KR102442807B1 - Initial burst controlled depot composition and a method thereof - Google Patents

Abstract

The present invention relates to a depot composition comprising a hydrophobic amino acid inside microspheres, and it is possible to control the initial release of an excessive amount of an active ingredient in a formulation containing a biodegradable polymer and an active ingredient, and has excellent suspending ability, so that the general user Even when used as an injection, the effect of the active ingredient can be obtained uniformly and continuously.

Description

Initial release controlled depot composition and manufacturing method thereof

The present invention relates to a depot composition, specifically, by including a hydrophobic amino acid inside the microspheres to control the initial release, and provide high user convenience through excellent resuspension properties.

Depot is one of the injection formulations that make the drug last for a long time, and it is a formulation that can be mainly used for long-term administration of hormones and the like. Depot formulations, which are sustained-release formulations, have the advantage of minimizing the number of injections to a patient, but it is not easy to implement constant and continuous release of the drug during the entire release period after injection. In particular, if the initial release of the drug in the depot in excess is high at the beginning of the injection, side effects due to the excess drug may occur, and the sustained effect of the drug for a specified period may decrease, so controlling the initial release from the depot is difficult. very important.

GLP-1 agonists, such as liraglutide and semaglutide, were first developed to lower blood sugar in diabetic patients, and were used in the treatment of diabetes and obesity by confirming that these drugs are also effective in reducing weight in obese patients. It is a drug that becomes It is an analog of the incretin hormone secreted by the intestines, and increases the amount of insulin secreted after a meal, and increases the time for excretion of the contents of the stomach so that the food passes slowly from the stomach to the small intestine. In addition, it can help lower blood sugar and reduce weight through various actions that suppress appetite by acting on the central nervous system.

However, when the body is exposed to an excessive amount of liraglutide, side effects such as nausea, diarrhea, increased heart rate, hypoglycemia or headache may appear, so the control of initial release from depot agents containing liraglutide as an active ingredient is more difficult. one of the important tasks.

On the other hand, Japanese Patent No. 5681626 discloses that a sustained-release formulation containing microspheres and phospholipid components in which a lipid component containing a sterol and a polylactide-co-glycolide (PLGA) polymer are bound can control the release rate. start However, in the case of lipids, it does not exhibit a sufficient effect to inhibit the initial release, and there is a disadvantage in that the properties of the microspheres are poor due to their strong hydrophobic properties. In addition, since the modification of the polylactide-co-glycolide (PLGA) polymer forming microspheres causes difficulties in permitting the use of the drug, there is a limit to the actual use of the composition by applying the composition to a depot agent.

In addition, in the case of a depot agent, in consideration of the storage safety of the drug, the microspheres may be stored in a freeze-dried state, and may be injected by suspending in water for injection. In this case, if the suspendability of the microspheres is lowered, less than a prescribed dose of the drug may be administered, and it is difficult to see a sufficient effect of the drug. In addition, due to the low suspendability, aggregation and sedimentation of the microspheres may be formed, and the injection may be difficult due to the clogging of the injection needle during injection.

As such, the conventional sustained-release microspheres have limitations in providing a depot agent with sufficiently controlled initial release, and therefore, development of a depot composition having appropriate resuspension properties and controlled initial release properties is still required.

1. Japanese Patent No. 5681626

The present invention is to solve the above problems, and an object of the present invention is to provide a depot composition having excellent resuspension ability and suppressed excessive initial release of a drug and a method for preparing the same.

As a result of the inventors of the present invention trying to develop a depot composition that can control the excessive initial release of the drug, the initial release of the drug can be inhibited by containing a hydrophobic amino acid as a release controlling material in the oil layer and the aqueous layer of the microspheres. was confirmed. In addition, in the case of the microspheres containing the hydrophobic amino acid, the properties were uniform and the suspending ability was excellent, it was confirmed that it has excellent formulation properties as a depot agent, and completed the present invention.

The present invention provides a depot composition comprising microspheres comprising an oil layer (O layer) comprising a biodegradable polymer and a hydrophobic amino acid.

The microspheres may further include a hydrophobic amino acid in the aqueous layer.

The microspheres may have a bulk density (BD) of 0.1 g/ml or more and a zeta potential of -8 mV to -30 mV.

The hydrophobic amino acid may be selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine and leucine.

The microspheres may be microspheres prepared by including less than 1.5 parts by weight of a hydrophobic amino acid in an oily solution based on 100 parts by weight of a biodegradable polymer.

The microspheres may be microspheres prepared by including 0.05% (w/v) to 25% (w/v) of hydrophobic amino acids with respect to the entire aqueous solution.

The biodegradable polymer may be a polymer including lactide and glycolide as monomers.

The biodegradable polymer may have an Inherent Viscosity of 0.35 dL/g to 0.65 dL/g.

In addition, the present invention prepares an oil phase (O) solution containing a hydrophobic amino acid and a biodegradable polymer or a first aqueous phase (Water phase 1: W1) solution containing a water-soluble solvent and the oil phase (O) solution preparing a W1/O emulsion by mixing; and adding the oil phase solution or W1/O emulsion to a second aqueous phase (Water phase 2: W2) solution to prepare an O/W2 emulsion or a W1/O/W2 emulsion. do.

The second aqueous phase (Water phase 2: W2) solution may further include a hydrophobic amino acid.

The hydrophobic amino acid may be included in an amount of less than 1.5 parts by weight based on 100 parts by weight of the biodegradable polymer in the oily solution.

The hydrophobic amino acid may be included in an amount of 0.05% (w/v) to 25.0% (w/v) based on the total amount of the second aqueous phase solution.

The method may further include recovering the microspheres by centrifuging the prepared O/W2 emulsion or W1/O/W2 emulsion after drying and/or filtration and centrifugation.

The microspheres of the depot composition of the present invention, including hydrophobic amino acids, can control the excessive release of the active ingredients contained in the microspheres at the beginning of the depot agent injection, and have excellent suspending ability, so that even when the user is administered as an injection, the active ingredient The effect can be obtained uniformly and continuously.

1A to 1G are SEM images of PLGA microspheres prepared according to an embodiment of the present invention. Specifically, FIGS. 1A to 1E are a case of using liraglutide as an API, and FIG. 1A shows microspheres that do not contain hydrophobic amino acids; Figure 1b is a microsphere comprising a hydrophobic amino acid in the first aqueous layer; Figure 1c is microspheres comprising a hydrophobic amino acid in the second aqueous layer; Fig. 1D shows microspheres containing hydrophobic amino acids in the oil layer, and Fig. 1E shows microspheres containing hydrophobic amino acids in the oil layer and the second aqueous phase. In addition, Figures 1f to 1g is a case of using semaglutide as API, Figure 1f is microspheres that do not contain a hydrophobic amino acid; And Figure 1g shows microspheres containing hydrophobic amino acids in the oil layer and the second aqueous layer.
2A to 2E are SEM photographs of PLGA microspheres prepared according to an embodiment of the present invention. FIG. 2A is microspheres containing valine; 2B shows microspheres comprising methionine; Figure 2c shows microspheres comprising phenylalanine; 2D shows microspheres comprising tryptophan; and Figure 2e shows microspheres comprising leucine.
3A and 3B are SEM photographs of PLGA microspheres prepared according to an embodiment of the present invention. Figure 3b shows microspheres coated with leucine without containing hydrophobic amino acids.
4A and 4B are SEM images of PLGA microspheres prepared according to an embodiment of the present invention. FIG. 4A is microspheres containing cholesterol; Figure 4b shows the microspheres containing the cationic lipid DOTAP (Dioleoyl-3-trimethylammonium propane).
5A to 5C are SEM photographs of PLGA microspheres including leucine in an oil layer prepared according to an embodiment of the present invention.
6a to 6d are SEM photographs of PLGA microspheres including leucine in the oil layer and the second aqueous layer prepared according to an embodiment of the present invention.
7 is a graph showing the results of DSC analysis of microspheres prepared according to an embodiment of the present invention.
8 is a graph showing the long-term release characteristics of microspheres prepared according to an embodiment of the present invention.
9 is a graph showing the results of confirming the drug release behavior in vivo of microspheres prepared according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

However, since the present invention can have various changes and can have various forms, the specific examples and descriptions described below are only for helping the understanding of the present invention, and are intended to limit the present invention to specific disclosed forms. it is not It should be understood that the scope of the present invention includes all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

The present invention provides a depot composition comprising microspheres comprising an oil layer (O layer) comprising a biodegradable polymer and a hydrophobic amino acid.

The depot composition of the present invention is a formulation capable of releasing a drug for a long period of time and corresponds to a sustained-release formulation, and more specifically, may be a sustained-release injection formulation. Sustained-release preparations should release the drug inside at an intended level slowly over a period of time. To this end, it is necessary to control the initial burst in which the internal drug is excessively released at the beginning of the injection of the depot agent, and to make the depot agent well suspended before injection. The present invention completed the depot composition by preparing microspheres with excellent suspending ability while controlling the initial release through mixing of hydrophobic amino acids.

The microspheres of the present invention may be in the form of O/W including a single aqueous layer (W layer) and a single oil layer (O layer). In addition, the microspheres of the present invention may be in the form of W/O/W including an aqueous layer (W layer), an oil layer (O layer), and an aqueous layer (W layer).

In the present specification, for convenience of explanation of two or more aqueous phases or aqueous solutions, the aqueous phase existing in the oil layer of microspheres or the aqueous solution for forming the same is used as the first aqueous layer (W1 layer) or the first aqueous solution, An aqueous layer formed outside the oil layer of the microspheres or an aqueous solution for forming the same may be referred to as a second aqueous layer (W2 layer) or a second aqueous solution, respectively. In this case, the aqueous phase (W layer) or aqueous solution in the microspheres of the O/W type is understood as a configuration corresponding to the second aqueous layer (W2 layer) or the second aqueous solution in the microspheres of the W1/O/W2 type. Therefore, the O/W emulsion may also be referred to herein as an O/W2 emulsion, and in the following description, referred to as the second aqueous layer (W2 layer), or the second aqueous solution, is in the form of O/W It should be understood as a description corresponding to the aqueous phase or aqueous phase solution for the microspheres.

The microspheres can be prepared by a single emulsification method of adding an oily solution containing a biodegradable polymer and a hydrophobic amino acid to an aqueous solution and emulsifying it to make an O/W emulsion. It can also be prepared by a double emulsification method in which a W/O emulsion is added to an aqueous phase solution and re-emulsified to make a W/O/W emulsion.

Specifically, the microspheres of the present invention are prepared by dissolving a hydrophobic amino acid and a biodegradable polymer in a fat-soluble solvent to prepare an oil phase (Oil phase: O) solution; Prepare a W1/O emulsion by mixing the first aqueous phase (Water phase 1: W1) solution prepared by dissolving the active ingredient in an aqueous solvent and the oil phase (O) solution prepared by dissolving the hydrophobic amino acid and biodegradable polymer in a fat-soluble solvent do;

It may be prepared by a method of preparing an emulsion by adding the oil phase solution or W1/O emulsion to a second aqueous phase (Water phase 2: W2) solution containing an aqueous solvent.

The oil-soluble solvent of the present invention is commonly used in the art, and any pharmaceutically acceptable carrier or solvent may be used. As an example, dichloromethane may be used, but is not limited thereto.

In the present invention, the water-soluble solvent is commonly used in the art of the present invention, and any pharmaceutically acceptable carrier or solvent may be used. As an example, polyvinyl alcohol (PVA) or sodium acetate may be used, but is not limited thereto. The second aqueous solution may further include an osmotic pressure adjusting agent, such as sodium chloride, in order to adjust the osmotic pressure of the solution during the emulsion preparation process.

The microspheres of the present invention may further include a hydrophobic amino acid in the aqueous phase. The receiving layer may be a second receiving layer. Specifically, the microspheres may be prepared by dissolving a hydrophobic amino acid in an aqueous solvent and adding the oily solution or W1/O emulsion to an aqueous solution prepared.

In the present invention, when hydrophobic amino acids are included in both the oil layer and the aqueous layer of the microspheres, the suspending ability of the depot composition is significantly improved, along with the initial release inhibitory effect of suppressing the excessive release of the active ingredient inside the microspheres. can be used effectively as

The microspheres of the present invention may have a bulk density (BD) value of 0.1 g/ml or more.

The bulk density (BD) refers to a density based on a volume including voids generated between particles when a powder or granular material is filled in a specific container. When the bulk density of the microspheres in the depot composition is less than 0.1 g/ml, the microspheres are too light to be used as an injection. In particular, due to the bulky nature of the microspheres, there is a problem of poor dispersion in water for injection during resuspension. The microspheres of the present invention have a bulk density of 0.1 g/ml or more, have excellent resuspension ability, and have excellent properties as injections.

The microspheres of the present invention may have a zeta potential value of -8 mV or less.

The zeta potential represents the magnitude of the repulsive force and attractive force between particles as a unit, and the microspheres of the present invention may have a zeta potential of -8 mV or less. When the zeta potential value is greater than -8 mV, the microspheres may be rapidly precipitated in water for injection due to reduced dispersibility.

Preferably, it has a zeta potential value of -8 mV to -40 mV, or -10 mV to -30 mV. When the above range is satisfied, the microspheres have excellent dispersibility and can be well suspended in water for injection.

The microspheres of the present invention further include an active ingredient. That is, since the depot composition of the present invention is administered into the body and serves to release the active ingredient into the body for a certain period of time, the active ingredient may be included in the depot composition. The active ingredient is especially contained in the microspheres and protected from the microspheres, and may remain in the body for a certain period of time.

The active ingredient may be included in the first aqueous layer (W1 layer) or the oil layer (O layer) of the microspheres of the present invention. In general, in the case of a single emulsion emulsion, the active ingredient may be included in the oil layer, and in the case of a double emulsion emulsion, it may be included in the first aqueous layer.

The active ingredient used for the purpose of release in the present invention may preferably be a drug for treating a specific disease, symptom or disease, more preferably a biopharmaceutical or a peptide drug. The drug may be, for example, liraglutide or semaglutide.

The liraglutide or semaglutide is a GLP-1 agonist involved in the action of GLP-1 (glucagon-like peptide-1; glucagon-like peptide-1), which is a hormone that lowers blood sugar, such as obesity or diabetes. can be used for treatment. When the body is exposed to excessive amounts of liraglutide or semaglutide, symptoms such as vomiting, nausea, hypoglycemia, and headache may occur.

Therefore, when the depot composition includes liraglutide or semaglutide as an active ingredient, it is very important to prevent excessive release of the active ingredient from the depot agent. In addition, liraglutide or semaglutide is a peptide drug that is difficult to store and store. In order to solve this, there is a method of drying a depot composition and storing it in a powder form. In this case, the powder should be suspended in water for injection for injection. If the suspending ability of the powder is poor, a uniform formulation cannot be formed, so that the uniformity of drug efficacy is reduced, and agglomeration and sedimentation of microspheres occur, or floating Since microspheres may occur, it may be difficult to secure the initial release control effect. Therefore, when including liraglutide or semaglutide as an active ingredient, it is very important to suppress the initial release and implement a formulation having excellent suspending ability.

In order to provide a depot composition having such an initial release control ability, the present invention includes a biodegradable polymer and a hydrophobic amino acid in the oil layer (O layer) of the microspheres.

The hydrophobic amino acid does not have a polar portion among amino acids, valine, methionine, alanine, phenylalanine, tryptophan. It may be selected from the group consisting of isoleucine and leucine. Preferably, it may be leucine. When a hydrophobic amino acid is included in the oil layer of the microspheres of the present invention, the pores on the surface of the microspheres are reduced by the hydrophobic amino acid, and the hydrophobic amino acid present in the O layer is present in the first aqueous layer or the oil layer due to the non-friendly nature of water. It is possible to control the initial release by partially inhibiting the active ingredient from coming out of the oil layer.

When the hydrophobic amino acid is included only in the first aqueous phase, it does not effectively inhibit the release of an excess active ingredient in the initial phase, and when it is included in only the second aqueous layer, the dissolution time increases due to the use of an excess of the raw material (hydrophobic amino acid) and There is a problem in the manufacturing process and process cost increase due to the increase in raw material cost. On the other hand, when it is included in the oil layer or the oil layer and the second aqueous layer, an effective initial release control effect can be obtained even with a low content of hydrophobic amino acids, and the suspending ability of the depot agent can be significantly improved. have an advantage

The microspheres may be prepared by including less than 1.5 parts by weight of a hydrophobic amino acid based on 100 parts by weight of a biodegradable polymer in an oily solution. When the hydrophobic amino acid is mixed in an amount of 1.5 parts by weight or more, there is a problem in that the uniformity of the depot drug is reduced due to the non-uniformity of the surface of the microspheres.

More specifically, the microspheres may be prepared by including more than 0.07 parts by weight to 1.1 parts by weight, or 0.3 parts by weight to 1.1 parts by weight of the hydrophobic amino acid based on 100 parts by weight of the biodegradable polymer in the oily solution. In this case, the effect of inhibiting the initial release of the active ingredient from the microspheres is excellent.

In this aspect, the microspheres of the present invention contain less than 1.5 parts by weight of hydrophobic amino acids in the oil layer based on 100 parts by weight of the biodegradable polymer; greater than 0.07 parts by weight to 1.1 parts by weight; Or 0.3 parts by weight to 1.1 parts by weight may be included. When the hydrophobic amino acid is contained in the oil layer in an amount of 1.5 parts by weight or more relative to 100 parts by weight of the polymer, the surface of the microspheres becomes non-uniform, and there is a problem in that the uniformity of the depot drug is reduced. In addition, when only hydrophobic amino acids are included in the oil layer alone, when the hydrophobic amino acids are included in an amount of 0.07 parts by weight or less, the initial release control effect by the hydrophobic amino acids is insignificant.

In addition, the microspheres of the present invention may be prepared by including 0.05% (w/v) to 25% (w/v) of hydrophobic amino acids with respect to the entire aqueous solution containing a water-soluble solvent and hydrophobic amino acids. In the aqueous layer, when the hydrophobic amino acid is contained in 0.005% (w/v) or less, the bulk density of the fine particles is too low, which is not suitable for use as an injectable formulation.

The biodegradable polymer has a property of being decomposed in vivo, and has a property of forming microspheres in the present invention, and as the polymer in the living body is gradually decomposed, the drug contained therein may be gradually released. That is, it serves to protect the drug inside during the drug release period and to control the drug release for a long period of time.

The biodegradable polymer specifically approved for use in injection preparations by the Ministry of Drug Safety of each country may be used without limitation in the present invention.

The biodegradable polymer may have an inherent viscosity of 0.35 dL/g to 0.65 dL/g, preferably 0.50 dL/g to 0.55 dL/g. When the intrinsic viscosity is less than 0.35 dL/g, drug release may be faster than the desired period, and if the intrinsic viscosity is more than 0.65 dL/g, drug release is slower than the desired period, so it may be difficult to obtain sufficient drug effect. In a preferred example, 0.53 dL/g viscosity polymer is used to maintain optimal drug durability in this case as a depot composition to maintain drug release properties for a long period of time, thereby making it bioavailable. The inhibitory viscosity may be measured according to the method for measuring the viscosity of poly(lactide-co-glycolide, PLGA) suggested by the manufacturer.

Specifically, the biodegradable polymer may be a polymer including lactide and glycolide as monomers. As long as the polymer contains lactide and glycolide as monomers, all of them are included in the present invention regardless of the polymerized form. In addition, it is meant to include a branched-polymer in which the end of the polymer is modified. Examples of the polymer include polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (Polylactide-co-glycolide, PLGA), and Glucose-PLGA It may be selected from the group, but is not limited thereto. In this respect, the microspheres of the present invention may be referred to as PLGA microspheres.

The biodegradable polymer may have the following molecular weight distribution for each molecular weight determined by gel filtration chromatography: 500 to 4,000 molecular weight range is 3% or more, 5,000 to 16,000 molecular weight range is 10 % or more and less than 30%, in the molecular weight range of 16,000 to 40,000, 20% or more and less than 50%. 40,000 or more is 20% or more.

The depot composition of the present invention may further include a pharmaceutically acceptable carrier or excipient. However, preferably, the depot composition of the present invention may not contain a stabilizer, a pH adjuster, or an oxidizing agent.

In addition, the present invention prepares an oil phase (O) solution containing a hydrophobic amino acid and a biodegradable polymer or a first aqueous phase (Water phase 1: W1) solution containing a water-soluble solvent and the oil phase (O) solution preparing a W1/O emulsion by mixing; and adding the oil phase solution or W1/O emulsion to a second aqueous phase (Water phase 2: W2) solution to prepare an O/W2 emulsion or a W1/O/W2 emulsion. do.

In addition, it may further comprise the step of recovering the microspheres by centrifugation after drying and / or filtration of the prepared emulsion.

The first aqueous phase (Water phase 1: W1) solution may be prepared by dissolving the active ingredient in an aqueous solvent. In the present invention, the water-soluble solvent is commonly used in the art of the present invention, and any pharmaceutically acceptable carrier or solvent may be used. As an example, polyvinyl alcohol (PVA) or sodium acetate may be used, but is not limited thereto.

The active ingredient means a drug included for the purpose of being released from the depot composition of the present invention, and may be used in the present invention without being limited to the type thereof. Specifically, it may be a water-soluble drug, and may be a biopharmaceutical or a peptide drug. For example, it may be liraglutide or semaglutide.

The oil phase (Oil phase: O) solution may be prepared by mixing and dissolving a biodegradable polymer and a hydrophobic amino acid in a fat-soluble solvent. In addition, the active ingredient may be mixed and dissolved in the oily solution. In particular, in the case of microspheres prepared by a single emulsification method, an emulsion can be prepared while adding the oily solution mixed with the active ingredient to the aqueous solution.

As a solvent for including the active ingredient in the oily solution, a solvent commonly used in the art of the present invention may be used.

The oil-soluble solvent of the present invention is commonly used in the art, and any pharmaceutically acceptable carrier or solvent may be used. As an example, dichloromethane may be used, but is not limited thereto.

The hydrophobic amino acid may be included in an amount of less than 1.5 parts by weight based on 100 parts by weight of the biodegradable polymer in the oily solution. When the hydrophobic amino acid is contained in an amount of 1.5 parts by weight or more in the oily solution, the surface of the microspheres becomes non-uniform, and there is a problem in that the uniformity of the drug depot agent is reduced. In addition, when only hydrophobic amino acids are included in the oil solution alone, when the hydrophobic amino acids are included in an amount of less than 0.07 parts by weight, the initial release control effect by the hydrophobic amino acids is insignificant.

The O/W emulsion may be prepared by mixing an oil phase with an aqueous phase and stirring with a homogenizer. The stirring speed and time may vary depending on the conditions for forming the emulsion and the amount of the sample.

The second aqueous phase (Water phase 2: W2) solution may be prepared by including an aqueous solvent.

In the present invention, the water-soluble solvent is commonly used in the art of the present invention, and any pharmaceutically acceptable carrier or solvent may be used. As an example, polyvinyl alcohol (PVA) or sodium acetate may be used, but is not limited thereto. The second aqueous solution may further include an osmotic pressure adjusting agent, such as sodium chloride, in order to adjust the osmotic pressure of the solution during the emulsion preparation process.

In addition, the water-soluble solvent is polyvinyl alcohol, methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyoxyethylene, olioxyethylene sorbitan fatty acid ester, polyoxyethylene perm oil derivatives and It may further include a mixture thereof.

In addition, the hydrophobic amino acid may be included in an amount of 0.05% (w/v) to 25.0% (w/v) with respect to the entire second aqueous phase solution. When the second aqueous solution contains hydrophobic amino acids in an amount of 0.005% (w/v) or less, the bulk density of the fine particles is too low, and thus it is not suitable for use as an injectable formulation.

In addition, the present invention provides a depot composition comprising the microspheres prepared by the above-described manufacturing method. In this case, it is possible to obtain a depot composition excellent in the suspending ability while excellent in the initial release inhibitory effect of the active ingredient in the microspheres.

Hereinafter, the present invention will be described in detail through Preparation Examples and Experimental Examples. The following Examples and Experimental Examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[Preparation Example] Preparation of test and preparation of microspheres

Preparation Example 1-1. Preparation of microspheres

As API, 252 mg of Liraglutide (polypeptide laboratories) or Semaglutide was dissolved in 1.2 mL of 1 wt% sodium acetate (Sodium acetate; Daejung Chemicals & Metals Co. Ltd.) solution to dissolve the first aqueous phase ( Water phase 1: W1) solution was prepared.

1350 mg of DL-lactic acid-glycolic acid copolymer (Poly D,L-lactide-co-glycolide; Resomer select 5545 DLG 5Glu, Evonik; Inherent Viscosity 0.53 dl/g) in 5 mL of dichloromethane (Dichloromethane, Honeywell) It was dissolved to prepare an oil phase (O) solution. The W1 and O solutions were mixed, and a W1/O emulsion was prepared by stirring at 9000 rpm for 2 minutes with a homogenizer.

Next, 0.5 g of sodium chloride (NaCl; Daejung Chemicals & Metals Co. Ltd) was dissolved in 100 mL of 1 wt% polyvinyl alcohol (PVA; Gohsenol EG-40P, Nippon Gohsei) solution to dissolve the second aqueous phase (Water phase) 2: W2) solution was prepared. A W1/O/W2 emulsion was prepared by injecting the W1/O emulsion onto W2 at a rate of 5 ml/min and stirring at 9000 rpm with a homogenizer. The prepared W1/O/W2 emulsion was dried in water at room temperature for 3 hours, filtered through a sieve with a mesh size of 75 μm, and centrifuged to recover microspheres. After redispersing the recovered microspheres in distilled water, the surface of the microspheres was washed three times by centrifugation. After washing, the microspheres were lyophilized to finally obtain drug-encapsulated microspheres.

The microspheres prepared by the above method were used as a control for the microspheres of the present invention in the following experiments.

Preparation 1-2. Preparation of Microspheres Containing Hydrophobic Amino Acids

For control of initial release in sustained-release formulations, microspheres containing hydrophobic amino acids were prepared, and the effect of controlling their release was confirmed. As a hydrophobic amino acid, valine, methionine, phenylalanine, tryptophan, or leucine was used.

Except for mixing the hydrophobic amino acid in the first aqueous phase (Water phase 1: W1) solution, the oil phase (O) solution, and/or the second aqueous phase (Water phase 2: W2) solution, the specific preparation method is Microspheres were prepared under the same conditions as in Preparation Example 1.

The mixing of the hydrophobic amino acids is performed in a 1:1 molar ratio with respect to the main component (liraglutide or semaglutide) when the hydrophobic amino acid is included in the first aqueous phase (Water phase 1: W1) solution or oil phase: O solution. The mixture was mixed, and when the hydrophobic amino acid was mixed in the second aqueous phase (Water phase 2: W2) solution, it was mixed with sodium chloride (NaCl) at the same concentration. The specific manufacturing prescription was different for each experiment.

[Test Methods]

1. Evaluation of Liraglutide or Semaglutide Content

Put 20 mg of microspheres in a 20 mL volumetric flask, dissolve it with 10 mL of acetonitrile, set the mark with 1 wt% sodium acetate solution, filter with a 0.45 syringe filter, and measure the amount of liraglutide or semaglutide by high performance liquid chromatography was quantified as At this time, the analysis conditions were measured at 215 nm with a flow rate of 1.0 mL/min at 215 nm using an Aegispak C18-L column using a solvent containing a mixture of 0.05M potassium phosphate monobasic and acetonitrile in a ratio of 53:47 as a mobile phase. did.

2. Emission test

20 mg of lyophilized microspheres were released in a DISTEK eluator (Dissolution system 2500) at 120 rpm and 37°C using 100 mL of phosphate buffer (1X PBS) containing 0.05% of Polysorbate 80. proceeded. After collecting a sample of 2 mL, the supernatant and microspheres were separated by centrifugation, and the amount of liraglutide or semaglutide present in the supernatant was quantified by high performance liquid chromatography. At this time, the analysis conditions were measured at 215 nm with a flow rate of 1.0 mL/min at 215 nm using an Aegispak C18-L column using a solvent containing a mixture of 0.05M potassium phosphate monobasic and acetonitrile in a ratio of 53:47 as a mobile phase. did.

The initial release of the drug from the microspheres confirmed the amount of liraglutide eluted for 1 hour in the above method, and the long-term release confirmed the amount of liraglutide eluted for 35 days.

3. SEM measurement

About 10 mg of the microspheres were fixed on an aluminum stub, and platinum was coated for 3 minutes under a vacuum degree of 0.1 torr and a high voltage (10 kV), then mounted on an SEM powder and the surface of the microspheres was observed using an image analysis program.

4. Particle size measurement

The size of the microspheres was measured using a Malvern Mastersizer. About 30 mg of the microspheres were suspended in 5 ml of distilled water, and the suspension was placed in a dispersion device and dispersed for 1 minute at 2800 rpm and 50% Ultrasound, and then the size was measured.

5. Determination of Suspension Density

When the microspheres of the present invention are resuspended in the injection solution, the density of the suspension was checked in order to check whether the microspheres were well dispersed. The density of the suspension was measured using a D5 from METTLER TOLEDO. 534 mg of the microspheres were suspended in 2 ml of the solvent and injected into the cell in the device to measure the density.

The reference value of the suspension density was confirmed based on a suspension density of 1.01 g/cm ³ of untreated microspheres.

6. Zeta-potential measurement

After dispersing 50 mg of microspheres in 3 ml of purified water, the zeta potential was measured using Malvern's Nano-ZS equipment.

7. Bulk Density Measurement

After filling the Eppendorf tube with 1 ml of microspheres, the mass of the filled microspheres was measured.

8. DSC Measurement

DSC measurements were performed on a differential scanning calorimeter. 5 to 10 mg of each microsphere was placed in an aluminum sample pan and measured from 25° C. to 250° C. at 5° C./min.

[Test Example 1] Confirmation of initial release control effect by mixing hydrophobic amino acids

Experiments were conducted to determine whether hydrophobic amino acids in microspheres could control the excessive release of drugs. According to the same method as the method for preparing the microspheres of Preparation Example 1, the first aqueous phase (Water phase 1: W1) solution, the oil phase: O solution, and/or the second aqueous phase (Water phase) of each step of the microsphere production 2: W2) Microspheres containing hydrophobic amino acids were prepared by mixing leucine as a hydrophobic amino acid in each solution, and preparing an emulsion. Except for mixing the hydrophobic amino acid in each solution, specific preparation conditions are the same as the microsphere preparation method of Preparation Example 1.

The mixing of the hydrophobic amino acids is performed in a 1:1 molar ratio with respect to the main component (liraglutide or semaglutide) when the hydrophobic amino acid is included in the first aqueous phase (Water phase 1: W1) solution or oil phase: O solution. The mixture was mixed, and when the hydrophobic amino acid was mixed in the second aqueous phase (Water phase 2: W2) solution, it was mixed with sodium chloride (NaCl) at the same concentration. The specific microsphere preparation recipe is shown in Table 1.

division Batch No. W1 Phase (mg) O Phase (mg) W2 Phase (%(w/v)) API amino acid PLGA amino acid NaCl amino acid Comparative Example 1 111 252 - 1350 - 0.5 - Comparative Example S-1 SMG21-008 252 - 1350 - 0.5 - Example 1-1 114 252 9
(Leucine) 1350 - 0.5 - Example 1-2 134 252 - 1350 - 0.5 0.5 Examples 1-3 139 252 - 1350 9
(0.67%*) 0.5 - Examples 1-4 137 252 - 1350 9
(0.67%*) 0.5 0.5 Example S-1 SMG21-009 252 - 1350 8.8
(0.65%*) 0.5 0.5 * Content of release control component compared to polymer (unit: % (w/w))

Microspheres containing no hydrophobic amino acid according to Table 1 (Comparative Example 1), microspheres mixed with leucine in the first aqueous solution (Example 1-1), and microspheres mixed with leucine in the second aqueous solution (Example 1) -2), the initial release inhibitory effect was confirmed in the microspheres mixed with leucine in the oil phase solution (Example 1-3) and the microspheres mixed with leucine in the oil phase solution and the second aqueous solution at the same time (Example 1-4) In order to do this, check the content and release rate of liraglutide (API) according to the conditions of Test Methods 1 and 2, observe the surface of microspheres using an SEM photograph according to the conditions of Test Methods 3 and 4, and average particle size of each microsphere The size (D[4,3]) was measured. The results are shown in Table 2 and FIGS. 1A to 1E below.

In addition, the initial release inhibitory effect in microspheres containing no hydrophobic amino acids by applying semaglutide (Comparative Example S-1), and in microspheres in which leucine was simultaneously mixed with an oil phase solution and a second aqueous solution (Example S-1) To confirm, check the content and release rate of semaglutide (API) according to the conditions of Test Methods 1 and 2, observe the surface of microspheres using an SEM photograph according to the conditions of Test Methods 3 and 4, and The average particle size (D[4,3]) was measured. The results are shown in Table 2 and FIGS. 1F to 1G below.

division Batch No. content
(%) release (1h)
(%) D[4,3]
(μm) SEM Characteristics Comparative Example 1 111 100.51 15.76 42.29 lots of pore Comparative Example S-1 SMG2I-008 101.54 11.88 61.61 lots of pore Example 1-1 114 102.90 16.21 40.77 lots of pore Example 1-2 134 105.21 1.61 35.65 no pores Examples 1-3 139 104.45 2.91 27.32 no pores Examples 1-4 137 106.94 3.14 39.60 no pores Example S-1 SMG2I-009 99.44 5.45 51.54 no pores

As shown in Table 2 and FIGS. 1A to 1G , many pores exist on the surface of the microspheres of Comparative Example 1 and Comparative Example S-1, and thus, it can be confirmed that the initial release of API is also large. In addition, even in the case of microspheres containing leucine in the first aqueous layer (Example 1-1), the effect of covering the pores could not be obtained, and thus the effect of inhibiting initial release could not be obtained.

However, it can be seen that when leucine is included in the oil layer (O) and/or the second aqueous phase layer (W2), the pores of the microspheres are covered, so that the pores on the surface are hardly recognized. Specifically, when leucine is included only in the oil layer (Example 1-3), the properties of the microspheres themselves are somewhat poor, but when leucine is included in both the oil layer and the second aqueous layer (Examples 1-4 and implementation) Example S-1), while having an initial release inhibitory effect of API, it was confirmed that the properties were also good.

[Test Example 2] Confirmation of initial release control effect according to the type of hydrophobic amino acid

In order to check whether hydrophobic amino acids other than leucine can equally inhibit the initial release of microspheres, release of liraglutide, an API, from microspheres prepared by mixing valine, methionine, phenylalanine, tryptophan and leucine in the microsphere preparation step, respectively did.

While preparing the microspheres in the same manner as in the method for preparing microspheres of Comparative Example 1, hydrophobic amino acids were mixed in each of the oil phase: O solution and the second aqueous phase (Water phase 2: W2) solution during the microsphere production step to make the microspheres hydrophobic. Microspheres containing amino acids were prepared. Except for mixing the hydrophobic amino acid in each solution, specific preparation conditions are the same as those of the microsphere preparation method of Comparative Example 1.

In the oil phase (O) solution, each hydrophobic amino acid was mixed in a molar ratio of 1:1 to the main component (liraglutide), and each hydrophobic amino acid was mixed in the second aqueous phase (Water phase 2: W2) solution at the same concentration as sodium chloride. was mixed. The specific microsphere preparation recipe is shown in Table 3 below.

division Batch No. W1 Phase (mg) O Phase (mg) W2 Phase (%(w/v)) API PLGA amino acid NaCl amino acid Comparative Example 1 111 252 1350 - 0.5 - Example 2-1 130 252 1350 8
(Valine 0.59%*) 0.5 0.5
(Valine) Example 2-2 131 252 1350 10
(Methionine 0.74%*) 0.5 0.5
(methionine) Example 2-3 132 252 1350 11
(Phenylalanine 0.81%*) 0.5 0.5
(Phenylalanine) Example 2-4 133 252 1350 14
(Tryptophan 1.04%*) 0.5 0.5
(tryptophan) Example 2-5 137 252 1350 9
(Leucine 0.67%*) 0.5 0.5
(Leucine) * Content of release control component compared to polymer (unit: % (w/w))

According to Table 3 above, microspheres not containing a hydrophobic amino acid (Comparative Example 1), microspheres containing valine (Example 2-1), microspheres containing methionine (Example 2-2), microspheres containing phenylalanine ( Example 2-3), microspheres containing tryptophan (Example 2-4), and microspheres containing leucine (Example 2-5) were subjected to the conditions of Test Methods 1 and 2 to confirm the inhibitory effect on initial release. Check the content and release rate of liraglutide (API) according to the conditions of Test Methods 3 and 4, observe the surface of microspheres using an SEM photograph, and measure the average particle size (D[4,3]) of each microsphere Thus, the results are shown in Table 4 and FIGS. 2a to 2e.

division Batch No. content
(%) release (1h)
(%) D[4,3]
(μm) Comparative Example 1 111 100.51 15.76 42.29 Example 2-1 130 110.19 2.21 37.94 Example 2-2 131 108.10 2.72 32.92 Example 2-3 132 108.40 3.53 37.53 Example 2-4 133 106.39 1.81 35.47 Example 2-5 137 106.94 3.14 39.60

As shown in Table 4 and 2a to 2e, compared to the surface and API release amount of the microspheres of Comparative Example 1 (FIG. 1a), all microspheres containing valine, methionine, phenylalanine, tryptophan, or leucine (Examples 2-1 to Example 2-1) In the case of Example 2-5), it was confirmed that the pores were reduced, and it can be seen that the initial release of API (liraglutide) was also inhibited. This means that when the microspheres are prepared so that the hydrophobic amino acids are included in the oil phase and the W2 phase, the hydrophobic amino acids can act to inhibit the release of the microspheres.

[Test Example 3] Confirmation of the difference in the initial release inhibitory effect with the coating of microspheres

In contrast to the case of including the hydrophobic amino acid inside the microsphere, it was confirmed whether the initial release could be inhibited even when the microsphere was prepared and coated with the hydrophobic amino acid.

Specifically, the preparation was according to Table 5, and microspheres were prepared according to the conditions and methods described in Preparation Example 1 (Comparative Example 2). The microspheres of Comparative Example 2 prepared above were again suspended in a leucine solution, and then lyophilized (Comparative Example 3). The leucine solution was prepared by mixing 0.5 g of leucine per 1 g of microspheres, maintained in suspension for 10 minutes, and then freeze-dried.

division Batch No. microsphere composition coating solution API PLGA W2 phase Comparative Example 2 100 520mg 2479mg NaCl 0.5% (w/w)
1% PVA - Comparative Example 3 100L Leucine 0.5% (w/w)

Accordingly, in order to confirm the initial release inhibitory effect on the microspheres of Comparative Examples 2 and 3 prepared above, the content and release rate of liraglutide (API) were checked according to the conditions of Test Methods 1 and 2, and the test According to the conditions of Methods 3 and 4, the surface of the microspheres was observed using an SEM photograph, the average particle size (D[4,3]) of each microsphere was measured, and the results are shown in Table 6 and FIGS. 3a and 3b below. .

division Batch No. content (%) Emission (1h) (%) D[4,3] (μm) Comparative Example 2 100 92.82 20.17 31.90 Comparative Example 3 100L 90.14 24.73 29.44

As shown in Table 6 and FIGS. 3a and 3b, when the microspheres are simply coated using a hydrophobic amino acid (Comparative Example 3), the effect of inhibiting the release even when compared to the non-coated microspheres (Comparative Example 2) It could not be obtained, but rather it can be confirmed that the emission amount is increased. Therefore, it can be seen that the release inhibitory effect obtained from the microspheres containing the hydrophobic amino acid of the present invention is a unique effect that can be obtained by controlling the pores and the surface of the microspheres in the emulsion preparation step of the hydrophobic amino acid.

[Test Example 4] Confirmation of initial release control effect according to the type of other hydrophobic materials

In order to check whether the effect of inhibiting the initial release from the microspheres of the hydrophobic amino acid of the present invention is an effect that can be obtained even when other substances are used, cholesterol as one of the hydrophobic substances and the cationic lipid DOTAP (Dioleoyl-3-trimethylammonium propane) was used to check whether the initial release of microspheres could be suppressed.

While preparing the microspheres in the same manner as in Comparative Example 1, cholesterol or DOTAP was mixed in the oil phase (O) during the microsphere production step to prepare microspheres containing cholesterol or DOTAP.

In the oil phase (O) solution, cholesterol or DOTAP was mixed in a molar ratio of 1:1 with respect to the main component (liraglutide), respectively. The specific microsphere preparation recipe is shown in Table 7 below.

division Batch No. W1 Phase O Phase W2 Phase API PLGA Emission Controlled Substances NaCl Comparative Example 1 111 252 mg 1350 mg - 0.5% (w/w) Comparative Example 4-1 115 252 mg 1350 mg 26 mg
(Cholesterol 1.92%*) 0.5% (w/w) Comparative Example 4-2 117 252 mg 1350 mg 47 mg (DOTAP 3.48%*) 0.5% (w/w) * Content % of release-controlling material compared to biodegradable polymer (w/w)

Liraglutide (API) in the microspheres (Comparative Example 1) not containing the hydrophobic amino acid according to Table 7, the microspheres containing cholesterol (Comparative Example 4-1) and the microspheres containing DOTAP (Comparative Example 4-2) In order to confirm the release of liraglutide (API) according to the conditions of test methods 1 and 2, the content and release rate of liraglutide (API) were checked, and the microsphere surface was observed using an SEM photograph according to the conditions of test methods 3 and 4, and each The average particle size (D[4,3]) of the microspheres was measured, and the results are shown in Table 8 and FIGS. 4A and 4B below.

division Batch No. content
(%) release (1h)
(%) average particle size
(μm) appearance Comparative Example 1 111 100.51 15.76 42.29 lots of pore Comparative Example 4-1 115 106.94 18.66 39.86 lots of pore Comparative Example 4-2 117 81.84 18.06 88.09 surface unevenness

As shown in Table 8 and FIGS. 4A and 4B, when compared with the surface and API release amount (FIG. 1A) of the microspheres of Comparative Example 1, Comparative Examples 4-1 and 4-2, which are microspheres containing cholesterol or DOTAP, were all It can be seen that the initial release of API (liraglutide) could not be suppressed or the content of API decreased. In addition, even when the surface is checked through SEM, it can be confirmed that a large number of pores of the microspheres exist or that the surface is not produced smoothly. Therefore, the above results show that the initial release inhibitory effect of the microspheres of the present invention is a specific effect of the hydrophobic amino acid in the PLGA microspheres.

[Test Example 5] Confirmation of release control inhibitory effect according to the content of hydrophobic amino acids

In order to confirm whether the effect of inhibiting the release of the drug inside the microspheres in the microspheres of the present invention is influenced by the content of the hydrophobic amino acid, the release of API from the microspheres and the properties of the microspheres according to the content of the hydrophobic amino acid were confirmed.

Leucine was used as a hydrophobic amino acid, and while the microspheres were prepared according to the method for preparing microspheres described in Comparative Example 1, only mixing of leucine was changed during the preparation of the oil phase and/or the second aqueous phase. In addition, microspheres (Comparative Example 5) not containing a hydrophobic amino acid were prepared and used as a control. The specific contents and mixing steps of the prescription and release-controlling ingredients for the preparation of microspheres for each experimental group are as shown in Table 9 below. The release-controlling component was expressed as a content % (w/w) of the release-controlling component relative to the content of the biodegradable polymer (PLGA) forming microspheres.

division Batch No. W1 O Phase W2 Phase (% (w/v)) API
(mg) PLGA
(mg) emission control
ingredient NaCl emission control
ingredient Comparative Example 5 LRG84I-145 252 1350 - 0.5 - Example 3-1 LRG84I-111 252 1350 0.07% (w/w)
(Leucine) 0.5 - Example 3-2 LRG84I-137 252 1350 0.36% (w/w)
(Leucine) 0.5 - Example 3-3 LRG84I-139 252 1350 1% (w/w) (Leucine) 0.5 - Example 4-1 LRG84I-146 252 1350 1% (w/w)
(Leucine) 0.5 0.5
(Leucine) Example 4-2 LRG84I-148 252 1350 1% (w/w) (Leucine) 0.5 2
(Leucine) Example 4-3 LRG84I-149 252 1350 1% (w/w)
(Leucine) 0.5 0.05
(Leucine) Example 4-4 LRG84I-150 252 1350 1% (w/w) (Leucine) 0.5 0.005
(Leucine)

Microspheres without hydrophobic amino acids according to Table 9 (Comparative Example 5), microspheres containing leucine in the oil layer (Examples 3-1, 3-2 and 3-2), and leucine in the oil layer and W2 layer In order to confirm the release of liraglutide (API) from microspheres (Examples 4-1, 4-2, 4-3 and 4-4) containing API) content and release rate were checked, and the microsphere surface was observed using an SEM photograph according to the conditions of Test Methods 3 and 4, and the average particle size (D[4,3]) of each microsphere was measured, and the result was reported as follows. It is shown in Table 10, FIGS. 5A to 5C, and FIGS. 6A to 6D.

division Batch No. API content (%) release (1h)
(%) average particle size
(μm) appearance Comparative Example 5 LRG84I-145 99.09 9.49 33.42 lots of pore Example 3-1 LRG84I-111 96.45 5.64 32.33 Slightly reduced pores Example 3-2 LRG84I-137 99.48 3.91 33.33 stomatal reduction Example 3-3 LRG84I-139 101.45 1.91 28.33 bad shape of microspheres Example 4-1 LRG84I-146 103.13 3.51 37.85 no pores
uniform microsphere shape Example 4-2 LRG84I-148 104.08 2.00 40.63 no pores
uniform microsphere shape Example 4-3 LRG84I-149 95.71 5.37 35.26 microsphere bulky Example 4-4 LRG84I-150 99.2 7.21 31.48 microsphere bulky

In addition, the physicochemical properties (bulk density (BD), suspension density, zeta potential) of each microsphere were measured according to Test Methods 5 to 8, and are shown in Table 11.

division Batch No. BD (g/ml) Suspension Density
(g/cm ³ ) Zeta potential
(mV) Comparative Example 5 LRG84I-145 0.34 1.06365
Good injection dispersibility -7.99 Example 3-1 LRG84I-111 0.33 1.06158
Good injection dispersibility -8.54 Example 3-2 LRG84I-137 0.32 1.06858
Good injection dispersibility -9.97 Example 3-3 LRG84I-139 0.31 1.05858
Good injection dispersibility -10.43 Example 4-1 LRG84I-146 0.175 1.00858
Good injection dispersibility -10.43 Example 4-2 LRG84I-148 0.36 1.064582
Good injection dispersibility -22.50 Example 4-3 LRG84I-149 0.085 0.9542
Poor injection dispersibility -5.41 Example 4-4 LRG84I-150 0.057 0.9254
Poor injection dispersibility -4.72

As shown in Table 10 and Table 11, respectively, in Examples 3-1 to 3-3 and Examples 4-1 to 4-4, leucine, a hydrophobic amino acid, was found in the microspheres included in the O layer or the O layer and the W2 layer. It can be confirmed that there is an effect of inhibiting the initial release of API. In particular, in the case of the microspheres of Example 3-2, it can be confirmed that the formulation is excellent in API release inhibition and has a uniform surface and shape of the microspheres. In addition, in this case, it can be seen that the injection dispersibility is good and the zeta potential is suitable, so that when the microspheres are suspended in water for injection, the dispersion is good, and it can be seen that it has suitable properties as an injection preparation.

However, when leucine was included in the O layer alone, when the content of leucine compared to the polymer was 0.07% (w/w), it was confirmed that the release control effect was somewhat decreased.

In addition, even when leucine is included in the O layer, when leucine is included in 0.05% (w/v) or less with respect to the entire W2 aqueous solution, the bulk density value (Bulk density, BD, g/ml) of the particles is too high low, indicating that it is difficult to use as an injection.

However, in the case of the microspheres of Examples 4-1 and 4-2, it is possible to better control the initial release of API by including both leucine in the O layer and the W2 layer, and provide a formulation with a very uniform shape of the microspheres. It can be confirmed that there is In particular, it can be seen that the suspension density is appropriate, so that the injection dispersibility is good, and it has a significantly excellent zeta potential value. This means that the dispersibility of the microspheres in water for injection is excellent, and the microsphere formulation of the present invention has significantly superior properties as a formulation for injection.

In addition, as shown in FIG. 7 , as a result of DSC analysis of the microspheres of Comparative Example 5, Comparative Example 4-2, and Example 4-1, changes in thermal behavior and crystal form of polymers and drugs even when leucine was included in the microspheres It was confirmed that there is no

And, as shown in FIG. 8, as a result of checking the long-term release behavior of the microspheres of Comparative Example 5, Example 3-2, and Example 4-2 for 35 days, the early dissolution of the microspheres of the present invention containing leucine was controlled. It was confirmed that the drug was continuously released in zero-order for 4 weeks while maintaining 5% or less within 1 day and 15% or less for 4 days. Through this, it can be seen that the PLGA microspheres containing leucine in the oil layer or the oil layer and the W2 layer of the present invention have very excellent initial release inhibition and sustained release maintenance effects, and have good properties for use as a depot agent.

[Test Example 6] Confirmation of initial release control and sustained-release effect in vivo

An experiment was performed to confirm the drug release behavior of microspheres in vivo.

Microspheres (28 mg/kg as liraglutide) prepared according to Examples 1-4 and Comparative Example 1 in Table 1 were subcutaneously administered to the backs of 5 8-week-old SD rats (male) with an average of 300 g, 0, 1, Blood was drawn at 2, 4, 8, 10, 12, 24, 48, 96, 168, 336, 504, 672, and 1008 hours.

Thereafter, the concentration of liraglutide in blood at each time in plasma samples from SD rats was measured using enzyme-linked immunosorbent assay (ELISA). In this case, GLP-1 (active) ELISA (IBL, Germany) was used as a kit.

The change in the concentration of liraglutide with time is shown in FIG. 9 . The results of FIG. 9 describe the average values for 5 mice used in the experiment.

As shown in FIG. 9 , the maximum blood concentration (Cmax) of the physiologically active substance was reduced by 4.4 times in the microspheres (Examples 1-4) containing hydrophobic amino acids compared to the microspheres of Comparative Example 1 in the form of general microparticles, It can be seen that the excellent sustained-release effect was exhibited up to 42 days.

Claims (13)

O/W2 type microspheres comprising an oil layer (O layer) and a second aqueous phase (W2 layer), or a first aqueous layer (W1 layer), an oily (O layer) and a second aqueous phase layer (W2) layer) comprising W1/O/W2 form microspheres,
The oil layer in the O/W2 form microspheres and the first aqueous layer in the W1/O/W2 form microspheres include an active ingredient,
The oil layer contains a biodegradable polymer and a hydrophobic amino acid,
The active ingredient is liraglutide or semaglutide,
The biodegradable polymer is polylactide (Polylactide, PLA), polyglycolide (Polyglycolide, PGA), poly (lactide-co-glycolide) (Polylactide-co-glycolide, PLGA), and Glucose-PLGA consisting of At least one selected from the group,
The hydrophobic amino acid is selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine and leucine,
The O/W2 form microspheres and the W1/O/W2 form microspheres each independently contain less than 1.5 parts by weight of hydrophobic amino acids in the oil layer based on 100 parts by weight of the biodegradable polymer, the depot composition.
According to claim 1,
The W1/O/W2 form microspheres will further comprise a hydrophobic amino acid in the second aqueous layer, the depot composition.
According to claim 1,
The microspheres have a bulk density (BD) of 0.1 g/ml or more and a zeta potential of -8 mV to -30 mV, the depot composition.
delete delete 3. The method of claim 2,
The microsphere is a depot composition prepared by including 0.05% (w / v) to 25% (w / v) of hydrophobic amino acids with respect to the entire aqueous solution.
delete According to claim 1,
The biodegradable polymer has an inhibitory viscosity (Inherent Viscosity/25° C.) of 0.35 dL/g to 0.65 dL/g, the depot composition.
Prepare an oil phase (O) solution containing a hydrophobic amino acid and a biodegradable polymer or mix the oil phase (O) solution with a first aqueous phase (Water phase 1: W1) solution containing a water-soluble solvent to obtain W1/ preparing an O emulsion; and
Including the step of preparing an O/W2 emulsion or a W1/O/W2 emulsion by adding the oil phase solution or W1/O emulsion to a second aqueous phase (Water phase 2: W2) solution,
In the oil phase solution and the W1/O emulsion, the first aqueous solution contains an active ingredient,
The active ingredient is liraglutide or semaglutide,
The biodegradable polymer is polylactide (Polylactide, PLA), polyglycolide (Polyglycolide, PGA), poly (lactide-co-glycolide) (Polylactide-co-glycolide, PLGA), and Glucose-PLGA consisting of At least one selected from the group,
The hydrophobic amino acid is selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine and leucine,
In the oil phase solution and the W 1 / O emulsion, the oil phase solution each independently contains less than 1.5 parts by weight of a hydrophobic amino acid based on 100 parts by weight of a biodegradable polymer, a method for producing a depot composition.
10. The method of claim 9,
The second aqueous phase (Water phase 2: W2) solution will further include a hydrophobic amino acid, the method for producing a depot composition.
delete 10. The method of claim 9,
The method for producing a depot composition, wherein the hydrophobic amino acid is contained in an amount of 0.05% (w/v) to 25.0% (w/v) with respect to the entire second aqueous phase solution.
10. The method of claim 9,
Further comprising the step of recovering the microspheres by centrifugation after drying and/or filtering the O/W2 emulsion or W1/O/W2 emulsion prepared above, the method for producing a depot composition.

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