Controlled initial release reservoir composition and method thereof

Abstract

This invention relates to a reservoir composition comprising hydrophobic amino acids in microspheres. This reservoir composition can control the initial burst release of excess active ingredient in formulations comprising biodegradable polymers and active ingredients, has excellent suspending ability, and can uniformly and continuously obtain the effect of the active ingredient, even when ordinary users use the composition as an injectable formulation.

Description

Technical Field

[0001] This invention relates to a reservoir composition, particularly by means of hydrophobic amino acids included in microspheres, which controls the initial burst release and provides high convenience to the user through good resuspension properties.

[0002] This invention relates to the technological development of a modified pharmaceutical product for patient-customized drug release control (long-acting injection technology) for entry into the Middle East and ASEAN markets for biotechnology development – ​​customized diagnostic and therapeutic products (Mission No.: 20014981). This development was supported by the Korea Evaluation Institute of Industrial Technology and funded by the Ministry of Trade, Industry and Energy. Background Technology

[0003] Depots are injectable formulations that allow for sustained drug efficacy and are primarily used when long-term administration of hormones and analogues is desired. Depot formulations are sustained-release formulations, offering the advantage of minimizing the number of injections required for the patient; however, they do not easily achieve a constant sustained drug release throughout the entire release period after injection. In particular, a high initial burst of drug release from the depot at the start of infusion can lead to side effects due to excessive drug release, and the sustained effect may be diminished over a specific period. Therefore, controlling the initial burst of drug release from the depot is crucial.

[0004] GLP-1 agonists (such as liraglutide and semaglutide) are drugs used to treat diabetes and obesity. They were initially developed to lower blood sugar in diabetic patients and have been shown to effectively reduce weight in obese patients. Analogs of the incretin hormone, secreted in the intestines, increase the amount of insulin secreted after meals and prolong the time it takes for gastrointestinal contents to pass through the stomach slowly into the small intestine. Additionally, their various effects on the central nervous system to suppress appetite can help lower blood sugar and reduce weight.

[0005] However, when the body is exposed to excessive amounts of liraglutide, side effects such as nausea, diarrhea, rapid heart rate, hypoglycemia, or headache may occur, and the control of the initial burst release of liraglutide as a reservoir of active ingredients is one of the more important issues.

[0006] Meanwhile, Japanese Patent No. 5681626 discloses a sustained-release formulation comprising microspheres of a phospholipid component, a lipid component including sterols, and a polylactic acid-co-glycolic acid (PLGA) polymer, which allows for controlled release rate. However, in the case of lipids, it does not exhibit a sufficient effect to suppress the initial burst release and has poor microsphere properties due to its strong hydrophobic nature. Furthermore, the modification of the PLGA polymer forming the microspheres makes it difficult to obtain drug approval, thus limiting the practical use of this composition in storage facilities.

[0007] Furthermore, in the case of storage, considering the safety of drug storage, microspheres can be stored in a freeze-dried state and suspended in water for injection. In this case, if the microspheres have poor suspension, it may be possible to administer less than one dose of the formulation drug, thus making it difficult to observe sufficient drug effect. In addition, low suspension capacity can lead to clumping and microsphere deposition, which can make injection difficult due to needle blockage during injection, and the suspension capacity of microspheres is also one of the most important factors.

[0008] In this regard, there remains a need to develop reservoir compositions with appropriate resuspension and initial burst release control properties, due to the limitations of conventional sustained-release microspheres in providing reservoirs with adequately controlled initial burst release.

[0009] [Related Technical Documents]

[0010] [Patent Literature]

[0011] 1. Patent Document 1: Japanese Patent No. 5681626 Summary of the Invention

[0012] The present invention aims to solve the above-mentioned problems, and the purpose of the present invention is to provide a reservoir composition with excellent resuspension properties and inhibition of excessive initial burst release of drugs, and a method for preparing the same.

[0013] The inventors of this invention attempted to develop a reservoir composition capable of controlling the initial burst release of an excessive amount of drug, and thus discovered that including hydrophobic amino acids as release-controlling substances in the oil and aqueous phases of microspheres can inhibit the initial burst release of the drug. Furthermore, it was confirmed that microspheres including hydrophobic amino acids possess uniform properties as a reservoir, excellent suspending ability, and excellent preparation characteristics, thus completing this invention.

[0014] The present invention provides a reservoir composition comprising microspheres, the microspheres comprising an oil layer (O layer) comprising a biodegradable polymer and a hydrophobic amino acid.

[0015] The microspheres may further include hydrophobic amino acids in the aqueous phase.

[0016] The microspheres may have a total density (BD) of at least 0.1 g / ml and a zeta potential between -8 mV and -30 mV.

[0017] Hydrophobic amino acids can be selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine, and leucine.

[0018] Microspheres may include less than 1.5 parts by weight of hydrophobic amino acids per 100 parts by weight of biodegradable polymer in an oil phase solution.

[0019] Microspheres can be prepared by means of hydrophobic amino acids comprising 0.05% (w / v) to 25% (w) based on the overall aqueous solution.

[0020] Biodegradable polymers can be polymers that include lactide and glycolide as monomers.

[0021] Biodegradable polymers can have an intrinsic viscosity ranging from 0.35 dL / g to 0.65 dL / g.

[0022] In addition, the present invention also provides a method for preparing a reservoir composition, comprising preparing an oil phase (O) solution comprising a hydrophobic amino acid and a biodegradable polymer or mixing the oil phase (O) solution with a first aqueous phase (aqueous phase 1: W1) solution comprising a water-soluble solvent to prepare a W1 / O emulsion; and introducing the oil phase solution or the W1 / O emulsion into a second aqueous phase (aqueous phase 2: W2) to prepare an O / W2 emulsion or a W1 / O / W2 emulsion.

[0023] The second aqueous phase (W2) solution may further include hydrophobic amino acids.

[0024] Hydrophobic amino acids can be expressed as a basis of 100 parts by weight of biodegradable polymer, with amounts less than 1.5 parts by weight included in the oil phase solution.

[0025] The content of hydrophobic amino acids can be from 0.05% (w / v) to 25.0% (w) based on the overall second aqueous phase solution.

[0026] The method further includes a step of centrifugation to recover the microspheres after drying and / or filtering the O / W2 emulsion or W1 / O / W2 emulsion prepared above.

[0027] The microspheres of the reservoir composition of the present invention, which include hydrophobic amino acids, can control the over-release of the active ingredients contained in the microspheres at the start of reservoir injection, can have excellent suspension ability, and can obtain the effect of the active ingredients uniformly and continuously, even when administered to the user by injection. Attached Figure Description

[0028] [Figures 1A to 1G] show SEM images of PLGA microspheres prepared according to specific examples of the present invention. Specifically, Figures 1A to 1E show the case where liraglutide is used as the API, wherein Figure 1A shows microspheres excluding hydrophobic amino acids; Figure 1B shows microspheres containing hydrophobic amino acids in the first aqueous phase layer; Figure 1C shows microspheres containing hydrophobic amino acids in the second aqueous phase layer; Figure 1D shows microspheres containing hydrophobic amino acids in the oil layer; and Figure 1E shows microspheres containing hydrophobic amino acids in both the oil layer and the second aqueous phase. Additionally, Figures 1F to 1G show the case where shimaglutide is used as the API, wherein Figure 1F shows microspheres excluding hydrophobic amino acids; and Figure 1G shows microspheres containing hydrophobic amino acids in both the oil layer and the second aqueous phase layer.

[0029] [Figures 2A to 2E] show SEM images of PLGA microspheres prepared according to specific examples of the present invention, wherein Figure 2A shows microspheres containing valine; Figure 2B shows microspheres containing methionine; Figure 2C shows microspheres containing phenylalanine; Figure 2D shows microspheres containing tryptophan; and Figure 2E shows microspheres containing leucine.

[0030] [Figures 3A and 3B] show SEM images of PLGA microspheres manufactured according to a specific example of the present invention, wherein Figure 3A shows microspheres that do not contain hydrophobic amino acids; and Figure 3B shows microspheres that do not contain hydrophilic amino acids and are coated with leucine.

[0031] [Figures 4A and 4B] show SEM images of PLGA microspheres prepared according to specific examples of the present invention, wherein Figure 4A shows microspheres comprising cholesterol; and Figure 4B shows microspheres comprising dioleoyl-3-trimethylammonium propane (DOTAP) (which is a cationic lipid).

[0032] [Figures 5A to 5C] show SEM images of PLGA microspheres prepared according to a specific example of the present invention, the microspheres comprising leucine in an oil layer.

[0033] [Figures 6A to 6D] show SEM images of PLGA microspheres manufactured according to a specific example of the present invention, the microspheres comprising leucine in an oil layer and a second aqueous phase layer.

[0034] [ Figure 7 [Graphic representation of DSC analysis results of microspheres prepared according to a specific example of the present invention.]

[0035] [ Figure 8 [Graphic representation showing the long-term release characteristics of microspheres prepared according to a specific example of the present invention.]

[0036] [ Figure 9[Graphic representation showing the results of identifying the in vivo drug release behavior of microspheres prepared according to a specific example of the present invention.] Detailed Implementation

[0037] The present invention will be described in detail below.

[0038] However, it should be understood that the present invention can take various modifications and alternative forms, and the specific examples and descriptions presented below are only intended to help understand the present invention and are not intended to limit the present invention to the specific forms disclosed. It should be understood that the scope of the present invention includes all modifications, equivalents, and alternatives that fall within the spirit and scope of the present invention.

[0039] The present invention provides a reservoir composition comprising microspheres, the microspheres comprising an oil layer (O layer) comprising a biodegradable polymer and a hydrophobic amino acid.

[0040] The reservoir composition of the present invention is equivalent to a sustained-release formulation, and more specifically, a sustained-release injectable formulation, capable of releasing a drug over a prolonged period of time. A sustained-release formulation should release the drug therein slowly at a desired level over a period of time. For this purpose, it is necessary to control the initial burst release of the drug at the start of reservoir injection and to prepare a good suspension before injection. The present invention has produced microspheres with excellent suspending ability, while controlling the initial burst release by mixing hydrophobic amino acids, to complete the reservoir composition.

[0041] The microspheres of the present invention can be in an O / W form comprising a single aqueous phase layer (W layer) and a single oil layer (O layer). Alternatively, the microspheres of the present invention can be in a W / O / W form comprising an aqueous phase layer (W layer), an oil layer (O layer), and an aqueous phase layer (W layer).

[0042] For ease of explanation regarding two or more aqueous phase layers or aqueous solutions, the aqueous phase layer or aqueous solution forming the oil layer within the microspheres may be referred to herein as the first aqueous phase (W1 layer) or first aqueous solution, and the aqueous phase layer or aqueous solution forming the oil layer outside the microspheres may be referred to herein as the second aqueous phase (W2) layer or second aqueous phase. In this case, the aqueous phase layer (W layer) or aqueous solution in the O / W type of the microspheres should be understood to correspond to the configuration of the second aqueous phase (W2 layer) or second aqueous solution in the W1 / O / W2 type of the microspheres. Therefore, the O / W emulsion may also be referred to herein as the O / W2 emulsion, and in the following description, the second aqueous phase layer (W2 layer) or second aqueous solution should be understood to correspond to the description of the aqueous phase layer or aqueous solution in the O / W type of the microspheres.

[0043] Microspheres can be prepared by a single emulsification method, wherein an oil phase solution comprising a biodegradable polymer and a hydrophobic amino acid is added to an aqueous phase solution and emulsified to form an O / W emulsion. Microspheres can also be prepared by a dual emulsification method, wherein a W / O emulsion is added to an aqueous phase solution and re-emulsified to form a W / O / W emulsion.

[0044] Specifically, the microspheres of the present invention are prepared by the following method, comprising: dissolving hydrophobic amino acids and biodegradable polymers in a lipid-soluble solvent to prepare an oil phase (O) solution; or by mixing a first aqueous phase (aqueous phase 1: W1) solution prepared by dissolving the active ingredients in a water-soluble solvent with an oil phase (O) solution prepared by dissolving the hydrophobic amino acids and biodegradable polymers in a lipid-soluble solvent to prepare a W1 / O emulsion; and adding the oil phase solution or W1 / O emulsion to a second aqueous phase (aqueous phase 2: W2) comprising an aqueous phase solvent.

[0045] The lipid-soluble solvents used in this invention are commonly used in the relevant technical field, and any pharmaceutically acceptable carrier or solvent can be used. One example is, but is not limited to, dichloromethane.

[0046] The water-soluble solvent used in this invention is commonly used in the relevant technical field, and any pharmaceutically acceptable carrier or solvent can be used. Examples include, but are not limited to, polyvinyl alcohol (PVA) or sodium acetate. The second aqueous phase solution may further include an osmotic pressure regulator, such as sodium chloride, used to adjust the osmotic pressure of the solution during emulsion preparation.

[0047] The microspheres of the present invention may further include hydrophobic amino acids in an aqueous phase layer. The aqueous phase layer may be a second aqueous phase layer. Specifically, the microspheres may be prepared by adding an oil phase solution or a W1 / O emulsion to an aqueous phase solution prepared by dissolving hydrophobic amino acids in an aqueous solvent.

[0048] In this invention, when both the oil layer and the aqueous phase layer of the microspheres contain hydrophobic amino acids, in addition to inhibiting the initial burst release inhibition effect of excessive release of active ingredients inside the microspheres, the suspension ability of the reservoir composition is also significantly improved, and therefore the composition can be effectively used as an injectable agent.

[0049] The microspheres of the present invention may have a total density (BD) value of 0.1 g / ml or greater.

[0050] Gross density (BD) refers to the density based on the volume including interparticle voids when powdered or granular materials are filled into a specific container. When the gross density of microspheres in a reservoir composition is less than 0.1 g / ml, the microspheres are too light to be used as injectable formulations. In particular, the large size of the microspheres causes poor dispersibility in water for injection during resuspension. The microspheres of the present invention have a gross density of 0.1 g / ml or greater, excellent resuspension properties, and excellent properties as injectable formulations.

[0051] The microspheres of this invention may have a ζ potential value of -8mV or less.

[0052] The zeta potential is expressed in units of the attractive and repulsive forces between particles, 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 of interest can exhibit reduced dispersibility and rapid deposition in water for injection.

[0053] The microspheres preferably have a zeta potential value of -8mV to -40mV or -10mV to -30mV. When the above ranges are met, the microspheres exhibit excellent dispersibility, allowing them to be well suspended in water for injection, and in this case, a homogeneous reservoir suspension can be formed, thereby providing excellent characteristics, particularly as an injectable formulation.

[0054] The microspheres of the present invention further comprise an active ingredient. That is, because the reservoir composition of the present invention is suitable for administration into the body to release the active ingredient into the body for a specific period of time, the active ingredient can be included within the reservoir composition. The active ingredient can be included in the microspheres and protected by the microspheres and retained in the body for a specific period of time.

[0055] The active ingredient may be included in the first aqueous phase layer (W1 layer) or oil layer (O layer) of the microspheres of the present invention. In the case of a single emulsion, the active ingredient is typically included in the oil layer, and in the case of a two-emulsion emulsion, the active ingredient is typically included in the first aqueous phase layer.

[0056] The active ingredient used in this invention for the purpose of release is preferably a drug for treating a specific disease or symptom, and more preferably a biopharmaceutical or peptide drug. Specific drugs may be liraglutide or hemaglutide.

[0057] Liraglutide or cimaglutide can be used to treat obesity or diabetes because GLP-1 agonists involve the action of glucagon-like peptide-1 (GLP-1), a hormone that plays a role in lowering blood sugar. When the body is exposed to excessive amounts of liraglutide or cimaglutide, symptoms such as vomiting, nausea, hypoglycemia, headache, and similar symptoms may occur.

[0058] Therefore, when liraglutide or cimarutide is included as the active ingredient in a reservoir composition, it is crucial that the active ingredient is not over-released from the reservoir. Furthermore, because liraglutide or cimarutide are peptide drugs and difficult to maintain or store, a method exists to address this issue by drying the reservoir composition and storing it in powder form. In this case, the powder should be suspended in water for injection, and when the powder has poor suspending ability, a homogeneous formulation cannot be formed. Therefore, the uniformity of drug efficacy is reduced, microsphere aggregation and deposition occur, or floating microspheres may form, making it difficult to ensure a controlled release effect. Therefore, when liraglutide or cimarutide is included as the active ingredient, it is crucial to inhibit the initial burst release and achieve a formulation with excellent suspending ability.

[0059] In order to provide a reservoir composition with this initial burst release control capability, the oil layer (O layer) of the microspheres contains biodegradable polymers and hydrophobic amino acids.

[0060] Among amino acids, hydrophobic amino acids do not have a polar portion and are selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine, and leucine. Leucine is preferred. When hydrophobic amino acids are included in the oil layer of the microspheres of the present invention, it is possible to control the initial burst release by reducing the pores on the surface of the microspheres and by partially inhibiting the release of some active ingredients present in the first aqueous or oil layer from the oil layer, because the hydrophobic amino acids present in the O layer are not water-sensitive.

[0061] When hydrophobic amino acids are included only in the first aqueous phase, the initial burst release of excessive active ingredients is not effectively controlled. When hydrophobic amino acids are included only in the second aqueous phase, the following problems arise: increased dissolution time due to the use of excessive raw materials (hydrophobic amino acids), and increased manufacturing and process costs due to increased raw material costs. On the other hand, when hydrophobic amino acids are included in the oil layer or in both the oil layer and the second aqueous phase, there is the advantage of achieving effective control of the initial burst release, even with only low concentrations of hydrophobic amino acids, and the suspension capacity of the reservoir can be significantly improved.

[0062] Microspheres may include less than 1.5 parts by weight of hydrophobic amino acids, based on 100 parts by weight of biodegradable polymer, in an oil phase solution. When the hydrophobic amino acids are mixed in amounts of 1.5 parts by weight or more, there is a problem of reduced drug uniformity in the reservoir due to the inhomogeneity of the microsphere surface.

[0063] More specifically, the microspheres may comprise, in an oil phase solution, greater than 0.07 to 1.1 parts by weight or 0.3 to 1.1 parts by weight of hydrophobic amino acids, based on 100 parts by weight of the biodegradable polymer. In this case, the microspheres exhibit excellent inhibition of the initial burst release of the active ingredient.

[0064] In this embodiment, the microspheres of the present invention may include less than 1.5 parts by weight, greater than 0.07 to 1.1 parts by weight, or 0.3 to 1.1 parts by weight of hydrophobic amino acids, based on 100 parts by weight of the biodegradable polymer, in the oil layer. When the hydrophobic amino acids are included in the oil layer in an amount of 1.5 parts by weight or more based on 100 parts by weight of the polymer, there is a problem that the surface of the microspheres becomes non-uniform and the uniformity of the drug reservoir is reduced. In addition, when the hydrophobic amino acids are included only in the oil layer, when the content of the hydrophobic amino acids is 0.07 parts by weight or less, the initial burst release control effect is small due to the hydrophilic amino acids.

[0065] Furthermore, the microspheres of the present invention can be those prepared to include 0.05% (w / v) to 25% (w) of hydrophobic amino acids relative to the overall aqueous solution comprising a water-soluble solvent and hydrophobic amino acids. In the aqueous phase, when the content of hydrophobic amino acids is 0.005% (w / v) or less, the overall density of the microspheres becomes too low to be suitable for use as an injectable formulation.

[0066] The biodegradable polymer has the property of degrading in vivo and forming the microspheres of the present invention, and the drug contained therein can be gradually released as the polymer gradually degrades in vivo. That is, it is suitable for protecting the drug inside during drug release and controlling the release of the drug over a long period of time.

[0067] Biodegradable polymers that have been specifically approved by the drug safety authorities of various countries for use in injectable formulations can be used in this invention without limitation.

[0068] Biodegradable polymers may have an intrinsic viscosity of 0.35 to 0.65 dL / g, preferably 0.50 to 0.55 dL / g. When the intrinsic viscosity is less than 0.35 dL / g, it may exhibit a faster drug release than desired, and when the intrinsic viscosity is greater than 0.65 dL / g, it may exhibit a slower drug release than desired, thus potentially failing to achieve a sufficient drug effect. In a preferred embodiment, a polymer with a viscosity of 0.53 dL / g is used to maintain drug release properties over a longer period, serving as an optimal reservoir composition for maintaining drug persistence and bioavailability. The intrinsic viscosity can be measured according to a method provided by the manufacturer for measuring the viscosity of poly(lactic-co-glycolic acid) (PLGA).

[0069] Specifically, the biodegradable polymer can be a polymer that includes lactide and glycolide as monomers. When lactide and glycolide are included as monomers, all such polymers are included in this invention, regardless of the polymerization form. This also means including terminally modified branched polymers. Examples of such polymers can be selected from, but are not limited to, the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic acid-co-glycolic acid (PLGA), and glucose-PLGA). In this context, the microspheres of this invention can be referred to as PLGA microspheres.

[0070] Biodegradable polymers may have the following molecular weight distributions as determined by gel filtration chromatography: 3% or more for molecular weights ranging from 500 to 4,000; 10% or more and less than 30% for molecular weights ranging from 5,000 to 16,000; 20% or more and less than 50% for molecular weights ranging from 16,000 to 40,000; and 20% or more for molecular weights of 40,000 or more.

[0071] The reservoir composition of the present invention may further include pharmaceutically acceptable carriers or excipients. However, the reservoir composition of the present invention preferably does not include stabilizers, pH modifiers, and oxidants.

[0072] In addition, the present invention also provides a method for preparing a reservoir composition, comprising preparing an oil phase (O) solution comprising a hydrophobic amino acid and a biodegradable polymer, or mixing the oil phase (O) solution with a first aqueous phase (aqueous phase 1: W1) solution comprising a water-soluble solvent to prepare a W1 / O emulsion; and introducing the oil phase solution or the W1 / O emulsion into a second aqueous phase (aqueous phase 2: W2) to prepare an O / W2 emulsion or a W1 / O / W2 emulsion.

[0073] The method may further include a step of centrifuging the prepared emulsion after drying and / or filtration to recover the microspheres.

[0074] The first aqueous phase (aqueous phase 1: W1) solution can be prepared by dissolving the active ingredient in a water-soluble solvent. Water-soluble solvents used in this invention are commonly used in the relevant technical field, and any pharmaceutically acceptable carrier or solvent can be used. Examples include, but are not limited to, polyvinyl alcohol (PVA) or sodium acetate.

[0075] The active ingredient refers to a drug included for the purpose of release from the reservoir composition of the present invention, and is not limited by type and can be used in the present invention. Specifically, it may be a water-soluble drug and may be a biopharmaceutical or peptide drug. An example may be liraglutide or hemaglutide.

[0076] The oil phase (O) solution can be prepared by mixing and dissolving a biodegradable polymer and a hydrophobic amino acid in a lipid-soluble solvent. Alternatively, active ingredients can be mixed and dissolved in the oil phase solution. In particular, when producing microspheres using a single emulsification method, an emulsion can be produced by adding the oil phase solution containing the mixed active ingredients to an aqueous phase solution.

[0077] Solvents commonly used in the technical field of this invention can be used as solvents in oil-phase solutions containing active ingredients.

[0078] The lipid-soluble solvents used in this invention are commonly used in the relevant technical field, and any pharmaceutically acceptable carrier or solvent can be used. One example is, but is not limited to, dichloromethane.

[0079] Hydrophobic amino acids can be included in the oil phase solution in amounts less than 1.5 parts by weight, based on 100 parts by weight of biodegradable polymer. When hydrophobic amino acids are included in the oil phase solution in amounts of 1.5 parts by weight or more, there is a problem of microsphere surface inhomogeneity and reduced drug homogeneity in the reservoir. Furthermore, when hydrophobic amino acids are included only in the oil phase solution, the initial burst release control effect of hydrophilic amino acids is not significant when the content is less than 0.07 parts by weight.

[0080] O / W emulsions can be prepared by mixing the oil phase in the aqueous phase and stirring with a homogenizer. The stirring rate and time that can be used depend on the emulsion formation conditions and the sample volume.

[0081] The second aqueous phase (W2) solution can be prepared by using a water-soluble solvent.

[0082] The water-soluble solvent used in this invention is commonly used in the relevant technical field, and any pharmaceutically acceptable carrier or solvent can be used. Examples include, but are not limited to, polyvinyl alcohol (PVA) or sodium acetate. The second aqueous phase solution may further include an osmotic pressure regulator, such as sodium chloride, used to adjust the osmotic pressure of the solution during emulsion preparation.

[0083] In addition, the water-soluble solvent may further include polyvinyl alcohol, methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyoxyethylene, oxyethylene dehydrosorbitol fatty acid ester, polyoxyethylene castor oil derivatives and mixtures thereof.

[0084] In addition, the content of the included hydrophobic amino acids can be from 0.05% (w / v) to 25.0% (w) based on the overall second aqueous solution. When the second aqueous solution contains 0.005% (w / v) or less of hydrophobic amino acids, the overall density of the microspheres becomes too low to be used as an injectable formulation.

[0085] The present invention also provides a reservoir composition comprising microspheres, which is prepared by the method described above. In this case, it is possible to obtain a reservoir composition having excellent suspending ability while exhibiting excellent initial burst release inhibition effect on the active ingredients in the microspheres.

[0086] The present invention will be described in detail below by way of preparation examples and experimental examples. The following examples and experimental examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[0087] Preparation example: Preparation of experiments and preparation of microspheres

[0088] Preparation Example 1-1: Preparation of Microspheres

[0089] Preparation of the first aqueous phase (aqueous phase 1: W1): 252 mg of liraglutide (Polypeptide Laboratories) or cimaglutide as the API was dissolved in 1.2 mL of 1 wt% sodium acetate (Daejung Chemicals & Metals Co. Ltd.) solution.

[0090] Preparation of the oil phase (O) solution: 1350 mg of DL-lactic acid-glycolic acid copolymer (polyD,L-lactic acid-co-glycolic acid; Resomer select 5545DLG 5Glu, Evonik; intrinsic viscosity 0.53 dl / g) was dissolved in 5 mL of dichloromethane (Honeywell). Preparation of the W1 / O emulsion: The W1 and O solutions were mixed and homogenized at 9000 rpm for 2 minutes.

[0091] Next, a second aqueous phase (Aqueous Phase 2: W2) solution was prepared: 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. The W1 / O / W2 emulsion was prepared: While stirring at 9000 rpm using a homogenizer, the W1 / O emulsion was injected into the W2 phase solution at a rate of 5 mL / min. The prepared W1 / O / W2 emulsion was dried in water at room temperature for 3 hours, filtered through a sieve with a 75 μm mesh size, and then centrifuged to recover the microspheres. The recovered microspheres were redispersed in distilled water and then centrifuged three times to wash the microsphere surface. After washing, the microspheres were lyophilized to obtain microspheres containing the encapsulated drug.

[0092] The microspheres prepared by this method were used as a control for the microspheres of the present invention in the following experiments.

[0093] Preparation Examples 1-2: Preparation of Microspheres Containing Hydrophobic Amino Acids

[0094] Microspheres comprising hydrophobic amino acids were prepared for initial burst release control in long-acting formulations to confirm their release control effect. Valine, methionine, phenylalanine, tryptophan, or leucine were used as the hydrophobic amino acids.

[0095] The specific preparation method is the same as that of Preparation Example 1, except that the hydrophobic amino acid is mixed in the first aqueous phase (aqueous phase 1: W1) solution, the oil phase (O) solution and / or the second aqueous phase (aqueous phase 2: W2).

[0096] When hydrophobic amino acids are mixed in the first aqueous phase (aqueous phase 1:W1) or the oil phase (O) solution, they are mixed with the main component (liraglutide or cimaglutide) at a 1:1 molar ratio. When hydrophobic amino acids are mixed in the second aqueous phase (aqueous phase 2:W2), they are mixed with sodium chloride (NaCl) at the same concentration. The specific preparation formulations vary depending on the experiment.

[0097] [Testing Method]

[0098] 1. Assessment of liraglutide or hemaglutide content

[0099] 20 mg of microspheres were placed in a 20 mL volumetric flask, dissolved in 10 mL of acetonitrile, and adjusted to the mark with 1 wt% sodium acetate solution. The solution was filtered through a 0.45 mm syringe filter, and the amount of liraglutide or hemaglutide was quantified by high-performance liquid chromatography (HPLC). An Aegispak C18-L column was used at a flow rate of 1.0 mL / min, and the mobile phase was a 53:47 mixture of 0.05 M potassium dihydrogen phosphate and acetonitrile at 215 nm.

[0100] 2. Release Test

[0101] 20 mg of lyophilized microspheres were subjected to a release assay in a DISTEK Dissolution system 2500 at 120 rpm and 37 °C using 100 mL of phosphate-buffered saline (1×PBS) containing 0.05% polysorbate 80. Two mL of the sample was collected and centrifuged to separate the supernatant and microspheres. The amount of liraglutide or hemaglutide present in the supernatant was quantified using high-performance liquid chromatography. Meanwhile, the analytical conditions were determined using an Aegispak C18-L column at a flow rate of 1.0 mL / min and 215 nm, using a mobile phase of 0.05 M potassium dihydrogen phosphate and acetonitrile in a 53:47 ratio.

[0102] For drugs released from microspheres in the initial burst, the amount of liraglutide that was dissolved after 1 hour using the above method was confirmed, and for drugs released over a long period, the amount of liraglutide that was dissolved after 35 days was confirmed.

[0103] 3. SEM measurement

[0104] Approximately 10 mg of microspheres were fixed on an aluminum stage and coated with platinum for 3 minutes under a vacuum of 0.1 Torr and a high voltage (10 kV). The microspheres were then mounted on SEM powder and their surface was observed using an image analysis program.

[0105] 4. Particle size measurement

[0106] The size of the microspheres was measured using a Mastersizer from Malvern. Approximately 30 mg of microspheres were suspended in 5 ml of distilled water, and the suspension was added to a dispersion device and then dispersed for 1 minute at 2800 rpm and 50% ultrasonic conditions to measure the size.

[0107] 5. Suspended density measurement

[0108] When the microspheres of the present invention were resuspended in the injection solvent, a well-dispersed suspension density was confirmed. The suspension density was measured using a D5 from Mettler Toledo. 534 mg of microspheres were suspended in a 2 ml solvent fraction and injected into the instrument cell to measure the density.

[0109] The reference value for suspended density is based on 1.01 g / cm³ for untreated microspheres. 3 The suspended density was used as a benchmark for determination.

[0110] 6. Zeta potential measurement

[0111] After dispersing 50 mg of microspheres in 3 ml of pure water, the zeta potential was measured using a Nano-ZS instrument from Malvern.

[0112] 7. Overall density measurement

[0113] 1 ml of microspheres were filled into a microcentrifuge tube (Eppendorf tube), and the mass of the filled microspheres was measured.

[0114] 8. DSC measurement

[0115] The DSC measurement was performed using a differential scanning calorimeter. 5 to 10 mg of microspheres were placed in aluminum and measured at 5 °C / min from 25 °C to 250 °C.

[0116] Experimental Example 1: Confirmation of the initial burst release control effect of mixed hydrophobic amino acids

[0117] The following experiments were conducted to determine whether the hydrophobic amino acid in the microspheres could control excessive drug release. Microspheres comprising the hydrophobic amino acid were produced using the same method as in Preparation Example 1: leucine, as the hydrophobic amino acid, was mixed in each of the first aqueous phase (aqueous phase 1: W1) solution, the oil phase (O) solution, and / or the second aqueous phase (aqueous phase 2: W2) at each stage of microsphere preparation, and an emulsion was prepared. The specific preparation conditions were the same as those in Preparation Example 1, except that the hydrophobic amino acid was mixed in each solution.

[0118] When the hydrophobic amino acid is mixed in the first aqueous phase (aqueous phase 1:W1) solution or the oil phase (O) solution, it is mixed with the main component (liraglutide or cimaglutide) at a 1:1 molar ratio. When the hydrophobic amino acid is mixed in the second aqueous phase (aqueous phase 2:W2), it is mixed with sodium chloride (NaCl) at the same concentration. The specific formulations for preparing the microspheres are shown in Table 1.

[0119] Table 1

[0120]

[0121] To confirm the initial burst release inhibition effect of the microspheres (excluding hydrophobic amino acids) according to Table 1 (Comparative Example 1), microspheres in which leucine is mixed in the first aqueous solution (Examples 1-1), microspheres in which leucine is mixed in the second aqueous solution (Examples 1-2), microspheres prepared by mixing leucine in the oil phase solution (Examples 1-3), and microspheres obtained by simultaneously mixing leucine with the oil phase solution and the second aqueous solution (Examples 1-4), the content and release rate of liraglutide (API) were determined according to the conditions of test methods 1 and 2, and the surface of the microspheres was observed using SEM images according to the conditions of test methods 3 and 4, and the average particle size (D[4,3]) of each microsphere was measured. The results are shown in Table 2 and Figures 1A to 1E below.

[0122] Furthermore, to confirm the initial burst release inhibition effect of microspheres containing cimaglutide (excluding hydrophobic amino acids, Comparative Example S-1) and microspheres containing leucine simultaneously mixed in an oil phase solution and a second aqueous phase solution (Example S-1), the content and release rate of cimaglutide (API) were determined according to the conditions of test methods 1 and 2, and the surface of the microspheres was observed using SEM images according to the conditions of test methods 3 and 4, and the average particle size (D[4,3]) of each microsphere was measured. The results are shown in Table 2 and Figures 1F to 1G below.

[0123] Table 2

[0124]

[0125] As shown in Table 2 and Figures 1A to 1G, it can be confirmed that numerous pores exist on the surface of the microspheres in Comparative Example 1 and Comparative Example S-1, and therefore the initial burst release of API is also greater. In addition, even when the microspheres containing leucine are included in the first aqueous phase (Example 1-1), the effect of covering the pores is not obtained, and therefore the initial burst release inhibition effect cannot be obtained.

[0126] However, when leucine is included in the oil layer (O) and / or the second aqueous phase (W2), the pores of the microspheres are covered and almost invisible on the surface. Specifically, in the case where leucine is only included in the oil layer (Examples 1-3), the properties of the microspheres themselves are somewhat poor, but in the case where leucine is included in both the oil layer and the second aqueous phase (Comparative Examples 1 to 4 and S-1), good properties are confirmed, along with the initial burst release control effect of API.

[0127] Experimental Example 2: Confirmation of the initial burst release control effect dependent on the type of hydrophobic amino acid

[0128] To confirm whether hydrophobic amino acids other than leucine could similarly inhibit the initial burst release of microspheres, the release of API liraglutide was confirmed in microspheres prepared by mixing valine, methionine, phenylalanine, tryptophan, and leucine respectively during the microsphere preparation step.

[0129] While preparing microspheres in the same manner as in Comparative Example 1, hydrophobic amino acids were mixed with oil phase (O) solution and second aqueous phase (aqueous phase 2: W2) respectively during the microsphere preparation stage to prepare microspheres containing hydrophilic amino acids. The specific preparation conditions were the same as those for the microspheres in Comparative Example 1, except that the hydrophobic amino acids were mixed in each solution.

[0130] In the oil phase (O) solution, the hydrophobic amino acids were mixed at a 1:1 molar ratio relative to the major component (liraglutide), and in the second aqueous phase (Aqueous Phase 2: W2), the hydrophobic amino acids were mixed with sodium chloride at the same concentration. The specific formulations for preparing the microspheres are shown in Table 3 below.

[0131] Table 3

[0132]

[0133] To confirm the initial burst release inhibition effect in the microspheres (excluding hydrophobic amino acids) listed in Table 3 (Comparative Example 1), microspheres including valine (Example 2-1), microspheres including methionine (Example 2-2), microspheres including phenylalanine (Example 2-3), microspheres including tryptophan (Example 2-4), and microspheres including leucine (Example 2-5), the content and release rate of liraglutide (API) were determined according to the conditions of test methods 1 and 2. The surface of the microspheres was observed using SEM images according to test methods 3 and 4, and the average particle size (D[4,3]) of each microsphere was measured. The results are shown in Table 4 and Figures 2A to 2E below.

[0134] Table 4

[0135]

[0136] As shown in Table 4 and Figures 2A to 2E, compared with the surface area and API release of the microspheres in Comparative Example 1 (Figure 1A), all microspheres (Examples 2-1 to 2-5) containing valine, methionine, phenylalanine, tryptophan, or leucine had reduced pore size, indicating that the initial burst release of API (liraglutide) was also inhibited. This means that when preparing microspheres, including hydrophobic amino acids in both the oil phase and the W2 phase allows the hydrophobic amino acids to function in inhibiting microsphere release.

[0137] Experimental Example 3: Confirming the difference in the initial burst release inhibition effect of microsphere coating.

[0138] Compared to cases where hydrophobic amino acids are included in the microspheres, it was determined whether the initial burst release could be suppressed even when the microspheres were coated with hydrophobic amino acids after preparation.

[0139] Specifically, the formulation system was prepared as shown in Table 5, and the 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 resuspended in a leucine solution and then lyophilized (Comparative Example 3). Preparation of leucine solution: 0.5 g of leucine was mixed with 1 g of microspheres, the suspension was maintained for 10 minutes, and then lyophilization was performed.

[0140] Table 5

[0141]

[0142] Therefore, in order to confirm the initial burst release inhibition effect of the microspheres prepared in Comparative Examples 2 and 3 above, the content and release rate of liraglutide (API) were determined according to the conditions of test methods 1 and 2, and the surface of the microspheres was observed using SEM images according to test methods 3 and 4. The average particle size (D[4, 3]) of each microsphere surface was measured, and the results are shown in Table 6 and Figures 3A and 3B below.

[0143] Table 6

[0144] Comparative Example 2 100 92.82 20.17 31.90 Comparative Example 3 100L 90.14 24.73 29.44

[0145] As shown in Table 6 and Figures 3A and 3B above, when the microspheres are simply coated with hydrophobic amino acids (Comparative Example 3), it can be seen that even compared with uncoated microspheres (Comparative Example 2), no release inhibition effect is obtained; on the contrary, the release amount increases. Therefore, it can be seen that the release inhibition effect obtained in the microspheres comprising hydrophobic amino acids of the present invention is a unique effect of controlling the pores of the microspheres and the hydrophobic amino acids available on the surface during the emulsion preparation step.

[0146] Experimental Example 4: Confirming the initial burst release control effect based on other hydrophobic material types

[0147] To confirm whether an initial burst release inhibition effect can be obtained in the hydrophobic amino acid microspheres of the present invention, even when using other materials, the initial burst release of the microspheres can be confirmed by using cholesterol (which is one of the hydrophobic materials) and DOTAP (which is a cationic lipid).

[0148] While preparing microspheres in the same manner as in Comparative Example 1, cholesterol or DOTAP was mixed in the oil phase (O) during the microparticle preparation stage to prepare microspheres containing cholesterol or DOTAP.

[0149] In an oil phase (O) solution, cholesterol or DOTAP was mixed at a 1:1 molar ratio relative to the major component (liraglutide). Specific formulations for the preparation of the microspheres are shown in Table 7 below.

[0150] Table 7

[0151]

[0152] To confirm the release of liraglutide (API) in microspheres excluding hydrophobic amino acids (Comparative Example 1), microspheres containing cholesterol (Comparative Example 4-1), and microspheres containing DOTAP (Comparative Example 4-2) according to Table 7, the content and release rate of liraglutide (API) were determined according to the conditions of test methods 1 and 2. The surface of the microspheres was observed using SEM images according to the conditions of test methods 3 and 4, and the average particle size (D[4,3]) of each microsphere surface was measured. The results are shown in Table 8 and Figures 4A and 4B below.

[0153] Table 8

[0154]

[0155] As shown in Table 8 and Figures 4A and 4B, when compared with the surface area and API release amount of the microspheres in Comparative Example 1 (Figure 1A), it can be seen that Comparative Examples 4-1 and 4-2, which contain microspheres including cholesterol or DOTAP, did not inhibit the initial burst release of API (liraglutide), or showed a decrease in API content. Furthermore, even when the surface was examined by SEM, it was confirmed that the microspheres contained numerous pores or could not produce a smooth surface. Therefore, the above results show that the initial burst release inhibition effect of the microspheres of the present invention is a specific effect of hydrophobic amino acids in PLGA microspheres.

[0156] Experimental Example 5: Confirmation of the release-controlled inhibition effect by measuring the content of hydrophobic amino acids

[0157] To determine whether the inhibitory effect of drug release inside the microspheres of the present invention is affected by the content of hydrophobic amino acids, the release of API in the microspheres and the nature of the microspheres are determined by the content of hydrophobic amino acids.

[0158] While preparing microspheres according to the microsphere preparation method described in Comparative Example 1, leucine was used as the hydrophobic amino acid, and the difference in the preparation of the oil phase and / or the second aqueous phase was only in the mixing of leucine. Additionally, microspheres excluding the hydrophobic amino acid (Comparative Example 5) were prepared and used as a control. The formulations for preparing microspheres for each experimental group, the specific contents of the release control components, and the mixing steps are shown in Table 9 below. The release control components are expressed as % (w / w) of the amount of biodegradable polymer (PLGA) forming the microspheres.

[0159] Table 9

[0160]

[0161] To confirm the release of liraglutide (API) in microspheres excluding hydrophobic amino acids (Comparative Example 5), microspheres containing leucine in the oil layer (Examples 3-1, 3-2, and 3-2), and microspheres containing leucine in the oil layer and W2 layer (Examples 4-1, 4-2, 4-3, and 4-4) according to Table 9, the content and release rate of liraglutide (API) were determined according to the conditions of test methods 1 and 2. The surface of the microspheres was observed using SEM images according to the conditions of test methods 3 and 4, and the average particle size (D[4,3]) of each microsphere surface was measured. The results are shown in Table 10, Figures 5A to 5C, and Figures 6A to 6D below.

[0162] Table 10

[0163]

[0164] In addition, the physicochemical properties of each microsphere (total density (BD), suspension density, zeta potential) were measured according to test methods 5 to 8 and are shown in Table 11.

[0165] Table 11

[0166]

[0167] As shown in Tables 10 and 11 above, leucine (the hydrophobic amino acid of Examples 3-1 to 3-3 and Examples 4-1 to 4-4) was confirmed to have an initial burst release inhibition effect on APIs contained in microspheres in the O layer or O and W2 layers. Particularly in the case of the microspheres of Example 3-2, it was confirmed to be a formulation with excellent API release inhibition and a uniform surface and microsphere shape. Furthermore, in this case, the injection dispersibility was good, the zeta potential was appropriate, and the dispersibility was excellent when the microspheres were suspended in water for injection; the composition was found to have characteristics suitable for use as a formulation for injection.

[0168] However, it was confirmed that the release control effect was slightly reduced when leucine was included in the O layer alone and when the leucine content was 0.07% (w / w) compared to the polymer.

[0169] Furthermore, even when leucine is included in the O layer, when the leucine content is 0.05% (w / v) or less relative to the overall W2 aqueous phase solution, the total density value of the particles (total density, BD, g / ml) is too low, and the particles are found to exhibit properties that make them unsuitable for use as injectable formulations.

[0170] However, in the case of the microspheres in Examples 4-1 and 4-2, it can be seen that the inclusion of leucine in both the O layer and W2 layer allows for better initial burst release control of the API and provides a formulation with a very uniform microsphere shape. In particular, it can be seen that the suspension density is appropriate, the injection dispersibility is good, and the zeta potential value is significantly excellent. This means that the microspheres have excellent dispersibility in water for injection, and the microspheres of the present invention have significantly superior properties as an injectable formulation.

[0171] Furthermore, such as Figure 7 As shown, DSC analysis of the microspheres of Comparative Example 5, Comparative Example 4-2 and Example 4-1 confirmed that the thermal behavior and crystal form of the polymer and the drug were not altered, even when leucine was included in the microspheres.

[0172] like Figure 8As shown, the long-term release behavior of the microspheres in Comparative Example 5, Examples 3-2, and Examples 4-2 was confirmed to be 35 days, thus confirming that the drug was continuously released in a zero-order manner for 4 weeks. Simultaneously, the initial dissolution control of the leucine-containing microspheres of the present invention was maintained at 5% or less within 1 day and 15% or less after 4 days. This demonstrates that the PLGA microspheres of the present invention, comprising leucine in the oil layer or oil layer and W2 layer, have an excellent effect in inhibiting initial burst release and maintaining sustained release, and possess reservoir properties.

[0173] Experimental Example 6: Determination of initial burst release inhibition and sustained release effects in vivo

[0174] This experiment was performed to confirm the in vivo drug release behavior of the microspheres.

[0175] Microspheres (28 mg / kg, liraglutide) prepared according to Examples 1-4 and Comparative Example 1 in Table 1 were administered subcutaneously to five 8-week-old male SD rats with an average body weight of 300 g, and blood was collected at 0, 1, 2, 4, 8, 10, 12, 24, 48, 96, 168, 336, 504, 672, and 1008 hours.

[0176] Subsequently, the concentration of liraglutide in the blood of SD rats at various time points was measured using an enzyme-linked immunosorbent assay (ELISA). The GLP-1 (active) ELISA (IBL, Germany) was used as a kit.

[0177] The change in liraglutide concentration over time is shown in Figure 9 In. Figure 9 The results show the average values ​​of the 5 mice used in the experiment.

[0178] like Figure 9 As shown, compared with the microspheres of Comparative Example 1 (which are in the form of general microspheres), it can be seen that the microspheres including hydrophobic amino acids (Examples 1 to 4) have a 4.4-fold lower maximum blood concentration (Cmax) of the physiologically active substances and still have an excellent sustained release effect until day 42.

[0179] Industrial applicability

[0180] The microspheres of the reservoir composition of the present invention, which include hydrophobic amino acids, can control the over-release of the active ingredients contained in the microspheres at the start of reservoir injection, can have excellent suspension ability, and can obtain the effect of the active ingredients uniformly and continuously, even when administered to the user by injection.

Claims

1. A reservoir composition comprising microspheres in the form of W1 / O / W2, the microspheres comprising a first aqueous phase (W1) layer, an oil (O) layer, and a second aqueous phase (W2) layer. The W1 layer of the W1 / O / W2 microspheres contains active ingredients. The O layer contains biodegradable polymers and hydrophobic amino acids. The biodegradable polymer is selected from the group consisting of polylactic acid lactide (PLA), polyglycolic acid (PGA), poly(lactic acid lactide-co-glycolic acid) (PLGA), and glucose-PLGA. The hydrophobic amino acid is selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine, and leucine. The microspheres in the W1 / O / W2 form contain less than 1.5 parts by weight of hydrophobic amino acids in the O layer, based on 100 parts by weight of the biodegradable polymer.

2. The reservoir composition of claim 1, wherein the microspheres in the W1 / O / W2 form further comprise the hydrophobic amino acid in the W2 layer.

3. The reservoir composition of claim 1 or 2, wherein the microspheres in the W1 / O / W2 form have a total density (BD) of 0.1 g / ml or greater and a zeta potential between -8 mV and -30 mV.

4. The reservoir composition of claim 2, wherein the microspheres in the W1 / O / W2 form contain 0.05% (w / v) to 25% (w / v) of hydrophobic amino acids based on the overall W2 layer.

5. The reservoir composition of claim 1 or 2, wherein the biodegradable polymer has an intrinsic viscosity of 0.35 to 0.65 dL / g at 25°C.

6. A method for preparing a reservoir composition, comprising: A W1 / O emulsion was prepared by mixing a first aqueous phase (W1) solution containing a water-soluble solvent with an oil phase (O) solution containing hydrophobic amino acids and a biodegradable polymer; and The oil phase solution or W1 / O emulsion is introduced into the second aqueous phase (W2) solution to form a W1 / O / W2 emulsion. The W1 solution of this W1 / O emulsion contains active ingredients. The O solution of this W1 / O emulsion contains less than 1.5 parts by weight of hydrophobic amino acids, based on 100 parts by weight of biodegradable polymer. The biodegradable polymer is selected from the group consisting of polylactic acid lactide (PLA), polyglycolic acid (PGA), poly(lactic acid lactide-co-glycolic acid) (PLGA), and glucose-PLGA. The hydrophobic amino acid is selected from the group consisting of valine, methionine, alanine, phenylalanine, tryptophan, isoleucine, and leucine.

7. The method of claim 6, wherein the W2 solution further comprises a hydrophobic amino acid.

8. The method of claim 7, wherein the content of the hydrophobic amino acid is from 0.05% to 25.0% (w / v) based on the total W2 solution.

9. The method of claim 6 or 7, further comprising the step of centrifuging to recover the microspheres after drying and / or filtering the W1 / O / W2 emulsion prepared above.

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