KR102237737B1 - Methods for preparation of donepezil loaded sustained-release PLGA microsphere - Google Patents

Abstract

The present invention relates to a method for preparing a donepezil-containing sustained-release PLGA microsphere, and more particularly, to a method for preparing a donepezil-containing sustained-release PLGA microsphere, which comprises the steps of: i) preparing microsphere droplets by injecting a dispersed phase containing donepezil or a pharmaceutically acceptable salt thereof, PLGA (poly(lactic-co-glycolic acid) and an organic solvent into a microfluidic chip; and ii) dispersing the microsphere droplets in an aqueous phase containing surfactant at 15 to 25℃. The method for preparing donepezil-containing sustained-release PLGA microspheres can prepare sustained-release microspheres with high production reproducibility while containing a high content of donepezil, and thus can be used for safe and stable preparation of therapeutic agents, etc.

Description

Method for preparing sustained-release PLGA microspheres containing donepezil {Methods for preparation of donepezil loaded sustained-release PLGA microsphere}

The present invention relates to a method for producing sustained-release PLGA microspheres containing donepezil, and specifically, in 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; And ii) dispersing the microsphere droplets in an aqueous phase of 15 to 25°C containing a surfactant. It relates to a method for producing sustained-release PLGA microspheres containing donepezil comprising the step.

Donepezil is a drug used in the treatment of Alzheimer's-type dementia, which has an acetylcholine esterase inhibitory effect, starting with 5 mg once a day in the form of donepezil hydrochloride at bedtime, 4-6. After weekly use and evaluation of clinical response, the dosage is increased to 10 mg and administered once a day. The drugs in circulation at the present time are oral tablet formulations containing donepezil hydrochloride and oral disintegrating film formulations, and patients with dementia have difficulty in taking regular medication on their own. Problems with digestive disorders such as diarrhea and nausea are emerging.

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

Representative biodegradable polymers used in the development of sustained-release microspheres include poly lactide-co-glycolic acid (PLGA) and poly lactic acid (PLA). Although most of them in vivo are decomposed into water and carbon dioxide, which are harmless to the human body by hydrolysis, they have different structures and thus have quite different properties. Polylactide-co-glycolide is a long bonded single polymer, and polylactide-co-glycolide is a copolymer in which lactide units and glycolide units are cross-linked. The physical properties of PLGA are those of lactide and glycolide. Depends on the composition ratio As the lactide ratio increases, the crystallinity increases, the strength increases, and the half-life in vivo increases. , It has different effects on the content and properties of microspheres.

According to the previously reported research results, PLGA as a representative biodegradable polymer used in the development of sustained-release microspheres of donepezil, although it has the advantage that it has a lower survival period in vivo compared to PLA, the content of the drug contained in the microspheres. It has been known to be unsuitable because it can reduce

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

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

Through this, in the case of microspheres containing donepezil, it is very important to control the content of donepezil inside the microspheres, considering an appropriate release rate and dosage, and sustained-release elution while containing donepezil in a high content. It can be seen that producing the representative PLGA microspheres is a technical challenge in the art. Accordingly, as a method of increasing the content of donepezil contained in the microspheres, a method of reducing the solubility of donepezil in the aqueous phase was investigated.

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 manufacture of microspheres forms a polymer matrix inside the microspheres and acts as a carrier for encapsulating the drug in the microspheres. If the aqueous phase is acidic or basic, the hydrolysis of PLGA serving as a carrier is accelerated and is therefore suitable. Since it cannot play the role of a carrier, the method of adjusting the pH of the aqueous phase is not suitable as a method for producing 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 a method of adjusting the surfactant concentration in the aqueous phase. For example, Open Pharmaceutical Science Journal, 2016, 3, 182-195 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 adsorbs to the interface between the aqueous phase and the oil phase, helping to form stable droplets. In this case, if the concentration of the surfactant increases, a stable droplet having a small size can be formed, but the solubility of the drug in the aqueous phase is increased, so that the drug is distributed from the inside of the microsphere to the outside, resulting in a decrease in the drug content. On the other hand, when the concentration of the surfactant decreases, colloidal stability decreases, resulting in unstable droplets, which may cause aggregation due to adhesion between droplets. Therefore, the 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 a method of adjusting the volume of the aqueous phase. For example, according to Arabian Journal of Chemistry (2012) 5, 103-108, the content and encapsulation rate of the drug may be adjusted according to the volume change of the aqueous phase. When the volume of the aqueous phase decreases during the manufacture of microspheres, collisions between droplets increase, causing agglomeration or expansion between droplets, thereby causing aggregation between droplets. On the other hand, the method of increasing the volume of the aqueous phase can reduce the occurrence of agglomeration by reducing collisions between droplets generated during stirring, but is a very counterproductive method considering the size and economy of the reaction tank in the scale-up step. For the above reasons, it is not preferable to control the volume of the aqueous phase as a method of reducing the solubility of donepezil in the aqueous phase.

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

On the other hand, known methods for producing 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 donepezil-containing sustained-release microspheres were mainly prepared using a solvent evaporation method or a membrane emulsification method (Korean Patent Publication No. 2017-0179678, Korean Patent Publication No. 2019-0064526).

In the solvent evaporation method, droplets are formed by mixing an aqueous phase and a dispersion (oil phase) by controlling the rotational speed of a homogenizer per minute. The droplets generated in this process are inevitably accompanied by agglomeration, expansion, and splitting due to collision due to high shear stress, and as a result, donepezil is easily exposed to the aqueous phase, reducing the content of the drug, or manufacturing between batches. Reproducibility may be low. In addition, in the membrane emulsification method, production problems may occur, such as when the dispersed phase passes through the micropores of the membrane, the polymer forms a film on the membrane surface, thereby lowering the manufacturing yield.

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

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

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

In addition, those of ordinary skill in the art can recognize or ascertain using only routine experimentation a number of equivalents to the specific aspects of the invention described herein. Also, such equivalents are intended to be included in the present invention.

One aspect of the present invention for achieving the above object is in the production method of 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; And ii) dispersing the microsphere droplets in an aqueous phase of 15 to 25°C containing a surfactant. It provides a method for producing a slow-release PLGA microsphere containing pegyl.

The term "donepezil" of the present invention means a compound having a structure represented by the following formula (1). Donepezil of the present invention may be used as a drug for treating Alzheimer's disease, but is not limited thereto.

[Formula 1]

In the present invention, it is intended to prepare PLGA microspheres specialized for donepezil, and specifically, it is characterized in producing PLGA microspheres that contain donepezil, particularly while containing donepezil in a high content, showing sustained release.

The term "pharmaceutically acceptable salt" of the present invention refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bound by electrostatic attraction. 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 alkandios. Non-toxic organic acids such as acid, aromatic acids, aliphatic and aromatic sulfonic acids and sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyro Phosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malo Nate, succinate, suberate, sebacate, fumarate, maleate, butine-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, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The term "PLGA" of the present invention refers to polylactide-co-glycolic acid, wherein monomers of a lactide unit and a glycolide unit are cross-linked. 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, and specifically, the target drug release period may be 75:25 to 85:15 when viewed from 1 week to 1 month.

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

The term "dispersed phase" of the present invention refers to a composition for constituting an internal water phase, in the case of water in oil microspheres, and an internal oil phase 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 inner primary water in oil emulsion or primary emulsion, the external phase of the composition for producing microspheres It refers to an inner phase excluding the outer phase, that is, a mixture in which a drug and a polymer are dissolved or dispersed. The dispersed phase of the present invention may contain 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 contained in the dispersed phase may be 15:85 to 40:60. Specifically, but not 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, there is a technical significance in that the production reproducibility of PLGA microspheres is high while containing a high content of donepezil of 15% or more as described above, and that sustained-release elution can be achieved.

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

The term "microfluidic chip" of the present invention refers to a reactor having microchannels having a size of a micrometer region, and means a reactor capable of continuously introducing a continuous phase and a dispersed phase that are not mixed with each other by a pump. If a microfluid can be prepared through the introduction 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 the sustained-release PLGA microspheres containing donepezil of the present invention, the dispersed phase in step i) may be injected into the microfluidic chip through separate passages at the same time as the continuous phase.

In the present invention, the "continuous phase" refers to a fluid containing a surfactant, which is injected into a microfluidic chip to produce 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 produced from the contact points of the dispersed phase and the continuous phase inside the microfluidic chip.

The term "droplets" of the present invention means droplets of a dispersed phase dispersed in a colloidal form in an aqueous phase. In the present invention, the terms “droplet” and “fine sphere droplet” have the same meaning and may be used interchangeably. The microsphere droplets of the present invention are intermediate materials obtained in the production step of sustained-release PLGA microspheres containing donepezil. 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 dispersing the prepared microsphere droplets in an aqueous phase containing a surfactant to Sustained-release PLGA microspheres containing pegyl can be prepared.

The term "surfactant" of the present invention is a substance that lowers the interfacial tension between the dispersed phase and the external aqueous phase, and serves to prevent merging and agglomeration through collision between the generated droplets. For example, anionic surfactants such as gelatin, sodium oleate, sodium stearate, sodium lauryl sulfate, sodium dodecyl sulfate (SDS), polyoxyethylene sorbitan fatty esters such as Tween 80 and Tween 60 (Polysorbate) , Nonionic surfactants such as polyoxyethylene castor oil derivatives, polyvinyl alcohol (PVA, polyvinyl alcohol), carboxymethylcellulose, lecithin, hyaluronic acid, or a mixture thereof, and specifically polyvinyl alcohol.

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

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

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 are rapidly cured, but the dichloromethane dissolved in the aqueous phase is evaporated and removed. Since the temperature of is too low, aggregation may occur due to merging 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, dichloromethane contained in the microsphere droplets diffuses into the aqueous phase and the droplet hardens rapidly, but the solubility of donepezil contained in the droplet increases at the same time. The final obtained microsphere content decreases.

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

The term "sustained release" of the present invention means that the drug is slowly released over a long period of time by controlling the mechanism of release of the drug. Specifically, the sustained release of the present invention is not controlled only by simple diffusion according to the general concentration gradient (Fick's law), but the mechanism of release of the drug by the simple diffusion and the dissolution control effect by the polymer mattress (Higuchi model) merge with each other. It may appear in the form of a When a drug is released only by simple diffusion, the drug release rate decreases in a linear fashion as time passes when the drug release rate per unit time is expressed as a log value. According to the basic principle of drug release of the sustained-release formulation, the sustained-release pharmaceutical formulation in the form of a polymer-based microsphere of the present invention has a relatively small initial burst release rate (%), and after drug release by initial simple diffusion. However, even if the drug release rate per unit time in the lag phase and the polymer degradation phase are expressed as log values, the value does not decrease linearly.

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

The term "fine sphere" of the present invention means spherical particles having a diameter of 1 mm or less.

In the present invention, the manufacturing method of the sustained-release PLGA microspheres containing donepezil may be characterized in that the content or encapsulation rate of donepezil contained in the sustained-release PLGA microspheres containing donepezil is uniform. Since donepezil, the drug of the present invention, has a significant difference in dissolution rate depending on its content, the internal content of the microspheres is very important, and its uniformity or manufacturing reproducibility is an important requirement. It was intended to be adjusted so that the rate is uniform.

Specifically, the error rate of the donepezil encapsulation rate compared to the average value may be 5% or less, but is not limited thereto. The uniform content or encapsulation rate of donepezil means that 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 donepezil to be added is 65 mg and the case of 110 mg, it was confirmed that the dissolution rate and the dissolution pattern are significantly different depending on the amount of donepezil to be introduced (Fig. 1 ), it can be seen that in order to secure the characteristics of the microspheres of sustained release, the uniformity of the donepezil content according to the production method of the microspheres must be guaranteed.

The "encapsulation rate" of the present invention means a percentage of a 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 the encapsulation rate of the sustained-release PLGA microspheres containing donepezil prepared using the microfluidic method and the solvent evaporation method are measured, and the error rate compared to the average value of the encapsulation rate according to the manufacturing method is calculated. I 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 significantly lower error rates compared to the average value of the encapsulation ratio (Table 6). ), it was confirmed that the manufacturing method using the microfluidic method has high manufacturing reproducibility between batches.

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

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

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 glass transition temperature of the prepared donepezil-containing sustained-release PLGA microspheres may be increased to 30 to 45°C, which is a temperature close to the boiling point of the organic solvent.

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

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

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

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

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

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

1 is a graph showing the cumulative dissolution rate according to the amount of donepezil added to the sustained-release PLGA microspheres containing donepezil.
2 is a diagram photographed with an optical microscope of sustained-release PLGA microspheres containing donepezil according to the temperature of an aqueous phase.
3 is a graph showing the encapsulation rate of sustained-release PLGA microspheres containing donepezil according to temperature.

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

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

200 mg of a biodegradable polymer PLGA (DLG85-7A, manufactured by Merck) and 65 mg of donepezil base (manufactured by Neuland Laboratories) were dissolved in 2 mL of a dichloromethane solvent to prepare a dispersed phase containing donepezil. The prepared dispersed phase was poured into a 1% polyvinyl alcohol aqueous solution, and stirred (1,500 rpm) using a homogenizer (Silverson, L5M-A) to form droplet-shaped microspheres. The dichloromethane solvent was removed from the obtained microsphere droplets 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 with a weight ratio of 35.5:64.5 of donepezil and PLGA contained in the dispersed phase (solvent evaporation method)

200 mg of a biodegradable polymer PLGA and 110 mg of donepezil base were dissolved in 2 mL of a dichloromethane solvent to prepare a dispersed phase containing donepezil. While injecting the prepared dispersed phase into a 1% polyvinyl alcohol aqueous solution, it was stirred (1,500 rpm) using a homogenizer to form droplet-shaped microspheres. The dichloromethane solvent was removed from the obtained microsphere droplets 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 lyophilized for 2 days to obtain powdery microspheres.

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

200 mg of biodegradable polymer PLGA and 65 mg of donepezil base were dissolved in 2 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then placed on a microfluidic chip (Dolomite, 3D focusing hydrophilic chip) at a flow rate of 0.01 mL/min. Injected. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 20° C. and 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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 with a weight ratio of 35.5:64.5 of donepezil and PLGA contained in the dispersed phase (microfluidic method)

200 mg of biodegradable polymer PLGA and 110 mg of donepezil base were dissolved in 2 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 20° C. and 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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 PLGA microspheres containing donepezil in which the temperature of the aqueous phase containing surfactant is 10°C (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, the aqueous phase (continuous phase) was a 1% polyvinyl alcohol aqueous solution, and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, and microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 10° C. and 150 rpm. 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 PLGA microspheres containing donepezil with a temperature of an aqueous phase containing a surfactant at 15°C (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 15° C. and 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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.

Fig. 2 shows the results of observing the obtained microsphere droplets with an optical microscope.

Example 4. Preparation of PLGA microspheres containing donepezil in which the temperature of the aqueous phase containing surfactant is 20°C (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 20° C. and 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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.

Fig. 2 shows the results of observing the obtained microsphere droplets with an optical microscope.

Example 5. Preparation of PLGA microspheres containing donepezil in which the temperature of the aqueous phase containing surfactant is 25°C (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 25° C. and 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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.

Fig. 2 shows the results of observing the obtained microsphere droplets with an optical microscope.

Comparative Example 4. Preparation of PLGA microspheres containing donepezil in which the temperature of the aqueous phase containing surfactant is 30°C (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 30° C. at 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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.

Fig. 2 shows the results of observing the obtained microsphere droplets with an optical microscope.

Comparative Example 5. Preparation of PLGA microspheres containing donepezil in which the temperature of the aqueous phase containing surfactant is 35°C (microfluidic method)

100 mg of biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of dichloromethane to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 35° C. at 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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.

Fig. 2 shows the results of observing the obtained microsphere droplets with an optical microscope.

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 to prepare a dispersed phase containing donepezil, and then injected into a microfluidic chip at a flow rate of 0.01 mL/min. At this time, 1% polyvinyl alcohol aqueous solution was used as the aqueous phase (continuous phase), and simultaneously injected with the dispersed phase at a flow rate of 0.08 mL/min, microsphere droplets formed inside the microfluidic chip were stirred at a temperature of 20° C. and 150 rpm. It was obtained by dispersing in a 1% polyvinyl alcohol solution. The dichloromethane solvent was removed from the obtained microsphere droplets 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)

100 mg of a biodegradable polymer PLGA and 55 mg of donepezil base were dissolved in 1 mL of a dichloromethane solvent to prepare a dispersed phase containing donepezil. The prepared dispersed phase was poured into a 1% polyvinyl alcohol aqueous solution and stirred (4,000 rpm) using a homogenizer to form droplet-shaped microspheres. The dichloromethane solvent was removed from the obtained microsphere droplets 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 sustained-release PLGA microspheres containing donepezil according to the content

Experimental Example 1-1. Measurement of drug content and encapsulation rate of 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 then filtered through a 0.45 μm PVDF syringe filter. Thereafter, a standard curve was obtained using HPLC-UV equipment according to the same conditions as in Table 1 below, and quantitatively analyzed.

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 20µl

From the quantitative analysis, the content of the drug was calculated by the following [Equation 1], and the content of donepezil 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 drug content * 100

Drug content (%) Drug encapsulation rate (%) Manufacturing method Comparative Example 1 21.0 85.7 Solvent evaporation Comparative Example 2 28.2 79.4 Use double evaporation method Example 1 18.9 77.1 Microfluidic method Example 2 29.2 82.3 Microfluidic method

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

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

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

First, it was found that there is an advantage in that the initial elution is slow when the microfluidic method of the Example is used compared to the solvent evaporation method of the comparative example.

In addition, apart from this, compared with [Comparative Example 1] and [Example 1] in which the content of donepezil to be added is 65 mg, and [Comparative Example 2] and [Example 2] in which the content of donepezil to be introduced is 110 mg. As a result, when the principle of the manufacturing method is the same, it was confirmed that the elution rate and the elution pattern greatly differ depending on the content of donepezil.

In other words, when the principle of the manufacturing method is the same, the difference in dissolution rate of donepezil-containing microspheres is remarkable according to the content of donepezil, and the uniformity of donepezil content is essential in the production of sustained-release PLGA microspheres containing donepezil. It was confirmed that it should be. In particular, the higher the content of donepezil to be added, the more promoted the elution of donepezil.In the production of sustained-release PLGA microspheres containing a high content of donepezil, it was confirmed that content uniformity and production reproducibility for the same are more important requirements.

In addition, both Examples 1 and 2, which are PLGA microspheres containing donepezil, provided from the method of dispersing the microsphere droplets in an aqueous phase of 15 to 25°C containing a surfactant, despite the high content of the drug is higher than 15%. Unlike conventional PLGA microspheres, it was confirmed to have 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 showing sustained-release elution while containing a high content of donepezil by combining the production conditions of a water-phase temperature setting of 15 to 25°C and a microfluidic method. Confirmed.

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

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

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

The shape of the microspheres prepared in each Example and Comparative Example is as shown in FIG. 2. [Comparative Example 3] dispersed in an aqueous phase at 10°C had a problem in that agglomeration between droplets occurred, and the shape of the sustained-release PLGA microspheres containing donepezil obtained in all Examples and Comparative Examples except for this was uniform without occurrence of aggregation. One microsphere was observed.

From this, it was confirmed that in the production of donepezil-containing PLGA microspheres, an aqueous phase temperature of less than 15°C is not suitable as a production condition for the 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 then filtered through a 0.45 μm PVDF syringe filter. Thereafter, the HPLC column, operating conditions and calculation method were the same as those of [Experimental Example 1-1].

Table 4 shows the content of donepezil and the 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 occurred due to merging between the microsphere droplets, and thus the content and encapsulation rate were excluded from measurement (FIG. 1).

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

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

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

Experimental Example 3. Reproducibility analysis between batches 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 then filtered through a 0.45 μm PVDF syringe filter. Thereafter, the HPLC column, operating conditions and calculation method were the same as those 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 sustained-release PLGA microspheres containing donepezil prepared using a microfluidic method and a 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 mean value of drug encapsulation rate (%) = standard deviation / mean value * 100

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

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

When comparing [Comparative Examples 6 to 8] and [Examples 6 to 8], compared to the solvent evaporation method, the microfluidic method has a uniform encapsulation rate and a significantly lower error rate compared to the average encapsulation rate. It was confirmed that the slow-release PLGA microspheres containing pegyl were prepared.

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

That is, the PLGA microspheres containing donepezil of the present invention set the temperature range of the aqueous phase and the conditions of the microfluidic method to solve the problem of the content of donepezil, which is a problem of the existing donepezil PLGA microspheres, and have a sustained-release effect and uniformity. It was confirmed that it has a very excellent effect in pharmaceutical terms of securing reproducibility in manufacturing.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (15)

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

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