KR20220152122A - mPEG-PCL block copolymer nanoparticles comprising hydrophobic drug and manufacturing method thereof - Google Patents

Abstract

The present invention relates to an effective method for manufacturing biodegradable polymer nanoparticles carrying a hydrophobic drug and controlling the drug release rate according to the size of the nanoparticles. The biodegradable polymer is a block copolymer of an A-B structure, A represents polyethylene glycol or a derivative thereof, and B represents polycaprolactone. During the preparation of the drug-loaded nanoparticles, the size of the nanoparticles can be effectively controlled by controlling the process temperature and stirring speed. Accordingly, the drug release rate of the nanoparticles can also be controlled.

Description

mPEG-PCL block copolymer nanoparticles containing hydrophobic drug and manufacturing method thereof {mPEG-PCL block copolymer nanoparticles comprising hydrophobic drug and manufacturing method thereof}

The present invention relates to a method for preparing biodegradable polymer nanoparticles carrying a hydrophobic drug and a method for controlling a drug release rate according to the size of the nanoparticles.

A drug delivery method using a biodegradable polymer carrying a drug has received a lot of attention because it has fewer side effects than traditional drug delivery methods and can effectively deliver a drug to a diseased area, and active research is still ongoing. In particular, studies using block copolymers of A-B, A-B-A, or B-A-B structures have been mainly conducted (A is a hydrophilic polymer block and B is a hydrophobic polymer block). Due to this chemical structure, biodegradable polymers form nanoparticles or micelle structures in aqueous solutions. At this time, the hydrophobic block forms the core of the nanoparticle and the hydrophilic block forms a corona shape. Since the hydrophobic core of the nanoparticle is suitable for carrying a hydrophobic drug and can slowly release the drug, research into carrying various kinds of drugs is being conducted.

In order to apply a biodegradable polymeric drug delivery system to the human body, it is very important to precisely control the drug release rate. It is known that the release rate of a drug is affected by various conditions such as chemical structure of a polymer and drug, molecular weight, drug-polymer interaction, polymer concentration, drug concentration, particle size, temperature, and pH. Temperature and pH cannot be arbitrarily changed when used in the human body, and the drug release rate can be controlled through the remaining conditions. In particular, the size of the particles can have a very large effect on drug release, and the smaller the particle size, the faster the drug release. This is because the drug can diffuse more easily as the contact area with the outside of the particle (blood or body fluid) widens, and the drug can be easily released as the decomposition rate of the biodegradable polymer increases. Therefore, a method for effectively controlling the particle size is very important in designing a drug delivery system.

Commonly known methods for preparing drug-loaded nanoparticles include a solvent displacement method, a cosolvent evaporation method, and a dialysis method. An appropriate method is selected according to the chemical structure of the polymer used, molecular weight, and type of drug. However, since the above methods perform the process in a state where the polymer is dissolved in an organic solvent, an additional process of removing the organic solvent after the particles are formed is absolutely necessary. In order to remove the organic solvent, dialysis or vacuum evaporation is required, so the process is prolonged and the drug may be slowly released while the organic solvent is removed, so there may be a limit to adjusting the required amount of the drug loaded. In addition, it is difficult to make a high-concentration nanoparticle dispersion, and an additional concentration process is required to increase the concentration. In addition, it is not easy to control the size of nanoparticles by conventional methods. In general, rather than controlling the size through process conditions (time, temperature, stirring speed, etc.), the particle size is controlled by adjusting the molecular weight of the input polymer or the ratio of hydrophilic/hydrophobic polymer blocks. As a result, in order to control the particle size, it is inconvenient to synthesize a new polymer every time.

Korean Patent Application No. 10-2001-0025860 relates to a method for preparing polymeric micelles by phase separation, and provides a polymeric micelle composition using a hydrophilic block of polyalkylene glycol and a hydrophobic block such as polycaprolactone. However, research or description of the technique of controlling micelles through temperature or stirring speed control using polyethylene glycol and polycaprolactone and the resulting drug release effect has not been disclosed.

Accordingly, the present inventors have made intensive efforts to provide a method for reducing the nanoparticle manufacturing process time and producing the particle size under desired conditions, and as a result, using polyethylene glycol or a derivative thereof and polycaprolactone to control the temperature and stirring speed In the case of manufacturing, it was confirmed that the size of the nanoparticles carrying the drug can be effectively controlled and the release rate of the drug can be controlled accordingly, and the present invention was completed.

Accordingly, an object of the present invention is to provide a method for preparing drug-loading nanoparticles comprising reacting polyethylene glycol or a derivative thereof with polycaprolactone by controlling temperature and stirring speed.

The present invention is i) polyethylene glycol or a derivative thereof; and lactones; reacting;

ii) obtaining a polymer by removing unreacted materials from step i) and precipitating the reactants;

iii) obtaining a drug-supported polymer film by mixing the polymer obtained in step ii) with the drug; and

iv) dissolving the film obtained in step iii) in water and stirring at 50° C. to 100° C. at 100 to 2,000 rpm to obtain nanoparticles;

It provides a method for producing nanoparticles for carrying drugs comprising a.

According to a preferred embodiment of the present invention, methanesulfonic acid, triflic acid, p-toluenesulfonic acid, benzenesulfonic acid as catalysts in step i) , Hydrochloric acid, sulfuric acid, and phosphoric acid may be added at least one selected from the group consisting of.

According to a preferred embodiment of the present invention, after the reaction of step i), triethylamine, diethylamine, 4-dimethylaminopyridine, 1 Adding at least one selected from the group consisting of ,8-bis (dimethylamino) naphthalene (1,8-Bis (dimethylamino) naphthalene) and N, N-diisopropylethylamine (N, N-Diisopropylethylamine) can

According to a preferred embodiment of the present invention, the polymer of step ii) may have a weight average molecular weight of 2,100 to 2,600 g/mol.

According to a preferred embodiment of the present invention, the drug of step iii) may be a hydrophobic drug.

According to a preferred embodiment of the present invention, the average diameter of the nanoparticles may be 10 to 200 nm.

In addition, the present invention provides biodegradable polymer nanoparticles containing a hydrophobic drug prepared by the above preparation method.

According to a preferred embodiment of the present invention, the hydrophobic drug may be at least one selected from the group consisting of hydrophobic steroids, anti-inflammatory drugs and anticancer drugs.

In addition, the present invention provides a composition for a drug release system using the nanoparticles.

The nanoparticle manufacturing method of the present invention is short in process time without using an organic solvent, can minimize drugs that are inevitably released during the process, and can control the concentration or size of nanoparticles according to the temperature and stirring speed. Drug-loaded nanoparticles can be prepared.

1 shows the results of gel permeation chromatography (GPC) of the mPEG-PCL block copolymer of the present invention. It was confirmed that the number average molecular weight was 2,244 g/mol, the weight average molecular weight was 2,375 g/mol, and the degree of dispersion was 1.06.
Figure 2 (A) shows the result of measuring the particle size distribution of the mPEG-PCL block copolymer of the present invention prepared at a stirring speed of 100 rpm by dynamic light scattering (DLS). When produced at 100 rpm, the average diameter of the produced particles was 105.8 nm.
(B) shows the result of measuring the particle size distribution of the mPEG-PCL block copolymer of the present invention prepared at a stirring speed of 200 rpm by dynamic light scattering (DLS). When manufacturing at 200 rpm, it was confirmed that the average diameter decreased to 73.4 nm.
(C) shows the result of measuring the particle size distribution of the mPEG-PCL block copolymer of the present invention prepared at a stirring speed of 500 rpm by dynamic light scattering (DLS). When prepared at 500 rpm, it was confirmed that the particle size became relatively uniform and small, and the average diameter was 65.2 nm.
(D) shows the particle size distribution measured by dynamic light scattering (DLS) of the mPEG-PCL block copolymer of the present invention prepared at a stirring speed of 1,000 rpm. When prepared at 1,000 rpm, the particle size became very uniform and smaller, and it was confirmed that the average diameter was 64.0 nm.
3 shows a standard curve of budesonide, one of the steroid agents, measured using high performance liquid chromatography (HPLC). Standards for the standard curve were prepared by diluting 1.0 mg/mL concentration by two times. A standard curve was obtained using the absorbance of the UV spectrum for the peak of the chromatogram for the measured standard solution. Based on this standard curve, the drug release rate of the nanoparticles of [Fig. 7] was compared and measured.
4 shows a standard curve of paclitaxel, one of the anticancer agents, measured using high performance liquid chromatography (HPLC). Standards for the standard curve were prepared by diluting 1.0 mg/mL concentration by two times. A standard curve was obtained using the absorbance of the UV spectrum for the peak of the chromatogram for the measured standard solution. Based on this standard curve, the drug release rate of the nanoparticles of [FIG. 8] was compared and measured.
5 shows a standard curve of sulfasalazine, one of the anti-inflammatory agents, measured using high performance liquid chromatography (HPLC). Standards for the standard curve were prepared by diluting 1.0 mg/mL concentration by two times. A standard curve was obtained using the absorbance of the UV spectrum for the peak of the chromatogram for the measured standard solution. Based on this standard curve, the drug release rate of the nanoparticles of [FIG. 9] was compared and measured.
Figure 6 shows the results of differential scanning calorimetry (DSC) for observing changes in thermal properties of the mPEG-PCL block copolymer of the present invention. The heating and cooling rates were measured in a nitrogen atmosphere at 5°C per minute.
Figure 7 shows the drug release rate (n = 3) of the mPEG-PCL block copolymer loaded with budesonide, one of the steroid agents. Error bars show standard deviation between samples. When preparing the nanoparticles, the temperature was slowly cooled from 80 ° C to room temperature. The stirring speed was 100, 200, 500, and 1,000 rpm.
Figure 8 shows the drug release rate (n = 3) of the mPEG-PCL block copolymer loaded with paclitaxel, one of the anticancer drugs. Error bars show standard deviation between samples. When preparing the nanoparticles, the temperature was slowly cooled from 80 ° C to room temperature. The stirring speed was 100, 200, 500, and 1,000 rpm.
Figure 9 shows the drug release rate (n = 3) of the mPEG-PCL block copolymer loaded with sulfasalazine, one of the anti-inflammatory drugs. Error bars show standard deviation between samples. When preparing the nanoparticles, the temperature was slowly cooled from 80 ° C to room temperature. The stirring speed was 100, 200, 500, and 1,000 rpm.
10 shows a schematic diagram of the system in which the rate of drug release was measured. 200 μl of the drug-loaded nanoparticle dispersion was placed on a transwell membrane and allowed to gel. Then, a buffer solution was filled at the bottom, and the concentration of the released drug was measured by collecting the buffer solution at predetermined intervals.
11 shows photographs of mPEG-PCL block copolymers of the present invention prepared at 50 and 100 rpm. In the case of the hydrogel prepared at 50 rpm, polymer lumps that are not dispersed and aggregated on the bottom surface can be confirmed.
FIG. 12 shows photographs of results obtained by slowly cooling the nanoparticles from 45° C., 50° C., and 55° C. to room temperature.

The present invention relates to an effective method for preparing biodegradable polymer nanoparticles carrying a hydrophobic drug and controlling the drug release rate according to the particle size. The biodegradable polymer is a block copolymer of A-B structure, A represents polyethylene glycol or a derivative thereof, and B represents polycaprolactone. During preparation of the drug-loaded nanoparticles, the size of the nanoparticles can be effectively controlled by controlling the process temperature and stirring speed, and the drug release rate can also be controlled according to the size of the nanoparticles. In addition, since the emulsification process was performed without an organic solvent, a dialysis process is not required.

Therefore, the present invention provides i) polyethylene glycol or a derivative thereof; and lactones; reacting;

ii) obtaining a polymer by removing unreacted materials from step i) and precipitating the reactants;

iii) obtaining a drug-supported polymer film by mixing the polymer obtained in step ii) with the drug; and

iv) dissolving the film obtained in step iii) in water and stirring at 50° C. to 100° C. at 100 to 2,000 rpm to obtain nanoparticles;

It is possible to provide a method for preparing nanoparticles for carrying drugs comprising a.

The lactone may be caprolactone.

According to a preferred embodiment of the present invention, methanesulfonic acid, triflic acid, p-toluenesulfonic acid, benzenesulfonic acid as catalysts in step i) , Hydrochloric acid, sulfuric acid, and phosphoric acid may be added at least one selected from the group consisting of.

According to a preferred embodiment of the present invention, after the reaction of step i), triethylamine, diethylamine, 4-dimethylaminopyridine, 1 To add at least one selected from the group consisting of ,8-bis (dimethylamino) naphthalene (1,8-Bis (dimethylamino) naphthalene) and N, N-diisopropylethylamine (N, N-Diisopropylethylamine) can More preferably, to terminate the reaction, triethylamine, diethylamine, 4-dimethylaminopyridine, 1,8-bis (dimethylamino) naphthalene (1,8-Bis (dimethylamino)naphthalene) and N,N-diisopropylethylamine (N,N-Diisopropylethylamine) may be added.

According to a preferred embodiment of the present invention, the polymer of step ii) may have a weight average molecular weight of 2,100 to 2,600 g/mol. More preferably, the weight average molecular weight may be 2,200 to 2,500 g/mol. The polymer may have a number average molecular weight of 2,000 to 2,500 g/mol, more preferably 2,100 to 2,400 g/mol.

According to a preferred embodiment of the present invention, the drug of step iii) may include all hydrophobic drugs commonly used as hydrophobic drugs, but are preferably selected from the group consisting of hydrophobic steroids, anti-inflammatory drugs and anticancer drugs. It may be any one or more, more preferably budesonide, dexamethasone, betamethasone, mometasone, fluticasone, paclitaxel, docetaxel, It may be one or more selected from the group consisting of sulfasalazine, tacrolimus, and doxorubicin.

Nanoparticles can be obtained by dissolving the film obtained in step iv) in an aqueous solution and stirring at 50 ° C. to 100 ° C. at 100 to 2,000 rpm, more preferably at 100 to 1,000 rpm to obtain nanoparticles. have. The aqueous solution may be deionized water or a buffer solution.

According to a preferred embodiment of the present invention, the average diameter of the nanoparticles may be 10 to 200 nm. Preferably, the average diameter of the nanoparticles may be 50 to 200 nm, more preferably 50 to 150 nm.

In addition, the present invention may provide a hydrophobic drug-supported biodegradable polymer nanoparticle prepared by the above preparation method or a composition for a drug release system using the nanoparticle.

Since the hydrophobic drug, the biodegradable polymer, and the nanoparticle are the same as the concept used in the method for preparing the drug-loading nanoparticle, the description is replaced with the above description.

The 'drug release system' of the present invention is a system that delivers a drug with therapeutic efficacy to a necessary part of the body, and efficiently delivers the drug to the tissue that needs the drug and the required amount of the drug. , compounding methods or drug formulations.

Hereinafter, examples will be described in detail in order to specifically describe the present specification. However, the embodiments according to the present specification may be modified in many different forms, and the scope of the present specification is not construed as being limited to the embodiments described below. The embodiments herein are provided to more completely explain the present specification to those skilled in the art.

Preparation of methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) block copolymer Methoxy polyethylene glycol (mPEG, 3.75 g, average molecular weight: 750 g/mol), caprolactone (ε-caprolactone, 8.31 ml), dichloro Methane (dichloromethane, 10 ml) was put into a well-dried 50 ml round flask and purged with nitrogen at room temperature. The solution was stirred using a magnetic bar, and when mPEG was completely dissolved, methanesulfonic acid (0.163 ml) as a catalyst was added. Polymerization proceeded for 2 hours, and triethylamine (0.39 ml) was added to terminate the reaction, followed by stirring for 30 minutes. To remove unreacted monomers and catalysts and to precipitate polymers, the reaction solution was placed in ether (diethyl ether, 180 ml). The precipitated polymer was filtered with filter paper and dried under reduced pressure to obtain a pure polymer. The molecular weight of the synthesized polymer was measured using gel permeation chromatography (GPC).

As a result, as shown in [Figure 1], the measured number average molecular weight was 2,244 g/mol, the weight average molecular weight was 2,375 g/mol, and the degree of dispersion (weight average molecular weight/number average molecular weight, poly dispersity index, PDI) was 1.06. was able to confirm that

Measurement of particle size distribution of mPEG-PCL nanoparticles

1 g of the methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) block copolymer synthesized in <Example 1> and 9 ml of deionized water were added thereto. This sample was placed in a constant temperature water bath set at 80°C and heated for 5 minutes. The sample was taken out of the constant temperature water bath, cooled slowly at room temperature, and stirred using a magnetic bar to prepare nanoparticles. The stirring speed was 100, 200, 500, and 1,000 rpm.

As a result, as shown in [Fig. 2], the particle size distribution of the mPEG-PCL block copolymer was measured by dynamic light scattering. According to the stirring speed of 100, 200, 500, and 1,000 rpm, the average particle size was measured to be 105.8 nm, 73.4 nm, 65.2 nm, and 64.0 nm, respectively. The following [Table 1] summarizes the particle size distribution of the mPEG-PCL block copolymer according to the stirring speed. D10, D50, and D90 represent sizes at the 10%, 50%, and 90% percentiles, respectively, in the analyzed particle size distribution.

stirring speed average diameter D10 D50 D90 100 rpm 105.8 nm 78.7 nm 94.7 nm 133.2 nm 200 rpm 73.4 nm 53.0 nm 65.2 nm 92.5 nm 500 rpm 65.2 nm 47.7 nm 57.5 nm 83.0 nm 1000 rpm 64.0 nm 46.3 nm 57.1 nm 82.2 nm

Preparation of budesonide-loaded mPEG-PCL nanoparticles

1 g of the methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) block copolymer synthesized in <Example 1> and budesonide (10 mg) were dissolved in 15 ml of acetone. Foreign substances in the solution were removed using a syringe filter (PTFE, 0.45 μm). Acetone was removed using a rotary evaporator to obtain a budesonide-supported mPEG-PCL film. 9 ml of deionized water was added thereto. This sample was placed in a constant temperature water bath set at 80°C and heated for 5 minutes. The sample was taken out of the constant temperature water bath, cooled slowly at room temperature, and stirred using a magnetic bar to prepare nanoparticles. The stirring speed was 100, 200, 500, and 1,000 rpm.

Preparation of mPEG-PCL nanoparticles loaded with paclitaxel

1 g of the methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) block copolymer synthesized in <Example 1> and 10 mg of paclitaxel were dissolved in 15 ml of acetonitrile. Foreign substances in the solution were removed using a syringe filter (PTFE, 0.45 μm). Acetonitrile was removed using a rotary evaporator to obtain an mPEG-PCL film supported with paclitaxel. 9 ml of deionized water was added thereto. This sample was placed in a constant temperature water bath set at 80°C and heated for 5 minutes. The sample was taken out of the constant temperature water bath, cooled slowly at room temperature, and stirred using a magnetic bar to prepare nanoparticles. The stirring speed was 100, 200, 500, and 1,000 rpm.

Preparation of mPEG-PCL nanoparticles loaded with sulfasalazine

1 g of the methoxy polyethylene glycol (mPEG)-polycaprolactone (PCL) block copolymer synthesized in <Example 1> and sulfasalazine (10 mg) were dissolved in 15 ml of tetrahydrofuran. Foreign substances in the solution were removed using a syringe filter (PTFE, 0.45 μm). Tetrahydrofuran was removed using a rotary evaporator to obtain an mPEG-PCL film supported with sulfasalazine. 9 ml of deionized water was added thereto. This sample was placed in a constant temperature water bath set at 80°C and heated for 5 minutes. The sample was taken out of the constant temperature water bath, cooled slowly at room temperature, and stirred using a magnetic bar to prepare nanoparticles. The stirring speed was 100, 200, 500, and 1,000 rpm.

Drug release from mPEG-PCL nanoparticles

200 μl of the drug-loaded mPEG-PCL nanoparticle dispersion solution (10% w/w) obtained in <Example 3> to <Example 5> was put into a transwell and heated to 37° C. with the lid closed. It was put in a set oven and gelled for 1 hour. After gelation, 1 ml of the drug release solution was put out of the transwell. The drug release solution was a mixed solution of phosphate buffer saline (PBS) at pH 7.4 and 0.5 wt% of Tween 80 (FIG. 11). The drug-releasing solution was collected at designated times, and 1 ml of the drug-releasing solution was added thereto. The concentration of the drug contained in the sampled solution was measured using HPLC, and the released concentration was calculated by comparing with a standard curve (Figs. 3 to 5).

As a result, it was confirmed that the stirring speed also affects the drug release rate of the nanoparticles, as shown in [FIGS. 7] to [FIG. 9]. The lower the stirring speed, the slower the drug release rate, and the faster the stirring speed, the faster the drug release rate. Compared to the results of [Fig. 2], the larger the particle size, the slower the release, and the smaller the particle size, the faster the release. Overall, it was confirmed that the agitation speed and temperature affect the drug release rate in the preparation of drug-loaded nanoparticles.

[Comparative Example 1]

Preparation of mPEG-PCL nanoparticles under conditions of 50 rpm or less

An mPEG-PCL nanoparticle dispersion solution (10% w/w) was prepared as described in <Example 2> at a stirring speed of 50 rpm. [Figure 11] is a photograph of the mPEG-PCL nanoparticle dispersion solution prepared at 50 and 100 rpm. The vials were laid on the floor and photographs were taken from the top and side views. When prepared at 50 rpm, mPEG-PCL block copolymer lumps that were not dispersed due to insufficient stirring speed were confirmed at the corner of the vial. On the other hand, when prepared at 100 rpm, it was confirmed that all nanoparticle dispersion solutions were made without the copolymer remaining on the bottom.

[Comparative Example 2]

Preparation of mPEG-PCL nanoparticles under conditions of 55 ° C or less

An mPEG-PCL nanoparticle dispersion solution (10% w/w) was prepared at a stirring speed of 500 rpm and a heating temperature of 45 °C, 50 °C, and 55 °C (see Example 2). [Figure 12] shows nanoparticle dispersion solutions prepared at each temperature. In the case of the sample prepared at 45 ℃, the turbidity is high and the turbidity decreases as the temperature increases. The reason for the high turbidity is that the aggregation of the particles is severe and the drug is not properly contained in the nanoparticles. The reason for this phenomenon is related to the melting point of mPEG-PCL. According to [Figure 5], the melting point of mPEG-PCL comes out at about 37 ℃ and 45 ℃ two points, the former is the melting point of the mPEG block, the latter is the melting point of the PCL block. In order to prepare a nanoparticle dispersion solution containing a drug, the process must be performed in a state in which the drug is sufficiently solubilized. mPEG-PCL is used as a drug carrier and solubilizer at the same time, but it is difficult to properly dissolve the drug because mPEG-PCL does not completely melt at low temperatures. Therefore, in order to prepare well-dispersed nanoparticles, it would be appropriate to prepare them above the melting point of mPEG-PCL (+ 10 °C or higher).

Claims (10)

i) polyethylene glycol or a derivative thereof; and lactones; reacting;
ii) obtaining a polymer by removing unreacted materials from step i) and precipitating the reactants;
iii) obtaining a drug-supported polymer film by mixing the polymer obtained in step ii) with the drug; and
iv) dissolving the film obtained in step iii) in water and stirring at 50° C. to 100° C. at 100 to 2,000 rpm to obtain nanoparticles;
A method for producing nanoparticles for carrying drugs comprising a.
The method of claim 1, wherein methanesulfonic acid, triflic acid, p-toluenesulfonic acid, benzenesulfonic acid, hydrochloric acid as catalysts in step i) acid), sulfuric acid, and phosphoric acid.
The method of claim 1, after the reaction of step i), triethylamine, diethylamine, 4-dimethylaminopyridine, 1,8-bis Nano, characterized by adding at least one selected from the group consisting of (dimethylamino) naphthalene (1,8-Bis (dimethylamino) naphthalene) and N, N-diisopropylethylamine (N, N-Diisopropylethylamine) Particle manufacturing method.
The method of claim 1, wherein the polymer of step ii) has a weight average molecular weight of 2,100 to 2,600 g/mol.
The method of claim 1, wherein the drug of step iii) is a hydrophobic drug.
The method of claim 1, wherein the nanoparticles have an average diameter of 10 to 200 nm.
Biodegradable polymeric nanoparticles bearing a hydrophobic drug prepared by the method of claim 1.
The nanoparticles according to claim 7, wherein the nanoparticles have an average diameter of 10 to 200 nm.
The nanoparticles according to claim 7, wherein the hydrophobic drug is at least one selected from the group consisting of hydrophobic steroids, anti-inflammatory drugs, and anti-cancer drugs.
A composition for a drug release system using the nanoparticles of claim 7.

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