidic chip were stirred at 150 rpm at a temperature of 20 ° C. It was obtained by dispersing in a 1% polyvinyl alcohol solution. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36° C. for 2 hours and solidified at a temperature of 20° C. for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

Comparative Examples 6 to 8. Preparation of PLGA microspheres containing donepezil for verification of content reproducibility between batches (solvent evaporation method)

A dispersed phase containing donepezil was prepared by dissolving 100 mg of the biodegradable polymer PLGA and 55 mg of donepezil base in 1 mL of dichloromethane solvent. While injecting the prepared dispersed phase into 1% polyvinyl alcohol aqueous solution, it was stirred (4,000 rpm) using a homogenizer to form microspheres in the form of droplets. From the obtained microsphere droplets, the dichloromethane solvent was removed at a temperature of 36° C. for 2 hours and solidified at a temperature of 20° C. for 1 hour to form microspheres. The formed microspheres were washed with purified water to remove polyvinyl alcohol, filtered through a membrane filter, and freeze-dried for 2 days to obtain powdery microspheres.

Experimental Example 1. Analysis of dissolution of donepezil-containing sustained-release PLGA microspheres according to the content

Experimental Example 1-1. Measurement of donepezil-containing PLGA microsphere drug content and encapsulation rate

1 mg of the final freeze-dried microspheres were dissolved in 1 mL of acetonitrile, 1 mL of this solution was diluted in 10 mL of acetonitrile, and filtered through a 0.45 μm PVDF syringe filter. Thereafter, a standard curve was obtained using HPLC-UV equipment under the same conditions as in Table 1 below, and quantitative analysis was performed.

Column	YMC-Triart C18 column, C18 (150 x 4.0mm ID), S-5㎛
Column temperature	35℃
mobile phase	A mixed solution of potassium dihydrogen phosphate aqueous solution (solution A) and acetonitrile (solution B) (solution A:solution B = 6.5:3.5)
Wavelength	224nm
Injection volume	20 μl

From the quantitative analysis, the drug content was calculated by [Equation 1] below, and the donepezil content in the donepezil-containing microspheres obtained in Comparative Examples and Examples is shown in Table 2.

[Equation 1]

Drug content (%) = concentration of drug quantified by HPLC / concentration of microspheres * 100

Drug encapsulation rate (%) = drug content / target content of drug * 100

	Drug content (%)	Drug Encapsulation Rate (%)	Manufacturing method
Comparative Example 1	21.0	85.7	solvent evaporation
Comparative Example 2	28.2	79.4	solvent evaporation
Example 1	18.9	77.1	microfluidic method
Example 2	29.2	82.3	microfluidic method

Experimental Example 1-2. Analysis of drug dissolution of donepezil PLGA microspheres according to the amount of injected donepezil

50 mg to 90 mg of donepezil-containing sustained-release PLGA microspheres were placed in 100 mL of pH 7.4 phosphate buffer containing 0.2% sodium lauryl sulfate, and left / It was shaken. At a preset time (0.3 days, 1 day, 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days), 1 mL of the eluate is taken and centrifuged at 9,000 rpm for 3 minutes. The supernatant was taken and quantitatively analyzed using HPLC-UV equipment. At this time, the HPLC column and operating conditions are the same as the analysis conditions of [Experimental Example 1-1].

Table 3 below shows the cumulative dissolution rate of donepezil-containing sustained-release PLGA microspheres according to the ratio of donepezil included in the dispersed phase, and a graph thereof is shown in FIG. 1 .

	Donepezil cumulative dissolution rate (%)
	0.3 days	1 day	3 days	7 days	10 days	14 days	17th	21 days	24 days	28 days
Comparative Example 1	<0.1	0.5	2.4	8.1	45.1	86.1	92.8	98.0	-	-
Comparative Example 2	<0.1	<0.1	2.0	56.3	78.1	89.3	92.2	-	-	-
Example 1	<0.1	<0.1	1.6	4.9	26.8	50.1	73.7	81.7	87.6	88.4
Example 2	<0.1	0.2	7.2	19.6	76.4	90.4	-	-	-	-

First, it was found that there is an advantage in that the initial dissolution is slow when the microfluidic method of Examples is used compared to the solvent evaporation method of Comparative Examples.

In addition, separate comparisons with [Comparative Example 1] and [Example 1] in which the injected donepezil content was 65 mg, and [Comparative Example 2] and [Example 2] in which the injected donepezil content was 110 mg, respectively As a result, it was confirmed that the dissolution rate and dissolution pattern greatly changed according to the content of donepezil when the principle of the manufacturing method was the same.

That is, when the principle of the production method is the same for the donepezil-containing microspheres, the dissolution rate differs significantly depending on the donepezil content. Therefore, the uniformity of the donepezil content is essential in the production of the donepezil-containing sustained-release PLGA microspheres. confirmed that it should be. In particular, since the dissolution of donepezil is accelerated as the amount of injected donepezil is increased, it was confirmed that content uniformity and production reproducibility for the production of sustained-release PLGA microspheres containing high content of donepezil are more important requirements.

In addition, Examples 1 and 2, which are donepezil-containing PLGA microspheres provided from a method of dispersing microsphere droplets in an aqueous phase of 15 to 25° C. containing a surfactant, both have a drug content higher than 15%, despite the high content It was confirmed that, unlike the conventional PLGA microspheres, they had a dissolution pattern corresponding to the sustained release of the present invention. That is, the PLGA microspheres containing donepezil of the present invention have a complex effect of showing sustained-release dissolution while containing a high content of donepezil by combining the production conditions such as the setting of the aqueous phase temperature of 15 to 25°C and the microfluidic method. Confirmed.

Experimental Example 2. Analysis of donepezil-containing PLGA microspheres according to the temperature of the external aqueous phase containing surfactant

Experimental Example 2-1. Observation of the morphology of donepezil-containing sustained-release PLGA microspheres

In order to analyze the morphological characteristics of the microspheres according to the temperature change of the aqueous phase containing the surfactant, some of the microsphere droplets obtained from the aqueous phase were collected and dispersed in a 1% polyvinyl alcohol solution, and then using an optical microscope to form the microspheres was observed.

The form of the microspheres prepared in each Example and Comparative Example is as shown in FIG. [Comparative Example 3] dispersed in an aqueous phase at 10 ° C had a problem in that aggregation occurred between the droplets, and the morphology of the donepezil-containing sustained-release PLGA microspheres obtained in all Examples and Comparative Examples except for this was uniform without aggregation. One microsphere was observed.

From this, it was confirmed that when preparing donepezil-containing PLGA microspheres, the temperature of the aqueous phase of less than 15° C. was not suitable as a production condition for donepezil-containing sustained-release PLGA microspheres.

Experimental Example 2-2. Measurement of drug content and encapsulation rate of sustained-release PLGA microspheres containing donepezil

1 mg of the final lyophilized microspheres were dissolved in 1 mL of acetonitrile, and 1 mL of this solution was diluted in 10 mL of acetonitrile and filtered through a 0.45 μm PVDF syringe filter. Thereafter, the HPLC column, operating conditions, and calculation method are the same as the analysis conditions of [Experimental Example 1-1].

	water temperature	Drug content (%)	Drug Encapsulation Rate (%)
Example 3	15℃	30.6	86.4
Example 4	20℃	30.7	86.5
Example 5	25℃	30.7	86.5
Comparative Example 4	30℃	29.2	82.2
Comparative Example 5	35℃	27.7	78.0

Table 4 shows the donepezil content and encapsulation rate of the sustained-release PLGA microspheres containing donepezil according to the temperature of the aqueous phase containing the surfactant, and a graph thereof is shown in FIG. 3 .

In the case of [Comparative Example 3], as mentioned above, in the process of dispersing the microsphere droplets in the aqueous phase, aggregation due to merging between the microsphere droplets occurred, and thus the content and the encapsulation rate were excluded from the measurement ( FIG. 1 ).

[Examples 3 to 5] are microspheres prepared by adjusting the temperature of the aqueous phase to 15 to 25 ° C. Under the above temperature conditions, the solubility of dichloromethane increases and the solubility of donepezil in the aqueous phase decreases, so that a high drug It was confirmed that the droplets were stably hardened while the microspheres having an encapsulation rate were manufactured.

In the case of [Comparative Examples 4 to 5], as the temperature of the aqueous phase increased, the organic solvent and donepezil contained in the droplets were simultaneously diffused into the aqueous phase, and it was confirmed that the encapsulation rate of the finally obtained donepezil in the microspheres decreased.

From the above results, it was confirmed that the aqueous phase temperature of 15 to 25° C. was most suitable for the production of donepezil-containing sustained-release PLGA microspheres having a high drug encapsulation rate.

Experimental Example 3. Batch-to-batch reproducibility analysis using microfluidic method and solvent evaporation method

1 mg of the final lyophilized microspheres were dissolved in 1 mL of acetonitrile, and 1 mL of this solution was diluted in 10 mL of acetonitrile and filtered through a 0.45 μm PVDF syringe filter. Thereafter, the HPLC column, operating conditions, and calculation method are the same as the analysis conditions of [Experimental Example 1-1].

	Drug content (%)	Drug Encapsulation Rate (%)
Comparative Example 6	28.2	79.4
Comparative Example 7	19.0	53.7
Comparative Example 8	21.8	61.4
Example 6	29.2	82.3
Example 7	31.0	87.4
Example 8	30.7	86.5

Table 5 shows the content and encapsulation rate of donepezil-containing sustained-release PLGA microspheres prepared using the microfluidic method and the solvent evaporation method.

From the obtained encapsulation rate, using [Equation 2], the error rate compared to the average value of the encapsulation rate according to the manufacturing method (microfluidic method or solvent evaporation method) is shown in Table 6 below.

[Equation 2]

Error rate (%) compared to the average value of drug encapsulation rate = standard deviation / average value * 100

	microfluidic method	solvent evaporation
Average drug encapsulation rate	85.40	64.83
Standard deviation of drug encapsulation rate	2.72	13.19
Error rate (%) compared to the average value of drug encapsulation rate	3.19	20.34

According to [Experimental Example 1] of the present invention, the dissolution rate of the donepezil-containing sustained-release PLGA microspheres is very sensitive to the donepezil content (FIG. 1), so content uniformity according to the preparation method must be guaranteed.

When comparing [Comparative Examples 6 to 8] and [Examples 6 to 8], the microfluidic method has a uniform encapsulation rate and a significantly lower error rate compared to the average encapsulation rate compared to the solvent evaporation method, resulting in high production reproducibility between batches It was confirmed that pegil-containing sustained-release PLGA microspheres were produced.

Therefore, it was confirmed that the microfluidic method was most suitable for the method for producing donepezil-containing sustained-release PLGA microspheres provided by the present invention considering the uniformity of the donepezil content or encapsulation rate.

That is, the PLGA microspheres containing donepezil of the present invention solve the problem of donepezil content, which is a problem of the existing donepezil PLGA microspheres, by setting the temperature range of the aqueous phase and the microfluidic method, and at the same time, have a sustained release effect and uniformity. It was confirmed that it has a very good pharmaceutical effect of securing manufacturing reproducibility.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (15)

1. In the production method of donepezil-containing sustained-release PLGA microspheres,

   i) preparing microsphere droplets by injecting a dispersed phase containing donepezil or a pharmaceutically acceptable salt thereof, poly (lactic-co-glycolic acid) (PLGA) and an organic solvent into a microfluidic chip; and

   ii) The method for producing sustained-release PLGA microspheres containing donepezil, comprising dispersing the microsphere droplets in an aqueous phase of 15 to 25° C. containing a surfactant.
2. The method according to claim 1, wherein the weight ratio of donepezil or a pharmaceutically acceptable salt thereof and PLGA in the dispersed phase is 15:85 to 40:60.
3. The method according to claim 1, wherein the content or encapsulation rate of donepezil contained in the donepezil-containing sustained-release PLGA microspheres is uniform.
4. The method of claim 3, wherein the donepezil encapsulation rate has an error rate of 5% or less compared to the average value.
5. The method of claim 1, wherein in step i), the dispersed phase is injected into the microfluidic chip through separate passages at the same time as the continuous phase.
6. The method of claim 1, wherein the organic solvent is dichloromethane.
7. The method of claim 1, further comprising the step of iii) removing the organic solvent from the dispersed microsphere droplets.
8. The method of claim 7, wherein the removal of the organic solvent in step iii) is by increasing the temperature of the aqueous phase, donepezil-containing sustained-release PLGA microspheres.
9. The method of claim 8, wherein the temperature of the aqueous phase in step iii) is increased to 30 to 45°C.
10. The method of claim 7, further comprising replacing the aqueous phase in step iii).
11. The method of claim 7, further comprising the step of iv) curing the microsphere droplets from which the organic solvent has been removed.
12. The method of claim 11, wherein the curing of step iv) is performed at a temperature of 15 to 25°C.
13. The method of claim 11, further comprising v) washing, filtering and drying the cured microspheres.
14. The method of claim 13, wherein the filtration in step v) uses a membrane filter.
15. The method of claim 14, wherein the drying in step v) is freeze-drying, donepezil-containing sustained-release PLGA microspheres.

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