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

Abstract

The present invention relates to sustained-release microspheres and sustained-release formulations containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof. More specifically, the present invention relates to sustained-release microspheres containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g, and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g. This enables long-term sustained release without the problems of initial over-release and delayed release of the drug, and provides excellent bioavailability.

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 sustained-release formulations containing the same, and more particularly to a method for producing the same and their use in the treatment of diabetes, obesity, non-alcoholic steatohepatitis, or degenerative brain diseases. [Background technology]

[0002] Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are a class of drugs used in the treatment of type 2 diabetes mellitus (T2DM) that are highly effective in lowering blood glucose levels. They are also known to have weight loss effects, reduce hypertension, hypoglycemia, and / or hyperlipidemia, and have cardiovascular protection effects, making them increasingly important clinically. Compared with traditional insulin secretagogues such as sulfonylureas or meglitinides, they have the advantage of a lower risk of hypoglycemia. However, frequent injections are required, making them less convenient for patients with chronic diseases requiring long-term administration.

[0003] Therefore, there is a need for the development of long-acting drugs that can reduce the burden on patients, and drugs with longer administration cycles tend to be more convenient for patients and more competitive in the market. The first GLP-1 RA drug, Byetta (exenatide), was an injection administered twice a day. Subsequently, Victoza (liraglutide), an injection administered once a day, was developed and has dominated the market. Currently, once-a-week injections such as Ozempic (semaglutide) and Trulicity (dulaglutide) dominate the market.

[0004] The great success of GLP-1 RA drugs has led to an increased demand for drugs with an even longer duration of action, such as once-a-month or longer dosing intervals. However, due to technological limitations, no drugs with a longer duration of action than once-a-month dosing intervals have yet been developed.

[0005] In addition, sustained-release preparations using biodegradable polymers have been developed to achieve sustained release over a long period of time. However, these preparations use slow-releasing biodegradable polymers to extend the drug release duration and prevent initial excessive release. However, this results in little or no drug release for a considerable period of time after administration (as short as 2-3 weeks, or as long as more than a month), or a significant lag phase with a cumulative drug release rate of less than 30%, resulting in a gap in the efficacy of the drug. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide sustained-release microspheres with excellent bioavailability and sustained-release preparations containing the same.

[0007] The present invention aims to provide sustained-release microspheres and sustained-release formulations that do not have an initial burst of drug and that release the drug continuously over a long period of time without the problem of lag release, as well as methods for producing the same.

[0008] Another object of the present invention is to provide a pharmaceutical composition containing the sustained-release microspheres or sustained-release formulation, and a method for treating a disease comprising administering the same to a subject. [Means for solving the problem]

[0009] The present invention provides sustained-release microspheres comprising a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and poly(lactide-co-glycolide) (PLGA), wherein the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof accounts for 3 to 12 wt % of the total weight, and the PLGA is a mixture of one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g.

[0010] The weight ratio of the one type of low-viscosity PLGA to the two or more types of high-viscosity PLGA in the mixture may be 1:0.2-5.

[0011] The GLP-1 receptor agonist may be semaglutide or tirzepatide.

[0012] The two or more types of high-viscosity PLGA may be one or more types of PLGA having a lactide to glycolide molar ratio of 45-55:45-55 and one or more types of PLGA having a lactide to glycolide molar ratio of 60-80:20-40.

[0013] The poly(lactide-co-glycolide) may be contained in an amount of 88 to 97% by 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 poly(lactide-co-glycolide) in glacial acetic acid, spraying the solution using an ultrasonic spray nozzle, and then volatilizing the solvent using dry air.

[0014] The ultrasonic spray nozzle may have a frequency of 40 to 80 kHz. After administration of the sustained-release microspheres to SD rats or minipigs, the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof may be released at a rate of less than 5% within 24 hours, at a rate of 10% or more within one week, at a rate of 20% or more within nine days, or at a rate of 50% or more within two weeks.

[0015] The sustained-release microspheres can be administered every one to three months.

[0016] The sustained-release microspheres may have an average particle size of 15 to 25 μm.

[0017] The present invention further provides a sustained-release preparation comprising the sustained-release microspheres according to the present invention.

[0018] The present invention also provides a pharmaceutical composition for treating or preventing diabetes, obesity, non-alcoholic steatohepatitis, or degenerative brain diseases, which comprises the sustained-release microspheres or sustained-release formulation.

[0019] The degenerative brain disease can be any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lugelig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

[0020] The present invention provides a method for producing sustained-release microspheres, comprising the steps of: dissolving 3 to 12 wt % of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and 88 to 97 wt % of poly(lactide-co-glycolide) in a solvent to prepare a mixed solution; and spraying the mixed solution through an ultrasonic spray nozzle and then volatilizing the solvent with dry air to produce microspheres, wherein the poly(lactide-co-glycolide) is a mixture of one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g.

[0021] The solvent may be an organic solvent, preferably glacial acetic acid or an acetic acid solution, but is not limited thereto.

[0022] The present invention also provides a method for preparing a sustained-release formulation by dispersing the sustained-release microspheres prepared above in an aqueous solution containing polyvinyl alcohol and lysine hydrochloride, filtering, washing, and recovering the dispersion.

[0023] The method for preparing the sustained-release formulation may further include suspending the recovered product in a D-mannitol and lysine hydrochloride solution, followed by freeze-drying. [Effects of the Invention]

[0024] The sustained-release microspheres and sustained-release formulations containing the same of the present invention are capable of sustained release of drugs over a long period of time without initial excessive release, and are free from the problem of delayed release, have excellent bioavailability, and can be administered to subjects at intervals of one month or more, thereby improving the convenience of administration to patients. [Brief explanation of the drawings]

[0025] [Figure 1a] FIG. 1 is a graph showing the change in blood drug concentration over time after subcutaneous injection of sustained-release formulations containing microspheres into SD rats in Examples 1 to 10 (FIGS. 1a, 1b, and 1c) and Comparative Examples 1 to 4 (FIG. 1d). [Figure 1b] FIG. 1 is a graph showing the change in blood drug concentration over time after subcutaneous injection of sustained-release formulations containing microspheres into SD rats in Examples 1 to 10 (FIGS. 1a, 1b, and 1c) and Comparative Examples 1 to 4 (FIG. 1d). [Figure 1c] FIG. 1 is a graph showing the change in blood drug concentration over time after subcutaneous injection of sustained-release formulations containing microspheres into SD rats in Examples 1 to 10 (FIGS. 1a, 1b, and 1c) and Comparative Examples 1 to 4 (FIG. 1d). [Figure 1d] FIG. 1 is a graph showing the change in blood drug concentration over time after subcutaneous injection of sustained-release formulations containing microspheres into SD rats in Examples 1 to 10 (FIGS. 1a, 1b, and 1c) and Comparative Examples 1 to 4 (FIG. 1d). [Figure 2] 1 is a graph showing the change in cumulative drug release rate over time after sustained-release preparations containing microspheres were subcutaneously injected into SD rats in Example 3 and Comparative Examples 1 to 4. [Figure 3] 1 is a graph showing the change in blood drug concentration over time after a sustained-release formulation containing microspheres was subcutaneously injected into minipigs in Example 3. [Figure 4] 1 is a graph showing the change in cumulative drug release rate over time after a sustained-release formulation containing microspheres was subcutaneously injected into a minipig in Example 3. [Figure 5a] 5A and 5B are graphs showing the results of weight measurement over time (FIG. 5A) and the weight gain rate (FIG. 5B) while a sustained-release formulation containing microspheres was administered to a DIO mouse model for 8 weeks in Example 3. [Figure 5b] 5A and 5B are graphs showing the results of weight measurement over time (FIG. 5A) and the weight gain rate (FIG. 5B) while a sustained-release formulation containing microspheres was administered to a DIO mouse model for 8 weeks in Example 3. [Figure 6] 1 is a graph showing the change in food intake over time while a sustained-release preparation containing microspheres was administered to a DIO mouse model for 8 weeks in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides sustained-release microspheres and sustained-release formulations comprising a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0027] The present invention provides a sustained-release formulation containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, which contains sustained-release microspheres containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g, and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g. This formulation is free from the problems of initial excessive release and delayed release of the drug, enables long-term sustained release, and has excellent bioavailability.

[0028] The present invention provides sustained-release microspheres and sustained-release formulations, which comprise sustained-release microspheres containing a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and poly(lactide-co-glycolide), wherein the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof accounts for 3 to 12% by weight of the sustained-release microspheres, and the poly(lactide-co-glycolide) is a mixture of one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g.

[0029] The term GLP-1 receptor agonist refers to glucagon-like peptide-1 receptor agonists (GLP-1 RA).

[0030] The GLP-1 receptor agonist may be any one selected from the group consisting of exenatide, lixisenatide, albiglutide, dulaglutide, liraglutide, semaglutide, tirzepatide, retatortide, caglilintide, cotadutide, mazdutide and caglisema.

[0031] In one embodiment, the GLP-1 receptor agonist is semaglutide. Semaglutide (NN9536) is an analogue 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-propanoic acid), 34-L-arginine]human glucagon-like peptide 1(7-37). Semaglutide can be prepared as described in Example 4 of WO2006 / 097537.

[0032] In one embodiment, the GLP-1 receptor agonist is tirzepatide, which is described in Example 1 of U.S. Patent No. 9,474,780.

[0033] In the present invention, the term "pharmaceutically acceptable salt" refers to any salt that has a relatively non-toxic and harmless effective effect on patients at a concentration, and the side effects attributable to the salt do not reduce the beneficial efficacy of the GLP-1 receptor agonist, and may be an acid addition salt or a base addition salt.

[0034] The pharmaceutically acceptable salt of the GLP-1 receptor agonist may be an acid addition salt of the GLP-1 receptor agonist.For example, the 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, It may be digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, 2-hydroxyethanesulfonate (isethionate), nicotinate, 2-naphthalenesulfonate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, thiocyanate, or bicarbonate.

[0035] The pharmaceutically acceptable salt of the GLP-1 receptor agonist can be a base addition salt of the GLP-1 receptor agonist, such as an alkali metal salt, an alkaline earth metal salt, or a quaternary ammonium salt of the GLP-1 receptor agonist.

[0036] The sustained-release microspheres of the present invention comprise poly(lactide-co-glycolide) (PLGA) together with a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0037] The sustained-release microspheres contain at least one type of low-viscosity PLGA and two or more types of high-viscosity PLGA.

[0038] As used herein, "low-viscosity PLGA" refers to PLGA with an intrinsic viscosity of 0.14 to 0.24 dL / g.

[0039] "High-viscosity PLGA" refers to PLGA with an intrinsic viscosity of 0.32 to 0.60 dL / g. High-viscosity PLGA includes at least two types.

[0040] The low viscosity PGLA and high viscosity PLGA may be contained 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.

[0041] In one embodiment, the high-viscosity PLGA can be a mixture of one type of PLGA (high-viscosity PLGA-1) with a viscosity of 0.32 to 0.44 dL / g and one type of PLGA (high-viscosity PLGA-2) with a viscosity of 0.45 to 0.60 dL / g.

[0042] The weight of the sustained-release microspheres may be 20 mg to 6,000 mg, 20 mg to 4,000 mg, 100 mg to 6,000 mg, 100 mg to 4,000 mg, 500 mg to 6,000 mg, 500 mg to 4,000 mg, 1,000 mg to 6,000 mg, 1,000 mg to 4,000 mg, 1,500 mg to 6,000 mg, 1,500 mg to 4,000 mg, 2,000 mg to 6,000 mg, 2,000 mg to 4,000 mg, 2,400 mg to 6,000 mg, 2,400 mg to 4,000 mg, or 2,400 mg to 3,600 mg.

[0043] The GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof may be included in an amount of 3 to 12% by weight of the sustained-release microspheres.

[0044] In one embodiment, the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof may be present at 3% by weight or more but less than 12% by weight, 3% by weight or more but less than 11.8% by weight, 3% by weight or more but less than 10.8% by weight, 3% by weight or more but less than 10.5% by weight, 3% by weight or more but less than 10.2% by weight, 3% by weight or more but less than 10% by weight, or 3% by weight or more but less than 9% by weight, or may be present at 8% by weight or more but less than 12% by weight, 8% by weight or more but less than 11.8% by weight, 8% by weight or more but less than 10.8% by weight, 8% by weight or more but less than 10.5% by weight, 8% by weight or more but less than 10.2% by weight, 8% by weight or more but less than 10% by weight, or 8% by weight or more but less than 9% by weight. Preferably, the content may be 5% by weight or more but less than 12% by weight, 5% by weight or more but 11.8% by weight or less, 5% by weight or more but 10.8% by weight or less, 5% by weight or more but 10.5% by weight or less, 5% by weight or more but 10.2% by weight or less, 5% by weight or more but 10% by weight or less, or 5% by weight or more but 9% by weight or less, but not limited to these.

[0045] When the sustained-release microspheres or sustained-release formulations according to the present invention contain 3 to 12% by weight of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof, they exhibit excellent bioavailability and can be administered at intervals of one month or more. From the viewpoint of suppressing initial release, the sustained-release microspheres preferably contain 3 to 10% by weight of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof. From the viewpoint of bioavailability, the sustained-release microspheres preferably contain 5 to 12% by weight or 7 to 12% by weight of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0046] If sustained-release microspheres do not contain two or more types of high-viscosity PLGA, the initial release amount of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof may be excessively high, whereas if low-viscosity PLGA is not contained, the initial release amount of the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof may be low, but the lag phase may be long, resulting in a low total drug release amount and reduced bioavailability.

[0047] The sustained-release microspheres of the present invention contain a poly(lactide-co-glycolide) mixture of low-viscosity PLGA and two or more types of high-viscosity PLGA, preferably a mixture of low-viscosity PLGA and two or more types of high-viscosity PLGA at a weight ratio of 1:0.2-5, more preferably a mixture of low-viscosity PLGA and two or more types of high-viscosity PLGA at a weight ratio of 1:1.8-2.2, thereby enabling long-term sustained release of the GLP-1 receptor agonist without initial excessive release or lag phase problems, and exhibiting excellent bioavailability.

[0048] The intrinsic viscosity of PLGA is the viscosity measured at 25°C using an Ubbelohde viscometer after dissolving it in chloroform at a concentration of 0.1% (W / V).

[0049] The low-viscosity PLGA may have a molar ratio of lactide to glycolide of 40-80:20-60. In one embodiment, the molar ratio of lactide to glycolide is so-called 45-70:30-55, 55-70:30-45, or 45-55:45-55.

[0050] Commercially available low-viscosity PLGA products include RG502, RG502H, RG752H, and RG752S from Evonik, Inc. RG502H is also referred to herein as PLGA50A.

[0051] The high-viscosity PLGA may have a lactide to glycolide molar ratio of 40-80:20-60. In one embodiment, the lactide to glycolide molar ratio is 40-75:25-60, 45-70:30-55, 55-70:30-45, or 45-55:45-55. In one embodiment, the high-viscosity PLGA may be a mixture of one PLGA having a lactide to glycolide molar ratio of 45-55:45-55 and one or more PLGAs having a lactide to glycolide molar ratio of 60-80:20-40. In one embodiment, the high-viscosity PLGA may be a mixture of high-viscosity PLGA-1 and high-viscosity PLGA-2.

[0052] Commercially available high-viscosity PLGA products include RG503, RG503H, RG653H, RG753H, RG753S, and RG504H manufactured by Evonik. In this specification, RG503H will also be referred to as PLGA50B, RG504H as PLGA50C, RG653H as PLGA65B, and RG753H as PLGA75B.

[0053] The poly(lactide-co-glycolide) may comprise 88 to 97% by weight of the total weight of the sustained-release microspheres.

[0054] The sustained-release formulation according to the present invention may further contain coating materials, additives, excipients, etc. in addition to the active ingredient and poly(lactide-co-glycolide).

[0055] In one example, the sustained release formulation may comprise 3-12% by weight of semaglutide, tirzepatide, or a pharmaceutically acceptable salt of either of these, 88-97% by weight of poly(lactide-co-glycolide), and coating materials, additives, or excipients, etc.

[0056] The sustained-release microspheres may be coated with lysine, and the amount of lysine coated on the sustained-release microspheres may be 0.01 to 5, 0.01 to 3, 0.01 to 1, 0.1 to 1, 0.2 to 0.8, or 0.4 to 0.6 parts by weight per 100 parts by weight of the sustained-release microspheres before coating.

[0057] The sustained-release formulation of the present invention containing sustained-release microspheres can maintain its efficacy for at least one month after a single administration, depending on the subject. The sustained-release formulation of the present invention can be administered to a subject every 1 month, 1.5 months, 2 months, 2.5 months, or 3 months.

[0058] When the sustained-release microspheres or sustained-release formulations containing the same of the present invention are administered to Sprague Dawley rats (SD rats), the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof can be released at 5% or less within 24 hours, 10% or more within one week, 20% or more within nine days, or 50% or more within two weeks.

[0059] The sustained-release microspheres of the present invention may have a mean diameter of 5 to 50 μm, 5 to 40 μm, 5 to 30 μm, 10 to 50 μm, 10 to 40 μm, 10 to 30 μm, 10 to 25 μm, 15 to 50 μm, 15 to 40 μm, 15 to 30 μm, or 15 to 25 μm.

[0060] The sustained-release microspheres have a span value of 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.

[0061] The sustained-release microspheres or sustained-release preparations containing the same may further contain additives and excipients that are commonly used in the formulation of drugs in the technical field to which the present invention pertains.

[0062] The sustained-release formulation of the present invention may further contain one or more protective colloids selected from the group consisting of polyvinyl alcohol, albumin, polyvinylpyrrolidone, gelatin, etc. The protective colloid substance prevents aggregation of microspheres containing the GLP-1 receptor agonist and improves dispersibility. The protective colloid is preferably present in an amount of 0.02 to 1.0 wt % based on the total weight of the sustained-release formulation.

[0063] The present invention provides a pharmaceutical composition for treating or preventing diabetes, obesity, non-alcoholic steatohepatitis (NASH) or degenerative brain diseases, which comprises the sustained-release microspheres or a sustained-release formulation containing the same.

[0064] The degenerative brain disease may be any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lugelig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

[0065] The sustained-release microspheres can be prepared by dissolving a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and poly(lactide-co-glycolide) in glacial acetic acid, spraying the solution using an ultrasonic spray nozzle, and then volatilizing the solvent using dry air.

[0066] The present invention provides a method for producing sustained-release microspheres that are free from initial burst, enable sustained release of drugs over a long period of time, and are free from the problem of lag release, resulting in excellent bioavailability.

[0067] The method for producing sustained-release microspheres in the present invention includes the steps of dissolving a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and poly(lactide-co-glycolide) in an organic solvent to prepare a mixed solution; and drying the mixed solution to obtain sustained-release microspheres.

[0068] In the method for producing the sustained-release formulation, the type of poly(lactide-co-glycolide) 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.

[0069] In the method for producing sustained-release microspheres, the weight ratio of the GLP-1 receptor agonist to poly(lactide-co-glycolide) can be appropriately adjusted so that the content of the GLP-1 receptor agonist in the produced sustained-release microspheres is 3 to 12 wt %.

[0070] The organic solvent may be one or a mixture of solvents selected from the group consisting of chloroform, ethyl acetate, methylene chloride, methyl ethyl ketone, alcohols having 1 to 5 carbon atoms, glacial acetic acid, formic acid, dimethyl sulfoxide, and n-methylpyrrolidone, and is preferably glacial acetic acid.

[0071] In the method for producing the sustained-release preparation of the present invention, spray drying can be used as a method for drying the mixed solution to obtain microspheres.

[0072] The spray drying method may be carried out by supplying a mixed solution of semaglutide or a pharmaceutically acceptable salt thereof and poly(lactide-co-glycolide) to a spray dryer at a flow rate of 5 to 20 mL / min, 5 to 15 mL / min, or 5 to 10 mL / min, and supplying dry air at 100 to 200° C., 100 to 150° C., or 120 to 150° C. Furthermore, the frequency of the spray dryer may be 40 to 80 kHz, 50 to 70 kHz, or 55 to 75 kHz, and preferably 60 kHz.

[0073] The method for preparing a sustained-release formulation of the present invention may further include, after the step of drying the mixed solution to obtain microspheres, a step of suspending the dried microspheres in an aqueous solution containing lysine hydrochloride to coat them with lysine.

[0074] The aqueous solution in which lysine hydrochloride is dissolved may further contain 1% (W / V) polyvinyl alcohol.

[0075] The present invention provides a method for treating diabetes, obesity, non-alcoholic steatohepatitis, or degenerative brain disease, which comprises administering the sustained-release microspheres or a sustained-release formulation containing the sustained-release microspheres to a subject.

[0076] The degenerative brain disease may be any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lugelig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

[0077] The sustained-release microspheres or sustained-release formulations containing the same of the present invention can be administered orally or parenterally, preferably via parenteral routes such as intravenous, subcutaneous, intramuscular, or intraperitoneal administration.

[0078] The effective dose of the sustained-release microspheres or sustained-release formulations according to the present invention can be adjusted appropriately depending on the patient's age, the type and severity of the disease, the patient's condition, etc. For example, it may be 1 to 30 mg / week of the active ingredient. The above dose can be administered all at once or in divided doses.

[0079] The present invention will be described in more detail below with reference to examples.

[0080] Example 1. Manufacturing of sustained-release microspheres GLP-1 receptor agonist semaglutide or tirzepatide and PLGA shown in Table 1 below were prepared.

[0081] [Table 1]

[0082] Semaglutide or tirzepatide and PLGA were completely dissolved in glacial acetic acid to obtain a mixed solution, which was then spray-dried to produce sustained-release microspheres.

[0083] The spray-drying process was carried out by feeding the prepared mixture solution into a spray dryer (EIN System) equipped with an ultrasonic nozzle (Sonictopia, 60 kHz) at a flow rate of 7.5 mL / min using a liquid peristaltic pump (Masterflex, Peristaltic Pump). The power of the oscillator (Sonictopia, 60 kHz) was adjusted between 50 and 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 after filtration and washing with distilled water. They were then suspended in a solution of 10% D-mannitol (ROQUETTE) and 0.5% L-lysine hydrochloride (Merck), and lyophilized to obtain the sustained-release formulation of the present invention.

[0084] The compositions of semaglutide and PLGA in the sustained-release microspheres are the same as those in Examples 1 to 9 and Comparative Examples 1 to 4 in Table 2 below, and the compositions of tirzepatide and PLGA are as in Example 10 and Comparative Example 5. In Example 7, high-viscosity PLGA (C) was used, and PLGA65B and PLGA75B were each used in the same amounts as the low-viscosity PLGA (A).

[0085] [Table 2]

[0086] The ratios of LA and GA in the high-viscosity PLGA in the microspheres of Examples 1 to 10 and Comparative Examples 1 to 5, and the ratios of LA and GA in the entire PLGA, are shown in Table 3 below.

[0087] [Table 3]

[0088] 2. Measurement of particle size of sustained-release microspheres The mean particle size (MD) was quantitatively measured for Examples 1 to 10 and Comparative Examples 1 to 5. 180 mg of microspheres and 10 mL of 1% PVA were placed in a 50 mL conical tube, dispersed in an ultrasonic generator for 1 minute, and then placed in a particle size analyzer (CILAS, French) to measure the particle size, which is shown in Table 4.

[0089] [Table 4]

[0090] 3. Pharmacokinetic profile of sustained-release formulation in SD rats A sustained-release formulation containing semaglutide or tirzepatide according to the present invention was administered to 8-week-old SD rats (n=5), and the drug release pattern in the body was evaluated.

[0091] The sustained-release formulations containing microspheres from the Examples and Comparative Examples were subcutaneously injected (SC) into 8-week-old male SD rats at a drug concentration of 2 mg / kg (dose, 2 mpk). Blood concentrations of the drug (semaglutide or tirzepatide) were monitored 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 (a total of 17 time points) after injection and stored at -80°C. Analysis was then performed using LC-MS / MS.

[0092] Table 5 shows the relative AUC values ​​and cumulative release rates (ratio of cumulative release over time to total release). The area under the curve (AUC) was calculated from the blood concentration graph over time, and then bioavailability was evaluated using the relative AUC value, which was expressed as a percentage compared to the AUC value calculated when only the drug was administered. The initial burst and lag phase phenomena were evaluated using the cumulative release rate according to the following criteria:

[0093] *Evaluation criteria 1) Initial burst evaluation: If the initial burst is 5% or less within 1 day (24 hours), it is evaluated as having no initial burst. 2) Lag phase evaluation: If the release rate is 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), it is evaluated as having no delayed release problems. 3) Bioavailability assessment: If the Relatvie AUC value is 50% or higher, the bioavailability is evaluated as high. The sustained-release formulations of Examples 1 to 10 according to the present invention exhibited relative AUC values ​​of 50% or more (Table 5 and Figures 1a, 1b, and 1c), and were evaluated as having high bioavailability according to the above evaluation criteria. Furthermore, the initial release showed a value of 5% or less, with no initial excessive release, and the cumulative release rate up to 7 days was 10% or more, the cumulative release rate up to 9 days was 20% or more, and the cumulative release rate up to 14 days was 50% or more, confirming that the sustained-release formulations had no delayed-release issues (Table 5 and Figure 2).

[0094] However, in the case of the sustained-release microsphere formulation in Comparative Example 1, the bioavailability was high and there was no problem of delayed release, but unlike the Examples, there was a problem of excessively high initial release, and in the cases of Comparative Examples 2 to 5, there was no initial excessive release, but there was a problem of low bioavailability and delayed release occurring for more than one week after administration (Table 5 and Figure 1d).

[0095] [Table 5]

[0096] Furthermore, since Examples 1 to 10 showed similar release patterns, Example 3 was selected as a representative and is shown in Figure 2. It can be seen that the Examples according to the present invention did not have a high initial release and exhibited sustained release without the problem of delayed release, whereas Comparative Example 1, in which only low-viscosity PLGA was used, exhibited an excessive initial release and a short release period compared to the Examples, and Comparative Examples 2 to 4, in which only high-viscosity PLGA was used, exhibited a delayed release phenomenon of more than one week.

[0097] Subsequent experiments were also carried out by selecting Example 3 as a representative.

[0098] 4. Pharmacokinetic profile of sustained-release formulation in minipigs Next, for pharmacokinetic evaluation in large animals, the sustained-release formulation of Example 3 according to the present invention was administered to minipigs to evaluate the drug release pattern in microspheres in the body.

[0099] The blood concentrations of semaglutide were monitored over time after subcutaneous injection (SC) of 0.08 mg / kg of semaglutide (dose, mg / kg) into male SPF minipigs weighing 20-30 kg. Blood samples (2.5 mL each) were collected from the jugular vein at 1 hour, 6 hours, 12 hours, 24 hours, 2 days, 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 (a total of 23 time points) after injection. The samples were then stored at -80°C and analyzed by LC-MS / MS.

[0100] The results can be seen in Figures 3 and 4, where Figure 3 shows the blood concentration of semaglutide and Figure 4 shows the cumulative release rate of semaglutide. The results of the present invention confirmed that no initial excessive release or delayed release phenomena were observed even in minipigs, an animal model that is more similar to the human body than rats, and that the semaglutide release duration was longer in minipigs, which are larger than SD rats. This can also be inferred from this that the release duration will be longer when administered to the human body.

[0101] 5. Evaluation of anti-obesity efficacy The sustained-release preparation of Example 3 according to the present invention was administered to a DIO (diet-induced obesity) mouse model to confirm its anti-obesity efficacy.

[0102] Experimental animals were 16-week-old male C57BL / 6J and C57BL / 6J-DIO mice (The Jackson Laboratory (JAX), Salon Bio, Korea). After delivery, the mice were kept on a regular chow diet (5 kcal of fat, PicoLab, #5053, USA) or a high-fat diet (60 kcal of fat, Research diet, D12492, USA) with ample water. The mice were maintained at 22 ± 2°C and 50 ± 10% humidity, with an automatically controlled 12-hour light / dark cycle (7 pm to 7 am). After one week of adaptation, all mice were dosed and observed twice daily (morning dosing and afternoon monitoring) for clinical signs (activity, stool abnormalities, bleeding, wounds, malformations, skin, hair, and eye abnormalities).

[0103] To evaluate body weight and food intake, drugs were administered for 8 weeks under the conditions shown in Table 6 below. Body weight (FX-2000i, A&D Company, 0.01-2200g) and food intake (CSG201F, OHAUS, 0.1g-200g) were measured daily from the start of administration. All results are expressed as mean ± standard error, and statistical significance within the population was calculated by independent t-test between test substance administration groups. The results are shown in Table 6.

[0104] [Table 6]

[0105] The results of body weight measurement are shown in Figure 5a, and the weight gain rate (△Body weight from baseline (vs. Con(-)), %) is shown in Figure 5b. This is the result of calculating the body weight change rate (%) due to drug administration in comparison with the negative control group.

[0106] 5, it can be seen that the normal control group and the negative control group gained weight, while the positive control group and Example 3 (Q3D, QW, Q2W) showed a statistically significant (p<0.05) weight loss compared to the negative control group. In addition, referring to FIG. 5, it can be seen that the positive control group and Example 3 (Q3D, QW, Q2W) showed a statistically significant (p<0.05) weight loss rate compared to the negative control group.

[0107] The food intake (g) is shown in Figure 6. The food intake measured during the drug administration period showed a rapid decrease immediately after drug administration in the positive control group and all groups administered with the evaluated drugs, which gradually recovered, but the decreased food intake was maintained compared to the negative control group, showing a statistically significant decrease (p<0.05) compared to the negative control group.

[0108] These results indicate that when the sustained-release formulation of the present invention is administered to the positive control group, which receives daily administration, significant weight loss effects can be achieved even with administration only once every two weeks (based on mice).

[0109] In summary, it was confirmed that the sustained-release formulation of the present invention is free from the problems of initial excessive release and delayed release of the drug, is capable of sustained release over a long period of time, and is a formulation with high bioavailability.

Claims

1. a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and poly(lactide-co-glycolide) (PLGA); The GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof is contained in an amount of 3 to 12% by weight based on the total weight of the composition; The sustained-release microspheres are a mixture of one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g.

2. 2. The sustained-release microspheres according to claim 1, wherein the weight ratio of the one type of low-viscosity PLGA to the two or more types of high-viscosity PLGA in the mixture is 1:0.2-5.

3. The sustained-release microspheres according to claim 1, wherein the GLP-1 receptor agonist is semaglutide or tirzepatide.

4. 2. The sustained-release microspheres according to claim 1, wherein the two or more high-viscosity PLGAs are one or more PLGAs having a lactide to glycolide molar ratio of 45-55:45-55 and one or more PLGAs having a lactide to glycolide molar ratio of 60-80:20-40.

5. 2. The sustained-release microspheres of claim 1, wherein the poly(lactide-co-glycolide) is 88 to 97% by weight of the total weight of the sustained-release microspheres.

6. 2. The sustained-release microspheres according to claim 1, which are prepared by dissolving the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and the poly(lactide-co-glycolide) in glacial acetic acid to prepare a mixed solution; spraying the mixed solution through an ultrasonic spray nozzle, and then volatilizing the glacial acetic acid with dry air.

7. 7. The sustained-release microspheres according to claim 6, wherein the frequency of the ultrasonic atomizing nozzle is 40 to 80 kHz.

8. The sustained-release microspheres according to claim 1, wherein, after administration to S.D. rats or minipigs, the GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof is released at less than 5% within 24 hours, at least 10% within one week, at least 20% within nine days, or at least 50% within two weeks.

9. The sustained-release microspheres according to claim 1, which are administered every one to three months.

10. 2. The sustained-release microspheres according to claim 1, wherein the sustained-release microspheres have an average particle size of 15 to 25 μm.

11. A sustained-release preparation comprising 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 diseases, comprising the sustained-release microspheres according to any one of claims 1 to 10.

13. 13. The pharmaceutical composition of claim 12, wherein the degenerative brain disease is any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lugelig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

14. The method includes the steps of dissolving 3 to 12 wt % of a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof and 88 to 97 wt % of poly(lactide-co-glycolide) in a solvent to prepare a mixed solution; and spraying the mixed solution through an ultrasonic spray nozzle and then volatilizing the solvent with dry air to prepare microspheres, The poly(lactide-co-glycolide) is a mixture of one type of low-viscosity PLGA having an intrinsic viscosity of 0.14 to 0.24 dL / g and two or more types of high-viscosity PLGA having an intrinsic viscosity of 0.32 to 0.60 dL / g.

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