Sustained-release microspheres containing GLP-1 receptor agonist or pharmaceutically acceptable salt thereof and application thereof

Abstract

The present invention relates to a sustained release microsphere and a sustained release preparation comprising a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof. The present invention relates to a sustained release formulation, and more specifically, to a sustained release formulation comprising a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, the sustained-release microparticles comprise a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, one kind of low-viscosity polylactic acid-glycolic acid copolymer (PLGA) having an intrinsic viscosity of 0.14 dL / g to 0.24 dL / g, and two or more kinds of high-viscosity polylactic acid-glycolic acid copolymers having an intrinsic viscosity of 0.32 dL / g to 0.60 dL / g, so that the sustained-release microparticles do not have the problems of excessive release and delayed release in the initial stage of a drug. Long-term sustained release can be realized, and the bioavailability is excellent.

Description

Technical Field

[0001] The present invention relates to sustained-release microspheres containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and a sustained-release preparation containing the same, and in particular, to a method for preparing the same and use of the microspheres for treating diabetes, obesity, non-alcoholic steatohepatitis or degenerative brain diseases. Background Art

[0002] Glucagon-like peptide-1 receptor agonists (GLP-1 RA) are a group of drugs used to treat type 2 diabetes and are highly effective in lowering blood sugar levels. Furthermore, they have been shown to have weight loss effects, alleviate hypertension, hypoglycemia, and / or hyperlipidemia, and provide cardiovascular protection, leading to their increasing importance. While they offer the advantage of a lower risk of inducing hypoglycemia compared to long-standing insulin secretagogues such as sulfonylureas and meglitinides, they require frequent injections, making them less convenient for patients with chronic diseases who require long-term medication.

[0003] Therefore, there is a need to develop long-acting drugs that can reduce the burden on patients. Longer dosing cycles increase patient convenience, leading to increasing market competitiveness. The original GLP-1 RA drug, Byetta (exenatide), was a twice-a-day injection. Subsequently, the once-a-day injection Victoza (liraglutide) was developed and dominated the market. Currently, once-a-week injections such as Ozemopic (semaglutide) and dulaglutide hold a significant market share.

[0004] Due to the tremendous success of GLP-1 RA drugs, there is a growing demand for once-a-month or longer-lasting medications with even longer durations of action. However, due to technological limitations, there are no drugs with a sustained-action duration longer than once a week.

[0005] Meanwhile, sustained-release formulations using biodegradable polymers have been developed to achieve long-term, sustained-release effects. However, these formulations use biodegradable polymers that delay drug release to extend the duration of drug release and prevent excessive initial release. Consequently, for a considerable period of time after administration (ranging from 2-3 weeks to over a month), little drug is released, or the cumulative release rate is as low as 30% or less. This leads to a significant lag phase in drug release, resulting in a period of drug-effectiveness gap. Summary of the Invention

[0006] Technical issues

[0007] An object of the present invention is to provide sustained-release microspheres having excellent bioavailability and a sustained-release preparation containing the same.

[0008] The object of the present invention is to provide sustained-release microspheres, sustained-release preparations and methods for preparing the same, which do not release drugs excessively in the initial burst and do not cause delayed release during long-term sustained release.

[0009] The object of the present invention is to provide a pharmaceutical composition comprising the sustained-release microspheres or sustained-release preparation and a method for treating a disease comprising the step of administering the pharmaceutical composition to a subject.

[0010] Technical Solution

[0011] The present invention provides sustained-release microspheres comprising a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and a polylactic acid-glycolic acid copolymer (PLGA). The content of the GLP-1 receptor agonist or the pharmaceutically acceptable salt thereof is 3 to 12 weight percent of the total weight percent of the sustained-release microspheres. The polylactic acid-glycolic acid copolymer is a mixture of a low-viscosity polylactic acid-glycolic acid copolymer having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more high-viscosity polylactic acid-glycolic acid copolymers having an intrinsic viscosity of 0.32 to 0.60 dL / g.

[0012] The weight ratio of the one low-viscosity PLGA to the two or more high-viscosity PLGA in the mixture may be 1:0.2 to 5.

[0013] The GLP-1 receptor agonist may be semaglutide or tezetide.

[0014] The two or more high-viscosity PLGAs are one or more PLGAs having a molar ratio of lactide to glycolide of 45-55:45-55 and one or more PLGAs having a molar ratio of lactide to glycolide of 60-80:20-40.

[0015] The polylactic acid-glycolic acid copolymer may be present in an amount of 88 to 97 weight percent of the total weight of the sustained-release microspheres. The sustained-release microspheres may be prepared by dissolving the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and the polylactic acid-glycolic acid copolymer in glacial acetic acid, spraying the mixture using an ultrasonic spray nozzle, and then volatilizing the solvent using dry air.

[0016] The frequency of the ultrasonic spray nozzle can be 40 kHz to 80 kHz. After administering the sustained-release microspheres to SD rats or minipigs, the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof can be released at a rate of less than 5% within 24 hours, at a rate of more than 10% within 1 week, at a rate of more than 20% within 9 days, or at a rate of more than 50% within 2 weeks.

[0017] The sustained-release microspheres can be administered at intervals of 1 to 3 months.

[0018] The average particle size of the sustained-release microspheres may be 15 μm to 25 μm.

[0019] Furthermore, the present invention provides a sustained-release preparation comprising the sustained-release microspheres of the present invention.

[0020] Furthermore, the present invention provides a pharmaceutical composition comprising the sustained-release microspheres or sustained-release preparation for treating or preventing diabetes, obesity, non-alcoholic steatohepatitis or degenerative brain disease.

[0021] The degenerative brain disease may be one or more selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

[0022] The present invention provides a method for preparing sustained-release microspheres, comprising: dissolving 3 to 12 weight percent of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and 88 to 97 weight percent of a poly(lactic-co-glycolic acid) copolymer in a solvent to prepare a mixed solution; and spraying the mixed solution using an ultrasonic spray nozzle and then volatilizing the solvent using dry air. The poly(lactic-co-glycolic acid) copolymer is a mixture of a low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more high-viscosity PLGAs having an intrinsic viscosity of 0.32 to 0.60 dL / g.

[0023] The solvent may be an organic solvent, preferably, glacial acetic acid or acetic acid solution, but is not limited thereto.

[0024] Furthermore, the present invention provides a method for preparing a sustained-release preparation as follows: dispersing the prepared sustained-release microspheres in an aqueous solution containing polyvinyl alcohol and lysine hydrochloride, and then filtering and washing the microspheres and recovering the microspheres.

[0025] The method for preparing the sustained-release preparation further comprises the steps of suspending the recovered product in a D-mannitol and lysine hydrochloride solution and then freeze-drying the product.

[0026] Effects of the Invention

[0027] The sustained-release microspheres and sustained-release preparations containing them of the present invention eliminate excessive initial drug release and enable sustained release. Furthermore, they eliminate the issue of delayed release, exhibit excellent bioavailability, and can be administered to subjects at intervals of more than one month, improving patient convenience. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1a to 1d To illustrate the Figure 1a 、 Figure 1b 、 Figure 1c ) and Comparative Examples 1 to 4 ( Figure 1d ) is a graph showing the changes in blood concentration of the drug over time after subcutaneous injection of a sustained-release preparation containing microspheres into SD rats.

[0029] Figure 2 The graph shows the cumulative release rate of the drug over time after the sustained-release preparations containing microspheres of Example 3 and Comparative Examples 1 to 4 were subcutaneously injected into SD rats.

[0030] Figure 3 This is a graph showing the changes in blood concentration of the drug over time after subcutaneous injection of the sustained-release preparation containing microspheres of Example 3 into minipigs.

[0031] Figure 4 The graph shows the cumulative release rate of the drug over time after the sustained-release preparation containing microspheres of Example 3 is subcutaneously injected into miniature pigs.

[0032] Figure 5a to Figure 5b To confirm the results of body weight measurement over time during 8 weeks of administration of the sustained-release preparation containing microspheres of Example 3 to the DIO mouse model ( Figure 5a ) and weight gain rate ( Figure 5b ) curve graph.

[0033] Figure 6This is a graph showing changes in dietary intake over time during 8 weeks of administration of the sustained-release preparation containing microspheres of Example 3 to a DIO mouse model. DETAILED DESCRIPTION

[0034] The present invention provides sustained-release microspheres and sustained-release preparations comprising a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0035] The present invention provides the following sustained-release preparation containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof: the sustained-release microspheres contain the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, one low-viscosity PLGA having an intrinsic viscosity of 0.14 dL / g to 0.24 dL / g, and two or more high-viscosity PLGA having an intrinsic viscosity of 0.32 dL / g to 0.60 dL / g. Thus, the sustained-release microspheres do not have the problem of delayed release due to excessive initial release of the drug, and while enabling long-term sustained release, they also have excellent bioavailability.

[0036] The present invention provides sustained-release microspheres and a sustained-release preparation. The sustained-release preparation comprises the sustained-release microspheres, wherein the sustained-release microspheres comprise a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and a polylactic acid-glycolic acid copolymer, wherein the content of the GLP-1 receptor agonist or the pharmaceutically acceptable salt thereof is 3 weight percent to 12 weight percent of the total weight of the sustained-release microspheres, and the polylactic acid-glycolic acid copolymer is a mixture of a low-viscosity PLGA having an intrinsic viscosity of 0.14 dL / g to 0.24 dL / g and two or more PLGAs having intrinsic viscosities of 0.32 dL / g to 0.60 dL / g.

[0037] GLP-1 receptor agonists refer to glucagon-like peptide-1 receptor agonists (GLP-1 RA).

[0038] The GLP-1 receptor agonist may be one or more selected from the group consisting of exenatide, lixisenatide, albiglutide, dulaglutide, liraglutide, semaglutide, tezetide, retaglutide, canagliflozin, cotidulide, mazdutide, and cagrisema.

[0039] In one embodiment, the GLP-1 receptor agonist is semaglutide. Semaglutide (NN9536) is an analog of human glucagon-like peptide-1, GLP-1(7-37), with two amino acid substitutions (Ala8 is replaced by Aib8 [2-aminobutyric acid], and Lys34 is replaced by Arg34). The chemical name of semaglutide is N 6,26-{18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctanoyl}-[8-(2-amino-2-propionic acid), 34-L-arginine] human glucagon-like peptide-1 (7-37). Semaglutide can be prepared as described in Example 4 of WO2006 / 097537.

[0040] In one embodiment, the GLP-1 receptor agonist is tirzepatide, which is described in Example 1 of U.S. Patent No. 9,474,780.

[0041] The "pharmaceutically acceptable salt" of the present invention refers to any salt that has an effective effect at a concentration that is relatively non-toxic and harmless to patients and does not cause side effects that reduce the beneficial effects of the GLP-1 receptor agonist. It may be an acid addition salt or a base addition salt.

[0042] The pharmaceutically acceptable salt of the GLP-1 receptor agonist may be an acid addition salt of the GLP-1 receptor agonist. For example, it may be a hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, 2-hydroxyethanesulfonate (isethionate), nicotinate, 2-naphthalenesulfonate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, thiocyanate, or bicarbonate of a GLP-1 receptor agonist.

[0043] The pharmaceutically acceptable salt of the GLP-1 receptor agonist may be a base addition salt of the GLP-1 receptor agonist, for example, an alkali metal salt, an alkaline earth metal salt, or a quaternary ammonium salt of the GLP-1 receptor agonist.

[0044] The sustained-release microspheres of the present invention contain a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and a polylactic acid-glycolic acid copolymer.

[0045] The sustained-release microspheres contain at least one low-viscosity PLGA and two or more high-viscosity PLGAs.

[0046] In this specification, "low-viscosity PLGA" means PLGA having an intrinsic viscosity of 0.14 dL / g to 0.24 dL / g.

[0047] "High-viscosity PLGA" refers to PLGA with an intrinsic viscosity of 0.32 dL / g to 0.60 dL / g, and contains at least two high-viscosity PLGAs.

[0048] Low viscosity PGLA and high viscosity PLGA can be included in a weight ratio of 1:0.2-5, 1:0.2-3, 1:0.3-5, 1:0.3-3, 1:0.5-5, 1:0.5-3, 1:0.5-2, 1:1-5, 1:1-3, 1:1-2.5, 1:1.5-2.5, or 1:1.8-2.2.

[0049] In one embodiment, the high-viscosity PLGA may be a mixture of a PLGA having a viscosity of 0.32 dL / g to 0.44 dL / g (high-viscosity PLGA-1) and a PLGA having a viscosity of 0.45 to 0.60 dL / g (high-viscosity PLGA-2).

[0050] The weight of the sustained-release microspheres can be 20 mg to 6000 mg, 20 mg to 4000 mg, 100 mg to 6000 mg, 100 mg to 4000 mg, 500 mg to 6000 mg, 500 mg to 4000 mg, 1,000 mg to 6000 mg, 1,000 mg to 4000 mg, 1500 mg to 6000 mg, 1500 mg to 4000 mg, 2000 mg to 6000 mg, 2000 mg to 4000 mg, 2400 mg to 6000 mg, 2400 mg to 4000 mg, or 2400 mg to 3600 mg.

[0051] The content of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof can be 3 weight percent to 12 weight percent of the weight of the sustained-release microspheres.

[0052] In one embodiment, the composition may contain 3 weight percent or more and less than 12 weight percent, 3 weight percent or more and less than 11.8 weight percent, 3 weight percent or more and less than 10.8 weight percent, 3 weight percent or more and less than 10.5 weight percent, 3 weight percent or more and less than 10.2 weight percent, 3 weight percent or more and less than 10 weight percent, or 3 weight percent or more and less than 9 weight percent of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof; and may contain 8 weight percent or more and less than 12 weight percent, 8 weight percent or more and less than 11.8 weight percent, 8 weight percent or more and less than 10.8 weight percent, 8 weight percent or more and less than 10.5 weight percent, 8 weight percent or more and less than 10.2 weight percent, 8 weight percent or more and less than 10 weight percent, or 8 weight percent or more and 9 weight percent of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof. Preferably, the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof may be contained in an amount of 5 weight percent or more and less than 12 weight percent, 5 weight percent or more and less than 11.8 weight percent, 5 weight percent or more and less than 10.8 weight percent, 5 weight percent or more and less than 10.5 weight percent, 5 weight percent or more and less than 10.2 weight percent, 5 weight percent or more and less than 10 weight percent, or 5 weight percent or more and less than 9 weight percent, but is not limited thereto.

[0053] When the sustained-release microspheres or sustained-release preparations of the present invention contain a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof at a content of 3 to 12 weight percent, bioavailability is excellent, and administration can be performed at intervals of one month or more. From the perspective of initial release inhibition, preferably, the sustained-release microspheres contain 3 to 10 weight percent of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof. From the perspective of bioavailability, preferably, the sustained-release microspheres contain 5 to 12 weight percent or 7 to 12 weight percent of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0054] If the sustained-release microspheres do not contain two or more high-viscosity PLGAs, the initial release amount of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof will be too much. If the sustained-release microspheres do not contain low-viscosity PLGA, although the initial release amount of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof is small, the release delay will be prolonged and the total drug release amount will be reduced, thereby reducing the bioavailability.

[0055] The sustained-release microspheres of the present invention comprise a poly(lactic acid-glycolic acid) copolymer mixture of low-viscosity PLGA and two or more high-viscosity PLGAs. Preferably, the low-viscosity PLGA and the two or more high-viscosity PLGAs are mixed in a weight ratio of 1:0.2 to 5, and more preferably, the low-viscosity PLGA and the two or more high-viscosity PLGAs are mixed in a weight ratio of 1:1.8 to 2.2. This allows for long-term sustained release of the GLP-1 receptor agonist without causing an initial excessive release or delayed release, thereby exhibiting excellent bioavailability.

[0056] The intrinsic viscosity of PLGA is the viscosity measured at 25° C. using an Ubbelohde viscometer after dissolving PLGA in chloroform at a concentration of 0.1% (W / V).

[0057] The molar ratio of lactide to glycolide in the low-viscosity PLGA may be 40-80:20-60. For example, in one embodiment, the molar ratio of lactide to glycolide may be 45-70:30-55, 55-70:30-45, or 45-55:45-55.

[0058] Commercially available low-viscosity PLGAs include RG502, RG502H, RG752H, and RG752S from Evonik. In this specification, RG502H is also referred to as PLGA50A.

[0059] The molar ratio of lactide to glycolide in high-viscosity PLGA can be 40-80:20-60. For example, in one embodiment, the molar ratio of lactide to glycolide is 40-75:25-60, 45-70:30-55, 55-70:30-45, or 45-55:45-55. In one embodiment, a mixture of one PLGA having a molar ratio of lactide to glycolide of 45-55:45-55 and one or more PLGAs having a molar ratio of lactide to glycolide of 60-80:20-40 can be used as the high-viscosity PLGA. In one embodiment, a mixture of high-viscosity PLGA-1 and high-viscosity PLGA-2 can be used as the high-viscosity PLGA.

[0060] Commercially available high-viscosity PLGAs include RG503, RG503H, RG653H, RG753H, RG753S, and RG504H from Evonik. In this specification, RG503H is also referred to as PLGA50B, RG504H as PLGA50C, RG653H as PLGA65B, and RG753H as PLGA75B.

[0061] The content of the polylactic acid-glycolic acid copolymer can be 88 weight percent to 97 weight percent of the total weight percent of the sustained-release microspheres.

[0062] In addition to the active ingredient and the polylactic acid-glycolic acid copolymer, the sustained-release preparation of the present invention may further contain coating materials, additives, excipients, and the like.

[0063] In one embodiment, the sustained-release preparation may comprise 3 to 12 weight percent of semaglutide, tezetide, or a pharmaceutically acceptable salt thereof, 88 to 97 weight percent of poly(lactic-co-glycolic acid), and coating materials, additives, or excipients.

[0064] The sustained-release microspheres can be coated with lysine, and the lysine coated on the sustained-release microspheres can include 0.01 to 5 parts by weight, 0.01 to 3 parts by weight, 0.01 to 1 part by weight, 0.1 to 1 part by weight, 0.2 to 0.8 parts by weight, or 0.4 to 0.6 parts by weight, relative to 100 parts by weight of the sustained-release microspheres before coating.

[0065] Depending on the subject, the sustained-release preparation comprising sustained-release microspheres of the present invention can maintain therapeutic effects for more than one month after a single administration. The sustained-release preparation of the present invention can be administered to the subject at intervals of 1 month, 1.5 months, 2 months, 2.5 months, or 3 months.

[0066] After administration to Sprague Dawley rats (SD rats), the sustained-release microspheres of the present invention or the sustained-release preparation containing the same can release less than 5% within 24 hours, more than 10% within 1 week, more than 20% within 9 days, or more than 50% within 2 weeks.

[0067] The mean particle size (Mean Diameter) of the sustained-release microspheres of the present invention can be 5 μm to 50 μm, 5 μm to 40 μm, 5 μm to 30 μm, 10 μm to 50 μm, 10 μm to 40 μm, 10 μm to 30 μm, 10 to 25 μm, 15 to 50 μm, 15 μm to 40 μm, 15 μm to 30 μm or 15 μm to 25 μm.

[0068] The span value of the sustained-release microspheres is 2 or less, more specifically, 0.1 to 2, 0.1 to 1.8, 0.3 to 2, 0.3 to 1.8, 0.5 to 2, 0.5 to 1.8, 0.8 to 2, 0.8 to 1.8, 1 to 2, 1 to 1.8, or 1 to 1.6.

[0069] The sustained-release microspheres or the sustained-release preparations containing the same may further contain additives and excipients commonly used in the formulation of pharmaceutical agents in the technical field to which the present invention belongs.

[0070] The sustained-release preparation of the present invention may further comprise one or more protective colloids selected from the group consisting of polyvinyl alcohol, albumin, polyvinyl pyrrolidone, gelatin, and the like. The protective colloid prevents aggregation between the microspheres comprising the GLP-1 receptor agonist and improves dispersibility. Preferably, the protective colloid comprises 0.02 to 1.0 weight percent relative to the total weight of the sustained-release preparation.

[0071] The present invention provides a pharmaceutical composition comprising the sustained-release microspheres or a sustained-release preparation thereof for treating or preventing diabetes, obesity, non-alcoholic steatohepatitis (NASH) or degenerative brain diseases.

[0072] The degenerative brain disease may be one or more selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

[0073] The sustained-release microspheres can be prepared by dissolving the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and polylactic acid-co-glycolic acid in glacial acetic acid, spraying the solution using an ultrasonic spray nozzle, and then evaporating the solvent using dry air.

[0074] The present invention provides a method for preparing sustained-release microspheres which have no initial excessive release of drugs, can release drugs continuously for a long time, have no release delay problem and have excellent bioavailability.

[0075] The method for preparing sustained-release microspheres of the present invention comprises: dissolving a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and a polylactic acid-glycolic acid copolymer in an organic solvent to prepare a mixed solution; and obtaining the sustained-release microspheres by drying the mixed solution.

[0076] In the method for preparing the sustained-release preparation, the type of the polylactic acid-glycolic acid copolymer dissolved together with the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof is the same as that described above for the sustained-release microspheres.

[0077] In the method for preparing sustained-release microspheres, the weight ratio of the GLP-1 receptor agonist to the polylactic acid-co-glycolic acid copolymer can be appropriately adjusted so that the content of the GLP-1 receptor agonist in the prepared sustained-release microspheres is 3 to 12 weight percent.

[0078] The organic solvent may be one selected from the group consisting of chloroform, ethyl acetate, dichloromethane, methyl ethyl ketone, alcohols having 1 to 5 carbon atoms, glacial acetic acid, formic acid, dimethyl sulfoxide and n-methylpyrrolidone, or a mixed solvent thereof, preferably glacial acetic acid.

[0079] In the method for preparing the sustained-release preparation of the present invention, the method for obtaining microspheres by drying the mixed solution may utilize a spray drying method.

[0080] The spray drying method can be carried out as follows: a mixed solution containing semaglutide or a pharmaceutically acceptable salt thereof and poly(lactic acid-co-glycolic acid) is supplied to a spray dryer at a flow rate of 5 mL / min to 20 mL / min, 5 mL / min to 15 mL / min, or 5 mL / min to 10 mL / min, and dry air at 100° C. to 200° C., 100° C. to 150° C., or 120° C. to 150° C. is supplied. In addition, the frequency of the spray dryer can be 40 kHz to 80 kHz, 50 kHz to 70 kHz, or 55 kHz to 75 kHz, preferably 60 kHz.

[0081] After the step of obtaining microspheres by drying the mixed solution, the method for preparing the sustained-release preparation of the present invention may further include the step of suspending the dried microspheres in an aqueous solution containing lysine hydrochloride to coat the microspheres with lysine.

[0082] The aqueous solution containing lysine hydrochloride may further contain 1% (W / V) of polyvinyl alcohol.

[0083] The present invention provides a method for treating diabetes, obesity, non-alcoholic steatohepatitis or degenerative brain disease, comprising the step of administering the sustained-release microspheres or a sustained-release preparation containing the same to a subject.

[0084] The degenerative brain disease may be one or more selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

[0085] The sustained-release microspheres of the present invention or the sustained-release preparation containing the same can be administered orally or parenterally, preferably, can be administered by parenteral routes such as intravenous administration, subcutaneous administration, intramuscular administration, and intraperitoneal administration.

[0086] The effective dosage of the sustained-release microspheres or sustained-release preparation of the present invention can be appropriately adjusted according to the patient's age, the type and severity of the disease, the patient's condition, etc. For example, based on the amount of the active ingredient, it can be 1 mg / week to 30 mg / week. The amount can be administered once or divided into several doses.

[0087] Hereinafter, the present invention will be described in more detail with reference to examples.

[0088] Example

[0089] 1. Preparation of sustained-release microspheres

[0090] Prepare PLGA with semaglutide or tezetribute as a GLP-1 receptor agonist and the following Table 1.

[0091] Table 1

[0092]

[0093] After semaglutide or tezetide and PLGA are completely dissolved in glacial acetic acid to obtain a mixed solution, a spray drying process is performed to prepare sustained-release microspheres.

[0094] The spray drying process was carried out as follows: the prepared mixed solution was supplied to a spray dryer (EIN System) equipped with an ultrasonic nozzle (Sonictopia, 60 kHz) using a peristaltic pump (Masterflex) at a flow rate of 7.5 mL / min. The oscillator (Sonictopia, 60 kHz) power was adjusted to 50-80%, and dry air at 135°C was supplied. The microspheres prepared by the spray drying process were dispersed in an aqueous solution containing 1% (w / v) polyvinyl alcohol (Mitsubishi Chemical Corporation) and 0.5 M L-lysine hydrochloride (Merck) as a protective colloid, and stirred for 3 hours. The microspheres were then recovered by filtration and washed with distilled water, suspended in a solution of 10% D-mannitol (ROQUETTE) and 0.5% L-lysine hydrochloride (Merck), and then freeze-dried to obtain the sustained-release preparation of the present invention.

[0095] The compositions of semaglutide and PLGA in the sustained-release microspheres are shown in Examples 1 to 9 and Comparative Examples 1 to 4 in Table 2 below, and the compositions of tezetide and PLGA are shown in Example 10 and Comparative Example 5. In Example 7, PLGA65B and PLGA75B were used as high-viscosity PLGA (C) in the same amounts as low-viscosity PLGA (A).

[0096] Table 2

[0097]

[0098]

[0099] The ratios of LA and GA in the high-viscosity PLGA and the ratios of LA and GA in the total PLGA of the microspheres of Examples 1 to 10 and Comparative Examples 1 to 5 are shown in Table 3 below.

[0100] Table 3

[0101]

[0102]

[0103] 2. Measuring the particle size of sustained-release microspheres

[0104] The mean particle size (MD) of Examples 1 to 10 and Comparative Examples 1 to 5 was quantitatively measured. 180 mg of microspheres and 10 mL of 1% PVA were placed in a 50 mL conical tube and dispersed in an ultrasonicator for 1 minute. The tube was then placed in a dynamics analyzer (CILAS, France) to measure the particle size. The results are shown in Table 4 below.

[0105] Table 4

[0106] distinguish Particle size (μm) Example 1 21.3 Example 2 18.7 Example 3 19.3 Example 4 21.8 Example 5 18.7 Example 6 22 Example 7 21.7 Example 8 22.6 Example 9 20.6 Example 10 20 Comparative Example 1 18.5 Comparative Example 2 20.9 Comparative Example 3 21.2 Comparative Example 4 21.3 Comparative Example 5 20.1

[0107] 3. Pharmacokinetic characteristics of the sustained-release formulation in SD rats

[0108] After administering the sustained-release preparation containing semaglutide or tezetribide of the present invention to 8-week-old SD rats (n=5), the in vivo release pattern of the drug was evaluated.

[0109] 8-week-old male SD rats were subcutaneously injected (SC) with the sustained-release formulations containing microspheres of Examples and Comparative Examples at a drug concentration of 2 mpk (dose, mg / kg). The blood concentrations of the drug (semaglutide or tezetide) were observed over time. 0.5 ml of blood was collected from the jugular vein at 1 hour, 3 hours, 6 hours, 24 hours, 3 days, 5 days, 7 days, 9 days, 11 days, 14 days, 16 days, 18 days, 21 days, 24 days, 28 days, 31 days, and 35 days after injection and stored at -80°C. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was then performed.

[0110] Table 5 shows the relative area under the curve (AUC) values and cumulative release rate (ratio of cumulative release amount at different times to total release amount). The area under the curve (AUC) was calculated from the blood concentration curve over time. For the case of drug administration alone, the relative AUC value, expressed as a ratio compared to the calculated AUC value, was evaluated as bioavailability. The cumulative release rate was used to evaluate the delayed release of the initial excess release agent according to the following criteria.

[0111] *Evaluation Criteria

[0112] 1) Evaluation of initial excessive release: If the release rate was 5% or less within one day (24 hours), it was evaluated as no initial excessive release.

[0113] 2) Release delay evaluation: The cases where the release rate was 10% or more by 1 week (7 days), 20% or more by 9 days (or 25% or more by 10 days), and 50% or more by 2 weeks (14 days) were evaluated as having no release delay problem.

[0114] 3) Bioavailability evaluation: A relative AUC value of 50% or higher was considered to be high in bioavailability.

[0115] The sustained-release preparations of Examples 1 to 10 of the present invention showed relative AUC values of 50% or more (Table 5 and Table 6). Figure 1a 、 Figure 1b 、 Figure 1c ), and the bioavailability was evaluated as high according to the evaluation criteria. In addition, the initial release showed a value of less than 5%, with no initial excessive release. The cumulative release rate to the 7th day was more than 10%, the cumulative release rate to the 9th day was more than 20%, and the cumulative release rate to the 14th day was more than 50%, thus confirming that it was a sustained-release preparation without the problem of delayed release (Table 5 and Figure 2 ).

[0116] However, although the sustained-release microsphere preparation of Comparative Example 1 had high bioavailability and no delayed release problem, it had the problem of excessive initial release, unlike the examples. Comparative Examples 2 to 5 had no excessive initial release, but had low bioavailability and delayed release for more than one week after administration (Table 5 and Table 6). Figure 1d ).

[0117] Table 5

[0118]

[0119] Also, Examples 1 to 10 show similar release patterns, and Example 3 is typically selected and Figure 2As shown in the figure, the Examples of the present invention show sustained release without a high initial release and a problem of delayed release. In contrast, compared with the Examples, Comparative Example 1 using only low-viscosity PLGA shows excessive initial release and a short release period, and Comparative Examples 2 to 4 using only high-viscosity PLGA show delayed release of more than one week.

[0120] The subsequent experiments were also typically performed according to Example 3.

[0121] 4. Pharmacokinetic characteristics of sustained-release formulations in minipigs

[0122] Next, in order to evaluate the pharmacokinetics in medium and large animals, the sustained-release preparation of Example 3 of the present invention was administered to minipigs, and the in vivo release pattern of the drug in the microspheres was evaluated.

[0123] Following subcutaneous injection of 0.08 mpk (dose, mg / kg) into 20-30 kg male SPF minipigs, semaglutide blood concentrations were observed over time. 2.5 ml of blood was collected from the jugular vein at 1 hour, 3 hours, 6 hours, 24 hours, 3 days, 5 days, 7 days, 9 days, 11 days, 14 days, 16 days, 18 days, 21 days, 24 days, 28 days, 31 days, 35 days, 42 days, 49 days, 56 days, 63 days, and 70 days (23 points in total) after injection and stored at -80°C before LC-MS / MS analysis.

[0124] The results can be found in Figure 3 and Figure 4 Confirmed in Figure 3 The blood concentration of semaglutide is shown. Figure 4 The cumulative release rate of semaglutide is shown. The results of the present invention confirm that even in minipigs, an animal model more similar to humans than rats, there is no initial excessive release or delayed release. Furthermore, it can be confirmed that semaglutide release lasts longer in minipigs, which are larger than SD rats. Furthermore, these results suggest that the release duration will be further increased when administered to humans.

[0125] 5. Evaluate the effectiveness of anti-obesity

[0126] The anti-obesity effectiveness was confirmed by administering the sustained-release preparation of Example 3 of the present invention to a diet-induced obesity (DIO) mouse model.

[0127] Sixteen-week-old male C57BL / 6J and C57BL / 6J-DIO mice (The Jackson Laboratory (JAX), Saeronbio) were used as experimental animals. Upon arrival, the mice were provided with a normal diet (Regular chow diet, 5 kcal% fat, PicoLab, #5053, USA) or a high-fat diet (60 kcal% fat, Research Diets, D12492, USA) and water. The mice were maintained at a temperature of 22 ± 2°C, a humidity of 50 ± 10%, and a 12-hour light-dark cycle (dark: 7 pm to 7 am). The mice were acclimated in individual cages. After one week of acclimation, all mice were administered medications and observed twice daily (dose in the morning and monitoring in the afternoon) for clinical symptoms (activity, abnormal stools, bleeding, wounds, deformities, skin, fur, and eye abnormalities, etc.).

[0128] To assess body weight and dietary intake, the drugs were administered for 8 weeks under the conditions shown in Table 6 below. Starting from the start of drug administration, body weight (FX-2000i, A&D, 0.01-2200 g) and dietary intake (CSG201F, OHAUS, 0.1 g-200 g) were measured daily. All results are expressed as mean ± standard error. Statistical significance was calculated using independent t-tests between the experimental drug-administered groups. The results are shown in Table 6.

[0129] Table 6

[0130]

[0131] Weight measurement results in Figure 5a As shown in the figure, the body weight gain rate (ΔBody weight from baseline (vsCon (-)), %) Figure 5b This is the result calculated by comparing the body weight change (%) following drug administration with that of the negative control group.

[0132] As shown in Figure 5, the normal control group and the negative control group increased their weight. In contrast, the weight of the positive control group and Example 3 (Q3D, QW, Q2W) decreased statistically significantly (p < 0.05) compared to the negative control group. Furthermore, referring to Figure 5, the positive control group and Example 3 (Q3D, QW, Q2W) showed statistically significant (p < 0.05) weight loss rates compared to the negative control group.

[0133] And, the dietary intake (g) was as Figure 6 The dietary intake measured during drug administration showed that in the positive control group and all the evaluated drug administration groups, dietary intake decreased sharply immediately after administration and then gradually recovered. However, compared with the negative control group, the reduced dietary intake was maintained, and a statistically significant (p < 0.05) reduction in dietary intake was shown compared with the negative control group.

[0134] The above results show that the sustained-release preparation of the present invention can show a significant body weight reduction effect even when administered once every two weeks (as for mice) compared to the positive control group administered daily.

[0135] In summary, it can be confirmed that the sustained-release preparation of the present invention does not have the problem of delayed release due to excessive initial release, can release for a long time, and is a preparation with high bioavailability.

Claims

1. A sustained-release microsphere, characterized in that: Containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and a polylactic acid-glycolic acid copolymer, The content of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof is 3 weight percent to 12 weight percent of the total weight percent of the sustained-release microspheres. The polylactic acid-glycolic acid copolymer is a mixture of a low-viscosity polylactic acid-glycolic acid copolymer with an intrinsic viscosity of 0.14 dL / g to 0.24 dL / g and two or more high-viscosity polylactic acid-glycolic acid copolymers with an intrinsic viscosity of 0.32 dL / g to 0.60 dL / g.

2. The sustained-release microspheres according to claim 1, characterized in that The weight ratio of the low-viscosity polylactic acid-glycolic acid copolymer to the two or more high-viscosity polylactic acid-glycolic acid copolymers in the mixture is 1:0.2 to 5.

3. The sustained-release microspheres according to claim 1, characterized in that The GLP-1 receptor agonist is semaglutide or tezetide.

4. The sustained-release microspheres according to claim 1, characterized in that The two or more high-viscosity polylactic acid-glycolic acid copolymers are one or more polylactic acid-glycolic acid copolymers having a molar ratio of lactide to glycolide of 45-55:45-55 and one or more polylactic acid-glycolic acid copolymers having a molar ratio of lactide to glycolide of 60-80:20-40.

5. The sustained-release microspheres according to claim 1, characterized in that The content of the polylactic acid-glycolic acid copolymer is 88 weight percent to 97 weight percent of the total weight percent of the sustained-release microspheres.

6. The sustained-release microspheres according to claim 1, characterized in that The sustained-release microspheres are prepared by the following steps: dissolving the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and the poly(lactic-co-glycolic acid) copolymer in glacial acetic acid to prepare a mixed solution; and The step of spraying the mixed solution with an ultrasonic spray nozzle and then volatilizing the glacial acetic acid with dry air.

7. The sustained-release microspheres according to claim 6, characterized in that The frequency of the ultrasonic spray nozzle is 40 kHz to 80 kHz.

8. The sustained-release microspheres according to claim 1, characterized in that After administration to SD rats or minipigs, the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof releases less than 5% within 24 hours, more than 10% within 1 week, more than 20% within 9 days, or more than 50% within 2 weeks.

9. The sustained-release microspheres according to claim 1, characterized in that The doses are given at intervals of 1 to 3 months.

10. The sustained-release microspheres according to claim 1, characterized in that The average particle size of the sustained-release microspheres is 15 μm to 25 μm.

11. A sustained-release preparation, characterized in that: The method comprises the sustained-release microspheres according to any one of claims 1 to 10.

12. A pharmaceutical composition for treating or preventing diabetes, obesity, non-alcoholic steatohepatitis or degenerative brain disease, characterized in that: The method comprises the sustained-release microspheres according to any one of claims 1 to 10.

13. The pharmaceutical composition according to claim 12, characterized in that The degenerative brain disease is one or more selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, stroke and multiple sclerosis.

14. A method for preparing sustained-release microspheres or sustained-release preparations, characterized in that: include: dissolving 3 to 12 weight percent of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and 88 to 97 weight percent of a poly(lactic-co-glycolic acid) copolymer in a solvent to prepare a mixed solution; and The step of spraying the mixed solution with an ultrasonic spray nozzle and then volatilizing the solvent with dry air, The polylactic acid-glycolic acid copolymer is a mixture of a low-viscosity polylactic acid-glycolic acid copolymer with an intrinsic viscosity of 0.14 dL / g to 0.24 dL / g and two or more high-viscosity polylactic acid-glycolic acid copolymers with an intrinsic viscosity of 0.32 dL / g to 0.60 dL / g.

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