KR20210100986A - Method for preparing microspheres with high density and sustained release - Google Patents

Abstract

The present invention relates to a method for preparing high-density sustained-release microspheres, including the steps of: (a) dissolving a drug in an aqueous solution to prepare a first aqueous phase (W1); (b) dissolving a biodegradable polymer in an organic solvent to prepare an oil phase (O); (c) mixing and dispersing the first aqueous phase (W1) with the oil phase (O) to prepare W1/O emulsion; (d) mixing and dispersing the W1/O emulsion with a second aqueous phase (W2) containing an emulsifier to prepare W1/O/W2 emulsion; and (e) recovering microspheres from the W1/O/W2 emulsion, wherein the weight ratio of the first aqueous phase (W1) to the oil phase (O) is 0.04-0.5 : 1.

Description

Method for preparing microspheres with high density and sustained release

The present invention relates to a method for producing high-density sustained-release microspheres.

The present invention relates to a method for producing microspheres capable of continuously controlling drug release by encapsulating a drug in a carrier made of a biodegradable polymer.

Conventionally, as a general method for preparing sustained-release drug delivery system (DDS) formulations, coacervation method, emulsion phase separation method, encapsulation by spray drying, and solvent evaporation in organic or aqueous phase are known. Among these methods, the solvent evaporation method is the most used in the aqueous phase, and it is largely classified into a double emulsion evaporation method (W1/O/W2; Water/Oil/Water) and a single emulsion evaporation method (O/W2; Oil/Water).

In general, the W1/O/W2 method, which is mainly used for the encapsulation of water-soluble drugs such as peptides or proteins, disperses the drug-containing aqueous solution prepared by dissolving the drug in an aqueous solution in an organic solvent containing a biodegradable polymer to form a primary emulsion. (water in oil) and then dispersed in water.

In addition, the O/W2 method mainly used for encapsulation of fat-soluble drugs is a method of dissolving a drug and a biodegradable polymer together in an organic solvent or a mixture of organic solvents (oil), and then dispersing it in an aqueous phase.

In both of the above methods, the organic solvent is removed by extraction or evaporation while the polymer in the organic solvent phase is dispersed in the aqueous phase, so that the solubility of the polymer decreases, resulting in solidification, and as a result, microspheres are formed. In general, microspheres prepared by the W1/O/W2 method have a higher porosity than the microspheres prepared by the O/W2 method, so that the surface area is increased, and thus the initial release rate of the drug is relatively high.

Representative factors that determine the period of sustained drug release are determined by the chemical composition of the polymer, molecular weight, water affinity, composition of organic solvent, type and concentration of emulsifier or additive. and water affinity.

Polymers that are decomposed for a long period of time have problems such as hardly being released at any part in the beginning or in the middle. Therefore, it takes a lot of effort and time to produce sustained-release microparticles that can continuously release a drug for a required period (eg, 1 month, 2 months, 3 months, 6 months, etc.) with only one polymer. Therefore, the conventional manufacturing method is a method of mixing a polymer with a fast decomposition rate and a polymer with a slow decomposition rate and encapsulating a drug in them. It is not possible to accurately predict the amount of drug released from the microspheres encapsulated in the The reason is that two or more polymers with different properties exist on one microsphere. Due to the influence of degradation products derived from high-degradation polymers in vivo, the degradation of slow-decomposing polymers is faster than when they exist alone. tends to lose As a result, the in vivo release rate of the drug is also affected by the fast decomposition rate of the polymer, resulting in a result different from the average value of the release rate of the drug encapsulated in each polymer.

In order to overcome this disadvantage, a method of obtaining microspheres (e.g., U.S. Patent No. 4,897,268) from which the drug is continuously released for a desired period by mixing the microspheres obtained by encapsulating the drug in two or more types of polymers with different decomposition rates, respectively, in an appropriate ratio. Although introduced, this method has disadvantages in that it is difficult to manufacture two or more microspheres for commercialization of one formulation, and is economically inefficient.

On the other hand, in the case of polylactide-co-glycolides (PLGA) polymers, which are copolymers having different molar ratios of lactide and glycolide, as the molar ratio of lactide and its molecular weight increase, The decomposition rate is slowed down. Therefore, it is known that a polymer having a relatively higher lactide content or a higher molecular weight is used to prepare microspheres for sustained release of a drug for a longer period of time. Therefore, various attempts have been made to control the drug release rate using polylactide-co-glycolides (PLGA) polymers.

However, the size, shape, and drug release of microspheres manufactured using PLGA tend to be sensitively affected by various factors, resulting in low manufacturing efficiency and difficult to provide a uniform release effect of the drug over a long period of time. There are disadvantages. Therefore, in the case of microspheres developed so far, the characteristics required in this field are not sufficiently satisfied.

Korean Patent Registration No. 10-1172699

An object of the present invention is to provide a method for efficiently producing high-density sustained-release microspheres having a uniform shape using a biodegradable polymer.

In addition, an object of the present invention is to provide a method for producing high-density sustained-release microspheres capable of loading a drug with a high content and uniformly releasing the drug over a long period of 2 to 15 weeks.

the present invention

(a) dissolving the drug in an aqueous solution to prepare a first aqueous phase (W1);

(b) dissolving a biodegradable polymer in an organic solvent to prepare an oil phase (O);

(c) mixing and dispersing the first aqueous phase (W1) and the oil phase (O) to prepare a W1/O emulsion;

(d) mixing and dispersing the W1/O emulsion in a secondary aqueous phase (W2) containing an emulsifier to prepare a W1/O/W2 emulsion; and

(e) recovering the microspheres from the W1/O/W2 emulsion;

It provides a method for producing high-density sustained-release microspheres, characterized in that the weight ratio of the primary aqueous phase (W1) and the oil phase (O) is 0.04 to 0.5: 1.

The manufacturing method of the present invention provides a method for efficiently manufacturing high-density sustained-release microspheres having a uniform shape.

In addition, the preparation method of the present invention provides a method for preparing high-density sustained-release microspheres capable of loading a drug with a high content of 7% by weight or more and uniformly releasing the drug over 2 to 15 weeks.

1 is an SEM photograph of microspheres prepared in an embodiment of the present invention.
2 is a graph showing the dissolution test results for the microspheres prepared in Examples of the present invention.

the present invention

(a) dissolving the drug in an aqueous solution to prepare a first aqueous phase (W1);

(b) dissolving a biodegradable polymer in an organic solvent to prepare an oil phase (O);

(c) mixing and dispersing the first aqueous phase (W1) and the oil phase (O) to prepare a W1/O emulsion;

(d) mixing and dispersing the W1/O emulsion in a secondary aqueous phase (W2) containing an emulsifier to prepare a W1/O/W2 emulsion; and

(e) recovering the microspheres from the W1/O/W2 emulsion;

It relates to a method for producing high-density sustained-release microspheres, characterized in that the weight ratio of the primary aqueous phase (W1) and the oil phase (O) is 0.04 to 0.5: 1.

The weight ratio of the primary aqueous phase (W1) to the oil phase (O) is more preferably 0.08 to 0.3: 1, and even more preferably 0.2 to 0.3: 1.

If the ratio of the primary aqueous phase (W1) is less than 0.04, a disadvantage that a polymer may be precipitated may occur, and if it exceeds 0.5, a problem that a drug may be precipitated may occur, which is not preferable.

The oil phase (O) may further include an organic solvent and other conventional components, and may consist only of an organic solvent.

In one embodiment of the present invention, the biodegradable polymer concentration of the oil phase (O) in step (b) may be 5 to 20% by weight, preferably 10 to 15% by weight. If the concentration of the polymer is less than 5% by weight, the drug encapsulation rate may be lowered, and if it exceeds 20% by weight, the shape of the microspheres may become non-uniform. .

In one embodiment of the present invention, the microsphere may have a drug loading amount of 4 to 10% by weight, more preferably 6 to 10% by weight, even more preferably 7 to 9% by weight.

When the amount of the drug is less than 4% by weight, it is difficult to supply a sufficient drug, and when it exceeds 10% by weight, it is not preferable because it is difficult to form microspheres.

In one embodiment of the present invention, the aqueous solution in the first aqueous phase (W1) of step (a) may be at least one selected from sodium acetate aqueous solution, ammonium acetate aqueous solution, phosphate acetate aqueous solution, and sodium citrate aqueous solution. The concentration of the aqueous solution may be 0.5 to 3 w / v %, more preferably 0.7 to 1.5 w / v %.

In one embodiment of the present invention, the biodegradable polymer in step (b) is selected from the group consisting of polylactide (PLA), polyglycolide (PGA), and a copolymer of lactide and glycolide (PLGA). At least one selected from the group consisting of polylactide (PLA) and a copolymer of lactide and glycolide (PLGA) may be preferably used.

The copolymer of lactide and glycolide (PLGA) is a material whose biocompatibility is recognized because PLGA is finally decomposed into monomers of lactic acid and glycolic acid in the living body. It is well known and approved by the US FDA, and it is used as a sustained drug release formulation of LHRH homologue, which is already used as a treatment for prostate cancer, and human growth hormone, which is administered to patients with dwarfism. There are various types of PLGA according to the ratio of monomers lactic acid and glycolic acid, molecular weight, and physical properties such as water affinity, and each of them has the property of being decomposed in vivo for a short period of 2 weeks to a long period of several months.

In the copolymer of lactide and glycolide (PLGA), the molar ratio of lactide and glycolide is preferably 99.9:0.1 to 55:45, and the weight average molecular weight is 4,000 to 100,000, more preferably 10,000 to 60,000 may be preferably used.

Among the biodegradable polymers, polylactide (PLA) may have a weight average molecular weight of 6,000 to 25,000, more preferably 8,000 to 20,000.

In one embodiment of the present invention, the organic solvent in the oil phase (O) of step (b) is at least one selected from the group consisting of methylene chloride (DCM), ethyl acetate, acetonitrile, heptane, and hexane. may be used, and methylene chloride (DCM) may be preferably used.

In one embodiment of the present invention, the emulsifier in the secondary aqueous phase (W2) of step (d) is polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), sodium carboxymethyl cellulose (NaCMC), At least one selected from the group consisting of gelatin, polysorbate, polyethylene sorbitan monorate, and the like may be used. Among them, polyvinyl alcohol (PVA) may be preferably used, and the weight average molecular weight of PVA may be 30,000 to 200,000, more preferably 80,000 to 130,000.

The emulsifier may be included in an amount of 0.3 to 0.7% by weight, and more preferably in an amount of 0.4 to 0.6% by weight.

In one embodiment of the present invention, the secondary aqueous phase (W2) may further include at least one selected from the group consisting _{of NaCl, KCl, CaCl 2 and the like as a salt compound.} Among them, NaCl can be preferably used.

The salt compound may be included in an amount of 0.3 to 0.7 wt%, more preferably 0.4 to 0.6 wt%.

In one embodiment of the present invention, the drug in step (a) is an antidiabetic agent, an antiobesity agent, an anticancer agent, an antibiotic, an antipyretic agent, an analgesic, an anti-inflammatory agent, an expectorant, a sedative, a muscle relaxant, an epilepsy agent, an antiulcer agent, an antidepressant Anti-allergic agent, cardiac agent, antiarrhythmic agent, vasodilator, hypotensive diuretic, hyperlipidemia agent, anticoagulant, hemolytic agent, anti-tuberculosis agent, hormone, anesthetic antagonist, bone resorption inhibitor, bone formation promoter, angiogenesis inhibitor, physiologically active peptide , and at least one selected from the group consisting of proteins and the like may be used.

As the diabetes treatment agent, liraglutide may be preferably used.

In one embodiment of the present invention,

The dispersion in step (c) may be performed using, for example, a homogenizer or ultrasonic waves. At this time, the dispersion may be performed at 5,000 to 30,000 rpm, more preferably 8,000 to 12,000 rpm. At this time, the temperature is 5 to 40 ℃, more preferably carried out at room temperature, the dispersion time may be carried out for 0.5 to 5 minutes, more preferably 0.8 to 1.5 minutes.

The dispersion in step (d) may be performed using, for example, a homogenizer or ultrasound. At this time, the dispersion may be performed at 1,000 to 10,000 rpm, more preferably 2,000 to 6,000 rpm. At this time, the temperature may be carried out at 5 to 25 ℃, more preferably 12 to 17 ℃.

The step (e) includes (e1) an evaporation step of evaporating the organic solvent; (e2) centrifugation step; and (e3) freeze-drying; It may further include one or more steps selected from among. Preferably, all three steps may be performed.

In addition, after step (e1), the step of cooling the emulsion may be further performed.

After the (e2) centrifugation step, the step of washing the microspheres by a conventional method may be further performed. In addition, the microspheres may be dispersed in sugar alcohols such as mannitol and sorbitol for the lyophilization step after washing.

The (e1) evaporation step of evaporating the organic solvent may be performed using, for example, a magnetic stirrer. At this time, the stirring may be performed at 20 to 40 ℃ for 1 to 5 hours, but is not limited thereto.

The centrifugation step (e2) may be performed by a conventional method performed in this field, for example, may be performed at 2,000 to 3,000 rpm for 1 to 10 minutes.

The (e3) freeze-drying step may be performed by a conventional method performed in this field.

It goes without saying that each of the embodiments described above may be selectively or all combined. In addition, processes not described in the present invention may be performed by employing methods known in the art.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are provided to illustrate the present invention in more detail, and the scope of the present invention is not limited by the following examples. The following examples can be appropriately modified and changed by those skilled in the art within the scope of the present invention.

Examples 1-13: Preparation of emulsion for high-density long-acting injection

<Example 1>

A first aqueous phase (W1) was prepared by dissolving 0.16 g of liraglutide in 3.094 g of an aqueous sodium acetate solution having a concentration of 1 w/v%. An oil phase (O) was prepared by dissolving 1.840 g of PLGA (RESOMER® RG503H, M.W.: 24,000-38,000) in 34.96 g of methylene chloride (DCM). The primary aqueous phase (W1) was put into the oil phase (O) at room temperature, and dispersed for 1 minute with a homogenizer at 10,000 rpm to prepare a W1/O emulsion.

Next, the W1/O emulsion was put into a secondary aqueous phase (W2), which is an aqueous solution containing 5 w/v% of polyvinyl alcohol (PVA) and 5 w/v% of NaCl at 15° C., and a homogenizer at 4,000 rpm. ) was dispersed for 1 minute to prepare a W1/O/W2 emulsion.

Next, evaporation was performed while stirring the W1/O/W2 emulsion at a temperature of 34° C. using a magnetic stirrer at 300 rpm for 2 hours to remove the organic solvent.

Next, the emulsion having completed the evaporation was cooled while stirring at 350 rpm for 30 minutes at a temperature of 20° C. using a magnetic stirrer, and then centrifuged at 2500 rpm for 5 minutes to separate microspheres.

Next, the separated microspheres were washed 5 times with distilled water and freeze-dried to prepare microspheres.

<Examples 2 to 13>

Microspheres were prepared in the same manner as in Example 1, but specific manufacturing conditions were performed according to Tables 1 and 2 below to prepare microspheres.

Test Example 1: Size measurement and shape evaluation of microspheres

The size of the microspheres prepared in Examples 1 to 13 was measured by photographing with SEM. The measurement results are shown in Table 3 below. The shape of the microspheres was the same as the SEM photograph shown in FIG. 1 .

Example 1 Example 3 Example 6 Example 8 Example 10 Example 13 average diameter
(μm) 13.5 11.5 15.1 34.2 19.1 25.4

Test Example 2: Evaluation of the amount of drug contained in microspheres

The amount of drug encapsulation was evaluated by a high-performance liquid chromatography analysis method, and is shown in Table 4 below.

Example 1 Example 3 Example 6 Example 8 Example 10 Example 12 drug loading
(w/w%) 7.6 6.4 6.8 7 7.2 7.3

Test Example 4: Evaluation of loading efficiency

The drug loading efficiency was evaluated by a high-performance liquid chromatography analysis method, and the results are shown in Table 5 below.

Example 1 Example 3 Example 6 Example 8 Example 10 Example 12 drug loading rate
(w/w%) 95.3 80.2 85.1 87.5 90.2 91.5

Test Example 4: In vitro drug release test

An in vitro release test was performed on the microspheres prepared in Examples 1 to 13 of the present invention. Each 5 mg of lyophilized microspheres were dispersed in 13 test tubes each containing 0.033 M phosphate buffer (pH 7.0), and then released at 37±1°C. The test days were 0, 25, 1, 4, 7, 14, 21, and 28, and for each test day, samples were collected from 5 test tubes, and the microspheres obtained by centrifugation were treated with a methylene chloride/acetate buffer (1:1, v/ The amount of liraglutide transferred to the acetate layer by extraction with v) was determined by HPLC. At this time, the analysis conditions are shown in FIG. 2 by measuring at a flow rate of 1.0 ml/min at 280 nm using a 28% aqueous solution of acetonitrile containing 0.1% trifluorinated acetic acid as a mobile phase.

Claims (15)

(a) dissolving the drug in an aqueous solution to prepare a first aqueous phase (W1);
(b) dissolving a biodegradable polymer in an organic solvent to prepare an oil phase (O);
(c) mixing and dispersing the first aqueous phase (W1) and the oil phase (O) to prepare a W1/O emulsion;
(d) mixing and dispersing the W1/O emulsion in a secondary aqueous phase (W2) containing an emulsifier to prepare a W1/O/W2 emulsion; and
(e) recovering the microspheres from the W1/O/W2 emulsion;
The method for producing high-density sustained-release microspheres, characterized in that the weight ratio of the primary aqueous phase (W1) to the oil phase (O) is 0.04 to 0.5: 1. According to claim 1,
The method for producing high-density sustained-release microspheres, characterized in that the biodegradable polymer concentration of the oil phase (O) in step (b) is 5 to 20% by weight. 3. The method of claim 2,
The method for producing high-density sustained-release microspheres, characterized in that the microspheres contain 4 to 10% by weight of the drug. According to claim 1,
The dispersion in step (c) is made at a stirring speed of 5,000 to 30,000 rpm,
The dispersion of step (d) is a method for producing high-density sustained-release microspheres, characterized in that made at a stirring speed of 2,000 to 50,000 rpm. According to claim 1,
The step (e) includes (e1) an evaporation step of evaporating the organic solvent; (e2) centrifugation step; and (e3) freeze-drying; Method for producing high-density sustained-release microspheres, characterized in that it further comprises one or more steps selected from. According to claim 1,
The aqueous solution in the first aqueous phase (W1) of step (a) is a method for producing high-density sustained-release microspheres, characterized in that at least one selected from sodium acetate aqueous solution, ammonium acetate aqueous solution, phosphate acetate aqueous solution, and sodium citrate aqueous solution. According to claim 1,
The biodegradable polymer of step (b) is a high-density sustained-release, characterized in that at least one selected from the group consisting of polylactide (PLA), polyglycolide (PGA), and a copolymer of lactide and glycolide (PLGA). A method for producing exuded microspheres. According to claim 1,
In the oil phase (O) of step (b), the organic solvent is at least one selected from the group consisting of methylene chloride (DCM), ethyl acetate, acetonitrile, heptane, and hexane. Method for producing high-density sustained-release microspheres . According to claim 1,
In the secondary aqueous phase (W2) of step (d), the emulsifier is selected from the group consisting of polyvinyl alcohol (PVA), sodium carboxymethyl cellulose (NaCMC), gelatin, polysorbate, and polyethylene sorbitan monorate. A method for producing high-density sustained-release microspheres, characterized in that more than one species. 10. The method of claim 9,
The method for producing high-density sustained-release microspheres, characterized in that the emulsifier in the secondary aqueous phase (W2) of step (d) is comprised in an amount of 0.3 to 0.7% by weight. 11. The method of claim 10,
The secondary aqueous phase (W2) as a salt compound NaCl, KCl and CaCl ₂ Method for producing high-density sustained-release microspheres, characterized in that it further comprises 0.3 to 0.7% by weight of at least one selected from the group consisting of. 8. The method of claim 7,
The biodegradable polymer is a copolymer of lactide and glycolide (PLGA), and the copolymer of lactide and glycolide has a lactide and glycolide molar ratio of 99.9:0.1 to 55:45 and a weight average molecular weight. Method for producing high-density sustained-release microspheres, characterized in that 4,000 to 100,000. 8. The method of claim 7,
The biodegradable polymer is polylactide (PLA), and the weight average molecular weight of the polylactide is 6,000 to 25,000. According to claim 1,
The drug of step (a) is an anti-diabetic agent, an anti-obesity agent, an anticancer agent, an antibiotic, an antipyretic agent, an analgesic, an anti-inflammatory agent, an expectorant, a sedative, a muscle relaxant, an epilepsy agent, an anti-ulcer agent, an antidepressant, an anti-allergic agent, a cardiac agent, an antiarrhythmic agent , vasodilators, hypotensive diuretics, hyperlipidemia agents, anticoagulants, hemolytic agents, anti-tuberculosis agents, hormones, anesthetic antagonists, bone resorption inhibitors, bone formation promoters, angiogenesis inhibitors, physiologically active peptides, and proteins 1 selected from the group consisting of A method for producing high-density sustained-release microspheres, characterized in that more than one species. According to claim 1,
The method for producing high-density sustained-release microspheres, characterized in that the drug of step (a) is liraglutide.

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